Association between maternal blood lipids during pregnancy and offspring growth trajectories in a predominantly macrosomic cohort: findings from the ROLO longitudinal birth cohort study.

The purpose of this study is to examine associations between maternal lipid profiles in pregnancy and offspring growth trajectories in a largely macrosomic cohort. This is a secondary analysis of the ROLO birth cohort (n = 293), which took place in the National Maternity Hospital, Dublin, Ireland. Infants were mostly macrosomic, with 55% having a birthweight > 4 kg. Maternal mean age was 32.4 years (SD 3.9 years), mean BMI was 26.1 kg/m2 (SD 4.4 kg/m2) and 48% of children born were males. Total cholesterol, high density lipoprotein cholesterol (HDL-cholesterol), low density lipoprotein cholesterol (LDL-cholesterol) and triglycerides were measured from fasting blood samples of mothers at 14 and 28 week gestation. The change in maternal lipid levels from early to late pregnancy was also examined. Offspring abdominal circumference and weight were measured at 20- and 34-week gestation, birth, 6 months, 2 years and 5 years postnatal. Linear spline multilevel models examined associations between maternal blood lipid profiles and offspring growth. We found some weak, significant associations between maternal blood lipids and trajectories of offspring growth. Significant findings were close to the null, providing limited evidence. For instance, 1 mmol/L increase in maternal triglycerides was associated with faster infant weight growth from 20- to 34-week gestation (0.01 kg/week, 95% CI - 0.02, - 0.001) and slower abdominal circumference from 2 to 5 years (0.01 cm/week, 95% CI - 0.02, - 0.001). These findings do not provide evidence of a clinically meaningful effect.    Conclusion: These findings raise questions about the efficacy of interventions targeting maternal blood lipid profiles in pregnancies at risk of macrosomia. New studies on this topic are needed. What is Known: • Maternal fat accumulation during early pregnancy may potentially support fetal growth in the third trimester by providing a reserve of lipids that are broken down and transferred to the infant across the placental barrier. • There are limited studies exploring the impact of maternal lipid profiles on infant and child health using growth trajectories spanning prenatal to postnatal life. What is New: • Maternal blood lipid profiles were not associated with offspring growth trajectories of weight and abdominal circumference during pregnancy up to 5 years of age in a largely macrosomic cohort, as significant findings were close to the null, providing limited evidence for a clinically meaningful relationship. • Strengths of this work include the use of infant growth trajectories that span prenatal to postnatal life and inclusion of analyses of the change of maternal lipid levels from early to late pregnancy and their associations with offspring growth trajectories from 20-week gestation to 5 years of age.

Review: Sperm cryopreservation in wild small ruminants: morphometric, endocrine and molecular basis of cryoresistance.

Reproductive technologies can help to protect wild ruminant species from becoming extinct. In addition, the decline in some wild game species has also raised interest in reproductive technologies to increase the number of animals that can be produced. Most biobanking efforts have focused on developing effective protocols for preserving sperm, oocytes, and embryos. Cryopreservation of sperm remains the least invasive method and the cheapest procedure for germplasm storage. Over the last few years, several reproductive biotechnologies have been developed beyond the conventional freezing of spermatozoa. These include ultra-rapid freezing techniques. Nevertheless, fertility results after artificial insemination using frozen-thawed spermatozoa are not always acceptable in wild small ruminants. Moreover, these technological efforts have met variable success related to the sample's origin (epididymal retrieved postmortem or ejaculated) and the season of sperm sample collection and storage. Epididymal sperm shows higher cryoresistance than ejaculated sperm. Changes in sperm proteome between epididymal and ejaculated sperm seem to contribute to this different cryotolerance. The role of endocrine status has been studied in some wild species to better understand the underlying mechanism of the annual variation in ruminant sperm cryoresistance. Seasonal changes in testosterone and prolactin are involved in sperm cryoresistance; sperm recovery and cryopreservation are recommended around the end of the rutting season, when good quality sperm samples can still be obtained, testosterone levels have already decreased, and prolactin concentrations remain low. The mechanisms of hormone action on sperm freezability are not well known. Still, it has been suggested that testosterone affects cell proliferation in the testis, during spermatogenesis, and membrane properties of sperm cells during their transit through the reproductive tract, which might influence their cryotolerance. Recent studies have revealed that the expression of aquaporins in the sperm cells of small wild ruminants could also be involved in the androgen-related seasonal variation of sperm cryoresistance. Along with epididymal and ejaculated spermatozoa, the cryopreservation of testicular tissue may provide a suitable source of male gametes, becoming an alternative for establishing germplasm banks when semen cannot be collected for whatever reason.

Protective effects of Saccharomyces boulardii CNCM I-745 in an experimental model of NSAID-induced enteropathy.

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce a broad spectrum of gastro-intestinal adverse effects, including ulceration and bleeding. The pathophysiology of NSAID enteropathy is complex and incompletely understood, but some evidence showed that NSAIDs impair the intestinal barrier and cause a gut dysbiosis. Identifying new treatments aiming to reverse or attenuate NSAID-induced adverse effects would have a significant impact on a high number of patients. The aim of this work is to assess the effects of the probiotic yeast Saccharomyces boulardii CNCM I-745 (Sb) on a model of NSAID-induced enteropathy. Four groups of mice were tested: Control, Indomethacin, Sb, and Sb + Indomethacin. A clinical score was evaluated throughout the experiment. Faecal calprotectin, microbiota and haemoglobin analyses were performed. At the end of the treatments, the small intestine, colon, and caecum lengths, and intestinal permeability were measured. Sections of ileum and jejunum were observed to assess a histological score and ileal cytokines were measured by immunoassay. Indomethacin-treated animals showed an increase in their clinical scores, reflecting a worsening of their general state. Mice co-treated with Sb and indomethacin displayed an improvement of their clinical score in comparison with mice treated with indomethacin alone. Sb prevented the indomethacin-induced shortening of the small intestine and caecum, and significantly attenuated the severity of intestinal lesions. Sb also prevented the increase in faecal calprotectin, reduced faecal haemoglobin, and prevented the increase of intestinal permeability in mice treated with indomethacin. Sb also counteracted the increase of faecal bacteria associated with the pathogenesis of NSAID-enteropathy. In conclusion, our results show a protective effect of Sb in a model of indomethacin-induced enteropathy. Sb improved the intestinal barrier function and exerted a positive action on gut microbiota composition.

A Comparison of Intubation and Airway Complications Between COVID-19 and Non-COVID-19 Critically Ill Subjects.

Introduction The number of subjects infected with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throughout the western hemisphere increased exponentially in the later months of 2020. With this increase in infection, the number of subjects requiring advanced ventilatory support increased concomitantly. We decided to compare the survival rates between coronavirus disease 2019 (COVID-19) subjects versus non-COVID-19 subjects undergoing intubation in the intensive care unit (ICU). We hypothesized that COVID-19 subjects would have lower rates of survival post-intubation. Methods We screened all subjects admitted to the adult critical care unit between January 2020 and June 2020 to determine if they met the inclusion criteria. These subjects were required to be spontaneously ventilating upon admission and eventually required intubation. Subjects were selected from our electronic health record (EHR) system EPIC© (Epic Systems, Verona, WI) through a retrospective ICU admission analysis. We identified and included 267 non-COVID-19 subjects and 56 COVID-19 subjects. Our primary outcome of interest was intubation-related mortality. We defined intubation mortality as unexpected death (within 48 hours of intubation). Our secondary outcomes were the length of stay in the ICU, length of time requiring ventilator support, and proportion of subjects requiring tracheostomy placement. Results Compared to non-coronavirus disease (COVID) subjects, COVID subjects were more likely to be intubated for acute respiratory distress. COVID subjects had longer stays in the ICU and longer ventilator duration than non-COVID subjects. COVID-positive subjects had a decreased hazard ratio for mortality (HR = 0.42, 95% CI: 0.20-0.87, P < 0.05) and increased chances of survival compared to non-COVID subjects. Conclusions We showed the rates of intubation survival were no different between the COVID and non-COVID groups. We attribute this finding to intubation preparation, a multidisciplinary team approach, and having the most experienced provider lead the intubation process.

Health professionals' experiences of and attitudes towards mental healthcare for migrants and refugees in Europe: A qualitative systematic review.

Migrants living in Europe constitute over half of the world's international migrants and are at higher risk of poor mental health than non-migrants, yet also face more barriers in accessing and engaging with services. Furthermore, the quality of care received is shaped by the experiences and attitudes of health professionals. The aim of this review was to identify professionals' attitudes towards migrants receiving mental healthcare and their perceptions of barriers and facilitators to service provision. Four electronic databases were searched, and 23 studies met the inclusion criteria. Using thematic synthesis, we identified three themes: 1) the management of multifaceted and complex challenges associated with the migrant status; 2) professionals' emotional responses to working with migrants; and 3) delivering care in the context of cultural difference. Professionals employed multiple strategies to overcome challenges in providing care yet attitudes towards this patient group were polarized. Professionals described mental health issues as being inseparable from material and social disadvantage, highlighting a need for effective collaboration between health services and voluntary organizations, and partnerships with migrant communities. Specialist supervision, reflective practice, increased training for professionals, and the adoption of a person-centered approach are also needed to overcome the current challenges in meeting migrants' needs. The challenges experienced by health professionals in attempting to meet migrant needs reflect frustrations in being part of a system with insufficient resources and without universal access to care that effectively stigmatizes the migrant status.

Temporary Epicardial Pacing After Cardiac Surgery.

Temporary epicardial pacing frequently is employed after cardiac surgery, and can have a significant impact on a patient's hemodynamics, arrhythmias, and valvulopathies. Given that anesthesiologists often are involved intimately in the initial programming and subsequent management of epicardial pacing in the operating room and intensive care unit, it is important for practitioners to have a detailed understanding of the modes, modifiable intervals, and potential complications that can occur after cardiac surgery. Because this topic has not been reviewed recently in anesthesia literature, the authors attempted to review relevant epicardial pacemaker specifics, discuss modes and parameters that apply to the perioperative period, present an algorithm for mode selection, describe the potential effects of epicardial pacing on valvulopathies and hemodynamics, and, finally, discuss some postoperative considerations.

Disturbance to biocrusts decreased cyanobacteria, N-fixer abundance, and grass leaf N but increased fungal abundance.

Interactions between plants and soil microbes influence plant nutrient transformations, including nitrogen (N) fixation, nutrient mineralization, and resource exchanges through fungal networks. Physical disturbances to soils can disrupt soil microbes and associated processes that support plant and microbial productivity. In low resource drylands, biological soil crusts ("biocrusts") occupy surface soils and house key autotrophic and diazotrophic bacteria, non-vascular plants, or lichens. Interactions among biocrusts, plants, and fungal networks between them are hypothesized to drive carbon and nutrient dynamics; however, comparisons across ecosystems are needed to generalize how soil disturbances alter microbial communities and their contributions to N pools and transformations. To evaluate linkages among plants, fungi, and biocrusts, we disturbed all unvegetated surfaces with human foot trampling twice yearly from 2013-2019 in dry conditions in cyanobacteria-dominated biocrusts in the Chihuahuan Desert grassland and shrubland ecosystems. After 5 years, disturbance decreased the abundances of cyanobacteria (especially Microcoleus steenstrupii clade) and N-fixers (Scytonema sp., and Schizothrix sp.) by >77% and chlorophyll a by up to 55% but, conversely, increased soil fungal abundance by 50% compared with controls. Responses of root-associated fungi differed between the two dominant plant species and ecosystem types, with a maximum of 80% more aseptate hyphae in disturbed than in control plots. Although disturbance did not affect 15 N tracer transfer from biocrusts to the dominant grass, Bouteloua eriopoda, disturbance increased available soil N by 65% in the shrubland, and decreased leaf N of B. eriopoda by up to 16%, suggesting that, although rapid N transfer during peak production was not affected by disturbance, over the long-term plant nutrient content was disrupted. Altogether, the shrubland may be more resilient to detrimental changes due to disturbance than grassland, and these results demonstrated that disturbances to soil microbial communities have the potential to cause substantial changes in N pools by reducing and reordering biocrust taxa.

Intraoperative Renal Replacement Therapy: Practical Information for Anesthesiologists.

Previous publications regarding perioperative renal replacement therapy (RRT) have focused on the general care of the RRT-dependent patient and provided a broad overview of the various RRT modalities. The goal of this review article is to provide anesthesiologists with specific practical information regarding the possible intraoperative advantages and limitations of each modality, mandatory equipment to institute intraoperative therapy, and background knowledge necessary to communicate effectively with nephrologists and/or support staff regarding the intraoperative RRT goals.

Colonoscopy skills improvement training improves patient comfort during colonoscopy.

INTRODUCTION: We aimed to assess the effect of Colonoscopy Skills Improvement (CSI) training on patient comfort and sedation-related complications during colonoscopy. METHODS: This retrospective cohort study was performed on 19 endoscopists practicing in a Canadian tertiary care center who completed CSI training between October 2014 and May 2016. Data from 50 procedures immediately prior to, immediately after, and eight months following CSI training were included for each endoscopist. The primary outcome variable was intraprocedural comfort, and secondary outcomes included intraprocedural hypotension and hypoxia. Data were extracted from an electronic medical record and analyzed using SPSS version 20.0. Univariate analysis and stepwise multivariable logistic regression were performed to determine if there was an association between patient comfort and CSI training. Predictors of these outcomes including patient age, gender, sedation use and dosing, procedure completion, quality of bowel preparation, endoscopist experience, and specialty were included in the analysis. RESULTS: 2533 colonoscopies were included in the study. The mean dose of sedatives was reduced immediately following CSI training and at 8 months for both Fentanyl (75.4 mcg v. 67.8 mcg v. 65.9 mcg, p < 0.001) and Midazolam (2.57 mg v. 2.27 mg v. 2.19 mg, p < 0.001). The percentage of patients deemed to have a comfortable exam improved following endoscopist participation in CSI training and remained improved at 8 months (55.1% v. 70.2% v. 69.8%, p < 0.001). No significant change in rates of intraprocedural hypoxia or hypotension were noted following CSI training. CONCLUSION: CSI training is associated with improved patient comfort and reduced sedation requirements during colonoscopy.

Bone resorption and dietary calcium in pregnancy-a window to future maternal bone health.

BACKGROUND: Pregnancy is characterized by increased bone turnover and reversible loss of bone mineral density (BMD) to meet fetal calcium demands. The long-term effect of bone turnover and maternal diet in pregnancy on maternal bone is not well established. OBJECTIVE: We aimed to determine if an association exists between [1] bone resorption, [2] dietary calcium, and [3] serum 25-hydroxyvitamin D in pregnancy with maternal BMD 5-year postpartum. DESIGN: This is a prospective, longitudinal study of 107 women recruited to the ROLO low glycemic index dietary intervention trial in pregnancy and followed-up at 13, 28, and 34 weeks' gestation and 5 years' postpartum. At 13 and 28 weeks' gestation, a biomarker of bone resorption, urine cross-linked N-telopeptide of type I collagen (uNTX), was measured. At the 5-year follow-up BMD was measured using dual-energy X-ray absorptiometry. Anthropometry, dietary intakes, and serum 25-hydroxyvitamin D were measured in pregnancy and at 5 years. Multiple linear regression, controlling for confounders, was used for analysis. RESULTS: Mean BMD at 5 years was 1.208 g/cm2. In pregnancy, 24-34% reported dietary calcium intakes <800 mg/day. Vitamin D deficiency (< 30 nmol/L) was observed in 38-41% of women in pregnancy and in 29% of women at the 5-year follow-up. At 13 and 28 weeks' gestation, uNTX levels greater than the median were associated with 0.060 and 0.050 g/cm2 lower BMD 5 years later, respectively. Dietary calcium <800 mg/day in trimester 3 was associated with 0.072 g/cm2 lower BMD 5 years later. Vitamin D deficiency at 5 years, but not in pregnancy, was associated with lower BMD. CONCLUSION: Higher bone resorption and low dietary calcium in pregnancy were associated with lower BMD 5 years later. These findings could enable the identification of women at risk of declining of BMD in later life, but further research is needed. Adequate dietary calcium should be advised in the antenatal setting to promote lifelong maternal bone health.

Measurement of 227Ac impurity in 225Ac using decay energy spectroscopy.

225Ac is a valuable medical radionuclide for targeted α therapy, but 227Ac is an undesirable byproduct of an accelerator-based synthesis method under investigation. Sufficient detector sensitivity is critical for quantifying the trace impurity of 227Ac, with the 227Ac/225Ac activity ratio predicted to be approximately 0.15% by end-of-bombardment (EOB). Superconducting transition edge sensor (TES) microcalorimeters offer high resolution energy spectroscopy using the normal-to-superconducting phase transition to measure small changes in temperature. By embedding 225Ac production samples in a gold foil thermally coupled to a TES microcalorimeter we can measure the decay energies of the radionuclides embedded with high resolution and 100% detection efficiency. This technique, known as decay energy spectroscopy (DES), collapses several peaks from α decays into single Q-value peaks. In practice there are more complex factors in the interpretation of data using DES, which we will discuss herein. Using this technique we measured the EOB 227Ac impurity to be (0.142 ± 0.005)% for a single production sample. This demonstration has shown that DES is a useful tool for quantitative measurements of complicated spectra.

Assessment of enteric viruses during a hepatitis outbreak in Detroit MI using wastewater surveillance and metagenomic analysis.

AIMS: This study investigates enteric viruses in wastewater during an outbreak of acute hepatitis caused by hepatitis A virus (HAV) in a large metropolitan area. Emphasis is given to caliciviruses and HAV. METHODS AND RESULTS: Metagenomic analysis was performed to characterize enteric viruses excreted by the population of Detroit MI, during a hepatitis A outbreak that occurred in 2017 and 2018. Additionally, HAV, norovirus GII, and sapovirus were quantified, using qPCR, in 54 untreated wastewater samples collected over the course of 4 months. Correlation analysis was performed to identify associations between the number of disease cases and HAV concentrations in wastewater. HAV obtained the highest relative abundance among other enteric viruses detected in wastewater metagenomes. Metagenomic analysis also detected several other enteric viruses including astrovirus, enterovirus and hepatitis E virus. Average sapovirus concentrations of 1·36 × 106  gc l-1 were significantly greater than norovirus GII concentrations (2·94 × 104  gc l-1 ). Additionally, norovirus GI and GII along with sapovirus GI.1 were detected using metagenomics. HAV loads in wastewater were significantly correlated with the number of disease cases reported 1 week after wastewater sampling. CONCLUSIONS: Surveying untreated wastewater is a promising method for detecting early signs of hepatitis A outbreaks and for routine environmental monitoring of enteric viruses circulating in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Authors demonstrate the usefulness of metagenomics for genogrouping and enteric viral surveillance.

Cervical cerclage, pessary, or vaginal progesterone in high-risk pregnant women with short cervix: a randomized feasibility study.

Objective: To assess feasibility for a definitive randomized controlled trial (RCT) comparing three treatments for short cervix in a population at high risk for spontaneous preterm birth (sPTB) over a 1-year period.Design: Three arm, open label feasibility randomized clinical study.Methods: Women with singleton pregnancy with risk factors for sPTB (history of sPTB or prelabor premature rupture of membranes (PPROM) <34 weeks or significant cervical surgery), and short cervix on transvaginal ultrasound scan detected between 16+0 and 24+6 weeks gestation were randomized to receive either cervical cerclage, vaginal pessary, or vaginal progesterone 200 mg nocte. Pregnancy outcomes and treatment costs were collected from hospital records, NHS Reference costs, and British National Formulary costs.Main outcome measures: Feasibility targets were defined as (i) at least 55% of eligible women randomized; (ii) maximum 5% failure to adhere to the protocol per arm; (iii) maximum 5% loss to short-term follow-up.Results: Of 417 women screened between October 2015 and 2016, 25 (6%) were eligible for trial inclusion, of whom 18 (72%) agreed to participate at the rate 0.75 participants/site/month. Adherence to protocol was 100% in pessary and cerclage arms and 80% in vaginal progesterone arm (95% CI 24-100%). No participants were lost to follow up. Cost of interventions accounted for 6% (95% CI 2-10%) of overall health care expenditure.Conclusions: A definitive clinical trial comparing treatments for prevention of sPTB in high-risk women with short cervix is feasible but will be challenging due to small numbers of eligible participants.

Fetal Growth Trajectories and Their Association with Maternal, Cord Blood, and 5-year Child Adipokines.

BACKGROUND: The growth of the fetus is a complex process influenced by multiple factors. Studies have highlighted the important role of biochemical growth markers such as leptin and adiponectin on fetal growth. OBJECTIVE: To compare fetal growth trajectories with biochemical growth markers from maternal blood samples at 28 weeks' gestation, cord blood samples at birth, and in child blood samples at 5 years of age from mother-infant pairs who were part of the longitudinal ROLO study. METHODS: 781 mother-infant pairs from the ROLO and ROLO Kids study were included. Ultrasound measurements and birth weight were used to develop fetal growth trajectory groups for estimated abdominal circumference and estimated weight. Blood serum levels of leptin, adiponectin, insulin, TNF-alpha, and IL-6 from maternal, cord, and 5-year child samples were recorded. ANOVA and chi-square tests were applied to test the associations between fetal growth trajectory membership and maternal and child biochemical growth indicators. The influence of child sex was also investigated. RESULTS: Male sex was associated with a faster weight trajectory compared to females (p=0.001). At 28 weeks' gestation, maternal leptin levels were significantly higher in mothers with a fetus on a slower estimated abdominal circumference trajectory compared to fast (25616 [IQR: 11656.0 to 35341.0] vs. 14753.8 [IQR: 8565.4 to 24308.1], p < 0.001) and maternal adiponectin levels were lower in fetuses on a slower estimated abdominal circumference trajectory compared to a fast trajectory (22.4 [IQR: 13.6 to 35.9] vs. 27.6 [IQR: 17.6 to 46.3], p=0.027). No associations were noted with inflammatory markers. No associations were identified between fetal growth trajectories and growth markers at 5 years of age. CONCLUSIONS: This study shows that male sex is associated with an accelerated estimated weight trajectory. Furthermore, high leptin and low adiponectin in maternal serum in late gestation are associated with a slower fetal growth trajectory. No associations were identified with blood growth markers after pregnancy.

Suboptimal sleep and incident cardiovascular disease among African Americans in the Jackson Heart Study (JHS).

BACKGROUND: Suboptimal sleep, including insufficient/long sleep duration and poor sleep quality, is a risk factor for cardiovascular disease (CVD) common but there is little information among African Americans, a group with a disproportionate CVD burden. The current study examined the association between suboptimal sleep and incident CVD among African Americans. METHODS: This study included 4,522 African Americans without CVD at baseline (2000-2004) of the Jackson Heart Study (JHS). Self-reported sleep duration was defined as very short (<6 h/night), short (6 h/night), recommended (7-8 h/night), and long (≥9 h/night). Participants' self-reported sleep quality was defined as "high" and "low" quality. Suboptimal sleep was defined by low quality sleep and/or insufficient/long sleep duration. Incident CVD was a composite of incident coronary heart disease and stroke. Associations between suboptimal sleep and incident CVD were examined using Cox proportional hazards models over 15 follow-up years with adjustment for predictors of CVD risk and obstructive sleep apnea. RESULTS: Sample mean age was 54 years (SD = 13), 64% female and 66% reported suboptimal sleep. Suboptimal sleep was not associated with incident CVD after covariate adjustment [HR(95% CI) = 1.18(0.97-1.46)]. Long [HR(95%CI) = 1.32(1.02-1.70)] and very short [HR(95% CI) = 1.56(1.06-2.30)] sleep duration were associated with incident CVD relative to recommended sleep duration. Low quality sleep was not associated with incident CVD (p = 0.413). CONCLUSIONS: Long and very short self-reported sleep duration but not self-reported sleep quality were associated with increased hazard of incident CVD.

Optimal Timing of CT Scanning in the Rapid Access Lung Cancer Clinic.

Aims To investigate whether a 'CT first' approach to Rapid Access Lung Cancer Clinic (RALC) review could be feasible in an Irish context. Methods A retrospective review of our institution's Lung Cancer Database was performed. All RALC first attendances from 2012-2018 were identified. Timing of CT was assessed as well as CT imaging findings. Results Total first attendances in this period were 2372, of whom 91% had CT thorax as part of their evaluation. 866 patients (37%) were diagnosed with lung cancer, all had an abnormal CT. 1290 patients (54%) underwent CT but did not have lung cancer after clinical work up. 34% of patients diagnosed with Lung Cancer had their CT scan post RALC. Time to diagnosis was longer in those who had post RALC CT (34.5 versus 21 days) Conclusion CT scanning plays a vital role in the RALC pathway. Initial delays in obtaining CT can result in delayed time to diagnosis. These findings warrant close consideration when devising future national lung cancer policy.

Combined Selective Plane Illumination Microscopy and FRAP Maps Intranuclear Diffusion of NLS-GFP.

Since its initial development in 1976, fluorescence recovery after photobleaching (FRAP) has been one of the most popular tools for studying diffusion and protein dynamics in living cells. Its popularity is derived from the widespread availability of confocal microscopes and the relative ease of the experiment and analysis. FRAP, however, is limited in its ability to resolve spatial heterogeneity. Here, we combine selective plane illumination microscopy (SPIM) and FRAP to create SPIM-FRAP, wherein we use a sheet of light to bleach a two-dimensional (2D) plane and subsequently image the recovery of the same image plane. This provides simultaneous quantification of diffusion or protein recovery for every pixel in a given 2D slice, thus moving FRAP measurements beyond these previous limitations. We demonstrate this technique by mapping both intranuclear diffusion of NLS-GFP and recovery of 53BP1-mCherry, a marker for DNA damage, in live MDA-MB-231 cells. SPIM-FRAP proves to be an order of magnitude faster than fluorescence-correlation-spectroscopy-based techniques for such measurements. We observe large length-scale (>∼500 nm) heterogeneity in the recovery times of NLS-GFP, which is validated against simulated data sets. 2D maps of NLS-GFP recovery times showed no pixel-by-pixel correlation with histone density, although slower diffusion was observed in nucleoli. Additionally, recovery of 53BP1-mCherry was observed to be slowed at sites of DNA damage. We finally developed a diffusion simulation for our SPIM-FRAP experiments to compare across techniques. Our measured diffusion coefficients are on the order of previously reported results, thus validating the quantitative accuracy of SPIM-FRAP relative to well-established methods. With the recent rise of accessibility of SPIM systems, SPIM-FRAP is set to provide a straightforward means of quantifying the spatial distribution of protein recovery or diffusion in living cells.