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Total and regional deposition of inhaled electronic cigarette (E-cig) particles in the respiratory tract (RT) depends on both physical properties of the inhaled particles and biological factors of users, for example, breathing pattern or puff profile, airway anatomy, and regional ventilation. Accurate particle sizing of E-cig aerosols is essential for predicting particle deposition in the RT. Studies using a variety of sizing methods have shown mass median aerodynamic diameters ranging from 0.2 to 1.2 um and secondary count diameters in the ultrafine range (0.1 μm). Incorporating these particle sizes into a multiple-path particle dosimetry (MPPD) model shows 10% to 45% total lung deposition by mass and 30% to 80% for ultrafine particles depending on the breathing patterns. These predictions are consistent with experimental measures of deposition fraction of submicron and ultrafine particles. While box-mod-type E-cig devices allow for full "direct-lung" inhalations of aerosol, the more recent pod-based, and disposable E-cigs (e.g., JUUL, Puff Bar, Stig) deliver the aerosol as a "mouth-to-lung" puff, or bolus, that is inhaled early in the breath followed to various degrees by further inhalation of ambient air. Measurement of realistic ventilation patterns associated with these various devices may further improve deposition predictions. Finally, while in vivo measures of RT deposition present a challenge, a recent methodology to radiolabel E-cig particles may allow for such measurements by gamma scintigraphy. Supported by NIH/NHLBI R01HL139369. © 2022 American Physiological Society. Compr Physiol 12: 1-10, year.

Three of the most prevalent developmental disorders (DDs) are autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD) and fetal alcohol spectrum disorder (FASD). As part of a study screening for DDs in Greater Manchester, UK, a unique opportunity was taken to explore parents' experiences of receiving reports about their child's previously unrecognised DD. Six parents out of a possible 16 took part in interviews, which were analysed thematically. Findings drawn from parental responses revealed a number of barriers to accessing support for their child's additional needs, including perceived resistance from schools, particularly for quiet, well-behaved girls, and difficulty in accessing assessment or referrals. There needs to be greater awareness of additional needs in children without externalising behaviours, the presence of gender-specific differences in the presentation of DDs, and FASD as a commonly occurring DD. Ultimately, better support for children with DDs would reduce the burden of unmet needs for the children and their families, and for wider services.

The dearth of ecological data from protected areas at relevant scales challenges conservation practice in West Africa. We conducted the first camera survey for Burkina Faso and Niger to elucidate interactions between resource users and mammals in the largest protected area complex in West Africa (W‐Arly‐Pendjari, WAP). We differentiated direct (e.g., poaching) and indirect (e.g., domestic animals) human activities to determine their effects on species richness, composition, and behavior. Livestock was the dominant human pressure while gathering was the most prevalent direct human activity. Human pressure did not influence species richness or composition, but reduced mammal activity with greater consequences from indirect activities. We also found distinct differences among guilds in their behavioral responses to human pressures as wild ungulates exhibited the greatest sensitivities to livestock presence. Our findings, that aggregated socioecological data, transition the WAP complex from the singular mandate of nature conservation to a dynamic coupled human‐natural ecosystem.

Objective The objective of this study was to understand the biological pathways involved in twin–twin transfusion syndrome (TTTS) by performing global gene expression analysis of amniotic fluid (AF) cell-free RNA. Methods A prospective whole transcriptome microarray study analyzing cell-free RNA in AF from TTTS recipient twins and singleton controls was carried out. Significantly differentially regulated genes in TTTS cases (N = 8) versus matched controls (N = 8) were identified and pathways analyses performed. Significant gene expression differences between stage II TTTS recipients (N = 5) and stage III TTTS recipients with abnormal Doppler measurements (N = 5) were also analyzed. Results Analysis of paired data from TTTS cases and controls revealed differential expression of 801 genes, which were significantly enriched for neurological disease and cardiovascular system pathways. We also identified cardiovascular genes and pathways associated with the presence of critically abnormal Doppler measurements in stage III TTTS recipients. Conclusions This study provides the first transcriptome-wide data on the impact of TTTS on fetal development. Our results show that gene expression involving neurological and cardiovascular pathways are altered in recipient fetuses prior to surgical treatment. This has relevance for the origins of long-term complications seen in survivors and for the development of future fetal biomarkers. © 2013 John Wiley & Sons, Ltd.

Remodeling of adipose tissue is required to support the expansion of adipose mass. In obesity, an increased death of adipocytes contributes to the accelerated cellular turnover. We have shown that obesity in pregnancy is associated with metabolic and immune alterations in the adipose tissue. In this study, we characterized the mechanisms responsible for increased death of adipose cells of pregnant obese women and its functional consequences. We postulated that a higher turnover of dead cells in white adipose tissue of obese women would translate into release of cell-free DNA (cfDNA) into their systemic circulation. Increase in adipose mass of obese compared to lean women results from a lesser number of hypertrophic adipocytes and an accumulation of macrophages in the stromal vascular fraction (SVF). The adipocytes of obese displayed enhanced necrosis with a loss of perilipin staining at the plasma membrane. Apoptosis was prominent in SVF cells with an increased expression of caspase 9 and caspase 3 and a higher rate of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) positive CD68 macrophages in obese vs. lean. Whereas circulating fetal cfDNA concentrations were not changed, there was a twofold increase in circulating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cfDNA and adipose tissue GAPDH mRNA in obese women. The maternal systemic GAPDH cfDNA was positively correlated with BMI and gestational weight gain. These data suggest that the active remodeling of adipose tissue of obese pregnant women results in an increased release of cfDNA of maternal origin into the circulation.

Background Acute exacerbations in allergic asthmatics may lead to impaired ability to clear mucus from the airways, a key factor in asthma morbidity.

During the last century the global biogeochemical cycles of carbon (C) and nitrogen (N) have been drastically altered by human activities. A century of land-clearing and biomass burning, followed by fossil fuel combustion have increased the concentration of atmospheric CO 2 by approximately 20%, and since the mid-1900s, the use of agricultural fertilizers has been the primary driver of an approximate 90% increase in bioavailable N. Geochemical records obtained through stable isotope analysis of terrestrial and marine biota effectively illustrate rising anthropogenic C inputs. However, there are fewer records of anthropogenic N, despite the enormous magnitude of change and the known negative effects of N on ecosystem health. We used stable isotope values from independent octocorals (gorgonians) sampled across the Western Atlantic over the last 143 years to document human perturbations of the marine C and N pools. Here, we demonstrate that in sea plumes δ 13 C values and in both sea plumes and sea fans δ 15 N values declined significantly from 1862 to 2005. Sea plume δ 13 C values were negatively correlated with increasing atmospheric CO 2 concentrations and corroborate known rates of change resulting from global fossil fuel combustion, known as the Suess effect. We suggest that widespread input of agricultural fertilizers to near-shore coastal waters is the dominant driver for the decreasing δ 15 N trend, though multiple anthropogenic sources are likely affecting this trend. Given the interest in using δ 15 N as an indicator for N pollution in aquatic systems, we highlight the risk of underestimating contributions of pollutants as a result of source mixing as demonstrated by a simple isotope-mixing model. We conclude that signals of major human-induced perturbations of the C and N pools are detectable in specimens collected over wide geographic scales, and that archived materials are invaluable for establishing baselines against which we can assess environmental change.

Background This report evaluated the association between surviving pediatric cancer and receiving a diagnosis of a stress-related mental disorder (SRMD) (i.e., post-traumatic stress disorder (PTSD), acute stress disorder, or adjustment disorders). Procedure The dataset comprised a cohort of Medicaid eligible children, ages birth to 15 years during baseline years 1996–2001 and followed at least 1 year until age 19 years or the end of 2003. Childhood cancer survivors (N = 390) identified from the SC Central Cancer Registry were frequency matched within age groups at each baseline year to children with no history of malignancy (N = 1,329). Survival curves and cumulative incidence of SRMD were estimated using the Kaplan–Meier method. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for pediatric cancer survival and selected covariates. Results The 8-year incidence of SRMD was 18.6% (95% CI: 12.47, 24.8) among childhood cancer survivors and 7.3% (5.0, 9.6) among children with no history of malignancy, HR = 3.22 (2.17, 4.76). Significant covariates for this group included race, sex, and previous mental disorder, adjusted HR = 3.00 (2.02, 4.45). Significant predictors among the childhood cancer survivors included cancer type, age group, treatment, and previous mental disorder. Conclusions Given the potential benefit of interventions for those with prior psychopathology, that children are less likely to verbalize emotional problems, and the detrimental implications of undiagnosed mental disorders, the health evaluations of childhood cancer patients and the follow-up visits for the survivors should incorporate assessment for mental disorders, especially SRMD. Pediatr Blood Cancer 2008;50:98–103. © 2007 Wiley-Liss, Inc.

Animal models are increasingly being used for the assessment of fetal cell microchimerism in maternal tissue. We wished to determine the optimal transgenic mouse strain and analytic technique to facilitate the detection of rare transgenic microchimeric fetal cells amongst a large number of maternal wild-type cells. We evaluated two strains of mice transgenic for the enhanced green fluorescent protein (EGFP): a commercially available, commonly used strain (C57BL/6-Tg(ACTB-EGFP)10sb/J) (CAG) and a newly created strain (ROSA26-EGFP) using three different techniques: in vivo and ex vivo fluorescent imaging (for whole body and dissected organs, respectively), PCR amplification of gfp, and flow cytometry (FCM). By fluorescent imaging, organs from CAG mice were 10-fold brighter than organs from ROSA26-EGFP mice (P < 0.0001). By PCR, more transgene from CAG mice was detected compared to ROSA26-EGFP mice (P = 0.04). By FCM, ROSA26-EGFP cell fluorescence was more uniform than CAG cells. A greater proportion of cells from ROSA26-EGFP organs were positive for EGFP than cells from CAG organs, but CAG mice had a greater proportion of cells with the brightest fluorescent intensity. Each transgenic strain possesses characteristics that make it useful under specific experimental circumstances. The CAG mouse model is preferable when experiments require brighter cells, whereas ROSA26-EGFP is more appropriate when uniform or ubiquitous expression is more important than brightness. Investigators must carefully select the transgenic strain most suited to the experimental design to obtain the most consistent and reproducible data. In vivo imaging allows for phenotypic evaluation of whole animals and intact organs; however, we did not evaluate its utility for the detection of rare, fetal microchimeric cells in the maternal organs. Finally, while PCR amplification of a paternally inherited transgene does allow for the quantitative determination of rare microchimeric cells, FCM allows for both quantitative and qualitative evaluations of fetal cells at very high sensitivity in a plethora of maternal organs.

Background Previously, we showed that analysis of amniotic fluid (AF) supernatant cell-free fetal (cff) DNA using DNA microarrays (array-CGH) allows for detection of whole chromosome differences between test and reference DNA. Subsequent technical advances have increased both the yield and quality of extracted cffDNA. Here we determined whether array-CGH using smaller volumes of both fresh and frozen AF cffDNA could identify fetal aneuploidy. Methods CffDNA was extracted from 10 mL of residual AF supernatant. The test AF samples (n = 10) included one with a normal karyotype, and nine with the following fetal aneuploidies: trisomies 13 (n = 1), 18 (n = 3), 21 (n = 2), trisomy 9 mosaicism (47,XX,+ 9[18]/46,XX[2]), triploidy (69,XXY) and Turner syndrome (45,X). Results Array-CGH using AF cffDNA from aneuploid fetuses, compared to euploid reference AF cffDNA, detected whole chromosome aneuploidy in 8 of 9 cases tested, including the case of trisomy 9 mosaicism. The case of triploidy was not detected. Conclusions CffDNA extracted from 10 mL AF supernatant can be analyzed using array-CGH to correctly identify human chromosome abnormalities. This technology allows for rapid screening of AF samples for whole chromosomal changes by using routinely discarded supernatant, and may augment standard prenatal karyotyping techniques by providing additional molecular information. Copyright © 2007 John Wiley & Sons, Ltd.

Analysis of cell-free fetal DNA (fDNA) and RNA in maternal plasma could be useful in the diagnosis and management of complications of pregnancy. In this review, we discuss our studies to investigate the potential of fetal nucleic acid measurement in maternal plasma as a marker of fetomaternal hemorrhage (FMH) after elective first-trimester termination of pregnancy (TOP). Using quantitative real-time PCR amplification of the DYS1 sequence, elevation of plasma fDNA levels after TOP was observed, especially in the late first trimester. This corresponds with the functional development of the placental vascular structure and fetal hematopoiesis. This Y sequence-based PCR amplification assay, however, limits the analysis to pregnant women carrying male fetuses. Therefore, we also developed a real-time quantitative reverse-transcriptase PCR assay of the γ-globin transcript as a marker of fetal erythroid cells. Although plasma γ-globin mRNA levels were decreased after TOP in many patients, an elevation was observed in some patients at greater than 9 weeks' gestation, which is consistent with the increase in plasma fDNA levels. Our data suggest that fetal hematopoietic cells contribute to the pool of fetal nucleic acids in the maternal circulation. Measurement of cell-free fetal nucleic acid levels in maternal plasma may have clinical application as a novel marker of FMH after 9 weeks of gestation.

Maternal obesity affects 1 in 5 pregnant women in the United States1. Maternal obesity is associated with increased circulating total, but not fetal, cf DNA2. This may be a result of increased production or decreased clearance of cf DNA in obese women. It is more likely that this is due to increased production of total cfDNA, because decreased clearance would also likely lead to an increase in cell-free fetal cfDNA. In a prior study performed on obese pregnant women, we showed that active remodeling of adipose tissue via adipocyte necrosis and/or apoptosis of the stromal vascular fraction results in an increased release of cfDNA of maternal origin into the circulation3. The focus of the prior study was on the mechanisms underlying the release of the cfDNA. In this study we more closely examined the correlation between maternal weight and total DNA levels. This study was approved by the Institutional Review Boards at Tufts Medical Center and Metrohealth Medical Center. The samples are from the same cohort reported previously3, but the analysis is different. Briefly, sixteen obese (mean=39.2; pre-gravid BMI range 31–51) and 14 lean (mean 21.8; pre-gravid BMI range 17–24) women carrying male fetuses and 10 women carrying female fetuses (negative controls) were recruited at term (37–40 weeks) prior to an elective cesarean section. Written informed consent was obtained prior to obtaining the samples. Women with a multiple gestations, placenta previa or invasive placentation, labor, infection, fetal anomalies or aneuploidy, intrauterine growth restriction, or preeclampsia were excluded. Maternal peripheral venous blood was collected at MetroHealth Medical Center on admission to labor and delivery, prior to placement of an intravenous line for hydration. All subjects had the same instructions prior to admission and had nothing to eat or drink for 6–8 hours prior to the blood draw. The blood was collected in an EDTA tube and plasma was separated by centrifugation and kept frozen at −20°C prior to being shipped to Tufts Medical Center for further analysis. DNA was extracted from 400 uL of plasma using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA) according to the blood and body fluid protocol. DNA was eluted in 50 μL of the elution buffer. Real time quantitative PCR amplification was performed as previously described4 to amplify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS14 as markers of total and fetal DNA, respectively. All samples were analyzed in triplicate. Analysis was blinded, and a female processed and handled all samples so that there was no risk of contaminating samples with male DNA. Conversion of raw PCR data to genome equivalents per mL of plasma was performed using the methods of Lo et al.5 The levels of total and fetal cf DNA in plasma from lean and obese women were compared using the t test. In addition, the maternal plasma volume from both obese and lean subjects was adjusted for blood volume6 and compared using the t test, as prior experiments in our laboratory suggested an increased blood volume as a function of weight7. Finally, we performed a regression analysis between total and fetal cf DNA and maternal BMI. Analysis of the raw data from all subjects showed that there was a 1.7% increase in total cell-free DNA per BMI unit (kg/m2). Following adjustment for blood volume, there was a 3.2% increase per BMI unit (Table 1). When the subjects were categorized into lean and obese groups, however, there was a decrease in total cf DNA per BMI unit in lean women and an increase in obese women (Table 1 and Figure 1). Neither of these changes were statistically significant. However, this lack of significance may be a function of the small sample size. Further studies with more subjects are warranted to further characterize this relationship between cf DNA and BMI. Figure 1 Graph showing relationship between total cell-free DNA levels and body mass index [BMI]. The correlation coefficient between BMI and cfDNA for the lean population is -0.061 (p=0.41) and for the obese population is 0.23 (p=0.20). Table 1 Change in maternal cell-free DNA by body mass index In this study, we found a correlation between BMI and total DNA levels. In lean women, the decrease in total cf DNA likely reflects a dilutional effect seen in all pregnant women due to increasing plasma volume. Conversely, in obese women, the increased levels of total cf DNA may reflect the increased adipocyte necrosis3 and stromal vascular apoptosis that is significant enough to overcome the dilutional effect that occurs in pregnant women. Our results are similar to a previous study by Lapaire et al.2, in which a correlation was found between maternal BMI and total, but not fetal, second trimester cf DNA levels. There are several differences between our study and the Lapaire study, however. Firstly, the gestational ages of the study subjects were different. In the present study, maternal blood samples were drawn at term, in contrast to 20 to 21 weeks. Secondly, in the Lapaire et al. study there was no adjustment for maternal weight. We adjusted for the effect of maternal weight on blood volume using the method described by Lemmens et al.6 The present study and that of Wataganara et al.7 suggest that a correction factor for maternal weight is needed when cf DNA analyses are performed. It is standard practice to correct for maternal weight when performing serum screening for Down syndrome. Diagnostic laboratories typically derive their own regression curves for the relationship between serum analytes and maternal weight because of several factors, including differences in instrument sensitivity in the measurement of serum analytes, as well as differences in maternal weight distribution among centers8. Review of the literature, however, indicates that there is no consensus as to the best method to adjust serum markers, including cf nucleic acids, for maternal weight in cases of extreme obesity. We selected the Lemmens et al.6 formula because it correlated total blood volume with BMI, although that formula was used for quantification of blood volume in non-pregnant surgical patients. In summary, we show here that maternal BMI affects total cf DNA levels in both lean and obese pregnant women. In the future, raw DNA values may need to be adjusted for maternal BMI for adequate interpretation of clinical tests that involve assessment of the fetal fraction9. In addition, the presence of increased total cf DNA levels in obese pregnant women suggests that this analyte may be a biomarker for systemic problems during gestation that may impact both maternal and fetal health.

Delayed planting shortens the effective growing season for corn (Zea mays L.), increasing the risk of exposure to lethal cold temperatures late in the season before grain maturation. Consequently, growers often must decide whether to switch to early maturity hybrids to minimize this risk. The objective of this study was to determine whether delayed planting influenced the growing degree day (GDD) ratings of silking and kernel black-layer (BL) development of corn. The effects of delayed planting on the phenological responses of three corn hybrid maturities common to the eastern U.S. Corn Belt were investigated at four locations in Indiana and Ohio over 4 yr. Thermal time from planting to silk emergence decreased an average of 34 GDDs for June vs. early May plantings while the grain-fill period decreased an additional 110 GDDs with late plantings. The total decrease in GDDs from planting to BL was 144 GDDs for corn planted in early June compared with early May, equal to a linear response to delayed planting of 3.8 fewer GDDs per day of delayed planting. The three hybrids responded differently to delayed planting, with greater GDD decreases occurring with late-maturity hybrid. Linear rates of GDD decrease with delayed planting ranged from 4.5 to 3.2 GDDs per day of delayed planting for late- and early maturity hybrids, respectively. Delayed planting decreases the GDD requirements of corn hybrids, resulting in less risk to grain maturation for adapted hybrid maturities from late-season killing freezes than previously thought.

Objectives The National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) is a prospective, multicenter clinical project to develop non-invasive methods of prenatal diagnosis. The initial objective was to assess the utility of fetal cells in the peripheral blood of pregnant women to diagnose or screen for fetal chromosome abnormalities. Methods Results of fluorescence in situ hybridization (FISH) analysis on interphase nuclei of fetal cells recovered from maternal blood were compared to metaphase karyotypes of fetal cells obtained by amniocentesis or chorionic villus sampling (CVS). After the first 5 years of the study we performed a planned analysis of the data. We report here the data from 2744 fully processed pre-procedural blood samples; 1292 samples were from women carrying singleton male fetuses. Results Target cell recovery and fetal cell detection were better using magnetic-based separation systems (MACS) than with flow-sorting (FACS). Blinded FISH assessment of samples from women carrying singleton male fetuses found at least one cell with an X and Y signal in 41.4% of cases (95% CI: 37.4%, 45.5%). The false-positive rate of gender detection was 11.1% (95% CI: 6.1,16.1%). This was higher than expected due to the use of indirectly labeled FISH probes in one center. The detection rate of finding at least one aneuploid cell in cases of fetal aneuploidy was 74.4% (95% CI: 76.0%, 99.0%), with a false-positive rate estimated to be between 0.6% and 4.1%. Conclusions The sensitivity of aneuploidy detection using fetal cell analysis from maternal blood is comparable to single marker prenatal serum screening, but technological advances are needed before fetal cell analysis has clinical application as part of a multiple marker method for non-invasive prenatal screening. The limitations of the present study, i.e. multiple processing protocols, are being addressed in the ongoing study. Copyright © 2002 John Wiley & Sons, Ltd.

Objective Intact fetal cells isolated from maternal blood can be used for non-invasive gender determination and genetic diagnosis. Recent studies demonstrating a large amount of cell-free fetal DNA in maternal plasma suggest that the circulating fetal DNA may result from fetal cells undergoing apoptosis. In the present study we evaluated the potential role of Fas and Fas ligand (FasL) cell surface expression with respect to apoptosis induction in fetal cells isolated from maternal blood. Methods We flow sorted candidate fetal cells that were gamma chain-positive and Fas- or FasL-positive or -negative, and subsequently analysed them by fluorescence in situ hybridization (FISH) analysis using X and Y chromosome-specific probes. Results Among all gamma hemoglobin-positive cells, there was a significant difference in the percent of cells expressing Fas versus FasL (4.4 and 12.3, respectively). We found no significant correlation between the total number of fetal nucleated red blood cells (NRBCs) and gestational age or the presence of Fas- and FasL-positive cells. From approximately 7 ml of maternal peripheral blood, most of the confirmed fetal (XY) cells were found in the Fas- and FasL-negative sorted population; the average numbers were 12.8 and 15.7, respectively. Conclusion We conclude that fetal NRBCs express FasL more than Fas, although most fetal NRBCs in first trimester maternal blood samples do not express Fas or FasL. This suggests the absence of a functional Fas/FasL apoptotic system in fetal NRBCs, and that programmed cell death in these cells, which may lead to circulating fetal DNA in maternal plasma, probably occurs by another pathway. Copyright © 2002 John Wiley & Sons, Ltd.

Systemic lupus erythematosus (SLE) is a serious multisystem disease that has a striking propensity to affect women. The cause of SLE remains elusive. Fetomaternal cell trafficking, or the passage of fetal cells into the maternal circulation, is now a well-established phenomenon. In addition, fetal cells have been implicated in the development of preeclampsia and in the pathogenesis of scleroderma. We undertook this study to determine whether fetomaternal cell trafficking might also be involved in pathogenic processes in SLE. Fluorescence in situ hybridization analysis was performed using X and Y chromosome-specific probes on affected and unaffected tissue obtained at autopsy from a woman who had previously given birth to 2 males and who had died of complications of SLE. The goal of the analysis was to detect the presence of male cells of putative fetal origin. Male cells were found in every histologically abnormal tissue type that was examined, but were not found in histologically normal tissue. These data suggest that fetal cells may be associated with SLE. It is unclear whether their presence may be related to disease causation, an effect of disease progression, or unrelated to disease pathology. However, this case study is an important step toward understanding the potential relationship between fetomaternal cell trafficking and SLE pathology.

Fetal clonogenic erythroid cells may be present in maternal blood and serve as a source of fetal DNA for prenatal genetic diagnosis. Proliferating nucleated red cells in cultures from first and second trimester fetal blood contain only fetal haemoglobin (HbF; F+A− cells), whereas nucleated red cells from adult blood contain also adult haemoglobin (HbA; F+A+ or F−A+ cells). Thus, fetal red cells can be identified and flow sorted. However, a few adult cells are also F+A−, which reduces the purity of fetal cell isolation. Culture media optimized for erythropoiesis contain interleukin-3 (IL3). We show here that IL3 strongly stimulates the growth of F+ cells (both F+A− and F+A+) in cultures from adult blood but has only a comparatively small effect on F+A− cells in cultures from fetal blood. This difference is maintained in the unified conditions of co-cultures of adult and fetal cells, so that the purity of fetal cell sorts can be increased by omitting IL3 from the culture medium. We further show that IL3 accelerates the exhaustion of the long-term division potential of adult cells, allowing fetal secondary colonies to be identified by their size following a two-stage culture scheme. Thus, the choice to omit or include IL3 in the growth medium of maternal blood cultures should depend on whether fetal nucleated red cells are to be isolated by flow sorting after one week, or by picking secondary colonies after a later secondary culture stage. Copyright © 2000 John Wiley & Sons, Ltd.

We investigate methods for the construction of confidence intervals for a proportion in a stratified two-stage sampling design with few events occurring in a small number of large, unequal size strata. The critical aspect is the incorporation of the weighting scheme into the construction of a single overall confidence interval. With small numbers of events, the binomial based methods may be inadequate since the normal approximation is not valid. Computer simulations compare coverage probability and bias for five methods of obtaining confidence intervals for proportions by combining: (1) binomial variances; (2) confidence intervals based on the F-distribution approximation to the cumulative binomial; (3) the binomial variance method with exact confidence limits when a zero prevalence occurs in any stratum; (4) confidence intervals based on the F-distribution using a rescaling factor; and (5) the binomial variance method with exact confidence limits using a rescaling factor. The method that performs best in terms of coverage probability is the combination of stratum specific confidence intervals based on the F-distribution using a rescaling factor. The methods involving the binomial variance tend to be negatively biased and the methods based on the F-distribution tend to be positively biased. Application of these methods with data from a study of adolescent depression that employs a stratified two-stage sampling design is consistent with these results.

Local recurrences in the surgical bed after tumor resection may be due to residual tumor cells “dropping” into the wound. Irrigation with water is often used to remove these cells. We designed experiments to determine whether irrigation would prevent tumor recurrence. Surgical wounds of uniform size in C57BL/6 mice were seeded with 5 × 102, 5 × 103, 5 × 104, 5 × 105, or 5 × 106 viable syngeneic B16-F10 melanoma cells to test the hypothesis that irrigation with water would decrease local tumor recurrence. The tumor-contaminated wounds were irrigated with distilled water or with saline (0.9% NaCl) immediately or 5, 30, 60, 120, or 240 min after seeding. Control wounds were seeded but not irrigated. The technique of irrigation was altered in a second group of experiments such that the amount of time the tumor cells were exposed to the water or saline was 5, 10, or 15 min. To determine the rapidity and durability of tumor cell attachment to host tissue, 1 × 104 viable B16-F10 tumor cells were seeded in vitro onto freshly cut disks of syngeneic mouse dermis. The tissue was irrigated with saline or distilled water 0, 2, 5, 10, 15, 30, 60, 120, or 240 min later. Tumor growth was observed in all the mice and neither the mechanical action of irrigation nor the hypotonic effect of distilled water changed the rate of growth. Scanning electron microscopy (SEM) demonstrated stable and firm attachment to mouse tissue within seconds of seeding with no noticeable dislodgement or cytotoxicity by either saline or water irrigation. The data suggest that the commonly used technique of irrigating the bed of the resected tumor may not be of value in preventing local recurrences. © 1993 Wiley-Liss, Inc.

Fetal cell-free mRNA in amniotic fluid (AF) is a valuable source of information regarding the developing fetus. Nucleic acids isolated from AF provide novel information compared to other sources of cell-free nucleic acids, such as maternal plasma (Zhong et al., 2006). Genomic and transcriptomic information from AF is of special interest because it derives solely from the fetus, and may therefore offer unique perspectives. The discovery-driven approach of using transcriptomic analysis has contributed novel information about the pathogenesis of several genetic disorders, such as Down syndrome and polyhydramnios in the setting of twin–twin transfusion syndrome (Larrabee et al., 2005; Slonim et al., 2009). The successful extraction of fetal RNA from cell-free AF supernatant and its subsequent hybridization to oligonucleotide microarrays has already demonstrated the wealth of new information this nucleic acid source has to offer (Hui and Bianchi, 2010). A possible impediment to the expanded utilization of AF is the cost and labor associated with extraction of DNA and RNA. A method using the combination of Trizol and the RNeasy Maxi Kit (QIAGEN, Hilden, Germany) has been used in our laboratory to successfully extract RNA from AF supernatant. However, this method does not allow the simultaneous processing of large numbers of samples and includes several possible sources of error, such as the division of samples into smaller aliquots and a precipitation step. Recently, a new kit was made commercially available to extract nucleic acids from human plasma or serum, the QIAamp Circulating Nucleic Acid Kit (QIAGEN). This kit utilizes a vacuum extraction method that allows throughput of large volumes on one mini column and eliminates the need for precipitation of the final sample. The purpose of this study was to compare RNA extraction from AF using this new method to our laboratory’s previously reported standard methods (Larrabee et al., 2005; Slonim et al., 2009). This study was approved by the Tufts Medical Center Institutional Review Board. Five residual samples of AF supernatant that were obtained for routine clinical indications at Tufts Medical Center were used [46,XY (n = 2) and 46,XX (n = 3)]. The gestational ages ranged from 15 4/7 weeks to 17 3/7 weeks. After the initial collection of AF, samples were processed and frozen at −80 °C within 12 h. The samples had been stored at −80 °C for up to 9 months before RNA extraction was performed. The samples of ≥ 10 mL were each split into two 5 mL aliquots for the two different extraction methods. For the standard approach, total RNA was extracted from one 5 mL aliquot of each AF sample using 15 mL of Trizol LS Reagent (Invitrogen) and 4 mL of chloroform per sample. Subsequent reactions were divided into smaller fractions, consisting of 750 μL of Trizol, 250 μL of AF supernatant sample, and 200 μL of chloroform per reaction in order to utilize the microcentrifuge necessary for the protocol. This method resulted in approximately 20 different reactions per 5 mL sample. Extracted RNA was then purified using the RNeasy Maxi Kit (QIA-GEN) as per the manufacturer’s protocol. RNA was precipitated from the original volume of approximately 650 μL and then rehydrated in 20 μL of RNase-free water. Briefly, 3 M NaOAc and 100% ethanol were added to the sample, the sample incubated at −20 °C for 4 h, the RNA was pelleted by centrifugation and the precipitated RNA was washed by adding 80% ethanol, followed by centrifugation and rehydration of the RNA. For the new protocol, total RNA was extracted from the second 5 mL aliquot from all samples using the QIAamp Circulating Nucleic Acid Kit (QIAGEN), according to the ‘Isolation of Circulating Nucleic Acids from Plasma or Serum’ protocol as recommended by the manufacturer. Briefly, the sample was lysed and then applied to a column via a vacuum pump. The RNA (bound to the spin column) was then washed three times and eluted with 20 μL of RNase-free water. On-column DNase digestion (RNase-Free DNase Set, QIAGEN) was performed during extraction in both methods. All extracted RNA samples were then stored at −80 °C for approximately one week prior to real-time quantitative reverse transcriptase (qRT) PCR amplification. Real time qRT-PCR was performed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with primer and probe sequences as previously reported (Maron et al., 2008), using an ABI 7900 Sequence Detection System with TaqMan One-Step RT-PCR Master Mix reagents kit (Applied Biosystems, Foster City, CA, USA). All qRT-PCR experiments were performed in duplicate with negative controls on each plate. The quality of total RNA in extracted RNA samples was assessed by an Agilent Bioanalyzer 2100 (Foster City, CA, USA). All statistical analyses were performed with SPSS 11.5 software (SPSS Inc., Somers, NY, USA). Independent t-tests were performed between the mean cycle of amplification of GAPDH between the samples extracted using the two different methods to determine if there were statistically significant expression differences. Statistical significance was set at a threshold level of p < 0.05. Our results demonstrated that RNA extracted using the new method amplified at a significantly earlier cycle threshold than RNA extracted using the standard method (p = 0.001, Figure 1). This demonstrates significantly improved quantity of extracted RNA using the newer method. In order to gather qualitative data, the bioanalyzer results for both extraction methods were analyzed. These results were inconclusive, with similar quality RNA obtained from both methods. We speculate this is due to the overall low concentration and degraded nature of the RNA obtained from both methods. These findings demonstrate that neither method has a significant effect on the quality of isolated RNA. This impeded qualitative comparisons but still allowed qRT-PCR amplification. The new extraction method also lowered the risk of pipetting error by eliminating the step of splitting the samples into 1.5 mL tubes and a precipitation step. The protocol changes also significantly decreased the time to process the samples, with the new method requiring only 2.5 h, whereas the standard method requires 8.5 h. The new method is also more cost effective, with the total cost of the standard method being $53.33/sample compared to $21.11/sample for the new method ($USD). Figure 1 Comparison of the mean Ct values of each extraction method. RNA extracted using the QIAamp Circulating Nucleic Acid Kit (CNA Kit) amplified significantly earlier than RNA extracted using Trizol and the Maxi Kit (p = 0.001). Bars represent standard deviation ... In summary, the new method of isolating cell-free RNA from AF samples using the QIAamp Circulating Nucleic Acid Kit represents overall improvement in many ways. It is both more cost effective and less labor intensive than the standard method of RNA extraction from AF. By increasing the quantity of extracted RNA this new method makes downstream applications more accessible and robust, thereby offering an improved workflow to further understand both normal fetal development and the pathogenesis of many genetic disorders.

The toxicity of oxytetracycline (OTC) was evaluated in adult grass shrimp, Palaemonetes pugio. Initially, static acute (96 h) toxicity tests were conducted with shrimp exposed from 0 to 1,000 mg/L OTC. A calculated lethal concentration 50% value of 683.30 mg/L OTC (95% confidence interval 610.85-764.40 mg/L) was determined from these tests, along with a lowest-observable-effect concentration of 750 mg/L and no-observable-effect concentration of 500 mg/L. Moreover, chronic sublethal effects of OTC exposure on grass shrimp intestinal bacterial population were assessed using doses from 0 to 32 mg/L OTC. The total viable counts in digestive tract content had levels between 5.2 and 1x10(4) colony-forming units per gram of tissue at times 0 and 96 h, respectively. Aeromonas hydrophila were the most resistant isolates (27.78%) to OTC exposure. Vibrio alginolyticus showed significant positive growth following exposure to OTC, whereas other bacterial species abundance declined over time. A total of 268 bacterial isolates were screened using antibiotic resistance analysis from a library containing 459 isolates. Among the tested isolates from the OTC treatments, 15.4% were resistant to OTC and 84.6% were OTC sensitive. Oxytetracycline was generally not consistently quantifiable with liquid chromatography-mass spectroscopy technique in shrimp homogenates. The only peak detected was at the 32 mg/L dose of OTC at 96 h. Nevertheless, OTC had a significant biological effect on the bacterial population. Antibiotic resistance to five other antibiotics (penicillin G, sulfathiazole, trimethoprim, trimethoprim and sulfamethoxazole, and tetracycline) was strongly associated with OTC exposures. The present study indicates that OTC toxicity effects in P. pugio and changes in the shrimp microbial community would only be expected under special circumstances.