108 research outputs found
Nitric oxide modulates sympathetic neurotransmission at the prejunctional level
No full textIn spite of accumulating evidence for a modulation of sympathetic neurotransmission by endogenously produced nitric oxide (NO), it remains unclear in which parts of the vascular system and at what level this interaction takes place. The aim of the present study was to investigate the distribution of endothelial and neuronal NO synthase (NOS) along the vascular tree of the heart at the light and electron microscopic level using NADPH-diaphorase (NADPH-d) staining as a marker for NOS. In addition, the functional effects of exogenous NO on coronary vascular resistance and cardiac adrenergic nerves was studied using the isolated perfused rat heart as a model. The intraaxonal catecholamine content of adrenergic nerve fibers was visualised and morphometrically assessed by applying glyoxylic acid-induced histofluorescence. The expression of endothelial NOS in the heart was found to depend on the diameter of the blood vessel. Arteries >100 ?m always showed intense staining, whereas staining in smaller arteries and veins was considerably weaker. Smooth-muscle free vessels were essentially devoid of NADPH-d activity. In atrial and ventricular myocardium, neuronal NOS localised in autonomic nerve fibers along the entire vascular tree. Ultrastructurally, NADPH-d staining revealed adjacent localisation of NOS-positive and -negative axons, suggesting an interaxonal modulation of adjacent autonomic nerve fibers by NO. In isolated perfused rat hearts, the intracoronary application of 10?8 M NO produced a marked decrease of coronary perfusion pressure, which was accompanied by a distinct increase in intraaxonal catecholamine levels of intramural adrenergic nerve fibers. These results suggest that the entire vascular system from arteries to veins is under the influence of NO and implies that two independently operating NO-driven processes are involved in the modulation of blood vessel tone: the well-known pathway of endothelium-derived NO acting directly on smooth muscle, and a second indirect pathway that inhibits noradrenaline release from perivascular nerve endings by endothelially or neuronally produced NO. The uneven distribution of endothelial NOS furthermore suggests that the latter mechanism predominates when the size of the blood vessel decrease
Addicks and Barker Dams: An optimization to minimize damage due to flooding
No full textThe Addicks and Barker Reservoirs, built in the forties, are located in Houston and collect precipitation and run-off from upstream areas to reduce flood risks along Buffalo Bayou to protect downtown Houston. During Hurricane Harvey (August 25 - August 30, 2017), the precipitation reached a new record of 910 mm [36.2 inches] in a 4 day period in Houston. The gates of Addicks and Barker Reservoirs were opened during the night of 27-28 August which led to major damages due to downstream flooding. Besides, non-government owned land upstream was flooded due to high water levels in the reservoirs.In this report, new design water levels for Addicks and Barker Reservoir are calculated based on inflowing discharge into the reservoirs and precipitation directly onto the reservoirs, including data of Hurricane Harvey. These calculated design water levels are compared with the critical water levels calculated based on the failure mechanisms of the dams. This study shows that the original design water level of the dams, based on the Probable Maximum Flood, are 2.83 m and 1.01 m higher than the critical water level for which failure of the dams can occur due to piping for Addicks and Barker Reservoir. However, the maximum allowed water level which is currently maintained by the United State Army Corps of Engineers, is 2.19 m and 2.46 m below the calculated critical water level. During Hurricane Harvey, these maximum allowed water levels were exceeded with 3.46 m and 1.93 m.The damage of residential properties upstream and downstream of the reservoirs are minimized based on the distribution of excess volume from the inflow of creeks and precipitation onto the reservoirs. The ratio of the amount of volume which should remain upstream of the dams and the volume discharged into the Buffalo Bayou is calculated for every considered event with its duration and return period. The ratio of Addicks Reservoir is the dominant ratio, which should be used for both reservoirs. Run-off alone already produces damage, especially for the 12h and 24h precipitation, so the Addicks and Barker Reservoirs should not release discharge into the Buffalo Bayou for small durations. For events with a longer duration, it would cause less damage to open the outlets of the reservoirs than to keep them closed. However, if the water level in the reservoir exceeds the critical water level for piping, it is advised to discharge more to the downstream area to prevent breaching of the dams. Since the critical water level is reached for approximately 25% of the events at Addicks Reservoir, mitigations against piping should be taken to improve the minimization of damage. For Barker Reservoir, the critical water level is not reached in the optimization. During big events, people living upstream will be more affected by the flooding than people living downstream since this optimization is based on the damage minimization of residential properties.MP225Master Project Repor
[Causes of subintimal hyperplasia in organ transplantation]. FT Ursachen der subintimalen Hyperplasie bei Organtransplantationen
No full textNephroprotective and antifibrotic potential of the vasopeptidase-inhibitor AVE7688 in a mouse model of progressive renal fibrosis
No full textRegulation of the L-type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition
No full textIn adult mammalian cardiomyocytes, stimulation of muscarinic receptors counterbalances the beta-adrenoceptor-mediated increase in myocardial contractility and heart rate by decreasing the L-type Ca2+ current (ICa) (1, 2). This effect is mediated via inhibition of adenylyl cyclase and subsequent reduction of cAMP-dependent phosphorylation of voltage-dependent L-type Ca2+ channels (3). Little is known, however, about the nature and origin of this pivotal inhibitory pathway. Using embryonic stem cells as an in vitro model of cardiomyogenesis, we found that muscarinic agonists depress ICa by 58 +/-3% (n=34) in early stage cardiomyocytes lacking functional beta-adrenoceptors. The cholinergic inhibition is mediated by the nitric oxide (NO)/cGMP system since it was abolished by application of NOS inhibitors (L-NMA, L-NAME), an inhibitor of the soluble guanylyl cyclase (ODQ), and a selective phosphodiesterase type II antagonist (EHNA). The NO/cGMP-mediated ICa depression was dependent on a reduction of cAMP/protein kinase A (PKA) levels since application of the catalytic subunit of PKA or of the PKA inhibitor PK) prevented the carbachol effect. In late development stage cells, as reported for ventricular cardiomyocytes (2, 4), muscarinic agonists had no effect on basal ICa but antagonized beta-adrenoceptor-stimulated ICa by 43 +/-4% (n=16). This switch in signaling pathways during development is associated with distinct changes in expression of the two NO-producing isoenzymes, eNOS and iNOS, respectively. These findings indicate a fundamental role for NO as a signaling molecule during early embryonic development and demonstrate a switch in the signaling cascades governing ICa regulation
Preemptive ramipril therapy delays renal failure and reduces renal fibrosis in COL4A3-knockout mice with Alport syndrome
No full textPreemptive ramipril therapy delays renal failure and reduces renal fibrosis in COL4A3-knockout mice with Alport syndrome.Background Alport syndrome (AS) is a common hereditary cause of end-stage renal failure in adolescence due to defects in type IV collagen genes. Molecular genetics allows early diagnosis, however, no preventive strategy can be offered. Using the COL4A3 -/- mouse, an animal model for human AS, we evaluated therapy with ramipril in mice.Methods One hundred and twenty-two Alport-mice were treated with 10 mg/kg/day ramipril added to drinking water. Proteinuria, serum-urea and lifespan were monitored. Renal matrix was characterized by immunohistochemistry, light- and electron microscopy, and Western blot.Results Untreated COL4A3 -/- mice died from renal failure after 71 ± 6 days. Early therapy starting at four weeks of age and continuing to death delayed onset and reduced the extent of proteinuria. Uremia was postponed by three weeks in treated animals. Lifespan increased by more than 100% to 150 ± 21 days (P < 0.01). In parallel, decreased deposition of extracellular matrix and lessened interstitial fibrosis as well as reduced amounts of renal transforming growth factor-beta1 (TGF-beta1) could be demonstrated. Late therapy starting at seven weeks decreased proteinuria, however, lifespan did not increase significantly.Conclusions The results indicate an antiproteinuric and antifibrotic nephroprotective effect of ramipril in COL4A3 -/- mice is mediated by down-regulation of TGF-beta1. This effect in mice is enhanced by initiation of therapy during pre-symptomatic disease. The data in COL4A3 -/- mice as an animal-model for Alport syndrome suggest that ramipril might as well delay renal failure in humans with AS. Early diagnosis and preemptive treatment also may be crucial in humans
DDR1-deficient mice show localized subepithelial GBM thickening with focal loss of slit diaphragms and proteinuria
No full textDDR1-deficient mice show localized subepithelial GBM thickening with focal loss of slit diaphragms and proteinuria.Background
Type IV collagen in basement membranes is a ligand for the receptor tyrosine kinase discoidin domain receptor 1 (DDR1). DDR1 is expressed in renal cells and regulates cell adhesion and proliferation ex vivo. The interaction between type IV collagen and cell surface receptors is believed important for normal renal function as well as significant in chronic renal diseases and we therefore analyzed mice with a targeted deletion of DDR1.Methods
Homozygous DDR1 knockout mice were compared to heterozygous and wild-type animals. The quantitative and qualitative amount of proteinuria was measured by urine-microelectrophoresis. Structural changes of the kidneys were determined by immunohistochemistry, light microscopy, and electron microscopy.Results
Compared to heterozygous littermates, adult DDR1 knockout mice showed a selective middle- to high-molecular proteinuria of up to 0.3 g/L and urinary acanthocytes. There was no evidence of uremia with no change in serum urea in the first 9 months of age. Little apparent change in renal morphology was detected using light microscopy. However, electron microscopy showed a localized, subepithelial, mushroom-like isodense thickening of the glomerular basement membrane (GBM). Within these areas, a focal loss of the podocytic slit diaphragms occurred.Conclusion
The loss of cell-matrix communication in DDR1-deficient podocytes appears to result in excess synthesis of basement membrane proteins leading to disturbed anchorage of foot processes and disruption of the slit diaphragm. Our data suggest that the interaction between type IV collagen and DDR1 plays an important role in maintaining the structural integrity of the GBM
Gene expression of PFAS exposed human liver spheroids and quality control
No full text<p>Per- and polyfluoroalkyl substances (PFAS) are a wide range of chemicals that are used in a variety of consumer and industrial products leading to direct human exposure. Many PFAS are chemically non-reactive and persistent in the environment, resulting in additional exposure from water, soil, and dietary intake. While some PFAS have documented negative health effects, data on simultaneous exposures to multiple PFAS (PFAS mixtures) are inadequate for making informed decisions for risk assessment. The current study leverages data from previous work in our group using Templated Oligo-Sequencing (TempO-Seq™) for high-throughput transcriptomic analysis of PFAS-exposed primary human liver cell spheroids; herein, we determine the transcriptomic potency of PFAS in mixtures. Gene expression data from single PFAS and mixture exposures of liver cell spheroids were subject to benchmark concentration (BMC) analysis. We used the 25<sup>th</sup> lowest gene BMC as the point of departure to compare the potencies of single PFAS to PFAS mixtures of varying complexity and composure. Specifically, the empirical potency of eight PFAS mixtures were compared to predicted mixture potencies calculated using the principal of concentration addition (i.e., dose addition) in which mixture component potencies are summed by proportion to predict mixture potency. In this study, for most mixtures, empirical mixture potencies were comparable to potencies calculated through concentration addition. This work supports that the effects of PFAS mixtures on gene expression largely follow the concentration addition predicted response and suggests that effects of these individual PFAS in mixtures are not strongly synergistic or antagonistic.</p><p>Funding provided by: Health Canada<br>Crossref Funder Registry ID: https://ror.org/05p8nb362<br>Award Number: </p><p><strong>Data was derived as follows, </strong></p>
<p>Text copied with permission from:</p>
<p>Addicks GC, Rowan-Carroll A, Reardon AJF, Leingartner K, Williams A, Meier MJ, Moffat I, Carrier R, Lorusso L, Wetmore BA, et al. 2023. Per- and polyfluoroalkyl substances (PFAS) in mixtures show additive effects on transcriptomic points of departure in human liver spheroids. <em>Toxicol Sci</em> 194: 38–52.</p>
<p><strong> </strong>DOI: 10.1093/toxsci/kfad044</p>
<p>https://academic.oup.com/toxsci/article/194/1/38/7169149</p>
<p><strong>Cell Culture </strong></p>
<p>3D InSightTM Human Liver Microtissues were purchased from InSphero (Brunswick, ME) in a 96 well format, with a single spheroid per well. These spheroids are a co-culture model from 10 different human liver donors including males and females and are a metabolically active system of hepatocytes and Kupffer cells. (Proctor et al. 2017; Rowan-Carroll et al. 2021). Upon arrival, culture media were replaced with InSphero Human Liver Maintenance Medium–Tox (InSphero (Brunswick, ME), and spheroids were acclimated at 37°C and 5% CO2 for 24 hours prior to PFAS exposures. PFAS were added to the media at the indicated concentrations and cells were exposed for either 24 hours or 10 days at 37°C and 5% CO2. For 10-day exposures, spent media were replaced every three days with new PFAS containing media. At the end of the exposures, media were collected and diluted 1:10 in storage buffer (200 mM Tris-HCl pH 7.3 containing 10% glycerol and 1% bovine serum albumin) then frozen at -80C to test the cytotoxicity. Spheroids were then washed once with Dulbecco's phosphate buffered saline (DPBS) (Thermo Fisher Scientific, Franklin, MA) and lysed with 5–7 µl of TempO-Seq™ lysis buffer (BioSpyder Technologies Inc, Carlsbad, CA). Samples were triturated, incubated for 10 min at Room Temperature and then stored at -80C.</p>
<p><strong>Chemical Preparation and Exposure Conditions </strong></p>
<p>Generation of raw transcriptomic data for single PFAS exposures that were used for comparison of mixture exposure data were the subject of our previous investigations (Reardon et al. 2021; Rowan-Carroll et al. 2021), with the details of PFAS purchase, preparation and concentration selection for individual PFAS exposures discussed therein. Details of individual PFAS exposures are briefly described herein for clarity. For individual PFAS exposures, the highest concentration of 100 µM was based on the EPA's ToxCast program's highest concentration. Additional exposures were selected to capture the response over three orders of magnitude and were based on the results obtained for PFOS in our first experiment (Rowan-Carroll et al. 2021). Single PFAS exposure levels were: 0.2, 2, 10, 20, 50, 100 µM unless otherwise stated (Table 1, bottom).</p>
<p>Specific mixture concentrations were selected with considerations of reducing operational complexity of exposure experiments while using exposures at a comparable range to single PFAS exposures for computational modeling of concentration-response. All mixtures contained equal molarities of each PFAS in the mixture. Mixtures, exposure concentrations (the total molarity of all combined PFAS in the mixtures) were of a similar range as the single PFAS exposures in our previous studies (Reardon et al. 2021). For each mixture, with the exception of Mixture 7, there were six exposure concentrations ranging from less than 1 µM up to 100 µM. Mixture 7 had five exposure concentrations with a top exposure of 40 µM. Concentration levels were 0.02, 0.2, 1, 2, 5 µM of each PFAS for high complexity mixtures (mixtures with more than three PFAS, (Mixture 2 – 5)) and 0.2, 1, 2, 10, 20 µM for each PFAS for low complexity mixtures (mixtures with two or three PFAS, (Mixture 1, 6 and 7)). All mixtures, with the exception of Mixture 7, also had a highest concentration level with the total combined molarities of component PFAS summing to 100 µM (Table 1). To allow comparisons of PFAS mixture potencies to single PFAS potencies, all mixture concentrations are reported as total molarity of all PFAS within the mixture. Thus, total combined molarity of PFAS for each mixture exposure level was dependent on the number of PFAS in each mixture; i.e., mixture 6, with three component PFAS (PFOA, PFOS, PFNA), had exposures of 0.6, 3, 6, 30, 60 and 100 µM total PFAS (0.2, 1, 2, 10, 20 and 33 µM of each PFAS). See also Table 1 for detailed summary of mixtures and the individual and total molarities of PFAS within the mixtures. See Figure 1 for a graphical depiction of the PFAS included in each mixture.</p>
<p>PFOS (95% purity CAS 1763-23-1), PFOA (95% purity CAS 335-67-1) and perfluorobutanesulfonic acid (PFBS) (95% purity CAS 375-73-5) were purchased from Sigma-Aldrich (Oakville, Ontario). The remainder of PFAS were obtained through a collaboration with the U.S. Environmental Protection Agency (EPA). These PFAS were procured under EPA contract (#EP-D-12-034) by Evotec Inc (Bradford, CT, USA) with a minimum target purity concentration of 95%. These were solubilized in dimethyl sulfoxide (DMSO) and monitored to ensure no precipitation was evident. If solubility was not an issue, solutions were prepared at 30 mM; otherwise, stocks were prepared at 10 or 20 mM. EPA PFAS stocks used passed an analytical quality evaluation and were deemed to be stable and free from contaminants (Smeltz et al. 2023) The full list of PFAS used in this study and the abbreviations used throughout the manuscript are summarized in Supplemental Table 1. PFAS were dissolved in DMSO (Sigma-Aldrich, Oakville, Ontario) to prepare working stock solutions up to 30 µM. Final DMSO concentrations in cell culture media were 0.1% for PFAS concentrations below 50 µM, 0.17% for 50 µM and 0.3% for 100 µM PFAS concentrations. PFAS exposures were matched to vehicle only (DMSO) time-matched and concentration-matched controls.</p>
<p>The overall design included twenty 96-well plates (10 plates per time- point, (24-hour and 10-day of exposures)) of liver cell spheroids. For each time point, four separate plates contained the complete range of PFAS exposure concentrations (i.e., each exposure had one replicate on each of four separate plates in the same experiment, resulting in a total of four replicates) alongside at least two matched DMSO controls (described below). Mixture exposures occurred in the same experiment along with all other PFAS except for PFOS, PFOA, PFBS, and PFDS from Rowan-Carroll et al. (2021), which were run separately and had duplicate exposures on each of two plates (i.e., each exposure had two replicates on two separate plates in the same experiment, with a total of four replicates). Within each plate there were 10 DMSO controls: two at 0.3% for 100 µM PFAS concentrations, two at 0.17% for 50 µM PFAS concentrations, and eight at 0.1% DMSO for all other PFAS concentrations at each timepoint (totaling eight, eight and 24, respectively, across all plates). DMSO controls for plates with PFOS, PFOA, PFBS, and PFDS exposures totaled 16 at 0.1%, 8 at 0.17% and 8 at 0.3% for each timepoint. All mention of replicates within the current manuscript and supplementary information refer to replicates of PFAS-exposed microtissues (as described above) with independent downstream processing (described below).</p>
<p><strong>Cytotoxicity Assessment and Cytotoxicity Exclusion Criteria</strong></p>
<p>Cytotoxicity was determined using a lactate dehydrogenase (LDH) assay (LDH-Glo, Promega J2380, Madison,WI), as per the manufacturer's instructions. Briefly, at the end of PFAS exposures cell culture media were collected and diluted 1:10 in storage buffer (200 mM Tris-HCl pH 7.3 containing 10% glycerol and 1% bovine serum albumin) and frozen at -80C. For analysis, samples were diluted 1:1 with LDH Detection Reagent and equilibrated for one hour at RT before reading relative luminescence units (RLU) (GloMax 96 Microplate Luminometer, Promega Corp, Madison,WI). Prior to analysis, RLU values from blank controls were subtracted from spheroid media RLU values. LDH ratios for each exposure were calculated by dividing experimental RLUs by the averaged RLU of respective DMSO controls. Means and standard deviations for each set of exposures (chemical, concentration, and timepoint) were then calculated from LDH ratios and expressed as fold change. Sample exclusion due to cytotoxicity was set at a 10-fold increase in LDH over controls (Rowan-Carroll et al. 2021). PFAS exposures at concentrations above the first cytotoxic concentration often resulted in decreased LDH signal, presumably due to cell death and spheroid degradation; therefore, all concentrations higher than the lowest concentrations yielding a 10-fold increase in LDH were also deemed cytotoxic. Samples that were determined to be cytotoxic based on the above metrics were excluded from all downstream analysis in order to ensure the fidelity of subsequent computational quality control of sequencing data (described below).</p>
<p><strong>TempO-Seq</strong>™<strong> Library Building and Next Generation Sequencing </strong></p>
<p>Gene expression was measured using the human TempO-Seq™ S1500+ panel (House et al., 2017; Mav et al., 2018) (BioSpyder Technologies Inc, Carlsbad, CA). This panel of approximately 3000 genes was selected by the National Institute of Environmental Health Sciences to cover a diverse set of biological pathways and provide a representative list of genes that captures transcriptomic variation and toxicological response with a high level of representation when compared to RNA-seq (Bushel et al. 2018) (National Toxicology Program 2015) ( https://www.federalregister.gov/documents/2015/04/15/2015-08529/list-of-environmentally-responsive-human-genes-selected-for-use-in-screening-large-numbers-of ). For TempO-Seq™ analysis, liver microtissues were lysed as described above using a volume of 2 x TempO-Seq™ lysis buffer equal to the residual volume of DPBS. Lysates and positive controls for sequencing reactions (1 x Human Universal Reference RNA— uhrRNA (Agilent Cat # 740000) and 1 x Human Brain Total RNA brRNA (ThermoFisher AM7962)), as well as 1 x negative controls for sequencing reactions (1 x TempO-Seq™ lysis buffer alone) were hybridized with detector oligo mix according to the manufacturer's protocol (Tempo-Seq™ Human Tox +Surrogate with a Standard Attenuation Transcriptome Kit (96 Samples)) (BioSpyder Technologies, Inc. Carlsbad, CA). Positive and negative controls were included on each plate. Hybridization was followed by nuclease digestion of excess oligos, detector oligo ligation, and amplification with tagged primers according to manufacturer's instructions. During amplification, each sample was ligated to sample-specific barcodes to allow identification of sample sequences after pooled sequencing reactions. Labeled and pooled amplicons were column purified using NucleoSpin Gel and PCR Clean-up kits, (Takara Bio USA, Inc, Mountain View, CA). Libraries were sequenced in-house, at Health Canada, using a NextSeq 500 High-Throughput Sequencing System (Illumina, San Diego, CA) using 50 cycles from a 75-cycle high throughput flow cell.</p>
<p><strong>Data Processing : Generation of Gene Expression Data</strong></p>
<p>Data processing was done with R v.3.6.1 (R. Core Team 2022).</p>
<p>To process TempO-Seq™ data, FASTQ files were generated from the BCL files using bcl2fastq v. 2.20.0.422 (Illumina, San Diego, CA). FASTQ files were processed using the TempO-SeqR script v 3.0 provided by BioSpyder (BioSpyder Technologies, Inc. Carlsbad, CA), as implemented within our transcriptomics data processing pipeline (https://github.com/R-ODAF/R-ODAF_Health_Canada). Briefly, reads from the FASTQ files were aligned to the TempO-Seq™ Human Surrogate+Tox Panel (S1500+) v2.0 probes reference sequences (Mav et al. 2018) using STAR 2.7.8a. The qCount function from QuasR (Gaidatzis et al., 2015) was used to extract feature counts specified in a GTF file (provided by BioSpyder) from the aligned reads. The result of this workflow is a table of counts per probe per sample. The gene expression data set is available through the NCBI Gene Expression Omnibus (series numbers GSE145239 and GSE144775).</p>
<p>Sequencing data for individual samples underwent several rounds of quality control for inclusion or exclusion from use in downstream toxicological modeling. The study-wide quality control workflow used in this study was adapted from the recommendations made by Harrill et al. (2021) and was used to assess the quality of the alignments and exclude samples if necessary. This data analysis pipeline was an improved implementation of that used in our prior articles (Reardon et al. 2021; Rowan-Carroll et al. 2021) with higher quality control (QC) stringency. Specifically, samples with read counts below 10% of our target depth of 1 M aligned reads (i.e., 100,000 reads) were removed; this quality control step flagged both PFAS exposed and control samples. Hierarchical clustering plots were generated (hclust function: default linkage function of hclust function in R; complete-linkage) for all the samples per time point using a distance metric defined as 1-Spearman correlation in order to identify potential outliers. Samples that clustered as singletons when cutting the dendrograms at the 0.1 dissimilarity were removed from the study. We also excluded samples based on the fraction of mapped reads (samples were excluded if the alignment rate was <40%). Additionally, for each sample, we calculated the number of probes with at least 5 uniquely mapped reads; the number of probes required to capture 80% of the signal in a given sample; and the Gini coefficient. For those three metrics, any samples identified as outliers based on Tukey's outer fence (3X interquartile range) were excluded (Harrill et al. 2021). Some samples were excluded based on multiple criteria. Further details on pipeline analysis, QA/QC, and exclusion criteria for all excluded samples are detailed in the Supplemental Rmd File 1 (<strong>provided here</strong>). Sample exclusion due to cytotoxicity and QA/QC is summarized in Figure 3. A minimum of two samples per chemical/mixture for each exposure concentration were required for benchmark concentration modeling (BMC), therefore any exposures with only one sample after QC exclusion were also excluded from BMC analysis.</p>
<p>For downstream analysis (e.g., BMD modeling), the count matrix (genes X samples) of all samples passing filters were log<sub>2</sub>-transformed and normalized by their library size scaling factor derived from the median-of-ratios method in DESeq2 to account for differences in the number of reads per sample (Love et al. 2014). To account for differences in the percentage of DMSO used for the dissolution of different chemical concentrations, the log<sub>2</sub>-transformed and DESeq2 normalized data for exposures to higher concentration PFAS that were dissolved in 0.17% and 0.30% DMSO were further normalized to their corresponding DMSO-matched controls and rescaled to the average of the DMSO 0.1% dose group. After normalizing to the controls, the DMSO 0.17% and the DMSO 0.30% controls were then removed.</p>
<p>Log<sub>2</sub>-transformed DESeq2 normalized gene counts in a format appropriate for analysis with BMDExpress v3 software package (Phillips et al. 2019), that is freely available at <a href="https://github.com/auerbachs/BMDExpress-3/releases">https://github.com/auerbachs/BMDExpress-3/releases</a> are <strong>provided here</strong>.</p>
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<p>Reardon AJF, Rowan-Carroll A, Ferguson SS, Leingartner K, Gagne R, Kuo B, Williams A, Lorusso L, Bourdon-Lacombe JA, Carrier R, et al. 2021. Potency Ranking of Per- and Polyfluoroalkyl Substances Using High-Throughput Transcriptomic Analysis of Human Liver Spheroids. <em>Toxicol Sci</em>. https://doi.org/10.1093/toxsci/kfab102 (Accessed October 19, 2021).</p>
<p>Rowan-Carroll A, Reardon A, Leingartner K, Gagné R, Williams A, Meier MJ, Kuo B, Bourdon-Lacombe J, Moffat I, Carrier R, et al. 2021. High-Throughput Transcriptomic Analysis of Human Primary Hepatocyte Spheroids Exposed to Per- and Polyfluoroalkyl Substances as a Platform for Relative Potency Characterization. <em>Toxicol Sci</em> <strong>181</strong>: 199–214.</p>
<p>Smeltz MG, Clifton MS, Henderson WM, McMillan L, Wetmore BA. 2023. Targeted Per- and Polyfluoroalkyl substances (PFAS) assessments for high throughput screening: Analytical and testing considerations to inform a PFAS stock quality evaluation framework.
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