Peak VO2 was determined by cardiopulmonary exercise testing (CPET), and lung function assessed using spirometry. Percentage predicted values were then calculated. HRR was defined as the decrease from peak HR to 1 min (HRR1) and 2 min of recovery (HRR2). Four-chamber echocardiography views were acquired at rest and exercise at 40% and 60% of CPET peak power. Change in left ventricular ejection fraction from rest to each work intensity was calculated (EFΔ40% and EFΔ60%) to estimate myocardial functional reserve. Peak VO2 and per cent of predicted peak VO2 were lower in preterm-born young adults compared with controls . HRR1 was similar between groups. HRR2 decreased less in preterm-born young adults compared with controls . In young adults born preterm, but not in controls, EFΔ40% and EFΔ60% correlated with per cent of predicted peak VO2 . Similarly, EFΔ60% correlated with HRR1 and HRR2 only in those born preterm .We recruited 101 normotensive young adults (2 and HRR in young adults born moderately preterm. Peak VO2 and HRR may aid risk stratification and treatment monitoring in this population.Impaired myocardial functional reserve underlies reductions in peak VO Prematurity is the leading cause of under-five child mortality,2) and heart rate recovery (HRR) are recognized as independent predictors of morbidity and all-cause mortality.,22 and HRR are seen in a range of conditions2 and HRR in individuals with a history of preterm birth.The rate of maximum oxygen uptake during exercise .We completed the Young Adult Cardiovascular Health sTudy (YACHT); an observational, case-control study with a primary outcome measure of cardiopulmonary exercise capacity to investigate cardiovascular structure and function, along with cardiopulmonary capacity and exercise stress response in preterm- and term-born young adults. Participants completed a detailed multimodal set of study measures including 24-h ambulatory blood pressure monitoring, cardiopulmonary exercise testing (CPET), and cardiac imaging. Ethical approval for YACHT was granted by the South Central Berkshire Research Ethics Committee (14/SC/0275). Study registration was completed via Table 1.A total of 149 participants aged 18–40 years were recruited through purposive active and passive recruitment as previously described.Participants were instructed to fast overnight for 12 h but to drink plenty of water prior to attending a study visit at the University of Oxford Centre for Clinical Magnetic Resonance (OCMR) and Oxford Cardiovascular Clinical Research Facility (CCRF) in the John Radcliffe Hospital in Oxford, UK. All measurements were completed by trained study investigators and clinical staff, including height, weight, venepuncture, and blood pressure as previously described see .151) and forced vital capacity (FVC), as well as per cent of predicted values according to Quanjer et al.Spirometry was performed according to standard guidelines by using the Cortex Metalyzer 3B .2 was calculated based on the Wassermann weight algorithm.Following spirometry, participants completed a peak CPET on a seated stationary cycle ergometer  using a validated incremental protocol with respiratory gases collected and measured using the same Metalyzer as used during spirometry. Heart rate was recorded using continuous electrocardiogram (ECG) monitoring and blood pressure was recorded every 4 min using a manual mercury sphygmomanometer . Participants maintained a rate of 60 revolutions per minute during the test, which began with one quiescent minute of resting measurements followed by a 2-min warm-up with a 20 watt workload. After the warm-up period, workload increased to 35 watts. To normalize test duration to ∼8–12 min, participants who reported higher activity or fitness levels had their workload increased to 75 watts after the warm-up period. Workload then incremented by 15 watts each minute and participants cycled continuously until exhaustion prevented them from maintaining at least 50 revolutions per minute or safety termination criteria were met.After completing CPET, participants had a 30-min recovery period during which they were offered water and a cereal bar. Sub-maximal exercise stress echocardiography was then performed on study subjects for 3 min at 40% and 60% of their peak exercise wattage achieved during CPET testing as previously described.Echocardiography analysis was completed offline in accordance with standard guidelines as previously described see , Methods1) and 2 min after exercise (HRR2).The ECG of each participant after the cessation of exercise was inspected by two researchers for valid readings at 1 and 2 min after exercise. Heart rate was smoothed within the Metalyzer Software  using the moving average  set to 15 s so that an average from the data points in this specified time interval (around the supporting point) was formed. The heart rates at 1 and 2 min after the cessation of exercise were then identified and extracted for each participant. HRR was defined as the absolute drop in heart rate from peak heart rate to 1 min . Peak VO2 measured in mL/kg/min was also lower in those born preterm . Heart rate at the ventilatory anaerobic threshold (VAT) was higher in the preterm group  but peak achieved heart rate was similar between groups. Ventilatory reserve at peak ventilation rate was similar between preterm-born and term-born individuals  (Table 2). As shown in Figure 2, HRR1was similar between groups  and remained similar after additional adjustment for per cent of predicted peak VO2 (P = 0.492). HRR2 was slower in preterm-born young adults , but no significant difference was found after additional adjustment for per cent of predicted peak VO2 (P = 0.813). Exercise characteristics are presented in Table 2.Peak cardiopulmonary exercise capacity was lower in preterm-born young adults, who on average, achieved 94% ± 20% of their predicted peak VO2 and HRR. As shown in Table S1, EFΔ40% as well as EFΔ60% were correlated with per cent of predicted peak VO2 only in preterm-born young adults  . EFΔ60% was correlated with HRR1 and HRR2 only in preterm-born young adults  .Bivariate correlations were carried out to investigate whether EFΔ, pulmonary, and physical activity measures were associated with per cent of predicted peak VO2 as well as HRR in preterm-born subjects were significantly influenced by pulmonary volume or dynamic pulmonary function, we completed multivariable linear regression analyses. Based on bivariable regression analyses, significant variables were carried forward to a multivariable model. To predict per cent of predicted peak VO2 in the preterm group, we included per cent predicted FVC, ventilatory reserve at peak VO2, and EFΔ40% and then EF60Δ% in two separate models. EFΔ40% and EFΔ60% remained the strongest predictors of per cent of predicted peak VO2  (Table 3). For the prediction of HRR1 and HRR2 in the preterm group, we included per cent of predicted FVC and EFΔ60% in the model. EFΔ60% remained the strongest predictor of HRR1  and HRR2 .To determine if the relationships between EFΔ and per cent of predicted peak VOThis study reports for the first time that normotensive, moderately preterm-born young adults exhibited reduced aerobic exercise capacity and slower HRR. These reductions were strongly related to their level of impairment in LV systolic function when going from rest to mild and moderate levels of exercise intensity. In addition, mechanical pulmonary function in this moderately preterm group was similar to controls, and exercise LV systolic response remained the strongest predictor of exercise capacity and HRR in preterm-born young adults after adjusting for pulmonary volume and dynamic function.,,,Several diseases including cardiac amyloidosis,We observed that impaired LV systolic response to exercise stress was associated with diminished exercise capacity in normotensive preterm-born young adults. However, because lung development continues into the third trimester, pulmonary function is often considered a risk area for deficits and vulnerability in preterm-born infants. In particular, the saccular and alveolar phases of development may be interrupted by premature delivery. In spite of this, spirometric values in our preterm-born group were similar to controls. Interestingly, this comports with a growing body of evidence that moderate to late preterm birth may be less impacting of pulmonary capacity later in life than once thought.2 slope in the preterm group indicates that exercise was limited by cardiac function. Such elevations most commonly occur secondary to deficits in cardiac performance, which drive a compensatory response of increased ventilation.2 slopes often exceed a value of 30. In the otherwise healthy, preterm-born young adults in this study, the presence of this compensatory increased ventilatory response relative to CO2 production and O2 uptake, combined with typical ventilatory reserve margin (23.3%) are consistent with cardiac limited exercise performance.In this study, the similar pulmonary volume and dynamic function between study groups and results from the multivariable analysis indicate that change in EF from rest to exercise, rather than mechanical ventilation, predicted exercise capacity. The elevated VE/VCOet al.In this article, we have shown that HRR is reduced at 2 min after exercise in normotensive, moderately preterm-born young adults and that this associated with impaired systolic function during exercise rather than pulmonary function. The metaboreflex is well known for its effect on blood pressure but the effect on post-exercise heart rate remains controversial.To our knowledge, apart from one study comparing HRR after sub-maximal exercise,2 and slower HRR adds further clinical relevance to the altered preterm cardiac phenotype. In addition, peak VO2 and HRR in this population may be important and beneficial for future risk stratification and treatment monitoring, acting as surrogate measures of reduced myocardial functional reserve. Continued investigation and longitudinal follow-up will be necessary to more fully understand the clinical implications of these findings.Overall, the moderately premature gestational age of the preterm-born group makes our results more broadly relevant, given that this reflects the demographic of the majority of preterm births.We designed YACHT as an observational study around a single, full-day study visit. As the exercise stress testing came towards the end of the visit in order not to confound other measures, not all individuals were willing to continue with the sub-maximal exercise testing and echocardiography stress testing component. Also, due to challenges with imaging quality, not all echocardiography scans were of sufficient quality for analysis. Finally, the overall sample size was modest with a lower percentage of males in the preterm cohort, and therefore, analyses were adjusted for sex where appropriate. Nevertheless, the dataset was sufficiently powered to make between-group comparisons for the YACHT primary outcome of cardiopulmonary aerobic fitness as well as LVEF at rest, 40%, and 60% of maximal exercise capacity using echocardiography.There was a wide gestational age range in our participants with the majority of our preterm-born individuals born moderate to late preterm, which reflects the demographic of the general preterm population.Preterm-born young adults have reduced exercise capacity and HRR, which relate to the preterm functional phenotype of reduced LV systolic response to physical exercise. Although below average, exercise capacity was within the normal range and did not reflect acute clinical deficits. Further research is needed to develop a more complete understanding of cardiopulmonary exercise capacity and function in the preterm-born population, as well as which mechanisms underlie reduced peak oxygen consumption and HRR in preterm individuals of varying degrees of prematurity.European Heart Journal - Cardiovascular Imaging online. jeaa060_Supplementary_DataClick here for additional data file.


Next generation sequencing has allowed the discovery of miRNA isoforms, termed isomiRs. Some isomiRs are derived from imprecise processing of pre-miRNA precursors, leading to length variants. Additional variability is introduced by non-templated addition of bases at the ends or editing of internal bases, resulting in base differences relative to the template DNA sequence. We hypothesized that some component of the isomiR variation reported so far could be due to systematic technical noise and not real.We have developed the XICRA pipeline to analyze small RNA sequencing data at the isomiR level. We exploited its ability to use single or merged reads to compare isomiR results derived from paired-end (PE) reads with those from single reads (SR) to address whether detectable sequence differences relative to canonical miRNAs found in isomiRs are true biological variations or the result of errors in sequencing. We have detected non-negligible systematic differences between SR and PE data which primarily affect putative internally edited isomiRs, and at a much smaller frequency terminal length changing isomiRs. This is relevant for the identification of true isomiRs in small RNA sequencing datasets.We conclude that potential artifacts derived from sequencing errors and/or data processing could result in an overestimation of abundance and diversity of miRNA isoforms. Efforts in annotating the isomiRnome should take this into account.The online version contains supplementary material available at 10.1186/s12859-021-04128-1. MicroRNAs (miRNAs), a class of small non-coding RNAs (ncRNAs), have an average length of 21–23 nucleotides (nt). They have been widely studied as endogenous regulatory molecules that modulate gene expression post-transcriptionally by inducing target mRNA silencing and decay . Target Most miRNA expression studies based on next generation sequencing (NGS) performed to this date have summarized all the reads mapping to a specific miRNA locus or miRNA sequence with or without mismatches and assign it to a single miRNA entity (a miRBase reference database entry). However this type of analysis neglects the fact that not all reads are identical to the mature reference sequence in miRBase . Small RImportantly, there is a lack of well established experimental validation methods complementary to NGS with isomiR level resolution. Commercial qPCR assays targeting miRNAs designed to recognize the canonical forms can have variable degrees of specificity for the different corresponding isomiR isoforms .The deviation in miRNA sequences from canonical references has been the focus of many studies correcting technical sources of variation, such as the effect on cross-mapping , or miniTo the best of our knowledge software tools developed to this date to detect isomiR diversity do not take PE into consideration. This is understandable given that most data is SR mainly due to sequencing cost constraints. We developed XICRA, a pipeline to analyze small RNA sequencing  data, which allows detecting and quantifying isomiR level variation in miRNA. As a prerequisite, the pipeline had to be capable of handling both SR and PE reads. It takes compressed fastq files as input, performs a quality check, trims and merges reads in the same pair, maps them using third party tools, such as miraligner , sRNAbenIn order to perform systematic analyses of miRNA expression at the isomiR level with error correction functionality based on paired end reads, we built a new pipeline in house named XICRA  type variants in SR analysis relative to PE analysis. Interestingly R1 data alone appeared to have similar or even enhanced sensitivity to detect isomiR variants but it was less precise, meaning that it led to more false positives than PE sequencing  or no mismatches  in the read pair merging step. Results showed that SR mode calls tend to have an overrepresentation of iso_snv related isomiRs relative to both PE mode calls. We then included two SR mode calls in the comparison  even though R2 reads on their own would hardly ever be used in real situations. Both SR mode calls yielded a higher proportion of non-canonical isomiR types. Allowing for mismatches led to a higher frequency of these classes possibly due to R2 reads having higher error rates than R1 reads Fig. : Fig. S4We went beyond a descriptive analysis by applying differential miRNA expression analysis methodology. Results showed many iso_snv-related variant classes to be differentially expressed in SR versus PE mode, both by DESeq and by GSEA Fig. , Table 1Searching the literature and genomic databases we were only able to identify a few public miRNA sequencing datasets with PE data. We tested if our observation could be made in data from a published study on non-alcoholic fatty liver disease  . Our results would support the notion that PE sequencing may lead to more exact profiling at the cost of not detecting a few variants that are detected by SR sequencing alone which may be in regions difficult to sequence, which often have even poorer sequence quality from the alternate strand.We tested two different conditions for PE read pair merging. The default of fastq-join is to allow up to 8% errors in the alignment between two paired reads (which for miRNAs would entail 1 or 2 nucleotide differences in ~ 21 bp). This could in principle lead to retention of sequencing errors in cases of ambiguity and low quality in both reads or with a call in R2 missing altogether (resulting in a conflicting position being incorrectly resolved in favor of the mismatch to the miRBase mature miRNA reference).Comparing SR1 versus SR2 performance showed that R2 accumulates many more errors and SR2 based leads to higher inferred isomiR diversity than SR1 mode analysis. Differences in quality along the length of the reads may explain why some particular isomiR type frequencies differed depending on the reads used. Illumina quality tends to dip the first few cycles due to the nature of the base caller calibration procedure used in Illumina sequencing by synthesis. Based on our results it is possible to hypothesize that R1 may be enriched in artifactual iso_snv isomiRs with SNVs in the seed region due to the slightly higher error rate in the first few bases. R2 on the contrary may tend to have errors in the 3′ end of the miRNA and isomiR sequences. Cancelling out these errors through PE_8 mode, with mismatch allowance, leads both to correcting iso_snv variants derived from R1 read errors, and to enhancing iso_3add and iso_3p variants (derived from R2 read errors). PE-0 mode, with zero mismatch tolerance in the joining step, would be the most conservative and closer to the truth and would be recommended at the cost of lower count yields.The mechanism triggering this may be that R1 reads mapping to iso_snv isomiRs may be intrinsically of poorer quality and may not have R2 read pairs of sufficient quality and are not eligible for fastq joining step. Alternatively and less likely R2 in the same read pair matching the canonical miRNA sequence should be of better quality than R1. This would support the hypothesis that inflated artifactual iso_snv counts are derived from sequencing errors in poor quality reads.GSEA analysis of isomiR type enrichment when comparing SR versus PE would suggest that the enrichment in iso_snv appears concomitant to depletion in canonical isoforms .Although in our particular results iso_snv isomiRs represented a fairly small proportion of differentially expressed isomiRs between male and female samples, in other studies it may still be worthwhile to test this in order to take into account those isomiRs that are artifactual, provided that samples have been sequenced in PE mode. Alternatives such as the use of unique molecular identifiers (UMIs) have been proposed to eliminate PCR duplicates and correct for sequencing errors but the cost involved by the inclusion of special adapters and additional required sequencing depth may justify using PE reads instead.Our work does not allow inferring conclusions on the impact of SR data prevalence in results in the literature on mature miRNA profiling and isomiRs reported as differentially expressed. It is possible to hypothesize that data derived from PE sequencing could help reduce noise and artifacts and prevent any false positive results that may have derived from SR data.Because the seed and central region of the isomiRs is the region that interacts with the mRNA, the finding that internal SNV isomiRs calls are most likely generated by sequencing errors could impact subsequent analyses and bias the derived conclusions regarding putative functional consequences. The results of our study are relevant because of the importance given to isomiRs with SNVs in the seed region for miRNA regulatory functions at the biological and mechanistic level. In addition, our findings could help to address the challenges that isomiR variation imposes on the development of validation and diagnostic assays for miRNA biomarker assessment by helping to define true isomiRs.Our study has limitations in that it only addresses noise due to sequencing errors revealed by mismatched read pairs, and is subject to biases in the software tools used. Besides and bioinformatic analysis data processing, additional sources of variation affecting isomiR profiling may come from key factors in library preparation such as enzymatic steps, adapter sequences and size selection procedures. Optimization of these different parameters is dependent on the use of controls and validation by alternative methodologies. Current state of the art makes isomiR data results inherently noisy and hard to verify. Widespread adoption of data formats and nomenclature such as miRTOP implemented in our pipeline XICRA will be required in order to be able to compare different studies. Reanalysis of older datasets may make it possible to recover and reinterpret previous work. Convergence to optimal analysis strategies and software standardization will be needed. Future work in the field should address all these issues. Concerted efforts should help to improve our understanding of miRNA biology and aid in identifying miRNA based biomarkers by exploiting and mining true isomiR diversity.We can conclude that PE sequencing improves isomiR calling in small RNA sequencing data.Internal variation isomiR calls are frequent artifacts in SR sequencing data. Gene set enrichment analysis by GSEA of isomiR classes pre-ranked by SR vs. PE log2 fold change shows significant iso_snv enrichment in SR data compared to PE data. Systematic differences between SR and PE mode sequencing affecting putative internally edited isomiR (iso_snv) levels exist. Many detectable internal sequence variant isomiRs in SR mode may be false positives.A significant fraction of differentially expressed miRNAs in serum in SR data are iso_snv isomiRs and can be filtered out using PE mode. Many of these are found at very low expression levels and often are removed by thresholding or universal filtering steps in DESeq2 DE analysis. In contrast, most differentially regulated canonical miRNA and length variant isomiR types  can be reliably detected in SR data. Many more isomiRs than canonical miRNAs are found regulated.Internal RNA editing miRNA results should be taken with caution. Most putative internal sequence variants detected in SR mode sequencing could be artifactual. This implies RNA editing may not be as prevalent in isomiR biogenesis as proposed. SR sequencing, with iso_snv filtering, may be sufficient for isomiR resolution. PE sequencing produces better quality reads. However, PE’s higher cost than SR precludes use in most studies. Filtering out iso_snv hits from SR data may be indicated for isomiR level analysis for most studies.Validation of isomiRs by alternative methods would be advisable especially for iso_snvs derived from SR datasets, but this may be technically unfeasible or inefficient because of the high false positive rate. Continually decreasing sequencing costs may allow adoption of PE sequencing as the preferred method in future miRNA studies focusing on isomiR variation.We extracted total RNA from 30 serum samples from healthy individuals, anonymous blood donors who consented to collection of a separate fraction for circulating RNA biomarker discoveries with the miRCURY RNA Isolation kit for Biofluids (Exiqon) spiked in with Exiqon RNA spike in mix (1:50) of recommended amount eluted in 50 µl. As RNA concentration was too low to be measured by spectrophotometry or fluorometry, quality of RNA was assessed with the microRNA QC PCR Panel, 96 well (V1.RO) from 5 µl of 1:50 dilution of cDNA obtained after reverse transcribing 2 µl of total RNA with miRCURY LNA™ Universal RT microRNA PCR (Exiqon) which uses external spiked in to control for miRNA yield and profiles specific miRNAs to estimate hemolysis. Quantitative PCR reactions were performed on Roche LightCycler 480 instrument (Roche) in 10 µl volume reactions. All samples included in the analysis had equivalent miRNA content and low levels of hemolysis (ΔCq (miR-23a–miR-451) ranging from 4.46 to 6.47, below the assay rejection limit value of 7). We constructed indexed libraries using the TruSeq Small RNA kit (Illumina) starting from all remaining RNA extracted from 200 µl . Library products were pooled together in equimolar amounts based on Bioanalyzer peak quantification at the expected size around 142 bp. A single pool with 30 libraries was size selected with the Pippin prep system (SAGE Science) with 3% agarose and dye free Marker F in the 115–165 bp broad range to enrich for miRNA products and minimize variation between samples in the size selection step. The sequencing data generated consisted of 2 × 50 nt reads resolved on a HiSeq 2500 system with v3 SBS reagents (Illumina). Two of the 30 samples were excluded because they were clear outliers by PCA exploratory analysis and had poor library yields. The final dataset consists of 28 samples.https://github.com/HCGB-IGTP/XICRA). It is developed in python, with multiple separate modules and it is available as a pip package (https://pypi.org/project/XICRA/). This pipeline is designed to take paired end reads in fastq format, trim adapters and low-quality base pairs positions, and merge read pairs (R1 & R2) that overlap. A mapping step to the reference genome (user defined) assigns joined reads to all major RNA biotypes including miRNA and isomiRs, tRNA fragments (tRFs) and piwi associated RNAs (piRNAs). Then, XICRA produces a miRNA analysis at the isomiR level using joined reads, with several choices of software that can be selected by the user with standardized output. Results are generated for each sample, analyzed and summarized for all samples in a single expression matrix. This information can be processed at the miRNA or isomiR level (single sequence) but also summarizing for each isomiR variant type. Statistical summaries can be easily accessed using the accompanied R package XICRA.stats (https://github.com/HCGB-IGTP/XICRA.stats). Although the pipeline is designed to take paired-end reads, it also accepts single-end reads. The workflow of the pipeline is described in Fig. To analyze small RNA-seq PE data we have developed the XICRA pipeline  that facilitates the retrieval of these matrices and parses the information included within each unique index name provided. The DE analysis can be done aggregating data at the mature miRNA level (i.e. hsa-miR-501-3p), by isomiR class (i.e. hsa-miR-501-3p_iso_5p), by specific length variant cluster (i.e. hsa-miR-501-3p_iso_3p:-2) or with the sequence of the read itself as the counting data. This is useful since different types of modification may coexist in a single sequence, and non-templated additions and internally edited sequences can differ leading to isomiRs that can fall into different categories or be derived from different mature miRNAs. DE analysis is performed outside of the tool with DESeq2 package in R .We adapted the Gene Set Enrichment Analysis (GSEA) tool  for miRNVenn diagrams depicting overlap between regulated genes were made using the Venn function in the gtools package in R .We searched the NCBI SRA database for any small RNA sequencing datasets generated using Illumina paired end reads. Very few datasets were found fulfilling our filtering criteria. We found a dataset belonging to a project  that studied miRNA biomarkers in nonalcoholic fatty acid liver disease (NAFLD). The design of the experiment contained 8 patients, classified as control or case, and the dataset was sequenced using paired-end NextSeq 500 paired-end libraries. See additional details in the original publication .We produced a full analysis at the isomiR level using XICRA and the three different software available  using paired-end information and single end reads . For the trimming process, we used as adapter sequences the corresponding NEBNext library adapters. Once the isomiR expression matrices were generated for each software and read type, we used R package XICRA.stats for parsing the information and DESeq2 in R  for the https://github.com/HCGB-IGTP/XICRA/tree/master/BMC_bioinformatics_paper/simulation).To illustrate the potential of paired-end reads at the isomiR level analysis we have generated computer simulations to test the impact of technical errors from single end or paired-end reads. We followed the guidelines previously described for isomiR computer simulations by Amsel et al. 2017 . We creaHomo sapiens mature and hairpin sequences in miRBase (v.21) [We created artificial miRNA isoforms from e (v.21) . We addiFor each biological dataset generated, we used ART  with IllWe evaluated the performance of using PE reads for miRNA isomiR analysis using the NGS simulation datasets and the pipeline XICRA. For each dataset, we used the miRNA module using paired-end mode and single end mode for the R1 and R2 reads. For the paired-end mode we initially joined reads using two different join percentage difference cutoff (fastq-join parameter) to test the effect of using 100% perfect R1 and R2 reads or allowing the default difference (8%) along the minimum default overlap length cutoff (6 bp). For the single-end mode, we used the total R1 reads simulated or the total R2, reversed complemented using seqtk software  respectiUsing the biological variation frequencies generated as true positives for each dataset, we evaluated the amount of isomiRs detected for each software and type of read. For paired-end reads, we also used a different percentage difference cutoff. For each isomiR, the detected counts were classified as: True positives (TP) when observed counts matched the expected counts; false positives (FP) when observed counts exceeded the expected counts and were wrongly assigned; and false negatives (FN) when observed counts did not get to the minimum expected counts. We calculated the sensitivity or recall as TP/(TP + FN) and the precision or specificity as TP/(TP + FP). We also reported True Negatives (TN) when expected counts were not observed and new generation isomiRs when new variants or miRNA appeared and were not expected. We plotted results using ggplot2  for eachAdditional file 1: Figure S1: Schematic summarizing the biogenesis of miRNA and isomiRs .Additional file 2: Figure S2 A: Simulation statistics for three different softwares, from left to right  and in colors for different read types: red, Paired-end (PE); green, Single-end R1 (R1) and blue, SE R2 (R2). Statistics are from top to bottom, Sensitivity, calculated as TP/(TP+FN); Precision or specificity, calculated as TP/(TP+FP); and New Generation as the total count of new non-expected isomiRs detected. We show results using PE reads joined with 0% fastq.Additional file 3: Figure S2 B: Simulation statistics for three different softwares, from left to right  and in colors for different read types: red, Paired-end (PE); green, Single-end R1 (R1) and blue, SE R2 (R2). Statistics are from top to bottom, Sensitivity, calculated as TP/(TP+FN); Precision or specificity, calculated as TP/(TP+FP); and New Generation as the total count of new non-expected isomiRs detected. We show results using PE reads joined with 0% fastq.Additional file 4: Figure S3 A: Classification and comparison of miRTOP sequences from simulations results using miraligner software, for each isomiR class and each category of analysis: PE_0 ; PE_8 ; SR1 (single end reads R1) and SR2 (SE reads R2). We represented the total average read counts (A) and the count of unique isomiRs detected (B).Additional file 5: Figure S3 B: Classification and comparison of miRTOP sequences from simulations results using miraligner software, for each isomiR class and each category of analysis: PE_0 ; PE_8 ; SR1 (single end reads R1) and SR2 (SE reads R2). We represented the total average read counts (A) and the count of unique isomiRs detected (B).Additional file 6: Figure S4: Venn diagrams of the overlapping isomiRs identified for each category of analysis and subclassified for each isomiR type category: A) canonical; B) iso_3p; C) iso_5p; D) iso_add3p; E) iso_snv; F) iso_snv_central; G) iso_snv_central_offset; H) iso_snv_central_supp; I) iso_snv_central_seed; J) mixed category.Additional file 7: Figure S5: Overlapping results for each isomiR class category showing of overall read cumulative read counts distribution into isomiR types recognized by each sequencing mode.Additional file 8: Figure S6 A: Classification and comparison of miRTOP sequences from GEO project GSE114923 using miraligner software, for each isomiR class and each category of analysis: PE_0 ; PE_8 ; SR1 (single end reads R1) and SR2 (SE reads R2). We represented the total average read counts (A) and the count of unique isomiRs detected (B).Additional file 9: Figure S6 B: Classification and comparison of miRTOP sequences from GEO project GSE114923 using miraligner software, for each isomiR class and each category of analysis: PE_0 ; PE_8 ; SR1 (single end reads R1) and SR2 (SE reads R2). We represented the total average read counts (A) and the count of unique isomiRs detected (B).Additional file 10: Table S1: Small RNA sequencing data processing statistics for all 28 serum samples from blood donor sequenced in house .Additional file 11: Table S2: Small RNA sequencing data processing statistics for all 16 plasma samples from NAFLD patients from a public dataset (GSE114923).


Chlamydomonas reinhardtii, and the free-living ciliate, Tetrahymena thermophila, have lent important new insights into the role of HAP2/GCS1 as a catalyst for gamete fusion in organisms ranging from protists to flowering plants and insects. Here we summarize the current state of knowledge around how mating types from these algal and ciliate systems recognize, adhere and fuse to one another, current gaps in our understanding of HAP2-mediated gamete fusion, and opportunities for applying what we know in practical terms, especially for the control of protozoan parasites.Most, if not all the cellular requirements for fertilization and sexual reproduction arose early in evolution and are retained in extant lineages of single-celled organisms including a number of important model organism species. In recent years, work in two such species, the green alga,  Chlamydomonas reinhardtii and Tetrahymena thermophila. Indeed, these simple to grow, genetically tractable systems have yielded important insights into the basic principles underlying gamete-gamete interactions culminating with membrane fusion. This is perhaps best exemplified in recent work on HAP2/GCS1, an ancient gamete fusogen that is now recognized as a catalyst for zygote formation in representative species across all of the major eukaryotic kingdoms of life  and continues to be an important if not essential part of the life cycle of organisms ranging from metazoans to single-celled protists . While s of life .HAP2/GCS1 in fertilization began with independent studies in Arabidopsis thaliana  membrane fusogen whose structural features closely mimic those of envelope proteins from Dengue, Zika and related viruses, as well as cell-cell fusion proteins (AFF-1 and EFF-1) from the nematode worm, C. elegans which adopt a similar fold . Additional resources for experimental work in these systems include well maintained genomic and transcriptomic databases  or minus (−) when deprived of nitrogen in the presence of blue light. These conditions bring mating type-specific genes into expression, enabling plus and minus mating types to eventually pair and then fuse to form zygotes (minus (−) gametes and is essential for initiating formation of a fusion pore that expands into a single contiguous membrane around both cells forming the zygote  locus following sexual conjugation. Individual mating types can be isolated as clonal lines that express a single mating type when sexual reproduction is activated (see below). A given mating type can mate with any of the other six mating types but not with itself.In the case of mpetence . Transiempetence . Followimpetence , 2015. SC. reinhardtii is induced by suspending vegetatively growing plus or minus cells in nitrogen-free medium for at least 6 h in the presence of light , along with the enzymatic machinery for conversion of peptidylglycine substrates into α-amidated products, was shown to be released from cells via ciliary ectosomes during gametogenesis  on plus and minus gametes named SAG1 and SAD1, respectively  contain hydroxyproline-rich repeats  inhibited cell wall loss without interfering with ciliary agglutination or tip activation .Following cell wall removal, membrane protuberances known as mating structures form at the anterior of cells between the cilia. Formation of the mating structures along with subsequent membrane events associated directly with gamete fusion in Tetrahymena thermophila, nutritional starvation is achieved by washing cells of different mating types into low ionic strength buffers for 70–75 min at 30°C  triggers a new round of RNA and protein synthesis involving up-regulation of >1,800 genes , many ofcombined . While tcombined . This maAlong with this “soluble” factor, different mating types must undergo multiple direct contacts between cells over a period of at least 20 min to activate the downstream events required for tight  pairing, which begins approximately 1 h after starved cells are mixed . InteresT. thermophila are still unknown, there is substantial indirect evidence that, as in the case of C. reinhardtii, activation is initiated through contacts between cilia  binding receptors at the plasma membrane become concentrated at the cell anterior, a phenomenon known as Con-A “tipping” . The addsolution , possiblsolution .Chlamydomonas and Tetrahymena are governed by genes at specific chromosomal regions known as the mating type loci. In C. reinhardtii, the mating type locus encompasses a genetically complex, 0.2–0.4 Mbp region on chromosome six that is rearranged between the two mating-type haplotypes and orchestrates the expression of genes for gamete recognition, adhesion, and fusion, as well as genes involved in sporulation, mitochondrial and chloroplast inheritance  that lie outside the locus itself  gametes of the related alga, V. carteri, leads to a pseudo-male gametic phenotype  as well as in land plants  of plus and minus cells that can easily be separated after hatching  , specifying different mating types and organized in a tandem array at the mat locus. After fertilization and karyogamy, developing macronuclei undergo a series of immunoglobulin-like genome rearrangements that give rise to one functional MTA/MTB gene pair at the mat locus, with each of the four progeny cells (karyonides) from a given mating having the potential to generate a different functional gene pair on a random basis (In the case of us (mat) . Neverthoccurred . The mol in 2013 . Brieflyom basis .MTA and MTB genes of T. thermophila are distantly related and gave rise to paralogs in different Tetrahymena species presumably through gene duplication and mutational drift , microvillus-shaped protrusions of the plasma membrane at the anterior ends of plus and minus gametes between the two cilia  just beneath the plasma membrane and extend ∼3 μM from the membrane at their maximal length , have no obvious cytoskeletal support, and bud from an electron dense patch of membrane with no underlying doublet zone  are incapable of adhering to those of minus gametes despite normal interactions between cilia  and activation  in minus gametes prevents the adherence of mating structures and subsequent fusion in crosses with wild type plus gametes but has no effect on ciliary adhesion and gamete activation.Co-immunoprecipitation studies have shown that MAR1 and FUS1 directly interact through their respective ectodomains. Furthermore, disruption of the mar1 minus cells. Furthermore, expression of the same construct can rescue adherence of mating structures when introduced into minus mutants carrying disruptions in both mar1 and hap2, demonstrating a key role for MAR1 in membrane adhesion per se  and wild type (plus) cells via their mating structures but is blocked in gamete fusion  and produce mating structures that are unable to adhere or fuse to wild type minus gametes. Interestingly, ectopic expression of a FUS1 transgene in the imp11 strain rescued the ability of these cells to undergo mating structure adhesion with wild type minus cells  specified by the plus mating type locus (which is not present in the imp11 mutant) likely contributes to efficient gamete fusion in Chlamydomonas.Aside from FUS1 and MAR1, mutant studies indicate that additional proteins may be involved in gamete adherence and fusion in e fusion . The gam10 cells . However pathway . Furtherus cells  but gameus cells . Since tplus gametes contain abundant actin filaments, plus cells pretreated with cytochalasin D can be activated and produce mating structures that lack F-actin when mixed with minus gametes. These actin-less plus mating structures make contact with the tips of the mating structures of minus cells but membrane fusion is strongly inhibited in these pairs , eventually fuse with the apposed membrane creating pores along the length of the junction that connect the two cells .Initially, junctional membranes are planar and continuous, but as pairs begin to form, out-pocketings appear on both membranes extending into the extracellular space and towards the apposed membrane on either side of the junction. These protuberances  have offered spectacular views of junctional membranes during pore formation and expansion, along with insights into the complex nature of these processes , 2018. FIn this regard, it is worth noting that mass spectrometric imaging studies of mating cells indicate that membranes at the conjugation junction are depleted in the abundant cylindrical lipid, phosphatidyl choline, and are enriched in cone-shaped lipids such as 2-aminoethylphosphonolipid compared with membranes on the cell body . This fiT. thermophila (see below), the complexities of pore formation and expansion revealed in ultrastructural studies clearly suggest that other proteins are involved in the initiation and resolution of membrane pores in this system. To identify such proteins, a variety of innovative approaches have been employed. Ethanol fixation and mechanical disruption of mating pairs by sonication has yielded structures remarkably similar in size and shape to the conjugation junction  genotypes that define pollen grain development and/or pollen tube growth and guidance in this species  in two widely diverged protists  underwent normal ciliary adhesion, gamete activation, and mating structure adhesion when mixed with plus gametes but failed to complete fertilization and form quadriciliated zygotes (minus cells where gamete fusion occurs (namely the minus mating structure) and ultrastructural studies demonstrated an inability of plus and minus mating structures to fuse in crosses between wild type plus and hap2-disrupted minus strains , while mutant cells deficient in HAP2 were able to attach but were unable to fuse with female gametes (see below). Subsequent studies in C. reinhardtii demonstrated that the HAP2 protein, along with the fertilization-essential membrane proteins, FUS1 and MAR1, were rapidly degraded after cell-cell fusion, showing tight regulation of the gamete membrane fusion machinery in a possible block to polygamy , one might predict that deletion of HAP2 from one cell of a mating pair would have little-to-no effect on mating efficiency in the Tetrahymena system. Nevertheless, in crosses between genetically marked WT and ΔHAP2 deletion strains, fertility was shown to decline by as much as 80% . As argued below (see Discussion), the most plausible explanation for these findings is that cooperative interactions between the fusion machinery on both membranes of a mating pair is required for efficient pore formation to occur in T. thermophila.Given that HAP2 is expressed in all seven mating types of h as 80% . This wah as 80% . In thes strains . Further crosses . Taken tHAP2/GCS1 in fertilization and its function downstream of gamete adhesion in a variety of different systems, clearly pointed to a role for HAP2 in membrane fusion. Nevertheless, until recently, large differences in primary amino acid sequence between HAP2 orthologs of different species, together with an absence of homologies to known membrane fusogens left open questions regarding HAP2’s precise function. Those questions were answered using a variety of analytical approaches. First, HAP2 orthologs were shown to assume a 3-dimensional structure closely resembling class II membrane fusogens of enveloped viruses . They also share weak homology overall but are identifiable as cousins through homology at the so-called HAP2/GCS1 domain (PFAM 10699), a stretch of ∼50 amino acids in the extracellular domain that, with few exceptions  have extended intracellular domains with 500 residues or more, while others have almost no cytoplasmic tail whatsoever  to either single amino acids or larger regions of protein structure . While iTetrahymena and Arabidopsis  were used by two laboratories to parse the structures of the HAP2/GCS1 ectodomains from bidopsis . At the solution . Both apectively , have vaC. reinhardtii and T. thermophila, and comparable regions of the E1 glycoprotein of Semliki Forest Virus  and the Epithelial Fusion Failure protein one of C. elegans (a CII protein required for cell-cell fusion in nematode worms) are shown in Models of HAP2/GCS1-mediated fusion). Finally, consistent with the known behavior of class II fusogens (see below), purified HAP2/GCS1 ectodomains form trimers when interacting with liposomes or detergent and their solved structures reflect the trimeric, post-fusion conformation of each protein with its fusion loop and transmembrane domain positioned as they would be in the fused membrane  fusion loop is bisected by a salt bridge connecting an arginine residue at position 185 in the loop (R185) with a glutamic acid residue at position 126 (E126)  was strictly dependent on the presence of the R185 residue.Along with these similarities, interesting differences between viral class II proteins and HAP2/GCS1 orthologs have also been noted. For example, the first crystal structure of the 6 (E126) . These ae fusion . FunctioChlamydomonas HAP2 ectodomain provided additional information about the location, orientation, and structural relationships of key residues within the HAP2 fusion loop, including R185  structure of the ing R185 . This neing R185 .Arabidopsis thaliana and Trypanosoma cruzi have shown that whereas the plant protein has a single fusion loop with an amphipathic helix (dubbed αF) that juts towards the membrane surface, the parasite ortholog has three small nonpolar loops  HAP2 forms trimers in vivo that are essential for fusion; 2) hydrophobic residues in the fusion loop are critical for enhancing trimerization; and, 3) mating structure adhesion is essential for HAP2 trimerization , while the rest remained as two ∼150 kDa monomer bands. Site-directed mutagenesis of leucine residues (L310E and L448E) designed to interfere with hydrophobic interactions present at the trimer core inhibited the trimerization of recombinant HAP2 ectodomain in vitro and blocked gamete merger in vivo, indicating that trimer formation is essential for fusion. Importantly, HAP2 trimer formation was also found to specifically require gamete membrane adhesion. When Chlamydomonas plus gametes lacking the Chlamydomonas membrane adhesion protein FUS1 were mixed with minus gametes, cells underwent wild-type levels of ciliary agglutination, gamete activation, and even mechanical contacts at their mating structures, but failed to form HAP2 trimers and failed to fuse, showing that gamete membrane adhesion is required for HAP2 trimer formation in vivo , which have the potential to interrupt the sexual phase of parasite’s life cycle that is required for disease transmission . Like otEimeria sicken birds and have a major impact on egg and meat production , one could easily envisage the use of recombinant subunit or nucleic acid vaccines encoding the HAP2 protein.With the idea that HAP2 plays a critical role in fertilization, vaccines . Followi oocysts . When tearasites , howeverEimeria spp. and Toxoplasma, HAP2 expression has been documented in several other coccidian species including Cystoisospora suis and Cryptosporidium parvum  resulted in similar findings and, more importantly, showed that antibodies against conserved HAP2 peptides significantly reduced the in vitro formation of zygotes from sexual forms , an important group of apicomplexan parasites that cycle between vertebrate and arthropod hosts. Within this group, industry . Recent arasites . Consistk midgut . In 2017al forms . AlthougPlasmodium can also infect humans and animals but is most well known in the context of human malaria, a disease responsible for ∼400,000 deaths (the majority in children) and over a million cases each year primarily in sub-Saharan Africa , has been recommended by the World Health Organization for widespread use. This 3-dose recombinant subunit formulation contains the major surface antigen on infectious sporozoites, namely, CSP, and provides ∼34% efficacy in preventing severe disease in children aged 5–17 months  but are nevertheless exposed to antibodies taken up with the blood meal. Indeed, there is now considerable evidence for the effectiveness of immunization with sexual stage antigens in blocking parasite transmission within closed laboratory settings demonstrating proof-of-concept for the use of this approach in the field  expands quickly to completely fuse the two gamete cells.Regardless of any hypothetical requirement for additional facilitators, a basic model for HAP2/GCS1-mediated gamete fusion emerges from work on ydomonas . As indi protein . Presuma protein . As fold protein . As tran protein . In ChlaTetrahymena, along with studies of AFF-1/EFF-1-mediated cell-cell fusion in C. elegans force a consideration of alternative models in instances where efficient pore formation requires that fusogens be present on apposed membranes. As described earlier, while a small percentage of Tetrahymena mating pairs can undergo fusion when HAP2 is expressed unilaterally on one membrane, the low efficiency of pore formation in crosses of wildtype cells with ΔHAP2 deletion partners strongly suggests that some type of bilateral interaction occurs between mating cells that is HAP2-dependent. Although one can only speculate as to what those interactions might be, it is reasonable to infer they involve either 1) heterotypic interactions between HAP2 and some hypothetical receptor(s) on apposed membranes that allows pores to form more readily (for example by enhancing membrane adhesion); 2) homotypic interactions between HAP2 monomers/dimer/trimers across apposed membranes that promote the initiation and/or opening of fusion pores between cells; or, 3) some combination of hetero- and homotypic interactions. Before speculating further, it is worth noting that massive overexpression of HAP2 in wild type cells (WT-OE) using a high-copy ribosomal DNA vector paired with a robust cadmium-inducible promoter, failed to increase the percent fusion observed in crosses between WT-OE and ΔHAP2 deletion strains  interactions between these proteins makes sense and various models for AFF-1/EFF-1-mediated pore formation involving monomer-dimer as well as trimer-trimer interactions have been proposed  had no effect on fusion in crosses between wildtype and mutant (HAP2-R164A) Tetrahymena strains (T. thermophila HAP2 does not have (or does not require) a fusion loop, it is entirely possible that the wildtype protein on one mating partner can rescue an otherwise defective HAP2-R164A on its mating partner through trans-interactions between HAP2 proteins on apposed membranes. To test that idea, it will be necessary to examine the effects of the HAP2-R164A mutation expressed in both cells of a mating Tetrahymena pair. Crosses between HAP2-R164A mutant cell lines would be expected to generate wildtype levels of fusion (if T. thermophila HAP2 can function in the absence of a fusion loop), while the same crosses would be expected to completely block fusion . Certainly, the latter outcome would argue the importance of bilateral trans-interactions between HAP2 proteins on both cells of a mating pair and necessitate a model for HAP2-mediated fusion in Tetrahymena that accommodates both trans-interactions of the fusogen as well as HAP2 fusion loop interactions with the membrane. More generally, these types of studies raise the possibility that class II fusion proteins can blend different aspects of these underlying unilateral and bilateral activities depending on the context in which they are expressed. A model consistent with the idea that Tetrahymena HAP2 contains a functional fusion loop and can function bilaterally across membranes is shown in a priori reason that the expression of HAP2, or any other gamete fusogen, should be restricted to a given mating type. Indeed, from an evolutionary perspective, the expression of HAP2/GCS1 in both mating partners in the case of Tetrahymena and other species may be a reflection of an ancestral isogamous state that was discarded in sexually dichotomous organisms  can be effective in natural settings are much anticipated. d humans , it willd humans  could po


Bleeding associated with left ventricular assist device (LVAD) implantation has been attributed to the loss of large von Willebrand factor (VWF) multimers to excessive cleavage by ADAMTS-13, but this mechanism is not fully supported by the current evidence. We analyzed VWF reactivity in longitudinal samples from LVAD patients and studied normal VWF and platelets exposed to high shear stress to show that VWF became hyperadhesive in LVAD patients to induce platelet microvesiculation. Platelet microvesicles activated endothelial cells, induced vascular permeability, and promoted angiogenesis in a VWF-dependent manner. Our findings suggest that LVAD-driven high shear stress primarily activates VWF, rather than inducing cleavage in the majority of patients. However, device-related complications remain common and often result in poor outcomes for patients.,Nearly all LVAD patients lose large VWF multimers in a condition called acquired von Willebrand syndrome (aVWS). aVWS develops soon after LVAD implantation, resolves rapidly after LVAD explantation, and is not observed in heart transplant recipients,1605-M1606 peptide bond cleaved by ADAMTS-13 in the A2 domain; and an integrin-binding RGD in the C-domain. Newly synthesized VWF multimers are either constitutively released  or stored in the Weibel-Palade bodies of ECs and the α-granules of platelets, where VWF is enriched in ultra-large multimers (ULVWF) that are intrinsically hyperactive in binding platelets and endothelial cells.,,,VWF is synthesized in megakaryocytes and endothelial cells (ECs) as a single-chain propolypeptide that dimerizes through C-terminal disulfide bonds and then multimerizes through N-terminal disulfide bonds. Each VWF monomer contains the binding sites for glycoprotein (GP) Ibα and collagen in the A1 and A3 domains, respectively; the Y1605-Met1606 scissile bond in the A2 domain to facilitate VWF cleavage and also to activate plasma VWF by exposing the GP Ibα-binding site in the A1 domain.HSS at similar levels has been shown to reduce VWF adhesive activity by exposing the cryptic TyrWe investigated VWF cleavage and activation under LVAD-driven circulation by: 1) studying longitudinal samples from LVAD patients; 2) identifying shear-induced structural changes of VWF in vivo and in vitro; and 3) dissecting the interplay between extracellular vesicles (EVs) and hyperadhesive VWF in promoting the aberrant angiogenesis that could be the cause of the angiodysplasia found in the mucosal tissue of LVAD patients.Blood samples were collected from 26 patients admitted to the Center for Advanced Heart Failure, the University of Texas Houston Health Science Center. All patients had New York Heart Association functional class IV symptoms and received HeartMate II LVADs (Abbott Laboratories). Patients with malignancies and autoimmune disease were excluded. All patients received standard post-implantation antithrombotic therapy: aspirin (81 mg/d), warfarin with a targeted international normalized ratio of 2 to 3, and dipyridamole . While some patients reported as regular smokers at the baseline, none reported smoking post-implantation because all patients were instructed to stop smoking as part of their post-LVAD care.Blood samples (20-30 mL) were collected at the time of admission (baseline), at discharge after LVAD implantation (mean 28 days), and at the time when a patient was readmitted for an adverse clinical event (bleeding or thrombosis) between discharge and the 3-month follow-up visit. The blood samples at clinical events were collected at presentation before any treatment was administered. Patient samples were coded before analyses to minimize experimental bias. Age- and gender-matched healthy volunteers were studied as control subjects. This study was approved by the review boards on human subject research of the University of Texas and the Bloodworks Research Institute.The data from patients with GI bleeding and thrombosis are presented separately in the results from directly analyzing the plasma samples of patients. For experiments in which patient EVs were tested for endothelial permeability, angiogenesis, and thrombus formation, the data from patients with thrombosis and those with bleeding were combined.6/mL)  euthanized under anesthesia were collected and dissected into 2-mm aortic vascular segments (AVSs). The AVSs were incubated in serum-free Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) overnight at 37°C and then placed in microwells coated with Matrigels (Corning Life Sciences) containing growth factor–poor medium (GPM) . They we6/mL)  or EV-fr2 of HSS . The AVSWe used multiple assays to comprehensively evaluate VWF, including: 1) VWF antigen (VWF:Ag) and VWF propeptide (VWF:pp) measured by enzyme-linked immunosorbent assay (Cat #: ab223864 [Abcam] and CS-MW1939 [Cell Sciences]); 2) thiols exposed on the surface of VWF multimers ; 3) rist2 of HSS for 5 minutes at 37 °C. The hemostasis of mice was evaluated using tail vein bleeding (Wild-type C57BL/6J mice and VWF-deficient mice on C57BL/6J background (The Jackson Laboratory) were infused with VWF multimers purified from human plasma cryoprecipitate or an equal volume of saline through the tail vein. VWF was tested at 30 μg/mL to be consistent with the amount found in LVAD patients and was infused before and immediately after exposure to 110 dynes/cmbleeding .t test was used to compare quantitative variables between 2 groups. Both paired and unpaired methods were used in the analyses, depending on the nature of the data. One-way analysis of variance or 1-way analysis of variance on ranks was used to compare more than 2 groups with Bonferroni post hoc test for multiple pairwise comparisons. A P value <0.05 was considered statistically significant.All data are presented as mean ± SEM for continuous variables or as percentages for categorical variables using SigmaPlot V11.2 (Systat). The Shapiro-Wilk normality test was performed to determine if the data followed a normal distribution. Student’s Among the 26 patients , 4 devel+/Annexin V+)   and to rexin V+) . Furtherexin V+) , whereas+/PS+) were significantly higher in patients at baseline than in the control subjects (+/VWF+ EVs from VWF-activated platelets (pEVs) were significantly increased after LVAD implants and increased further at clinical events , suggesting a significant increase in platelet microvesiculation in LVAD-driven blood flow. No significant difference was detected in the levels of pEVs between patients with bleeding and those with thrombosis. Consistent with these observations, more VWF was detected on platelets collected at bleeding or thrombotic events than on those collected outside of these events .The results reported in hat EVFP  but not hat EVFP  from prehat EVFP . The AVShat EVFP  similar hat EVFP , whereashat EVFP . The VWFhat EVFP  reduced hat EVFP . VWF . The CD31+ microvessels from AVSs cultured in pEV-supplemented GPM were small and often lacked intact vascular lumens (+ vessels was higher in AVSs cultured with pEV and was reduced by the VWF antibody (+/CD41a+) promoted the formation of endothelial networks in a VWF-dependent manner, whereas purified VWF did not  or erythrocytes (2 × 106/μL), but it was prevented by collagen (10 μg/mL). The cleavage was similarly induced under a turbulent flow generated in a vortex for 60 minutes.Exposing normal platelet-rich plasma to HSS for 5 minutes failed to induce VWF cleavage , but it hemicals . In conthemicals . The cle-/- mice infused with VWF and completely with the VWF that was exposed to HSS for 5 minutes at 37 °C  the HSS induced mouse SIPA  and promat 37 °C . Plateleed CD62p , developed CD62p  and gene (+ pEVs . These m (+ pEVs . These r,,,In contrast to the widely held belief, our data demonstrate that VWF in LVAD patients undergo conformational changes to bind and activate platelets and to produce VEGF-carrying pEVs that promote aberrant angiogenesis measured in both 2- and 3-dimensional angiogenesis assays . While t,,vβ3 integrin and CD62p on ECs,,First, the EVs from LVAD patients induced significant vascular permeability and aberrant angiogenesis in a VWF-dependent fashion  and 2. T,,,Second, we show that the VWF in LVAD patients was hyperadhesive, activating platelets  and enhaThe finding that VWF lost large multimers but remained hyperadhesive, albeit at reduced levels , defines1669 and C1670 in the A2 domain are reduced and is disassociated when they are oxidized.,Third, we provided several lines of evidence to show that VWF was activated in LVAD patients through structural changes. The first is that the A1 and A2 domains form a complex in plasma VWF during homeostasisThis study is limited by its small sample size of patients, which did not allow sufficient stratification to conclusively link VWF hyperadhesive activity to the clinical outcomes of LVAD patients. However, defining VWF phenotypes in LVAD patients and understanding their underlying causes would allow us to identify key markers linked to VWF reactivity and to study the causal relationship between the aVWS and hemostatic complications of LVAD patients. The patient recruitment is ongoing, with the follow-up period extended to 24 months, including multiple time points of clinical assessments and blood sampling. The ongoing recruitment will allow a sufficient sample size for developing a composite score system that combines VWF and other laboratory and clinical measurements to assess the bleeding risk of patients. Second, the patient study was conducted with blood samples collected from patients on axial-flow LVADs, whereas most new patients receive centrifugal LVADs. However, our ongoing recruitment will allow us to collect samples from patients on both types of LVADs in order to compare VWF profiles so that we will be able to more precisely define the impact of shear stress on VWF. Third, because of a limited plasma volume, we were unable to map the specific amino acids involved in forming intermultimer disulfide bonds or being oxidized. We are developing a new mass spectrometric protocol to overcome this technical difficulty. Finally, because of the lack of suitable research on LVADs in mouse models, we were unable to test our hypothesis of LVAD-induced GOF VWF and its synergistic actions with pEVs to promote angiogenesis in vivo. We can overcome this obstacle in experiments on large animals in the future.,PerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: VWF is an adhesive ligand critical for hemostasis, but it is also an acute phase reactant involved in inflammation, angiogenesis, and thrombosis. These diverse activities derive from the size and structure of VWF multimers. How hydrodynamic changes induced by LVAD-driven blood flow alter VWF structure remains poorly understood. This study shows that VWF is predominantly activated by HSS and oxidative stress, calling for more comprehensive study of VWF in the pathogenesis of LVAD-induced bleeding complications.TRANSLATIONAL OUTLOOK: The current study used extensive technologies to define the structure-and-function correlation of VWF multimers found in LVAD-driven blood flow. The study raises the question as whether VWF multimers are excessively cleaved by the metalloprotease ADAMTS-13 or activated to bind platelets, and more importantly, how the 2 opposing processes reach the equilibrium in patients on LVAD supports. This study identified shear-induced and VWF-mediated platelet microvesiculation as a potential key contributor to angiodysplasia and associated bleeding in patients on LVAD supports. The clinical and laboratory VWF variables obtained from this study could allow us to develop a composite score that predicts the bleeding risk and need for targeted prophylaxis of individual patients based on their VWF profiles.In summary, we demonstrate that VWF was not excessively cleaved but became hyperadhesive in the majority of LVAD patients. We identified platelet microvesiculation, resulting from the LVAD-driven HSS blood flow, as a key contributor to the hemostatic dysfunction, endotheliopathy, and aberrant angiogenesis found in LVAD patients. These findings could have a direct impact on the clinical management of patients on LVAD support. For example, aspirin, which is commonly prescribed after LVAD, may not be effective in preventing this VWF-mediated platelet activation and EV-driven angiogenesis because shear-induced platelet activation is insensitive to aspirin.The authors have reported that they have no relationships relevant to the contents of this paper to disclose.


Background: Chronic traumatic brain injury is a condition that predisposes the brain to activate B-cells and produce neural autoantibodies. Anti-adaptor protein 3, subunit B2 (AP3B2) autoantibodies have thus far been associated with diseases affecting the cerebellum or vestibulocerebellum. Through this case report, we aim to broaden the spectrum of anti-AP3B2-associated disease. Case description: We report on a 51-year-old woman with a brain injury approximately 28 years ago who recently underwent neuropsychological testing, magnetic resonance imaging of the brain (cMRI), and cerebrospinal fluid (CSF) analysis. Neural autoantibodies were determined in serum and CSF. Our patient suffered from mild cognitive impairment  with stable memory deficits and a decline in verbal fluency and processing speed within a two-year interval after the first presentation in our memory clinic. Brain MRI showed brain damage in the right temporoparietal, frontolateral region and thalamus, as well as in the left posterior border of the capsula interna and white matter in the frontal region. Since the brain damage, she suffered paresis of the upper extremities on the left side and lower extremities on the right side as well as gait disturbance. Our search for autoantibodies revealed anti-AP3B2 autoantibodies in serum. Conclusions: Our report expands the spectrum of symptoms to mild cognitive impairment in addition to a gait disturbance associated with anti-AP3B2 autoantibodies. Furthermore, it is conceivable that a prior traumatic brain injury could initiate the development of anti-AP3B2-antibody-associated brain autoimmunity, reported here for the first time. Neural autoantibodies in psychiatric  and neurThis 51-year-old business woman, married and mother of two children, presented in our memory clinic complaining of predominant and progressive memory disturbances. She initially came to our memory clinic two years ago with a cognitive impairment she believed had been getting even worse within the last years. She also reported a known but slightly worse gait disturbance. The gait disturbance originated from a head trauma followed by a coma lasting 10 days (caused by a car accident nearly 28 years earlier). Her coma involving consecutively diagnosed disorders affecting her memory, concentration, word-finding, and reading capabilities was the consequence of head trauma. Her cMRI shows a posttraumatic right temporoparietal, right frontolateral brain lesion in the thalamus and on the left posterior border of the capsula interna, as well as in left white matter in the frontal region. She developed structural epilepsy entailing secondary generalized seizures. Under treatment with lamotrigine (200 mg/d), she is currently seizure-free. Other comorbidities in her history are ferritin anemia, restless legs syndrome, and alcohol abuse previously (currently she is abstinent). She also suffers from neurodermitis and bronchial asthma. Her family anamnesis is inconspicuous concerning psychiatric or neurologic diseases. Psychopathological assessments revealed fluctuating mood and drive. Her depressive symptoms have been treated with citalopram (20 mg/d) and psychotherapeutical interventions. Neurological examination confirmed the known spastic paresis of the upper left-sided and lower right-sided extremities and a gait disturbance with upright stand and gait ataxia since her brain trauma. Furthermore, she exhibited bradydysdiadochokinesis and ataxic upper extremities, as well as bilateral square werve jerks.Cognitive screening with the Mini-Mental Status Examination (MMSE) and Clock Drawing Test (CDT) revealed normal results, but extensive neuropsychological testing was indicative of a mild cognitive impairment  with deficits in verbal fluency, processing speed, flexibility, verbal und figural memory .In comparison to neuropsychological testing in 2018, deficient memory performance remained stable over time without further deterioration, but impairments in verbal fluency and processing speed declined slightly. Concurrently, her depressive symptoms increased and might have caused or contributed to the mild worsening of attentional and executive dysfunctions. A lumbar puncture to enable a differential diagnosis had been declined two years ago but was consented to at her last visit in 2020. This lumbar puncture aimed to determine specific autoantibodies, as she had begun to reveal progressive memory impairment a few years ago and a “red flag”, i.e., the gait disturbance and slight cerebellar ataxia. The autoantibodies we analyzed were: via antibody blots  and immunofluorescence tests . Using fluroescence brain-tissue slides, we set a 1:10 intensity as the cut-off for positivity compared to the control slides. CSF testing revealed no destruction markers and no pleocytosis. Serum cell-based antibody analysis showed anti-AP3B2 antibodies with an intensity of 1:100 . 1:100 wOur case report highlights both the importance of a prior brain lesion that might predispose the brain to develop an autoimmune process and enlarges the clinical spectrum of AP3B2 antibodies. AP3A is a protein in nerve cell bodies that is important for the transport of proteins by regulating endosomes to synaptic vesicles, and for forming synaptic vesicles ,24. AutoA study by Goryunova et al.  in childHowever, antibodies against another NMDAR subunit, namely NR1, are known to be associated with ischemic lesions with the occurrence of neuropsychiatric symptoms entailing cognitive decline, as mentioned above . AutoantThe depressive syndrome only slightly worsened in the last years so that it cannot be excluded that depression might also be a manifestation of AP3B2 antibody-related immunity. However, it is also possible that the depressive symptoms have been stable in the long run and might be due to the postconcussive syndrome that developed after brain injury. However, it remains an open question whether traumatic brain injury triggers anti-AP3BS autoantibodies to result in a phenotype with cognitive dysfunction or whether these abnormalities are unrelated epiphenoma. Diagnostic lumbar puncture including neural autoantibody analysis in presence of additional hints for autoimmunity may help to clarify this and other autoantibodies—clinical phenotype associations. Furthermore, the increase in anti-AP3B2 antibodies in our patient’s serum after TBI, and her responsivity to immunosuppressive therapy argue for a link between these autoantibodies and damaged synaptic transmission processes.In conclusion, we report here the rare case of a woman with a chronic brain injury that might have triggered an autoimmune process entailing the production of AP3B2 autoantibodies. However, we emphasize that these autoantibodies’ aforementioned association does not prove a causative relationship between TBI and AP3B2 antibodies. Our report expands the clinical spectrum of AP3B2 disease to a manifestation characterized by a predominant amnestic MCI in multiple cognitive domains. Furthermore, this is the first time that AP3B2 autoantibodies have been demonstrated in association with chronic traumatic brain injury. They may eventually prove to be a novel biomarker target in TBI warranting further investigation.


Methanobacterium lineages were recovered from subsurface fracture waters of the Samail Ophiolite, Sultanate of Oman. Lineage Type I was abundant in waters with circumneutral pH, whereas lineage Type II was abundant in hydrogen rich, hyperalkaline waters. Type I encoded proteins to couple hydrogen oxidation to CO2 reduction, typical of hydrogenotrophic methanogens. Surprisingly, Type II, which branched from the Type I lineage, lacked homologs of two key oxidative [NiFe]-hydrogenases. These functions were presumably replaced by formate dehydrogenases that oxidize formate to yield reductant and cytoplasmic CO2 via a pathway that was unique among characterized Methanobacteria, allowing cells to overcome CO2/oxidant limitation in high pH waters. This prediction was supported by microcosm-based radiotracer experiments that showed significant biological methane generation from formate, but not bicarbonate, in waters where the Type II lineage was detected in highest relative abundance. Phylogenetic analyses and variability in gene content suggested that recent and ongoing diversification of the Type II lineage was enabled by gene transfer, loss, and transposition. These data indicate that selection imposed by CO2/oxidant availability drove recent methanogen diversification into hyperalkaline waters that are heavily impacted by serpentinization.Metagenome assembled genomes (MAGs) and single amplified genomes (SAGs) affiliated with two distinct  Briefly, candidate [NiFe]-hydrogenase catalytic subunits were compiled and combined with references for specific hydrogenase groups, as defined previously . The comDatabase  and BLASThe average nucleotide identities (ANIs) among SAGs and MAGs were calculated using the MUMmer (v4.0.0beta2) average_nucleotide_identity.py script in python . SAGs , as identified by BLASTp searches of the proteins encoded by each SAG. The amino acid identities of proteins encoded by the SAGs relative to proteins encoded in the NSHQ14C MAG were then averaged to calculate an overall average amino acid identity for each SAG.Orthologous protein groups among the SAGs were first defined as those proteins encoded in SAGs that were most closely related to a single protein from the reference genome (NSHQ14C Type II MAG), as determined by BLASTp. Among the most commonly identified SAG orthologs that exhibited differences in pairwise amino acid identity were transposases. Transposase orthologs were further classified using ACLAME v.0.4  and were3− and HCOO− to CH4 were measured via microcosm-based activity assays containing 14C-radiolabeled substates as previously described -hydrogenase domain (Mbh) -hydrogenases, Mvh and Frh, that supply reductant from H2 oxidation for methanogenesis. Metabolic reconstructions indicated that these functionalities were replaced by formate dehydrogenases that supply reductant from formate oxidation and that yield intracellular CO2 to allow for methanogenesis to proceed under otherwise DIC limited conditions. The genomic prediction that Methanobacterium Type II can couple H2 with formate but not DIC to drive methanogenesis via a pathway that is unique among characterized Methanobacteria was supported by microcosm-based radiotracer experiments revealing significant biological methane production from H2 + formate but not H2 + bicarbonate. The combination of phylogenetic and gene distribution data relative to other Methanobacteria indicated that the Type II lineage was derived from the Type I lineage, suggesting that the replacement of H2-based reductant by formate-based reductant is a derived trait. In turn, this suggested that the directionality of the diversification of Methanobacterium inhabiting the Samail Ophiolite was from circumneutral into hyperalkaline environments, the latter of which is likely to be limiting in DIC, but where formate could be supplied by serpentinization reactions. Thus, diversification may have taken place in a system where serpentinization was generating large amounts of formate which could act as a selective pressure to evolve the suite of physiological traits allowing for its use in key methanogenesis reactions.Reconstruction of two Methanobacterium into hyperalkaline environments appeared to have been facilitated, in part, by lateral gene transfer of a [NiFe]-hydrogenase complex from a bacterium, perhaps affiliated with an ancestor of Firmicutes. Single cell genomes of Type II Methanobacterium revealed evidence of gene rearrangement, possibly through transposition. Collectively, these results point to the importance of gene loss, gain, and transposition in the adaptation of methanogens to hyperalkaline conditions and provide strong, albeit indirect, evidence that cells are active and adapted to the polyextremophilic subsurface environment of the serpentinizing Samail Ophiolite.The recent diversification of Supplemental MaterialSupplemental Figure 2Supplemental Table 2Supplemental Table 4Supplemental Table 6Supplemental Table 7


Genes were further filtered by mean normalized read count with a cutoff≥10. We observed that mammary gland position had a significant impact on mammary gland gene expression with either LFD or HFD diet, with 1264 DEGs in LFD dams and 777 DEGs in HFD dams. We found that genes related to snRNP binding and translation initiation were most significantly altered between the TMG and IMG. Although we were not able to discern a molecular mechanism, many small nuclear RNAs and small nucleolar RNAs were differentially expressed between the TMG and IMG responsible for cellular functions such as splicing and ribosome biogenesis, which provides and interesting avenue for future research. Our study supports the hypothesis that collection of the mammary gland from a particular location influences mammary gland gene expression, thereby highlighting the importance for researchers to be vigilant in documenting and reporting which mammary gland they are using for their studies.Little attention has been given to the effect of positional variation of gene expression in the mammary gland. However, more research is shedding light regarding the physiological differences that mammary gland location can have on the murine mammary gland. Here we examined the differentially expressed genes between mammary gland positions under either a low-fat diet (LFD) or a high-fat diet (HFD) in the mid-lactation mammary gland . Three-week old WT C57BL/6 mice were randomly assigned to either a low-fat diet (LFD) or high fat diet (HFD) (n = 3/group) and either the right thoracic mammary gland (TMG) or inguinal mammary gland (IMG) was collected from each dam for a total of 12 unique glands. Within each diet, differentially expressed genes (DEGs) were first filtered by adjusted  Early studies focused on the mammary gland using a rabbit model led scientists to believe that mammary gland development was symmetrical, giving the impression that each gland was just a copy of the other , 2. HoweIn the mouse, the majority of mammary gland development occurs postnatally at approximately three weeks of age (puberty), and again during pregnancy and lactation . Our labS1 Table) in LFD fed dams. There were 503 genes upregulated in the TMG and 761 genes downregulated in the TMG when compared to the IMG. Notable biological processes that were upregulated in the TMG were those involved in circulatory system development , blood vessel morphogenesis , and RNA splicing  (S2 Table). Meanwhile, biological processes that were downregulated in the TMG when compared to the IMG included cellular amide metablic processes , organonitrogen compound biosynthetic processes , translation , and amide biosynthetic processes . Other notable biological processes upregulated in the TMG included processes related to purine ribonuleoside and nucleoside biosynthetic processes (S3 Table). Molecular functions upregulated in the TMG included binding , mRNA binding , protein binding , and nucleic acid binding . (S4 Table). Molecular functions downregulated in the TMG included structural constituents of ribosome , DNA binding transcription factor activity , and double-stranded DNA binding  (S4 Table) Reactome pathways that were downregulated in the TMG when compared to IMG included pathways involved in translation , formation of pool of free 40S subunits , and cap-dependent translation initiation  (S5 Table). A summary of top DEGs and GO biological processes affected by mammary gland position in dams fed a LFD are depicted in Figs 2.A total of 1264 genes were differentially expressed between the TMG and IMG (S6 Table). No GO biological processes., molecular functions, or reactome pathways were significantly upregulated in the TMG compared to the IMG in dams fed a HFD. GO biological processes downregulated in the TMG compared to the IMG included those involved in amide biosynthetic process , translation , and peptide biosynthetic processes  (S7 Table). Molecular functions significantly downregulated in the TMG compared to IMG included structural constituent of ribosome , electron transfer activity , and RNA binding . Reactome pathways that were downregulated in HFD TMG dams compared to IMG included metabolism of RNA , translation , and rRNA processing  (S8 Table). A summary of the differences between TMG and IMG in peak lactation dams fed a HFD is depicted in Figs 4.There were 777 DEGs in HFD dams between the TMG and IMG, 296 of which were upregulated in the TMG compared to the IMG, and 481 that were downregulated in the TMG compared to the IMG , we took our two comparisons, TMG and IMG LFD dams, and TMG and IMG HFD dams, and compared genes that were up or downregulated to determine shared genes that were differentially expressed as well as diet specific pathways affected. There were 228 genes commonly downregulated in the TMG in comparison to the IMG irrespective of diet. Notable GO processes downregulated in the TMG of both LFD and HFD dams included amide biosynthetic process  and translation , as well as multiple processes involved in purine ribonucleotide and nucleoside biosynthesis (S9 Table) Notable molecular functions that were downregulated in the TMG compared to the IMG included structural constituent of ribosomes , U1 snRNP binding , and NADH dehydrogenase activity . Reactome pathways that were significantly downregulated in the TMG compared to the IMG included translation , formation of a pool of free 40S subunits , and nonsense mediated decay , as well as multiple pathways related to rRNA processing, translation, and initiation (S10 Table). There were 85 genes exclusively upregulated in the TMG compared to IMG in dams irrespective of diet; however, no notable GO biological processes or reactome pathways were common amongst them. Fig 6 depicts reactome pathways that were downregulated in the TMG compared to IMG in both LFD and HFD fed dams. Interestingly, in this study we observed that small RNAs such as Snora73b, Rnu3b4, and Snord118 were differentially expressed between the TMG and IMG in both LFD and HFD fed dams, and is a topic addressed in our discussion.Using the bioinformatics and evolutionary genomics Venn diagram software , DNA binding transcription activity , and double-stranded DNA binding . Reactome pathways downregulated in the TMG compared to IMG related to membrane trafficking  and vesicle mediated transport .There were 211 genes exclusively upregulated and 253 genes exclusively downregulated in the mammary glands of HFD when comparing the TMG to IMG. Biological processes exclusively downregulated in the HFD TMG compared to IMG included ribosome biogenesis  and ribonucleoprotein complex biogenesis .This study supports the hypothesis that there are differences between the TMG and IMG in C57/BL6 mice during peak lactation. The stark changes observed within a diet between the TMG and IMG highlights the need for scientists to be vigilant in carefully documenting and reporting which mammary glands are being used for mammary gland studies. Our lab has previously documented the importance of dietary intake in regulating mammary gland gene expression , but havS8; p <<< 0.0001). One explanation for the differences in RNA metabolism might be due to noncoding RNAs such as small nuclear RNAs (snRNA) and small nucleolar RNAs (SnoRNA). In this study we observed that small RNAs such as Snora73b, Rnu3b4, and Snord118 were significantly different between the TMG and IMG in both LFD and HFD fed dams. This is particularly of interest, as pathways related to nonsense mediated decay (NMD) were demonstrated to be the most significantly different between the TMG and IMG in shared pathways between LFD and HFD fed dams . NMD eliminates eukaryotic RNAs with premature stop codons, and it has been demonstrated that genes that host large quantities of snoRNAs in their introns produce NMD sensitive splice variants [snord118, which is responsible for encoding the snoRNA U8, results in cerebral microangiopathy leukoencephalopathy [The most significant downregulated reactome pathway in the TMG when compared to the IMG was the metabolism of RNA  genes such as Igfbp5, Igfbp2, and Igfbp3. The IGF axis is crucial in mammary gland physiology and play a role in mammary epithelial cell differentiation [Igfbp5 is upregulated during involution [Igfbp5 may act as a tumor suppressor in breast cancer [Igfbp2 is thought to have an opposite effect of Igfbp5 [Igfbp2 in the IMG and a decrease of Igfbp2 in the IMG when compared to the TMG. Igfbp2 is thought to have a pro-tumorigenic effect [Igfpb expression between the TMG and IMG of dams fed a LFD indicate that there could be significant changes in mammary epithelial cell differentiation in dams fed a LFD between the TMG and IMG and may explain positional differences in breast cancer susceptibility between mammary gland position as well as the influence of mammary gland position on mammary involution.Interestingly, within each diet we observed differences in a multitude of insulin-like growth factor binding proteins (IGFBP), such as ntiation . It is ivolution  and it ht cancer , indicatf Igfbp5 , which ic effect , potentic effect . Thus, tThis research highlights the drastic gene expression differences mammary gland position can have. We have shown here that mammary gland position has a significant effect on gene expression under either a HFD or a LFD consumption. Thus, it is vital for scientists to begin documenting and reporting the mammary glands used in mammary gland related studies. Since the main goal of this study was to identify the transcriptome changes between mammary glands collected from different locations, we employed pairwise comparisons within each diet. More studies using a statistical model accounting for the impacts of diet, mammary gland location and the interaction of the two are required to comprehensively dissect the impacts of biological and environmental factors on mammary gland function. Further studies are also needed to assess the effect of time dependent gene expression pattern in mammary gland as well.ad libitum  for a total of 12 unique mammary glands used for analysis (S11 Table). Mouse feed was changed on a weekly basis. The HFD  consisted of 60% fat, 21.4% carbohydrates, and 18.3% protein at 5.1 kcal/g. The LFD  consisted of 9% fat, 44.9% carbohydrates, and 19.0% protein at 3.3 kcal/g. Mice were on their respective diets for four weeks prior to mating. Female mice were bred at 7 weeks of age, with date of vaginal plug denoted as embryonic day zero (E0) and date of parturition as lactation day zero (L0). Litters were not standardized, but litters sizes were not significantly different between treatment groups .The University of Wisconsin-Madison Research Animal Care and Use Committee (A005789) approved all animal protocols and experiments. All experiments were conducted according to proper protocol guidelines and regulations. The animal facility was maintained at 25°C at 50–60% humidity under a 12:12 light: dark cycle for the entirety of the study. Ten 3-week old WT C57BL/6 mice were randomized and divided into either a low-fat diet (LFD) or high fat diet (HFD) 2 asphyxiation and decapitation. Only one dam was euthanized at a time, to prevent mix ups in dams as well as to decrease suffering. Euthanasia chamber was cleaned prior and after use. For mice in all diet groups, either the entire right thoracic mammary gland or the entire right inguinal mammary gland was collected and snap frozen in liquid nitrogen for RNA extraction and sequencing. Thoracic and inguinal glands did not come from the same dam and is an issue that we have addressed in our discussion.Mice were euthanized the morning of lactation day 11  via COhttps://zenodo.org/record/4025240#.X1vhnWdKhsM.At total of 12 unique mammary glands underwent RNA extraction. Mammary gland RNA was extracted using the miRNeasy mini kit . RNA quantity was determined using a Qubit 3.0 fluorimeter . RNA quality was confirmed using the RNA 6000 Nano kit on the Agilent 2100 Bioanalyzer . All samples used for downstream analysis were of an RNA integrity score (RIN) of 7.8 or above. One microgram of total RNA per sample was used for RNA-sequencing library preparation. All samples were prepared using the TruSeq Stranded Total RNA sample preparation kit  per manufacturer’s instructions. Quality and quantity of RNA libraries were subsequently evaluated using the DNA 1000 kit using the Agilent 2100 Bioanalyzer . Prepared cDNA libraries were normalized using the Kapa library quantification kit . cDNA libraries were first sequenced using a MiSeq Nano kit . Further normalization of pooled library was done according to the index ratio obtained by the MiSeq to ensure even sequencing depth among pooled samples. Finally, pooled samples were sequenced on the NextSeq500  using 150 cycle high-output kit to generate paired-end reads (2x75bp). Raw read quality was verified using FastQC. All data is available at https://ccb.jhu.edu/software/tophat/igenomes.shtml) was used as a reference and Tophat2 [http://bioinformatics.psb.ugent.be/webtools/Venn/), we compared common DEGs and exclusive DEGs between inguinal and thoracic mammary glands when mice were fed either a LFD or HFD. Workflow for the RNA-sequencing portion of the study is depicted in Fig 7. To identify stably expressed genes regardless of mammary gland location or diet, we performed ANOVA (using the scipy.stats package in Python) analysis using the FPKM values. P-value >0.5 and Coefficient of variation <0.2 were used as a cutoff to determine stably expressed genes [S12 Table. We picked two of the stably expressed genes as reference genes in the RT-qPCR expression analysis.To align raw reads to the mouse genome, NCBI build 37.2 Mouse genome . All primers followed MIQE guidelines [2 of 0.96 or above. All samples were normalized to 1μg and reverse transcribed to cDNA using the Applied Biosciences High-Capacity cDNA Reverse Transcription Kit . cDNA was diluted in RNase free H2O in a 1:5 ratio. Reaction mixtures contained 6.5μL of SSoFast EvaGreen Supermix , 0.5μL of 10μM of each forward and reverse primers, 2.5μL of diluted cDNA, and 0.75μL of RNase free H2O. The following conditions were used: Step 1) 1 cycle at 95°C for 3 minutes, Step 2) 95°C for 10 seconds and Step 3) 60°C for 30 seconds. Steps 2 and 3 were repeated for 45 cycles. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using a Bio-Rad CFX96 Touch Real-Time PCR Detection System . Samples were run in duplicate, with a standard deviation less than 0.5. The geometric mean of Ribosomal Protein 9 (RPS9), Keratin 8  [Eef2) and NADH:ubiquinone oxidoreductase subunit B10 (Ndufb10) was taken to represent the reference gene [-ΔΔCt method [S13 Table.Primers were purchased from Integrated DNA Technologies . Primers were designed and verified using Primer-BLAST  , 33, Euknce gene . Fold cht method . RPS9 ant method . AdditioqRT-PCR was evaluated using a student’s t-test in GraphPad Prism v7 . Heat maps were generated in GraphPad Prism using the most significant DEGs. Reactome pathway figures were generated in R .S1 Table1264 DEGs when comparing thoracic and inguinal mammary glands of LFD fed L11 dams.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableGO Molecular Functions upregulated in thoracic mammary glands in L11 dams fed a LFD (top). GO Molecular Functions downregulated in thoracic mammary glands in L11 dams fed a LFD (bottom).(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table777 DEGs between the thoracic and inguinal mammary glands L11 dams fed a high fat diet.(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.S12 Table(XLSX)Click here for additional data file.S13 Table(XLSX)Click here for additional data file.