Native highlanders (e.g. Sherpa) demonstrate remarkable hypoxic tolerance, possibly secondary to higher levels of circulating nitric oxide (NO) and increased microcirculatory blood flow. As part of the Xtreme Alps study , we investigated whether dietary supplementation with nitrate could improve NO availability and microvascular blood flow in lowlanders. Plasma measurements of nitrate, nitrite and nitroso species were performed together with measurements of sublingual (sidestream dark-field camera) and forearm blood flow (venous occlusion plethysmography) in 28 healthy adult volunteers resident at 4559 m for 1 week; half receiving a beetroot-based high-nitrate supplement and half receiving an identically-tasting low nitrate ‘placebo’. Dietary supplementation increased plasma nitrate concentrations 4-fold compared to the placebo group, both at sea level  and at day 5 (D5) of high altitude . Dietary nitrate supplementation also significantly increased both plasma nitrite  and total nitroso product  levels both at sea level and at 4559 m. However, plasma nitrite concentrations were more than 50% lower at 4559 m compared to sea level in both treatment groups. Despite these significant changes, dietary nitrate supplementation had no effect on any measured read-outs of sublingual or forearm blood flow, even when environmental hypoxia was experimentally reversed using supplemental oxygen. In conclusion, dietary nitrate supplementation does not improve microcirculatory function at 4559 m. •Xtreme Alps is a randomised controlled field study of dietary nitrate at altitude.•Dietary nitrate significantly increased plasma nitrate, nitrite & nitroso species.•No changes in sublingual blood flow were seen in response to high dietary nitrate.•Dietary nitrate did not alter forearm blood flow under any experimental condition.•Dietary nitrate supplementation did not improve microcirculatory function at 4559 m This hypobaric hypoxia can cause many challenges to those visiting or living in highland regions, including serious illnesses which can prove fatal if left untreated ,2. Yet gThe microcirculation refers to the smallest blood vessels in the body, typically those less than 100 μm in diameter, and microcirculatory dysfunction is increasingly being implicated in the development of critical illnesses, sepsis and other shock states ,12. Novel-arginine by a family of isoenzymes known as ‘nitric oxide synthases’ (NOS)  in an oxygen-dependent process; an alternative pathway is thought to proceed through serial reduction of inorganic nitrate (NO3−) to nitrite (NO2−) and then finally to NO. This latter process is promoted by hypoxic conditions because oxygen normally inhibits the reduction process [NO is a ubiquitous cellular messenger and effector molecule  with potent vasoactive and anti-inflammatory properties . Physiol process , which m process . Dietary process . Inorgan process .The Xtreme Alps expedition was a double-blinded randomised placebo-controlled trial to investigate whether dietary nitrate supplementation could improve performance at altitude, and a full study methodology has been published elsewhere . The fir22.1Ethical approval for the Xtreme Alps study was obtained from Research Ethics Committees at both University College London, UK and the University of Turin, Italy, which manages a research laboratory inside the Capanna Regina Margherita (‘Margherita Hut’) on the summit of Monte Rosa  where the study was performed in August 2010. Complete methods of the Xtreme Alps expedition and protocols have been described elsewhere . HealthyBaseline measurements were taken in London . Participants were separated into 2 trekking groups (Trek 1 and Trek 2) based on their availability, with Trek 1 starting their ascent seven days before Trek 2. All trekkers initially flew into Milan (102 m) and remained there overnight before starting to ascend. The following day, participants travelled by road to Alagna (1205 m), ascended to Punta Indren (3250 m) using lifts and then walked to the Gnifetti Hut (3611 m). We planned for both trekking groups to acclimatise at 3611 m for 3 nights before continuing to ascend on foot to the Margherita Hut (4559 m). However, incoming storms forced Trek 1 to ascend to 4559 m one day earlier than Trek 2, i.e. after spending only two nights at 3611 m. After reaching the Margherita Hut, participants remained at 4559 m for the duration of the study  to receive either an intervention or placebo version of a custom formulated all-natural beetroot/fruit juice drink  starting 72 h before and continuing for the duration of each testing period . The low nitrate supplement (placebo group) contained only 11.5 μmol/kg per day  as the majority of the nitrate content of the beetroot portion had been removed by a microbial denitrification process; whilst in the high nitrate supplement (intervention/treatment) group the total daily nitrate content was approximately 0.10–0.18 mmol/kg/day  [Participants were allocated at random  . This inh meals) ,33. Parth meals) .2.3g for 15 min, and 1 ml of plasma and the red blood cell pellet were both aliquoted into individual cryovials and frozen at −40 °C for transport back to sea level where they were stored at −80 °C until later analysis.Fasted venous blood samples (5 ml) were taken at sea level and on the 1st, 3rd and 5th testing mornings  during the study at altitude, using EDTA-containing BD Vacutainer™ tubes. Each of these samples was immediately centrifuged at 800×Blood haemoglobin (Hb) concentrations were quantified in the field immediately after sampling using a handheld photometric device . Measurement ‘cuvettes’ for the HemoCue were always stored within a thermostatically controlled storage unit in accordance with the manufacturer's recommendations.2.4g for 10 min before undergoing analysis by high-pressure liquid chromatography (HPLC) using a dedicated nitrite/nitrate analyzer . All sample analysis was performed with repeated daily calibrations and staggered to ensure processing times were similar, and all reported values were corrected for background levels of nitrite/nitrate. Total nitroso product concentrations were quantified by group-specific denitrosation of NEM-treated EDTA plasma samples after injecting samples incubated with acidic sulfanilamide directly into an acidic triiodide-containing reaction chamber and measuring the NO produced (following reduction of protein nitroso-species) by gas phase chemiluminescence  as described previously [l-arginine were measured spectrophotometrically  using a commercial assay . Plasma arginase activity was also measured spectrophotometrically .All plasma biomarker concentrations were quantified after reaction with an excess of N-ethylmaleimide  immediately after frozen plasma aliquots were thawed. To quantify plasma nitrite and nitrate concentrations, NEM-treated samples were deproteinized with methanol (1:1) and centrifuged at 16,100×eviously . Circula2.5The sublingual microcirculation was imaged whilst resting in a supine position by sidestream dark field (SDF) imaging; a non-invasive hand-held video microscope  was placed under participants' tongues with the camera-tip pointing sublingually. The probe was moved around gently until clear images of the microcirculation were displayed, then retracted as much as possible without losing these images to minimise pressure-flow artefacts. Four repeat 20-s films were recorded from each subject, first at sea level and then again after residing at 4559 m for at least 4 days. These were assessed later using the Automated Vascular Analysis (AVA) 3.0 microcirculatory analysis software  by two trained and experienced investigators who were both blinded to the subjects’ identity and also to the location of each recording .Microcirculatory Flow Index (MFI) scores were calculated by vessel size in accordance with recognized guidelines . Initial. mm−2.The AVA 3.0 software was also used to calculate the vessel density. The position of blood vessel walls needs to be deduced in SDF imaging using changes in contrast between hemoglobin near the very edge of blood vessels and surrounding tissues. After image stabilization, time averaging of individual film frames filled interruptions in perceived vessel walls caused by plasma gaps and white blood cells. This slope of change in color was then used to define and trace out blood vessel edges automatically, allowing the software to calculate small vessel density , medium vessel density  and total vessel density , measured in mm 2.6All participants underwent a non-invasive assessment of forearm blood flow in their dominant arm using strain gauge venous occlusion plethysmography . An appropriately sized mercury-in-silastic strain gauge was placed just distal to the antecubital fossa, where the forearm circumference was largest, as the subject sat with their arm resting on styrofoam blocks elevated to the level of the right atrium. Initially, blood pressure cuffs placed around the wrist (inflated to 250 mmHg) and the upper arm (inflated to 50 mmHg) were used to exclude the hand circulation and occlude venous outflow from the forearm, and blood flow measurements as well as oxygen saturation levels were recorded at rest. Both cuffs were then deflated and after resting for 1 min, participants completed a standard forearm exercise protocol . Immediately following this handgrip exercise, both cuffs were re-inflated and subsequent circumference changes recorded. With venous outflow occluded, continuous arterial inflow led to linear increases in forearm circumferences, which are reported as the percentage changes of the slopes per minute generated. In accordance with classical venous occlusion plethysmography, five repeat values were recorded for each measurement. However, as described by our group previously , althougAfter taking measurements under ‘normal’ environmental conditions , all participants underwent further testing with these environmental conditions experimentally ‘reversed’. Specifically, after normal sea level measurements were taken, all participants were subjected to breathing a hypoxic gas mixture (12% oxygen) for 45 min before repeating the measurement protocol; and similarly, measurements were repeated at 4559 m after participants breathed 35% oxygen for 45 min. All responses were normalised to total forearm volume, determined individually by volume displacement.2.7http://www.stata.com) to account for the multiple time points at which measurements were taken at altitude and the different ascent profiles of each trek group. All p values report the treatment effect in response to taking the high nitrate dietary supplement unless otherwise specified separately in the text. All reported variables were either normally distributed, or converted into normally distributed data after logarithmic transformation . Nitrate supplementation also significantly increased both plasma nitrite (p = 0.03) and total nitroso product (p < 0.001) concentrations, both at sea level and at altitude. However, fasting plasma nitrite concentrations were more than 50% lower at 4559 m compared to sea level in both the treatment and the placebo groups see , Table 2l-arginine or plasma protein concentrations either (p = 0.73 and 0.90 respectively), but mean plasma arginase activity was significantly higher (p = 0.04) in the high nitrate treatment group , Proportion of Perfused Vessels (PPV) and Microcirculatory Flow Index (MFI) for all measured vessels, and specifically for the smallest measured blood vessels (defined as those < 25 μm in diameter), both at sea level and at altitude, are shown in Despite the significant biochemical increases seen in response to high nitrate dietary supplementation described above, no significant differences were seen in any measure of sublingual microcirculatory function in response to treatment with high nitrate supplementation.3.3The handgrip protocol successfully resulted in measureable increases in forearm blood flow after a standardised exercise protocol, both at sea level and at altitude. At sea level, large increases in blood flow were observed; from 3.3 (±1.5) %/min to 19.9 (±7.0) %/min in the placebo group and from 3.3 (±1.8) %/min to 19.1 (±8.0) %/min in the treatment group. Similar changes were also seen at 4559 m, although both the placebo and the treatment groups displayed more moderate increases in peak forearm blood flow with exercise at altitude see .Fig. 3FoAs with all reported measures of sublingual microcirculatory function above, the increase in forearm microcirculatory blood flow with exercise did not change with dietary nitrate supplementation in any measureable way, neither at sea level nor at altitude. Similarly, when environmental conditions were experimentally reversed by breathing 12% oxygen for 45 min at sea level or by inhaling 35% for 45 min at 4559 m altitude, there was still no significant difference in forearm blood flow between the placebo and treatment groups see . Further4Our findings show that dietary nitrate supplementation significantly increases circulating levels of nitrate, nitrite and nitroso product concentrations, both at sea level and at 4559 m altitude. However, despite these significant biochemical changes, there was no demonstrable improvement in any measure of sublingual microcirculatory function or forearm blood flow , suggesting that overall NO bioavailability remained unaltered both at sea level and at altitude. In a number of previous studies, dietary nitrate supplementation has been demonstrated to increase plasma nitrite (and/or nitrate) concentrations and result in improved running performance, walking performance, and cycling performance under conditions of normobaric hypoxia . However2) either despite increasing exhaled NO levels (PENO) by 24%, also supporting previous findings of the Xtreme Alps study [A similar study to ours has recently also reported no significant change to forearm blood flow during acute exposure to a simulated altitude of 4300 m, despite increasing plasma nitrate levels 10-fold . Interesps study . In anotps study , nor anyps study .Decreases in microcirculatory blood flow at higher altitudes in native lowlanders, similar to the findings reported here, have been well documented previously ,17,24. TWe cannot exclude that an even higher nitrate dose might have affected microvascular function. We are aware that more recent studies have used higher doses of nitrate and also investigated the dose-response curve of nitrate ; howeverOne possible explanation might be that the pharmacological effects of nitrate are critically dependent on the dosing regimen and thus not simply a function of the total dose administered and steady state-concentrations achieved. Unlike in other previous studies, the nitrate supplementation in our Xtreme Alps study was administered in three equally divided doses throughout the day with the aim of maintaining more consistent elevations in plasma concentrations of nitrate and related metabolic products . This maOur observations might also suggest that nitrite utilisation is enhanced in hypobaric hypoxia and/or that nitrite is being sequestered in other body compartments that have not been measured here. Equally little is known currently about the elimination and renal handling of nitrite and nitrate, although a recent study suggested that either one or both of these species may have direct effects on the renal tubular bed . Given t4.1To our knowledge, the Xtreme Alps study remains one of the largest studies to date investigating the effects of dietary nitrate supplementation under hypobaric hypoxia performed under field conditions at high altitude. Using intervention and placebo supplements  allowed the study to use a double-blinded design and added reliability despite the many challenges that performing a study of this nature carries. Space on the mountain limited overall sample sizes and therefore the power of the study; potentially both increasing the risk of type 2 errors, and also reducing the positive predictive value of statistically significant results  . We atte5Dietary nitrate supplementation at altitude can significantly increase levels of circulating nitrate and other NO-related metabolites. However, despite these marked biochemical changes, we did not identify any significant alteration to sublingual or forearm microcirculatory flow whilst living at 4559 m for over five days. This is consistent with the lack of effects on blood pressure, respiratory function and peripheral oxygen saturation reported by us previously in response to treatment with dietary nitrate supplementation . HoweverUniversity College Hospital NHS Foundation Trust as well as unrestricted research funding from Smiths Medical Ltd. and Deltex Medical Ltd. MP, LH and MF acknowledge support from the Faculty of Medicine, University of Southampton. Part of the work carried out at the University of Warwick was supported by funds from the Medical Research Council . None of the funding bodies or the institutions the authors are affiliated with had any role in the study design; collection, analysis and interpretation of data; manuscript preparation, or decision to publish. AC is funded by a Southampton NIHR BRC Fellowship.Xtreme Alps received charitable support from the Friends of MPWG serves on the medical advisory board of Sphere Medical Ltd and is a director of Oxygen Control Systems Ltd. He has received honoraria for speaking for and/or travel expenses from BOC Medical (Linde Group), Edwards Lifesciences and Cortex GmBH. MPWG leads the Xtreme- Everest Oxygen Research Consortium and the Fit-4-Surgery research collaboration. MPWG serves as the UK NIHR CRN national specialty group lead for Anaesthesia Perioperative Medicine and Pain and is an elected council member of the Royal College of Anaesthetists, and President of the Critical Care Medicine Section of the Royal Society of Medicine. DM has received lecture and consultancy fees from Siemens Healthineers and Edwards LifeSciences. MGM is a paid Consultant for Edwards Lifesciences; his University Chair is sponsored by Smiths Medical; he is the founding Editor of the Journal Perioperative Medicine and sits on the Editorial Board of the British Journal of Anaesthesia; he is the Editor-in-Chief of TopMedTalk.


Testicular germ cell cancer (TGCC), being the most frequent malignancy in young Caucasian males, is initiated from an embryonic germ cell. This study determines intratumour heterogeneity to unravel tumour progression from initiation until metastasis.In total, 42 purified samples of four treatment-resistant nonseminomatous (NS) TGCC were investigated, including the precursor germ cell neoplasia in situ (GCNIS) and metastatic specimens, using whole-genome and targeted sequencing. Their evolution was reconstructed.BRCA1 and BRCA2 genes.Intratumour molecular heterogeneity did not correspond to the supposed primary tumour histological evolution. Metastases after systemic treatment could be derived from cancer stem cells not identified in the primary cancer. GCNIS mostly lacked the molecular marks of the primary NS and comprised dominant clones that failed to progress. A BRCA-like mutational signature was observed without evidence for direct involvement of Our data strongly support the hypothesis that NS is initiated by whole-genome duplication, followed by chromosome copy number alterations in the cancer stem cell population, and accumulation of low numbers of somatic mutations, even in therapy-resistant cases. These observations of heterogeneity at all stages of tumourigenesis should be considered when treating patients with GCNIS-only disease, or with clinically overt NS. These cancers are clinically and histologically classified into two variants, being seminoma (SE) and nonseminoma (NS). Both arise from a common cancer stem cell, currently referred to as germ cell neoplasia in situ (GCNIS),3 which resembles totipotent primordial germ cells (PGCs)/gonocytes. Patients with proven GCNIS have a 70% chance of progression to TGCC (both SE and NS) within 7 years. SE consists of a homogeneous population of cells with similarity to GCNIS and PGC/gonocytes. About 50% of the TGCC patients present with a NS that can be composed of different histological elements, embryonal carcinoma (EC), teratoma (TE), yolk sac tumour (YST), and choriocarcinoma, either pure or mixed. The EC is the pluripotent stem cell component of NS, which can mimic normal early embryogenesis including the formation of so-called embryonal bodies (EBs), and thereby give rise to all differentiated components.5Malignant germ cell tumours of the adult testis, referred to as type II TGCTs of testicular germ cell cancer (TGCC), are the most frequent cancer in young Caucasian males.6 So far, detailed studies into the molecular profile of TGCCs and their progression stages were focused on specific genes, like KIT7 and TP53,10 and on the chromosomal constitution. Analyses have revealed many changes in the (relative) number of individual chromosomes in the different tumour components.16 Gain of the short arm of chromosome 12 is a hallmark of (invasive) TGCCs,17 but as yet no causative gene(s) have been identified. Information on driver mutations underlying the development of these NS using exome sequencing is scarce.20Although all TGCC, including mature TE, are in principle capable to metastasise, about 80–85% of the SE patients and 55–60% of the NS patients present with localised (stage I) disease. Patients with metastatic TGCC are generally cured by standard treatment regimens involving platinum compounds and additional surgery for residual TE, while only few patients show resistance to treatment.TM/LowPass WGS were interrogated to decipher the evolution of the disease.In order to unravel the molecular heterogeneity of NS, we extensively investigated four rare cases of primary therapy-resistant NS and performed WGS on the primary cancer and targeted sequencing analyses on 42 enriched histological components, precursor cell populations, and metastatic lesions after treatment Figs. . Focus wA brief description is provided here. Further details are provided in the NS samples of patients with established intrinsic resistance to standard first-line chemotherapy (detailed in 21 using laser capture micro dissection (Zeiss). Tumour and paired normal DNA samples were whole-genome sequenced (40 times coverage) and analysed at Complete Genomics Inc. (CG)  using NCBI build 36.3 as human reference genome and pipeline software version 2.0.2.22.22 Lists of putative somatic DNA variants (SNVs) were established from the WGS data as described in the 23 Characterisation of the mutational signature was done by comparison of the trinucleotide context of each SNV to the established COSMIC signatures using the MutationalPatterns R package (v1.0).24Purified tumour components  as previously described. and SNV .27 or at the Microarray unit of the Genomics and Proteomics Core Facility of the German Cancer Research Centre  strictly adhering to the Illumina EPIC protocols for the T6107-YSTmeta. CNA based on methylation intensities were resolved using the Conumee package.28 DEPArrayTM experiments on a T6107 metastatic sample, and GCNIS and YST samples of T618 were performed by Menarini Silicon Biosystems , essentially as described.29RNA samples of T6107 and T3209 were rRNA-reduced and Ion Proton sequenced  using the supplier’s protocols and reagents (ThermoFisher Scientific). Generation of methylation profiles of primary tumour DNA was performed as previously described30 In all cases  and SE (N = 18),14 also revealed this signature to be the most prominent .To address tumour heterogeneity and progression, WGS data from four primary chemo-naive NS were exploited. Comparison of the primary tumours with the matched normal provided a set of 1239 somatic putative DNA variants (SNV) for these cases Fig. . RNAseq,ses Fig. , signatuent Fig. . This siN = 42, Figs. 21 To evaluate the allelic imbalances and the presence of SNV in these enriched specimens, amplicons were designed across the genome containing a tumour-specific SNV and additional heterozygous SNPs . An evolutionary tree for this case  was markedly improved for the enriched EC sample, with increased read frequencies for most SNV Fig. . Absence17 Inspection of the LAF profiles showed increasing allelic imbalances up to LOH for chromosomes 4 and 5 in these GCNIS stages .35Our studies on multiple samples of four cases of therapy-resistant NS provided insight in the complexity of tumourigenesis Fig. . NS carr36 A typical example is provided by the T3209 EB23, which resembles an early developing embryo derived from a single EC with a different genomic make-up , suggesting that this mutational event preceded genome duplication  hardly ever exceeded the 50% level , although increased methylation of the BRCA1 gene promoter was observed in some NS without EC.20 It is tempting to speculate that other components of this homologous recombination repair pathway may be affected and responsible for the specific base substitution signature in the absence of direct involvement of BRCA genes.56 Of other genes linked to this repair pathway,32ATM, CHEK1 and MRE11 show aberrations (predominantly deep deletions) in approximately 10% of the NS,57 but complete gene deletions were not observed in our cases , which allows the tetraploid GCNIS to evade apoptosis and survive till puberty and subsequently progress to malignancy due to genomic destabilisation.59 The (epi)-genetic triggers for destabilisation of the tetraploid genome and initiation of the development of malignant clones remain as yet largely unknown, although the induction of mitogenic signalling by testosterone may contribute.Our data and the lack of recurrent driver mutations support the hypothesis that whole-genome duplication is the primary event in NS development Fig. , to be fses Figs  and S2 a60 In view of its tendency to progress in 70% of the patients within 7 years, novel predictive markers for the GCNIS progression are needed. The BRCA-like mutational signature in TGCC indicative for reduced homologous recombination repair, may be suggestive for combined use of poly ADP ribose polymerase (PARP) inhibitors and platinum-based therapy61 but requires additional support. Further studies into NS and SE cases, with GCNIS adjacent to the primary tumour and different histologies or localisation, are needed to identify reliable markers for GCNIS progression, to unravel the critical steps for malignancy and therapy resistance, and to decipher the origin of the BRCA-like mutational signature of TGCC.The observed heterogeneity in the primary tumour, metastases and precursor lesions of NS may impact on clinical decisions and treatment strategies. The occurrence of metastatic tumours with little molecular overlap with the primary lesion indicates that treatment of therapy-resistant recurrences should be targeted at their molecular properties according to the concepts of personalised medicine. The identification of abundant cancer stem cells GCNIS, which did not contribute to the development of the NS further complicates the clinical advice to patients with GCNIS-only disease.Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7Supplementary Table S8Supplementary MethodsInformation Supplementary Tables S1-S8Supplementary Figures S1-S10 and details


These molecular variations provide insight into the (1) evolution of the molecular structure of kerogen with increasing thermal maturity and, (2) the primary molecular contributors to HC generation. Our results also indicate that old model equations based on structural parameters of kerogen underestimate the thermal maturity and overestimate the HC generation potential of Marcellus Shale samples. This could primarily be due to the fact that the kerogen samples used to reconstruct old models were mostly derived from immature shales (VRo <1) acquired from different basins with varying depositional environments. We utilized the kerogen molecular parameters determined from the Marcellus maturity series samples to develop improved models for determining thermal maturity and HC potential of Marcellus Shale. The models generated in this study could also potentially be applied to other shales of similar maturity range and paleo-depositional environments.Kerogen is the insoluble component of organic-rich shales that controls the type and amount of hydrocarbons generated in conventional and unconventional reservoirs. Significant progress has recently been made in developing structural models of kerogen. However, there is still a large gap in understanding the evolution of the molecular components of kerogen with thermal maturation and their hydrocarbon (HC) generative potential. Here, we determine the variations in different molecular fragments of kerogen from a Marcellus Shale maturity series (with VRo ranging from 0.8 to 3) using quantitative  Kerogen formed in the diagenetic stage of burial later cracks to form oil and gas in catagenetic and metagenetic stages of burial. The type and amount of HC generated, its sorption/retention and release on hydraulic fracturing operations is controlled by the molecular structure of kerogen6. Therefore, it is critical to understand the chemical structure of kerogen at the molecular level. In addition, molecular parameters of kerogen can serve as a more robust proxy for determining thermal maturity and hydrocarbon potential in mature shales (with VRo >1) where traditional techniques such as vitrinite reflectance, SRA, and biomarker analysis fails.The advent of unconventional shale gas drilling has necessitated the need to develop a better understanding of the spatiotemporal variations in type and quality of organic matter in shale source rocks. Kerogen is a high molecular weight organic matter (OM) that serves as source and reservoir of all the hydrocarbons in these shales. Kerogen is formed by the degradation, condensation, and polymerization of biomolecules contributed by different sources of OM10. However, the results generated using pyrolytic experiments can be biased due to the interaction of products generated from the labile fraction of kerogen10 or because reactions taking place in laboratory conditions might not be representative of sedimentary basin conditions14. Due to these limitations, non-destructive methods such as Fourier transform infrared (FT-IR), Raman spectroscopy (RS), X-ray photoelectron spectroscopy (XPS), X-ray absorption near edge structure (XANES), and 13C solid-state nuclear magnetic resonance (13C NMR) have been employed for the qualitative, semi-quantitative, and quantitative measurements of kerogen21.Significant progress has been made in understanding the molecular structure of kerogen using destructive (pyrolytic) and non-destructive (spectroscopic) methods13C solid state NMR22. Numerous studies have been conducted using 13C solid-state NMR for kerogen characterization29. A few recent attempts have been made to develop realistic structural models of kerogen31. However, the evolution of different molecular components of kerogen on thermal maturation and the primary contributors to HC generation are still not well understood, especially for mature shales. A recent study by Agrawal and Sharma, 201819 indicated that kerogen structural parameters used in previous models for determining HC generative potential28 and thermal maturity32 could over or under estimate these values.The most reliable and robust tool for determination of fractions of molecular components of kerogen is 13C multiple CP/MAS and multiple CP/MAS plus dipolar dephasing technique detailed in Agrawal and Sharma, 201819. A total of 15 samples were obtained from six Marcellus shale wells  across a thermal maturity gradient in Appalachian basin . The variations in molecular parameters of kerogen were determined using quantitative MultiCP/MAS NMR and MultiCP NMR/DD (dipolar dephasing). Most of the previous investigations used CP/MAS NMR technique, a semi-quantitative technique that could not differentiate between non-protonated carbons (from protonated carbons) and mobile groups (from immobile groups). However, in this study, by using MultiCP/MAS NMR with and without dipolar dephasing, it was possible to determine the fraction of non-protonated carbons, protonated carbons, mobile groups and immobile groups , total aromatic carbon, alkyl substituted aromatic, O-substituted aromatic (phenol), carboxyl and amide, aldehyde and ketone were determined using the multiple CP method (without dipolar dephasing). However, the fractions of mobile (freely rotating) and immobile (restricted rotation) methyl, mobile and quaternary alkyl (without heteroatoms), methoxy, protonated aromatic, non-protonated aromatic bridgehead carbon (faB) were determined using dipolar dephasing method with multiple CP  and vitrinite reflectance measurement. However, Tmax may not be reliable in mature and over-mature samples19 are primarily controlled by thermal maturity. Similar observations have also been made for shales from other basins basin28. However, the cracking mechanism of different structural parameters of kerogen can vary because of the difference in their thermal stability. To determine the sensitivity of different kerogen structural parameters with thermal maturation, linear correlation plots were made using the structural parameters of kerogen and calculated vitrinite reflectance , followed by mobile and quaternary alkyl carbon chains (R2 = 0.61) and mobile methyl carbon chains . Immobile alkyl chains are mostly attached to the aromatic rings, restricting their rotation. The resistant nature of the immobile methyl group is possibly due to the instability of free radicals formed on thermal degradation , mobile (freely rotating), and alkyl-substituted aromatic carbons2 = 0.48) followed by protonated aromatic carbon (R2 = 0.06), O- substituted aromatic carbon (R2 = 0.06), and alkyl substituted aromatic carbon chains (R2 = 0.02). However, it is important to note that although individual aromatic parameters showed poor correlation with the vitrinite reflectance . This indicates that good correlation of total aromatic carbon with increasing maturity as determined by several previous studies is actually due to increase in the relative percentage of aromatic carbon due to the higher breakdown of aliphatic carbon chains rather than formation of new aromatic carbons. The similar mole fraction of bridgehead aromatic carbon (Xb) in all mature kerogen samples  was highest with R2 = 0.94  followed by alkyl chains  with R2 = 0.91 (95% CI of slope = ± 2.82), then by total aromatic carbon  and by total alkyl (without heteroatoms) with R2 = 0.87 (95% CI of slope = ±2.54) as shown in Fig. 2 >0.85). The correlation coefficents and 95% confidence interval (CI) of the slope of the equations proposed for determining VRo were determined using software XLSTAT. The equations are as follows:2n groups) with n >1.2 is fraction of total alkyl chains (CnH2n groups) with n >1.ar is fraction of total aromatic chains.2n groups) with n >0.Regression models have been developed to determine thermal maturity using aromatic carbon percentage, the mole fraction of bridgehead carbon (Xb), SP19 indicates that previously build kerogen models used for determining HC generative potential overestimate the S2 values at least by a factor of two. This can lead to underestimation of the total amount of HC generated in the reservoir. We determined primary contributors to HC generation by evaluating the correlation of different structural parameters of kerogen with S2. We observed that the carbon chains that had highest correlation coefficient with S2 were mobile and quaternary alkyl group , mobile methyl group  and immobile alkyl chains  , MA is mobile alkyl chains  and IA is immobile alkyl chains .A recent study done on kerogen from Marcellus Shale by Agrawal and Sharma, 20182) between the true S2 and modeled S2 was 0.98, and the RMS (root mean square) error of prediction was ± 0.70 mg hydrocarbon per gram of rock. It was also, observed that the by adding additional structural parameters in the multiple regression model, the coefficient of determination (R2) between true vs. modeled S2 did not improve. This observation further suggests that the structural parameters used in the model are the primary contributors of hydrocarbon potential.The coefficient of determination . S2org is calculated by the dividing the TOCorg by HIorg. TOCorg is calculated using equation given by Peters et al.42:f = 1 − , in wt %. After every five sample WFT Source Rock Standard 533 (P/N 810-141) was run. SRA analysis was performed at the National Energy Technology Laboratory and at IsoBioGEM lab in Morgantown.Approximately 80 mg of powdered (200 mesh) shale sample was weighed into a SRA crucible and placed in the autosampler and held isothermally at 300 °C for 3 minutes. The free hydrocarbons are volatilized during this isothermal heating which is quantitatively detected by the FID detector and reported as milligrams (mg) of S1 per gram of rock. The free COSupplementary Information


Type 2 diabetes (T2D) is a growing health concern with nearly 400 million affected worldwide as of 2014. T2D presents with hyperglycemia and insulin resistance resulting in increased risk for blindness, renal failure, nerve damage, and premature death. Skeletal muscle is a major site for insulin resistance and is responsible for up to 80% of glucose uptake during euglycemic hyperglycemic clamps. Glucose uptake in skeletal muscle is driven by mitochondrial oxidative phosphorylation and for this reason mitochondrial dysfunction has been implicated in T2D. In this review we integrate mitochondrial function with physiologic function to present a broader understanding of mitochondrial functional status in T2D utilizing studies from both human and rodent models. Quantification of mitochondrial function is explained both in vitro and in vivo highlighting the use of proper controls and the complications imposed by obesity and sedentary lifestyle. This review suggests that skeletal muscle mitochondria are not necessarily dysfunctional but limited oxygen supply to working muscle creates this misperception. Finally, we propose changes in experimental design to address this question unequivocally. If mitochondrial function is not impaired it suggests that therapeutic interventions and drug development must move away from the organelle and toward the cardiovascular system. A brief list of specific definitions is provided for clarity in conceptual aspects of this document. They are to be used in place of other meanings that may have several less stringent descriptions within the literature.MITO)—Functional capacity inherent to the organelle. Equivalent to mitochondrial function.Mitochondrial respiratory capacity —An indicator for the amount of mitochondria within a tissue. VMITO is the amount of volume mitochondria hold relative to the volume of the cell. This definition does not discriminate between hypotheses on mitochondrial networks or individual organelles. This measure is derived from the estimates of mitochondrial protein, respiratory enzyme activity, and other biochemical determinations .MITO and MITO: Muscle mitochondrial oxidative phosphorylation capacity (MOP Capacity)—Absolute capacity for muscle to produce ATP aerobically. This is a function of the VFasting blood glucose (FBG)—Refers to 12 h fasting conditions. +Nicotinamide adenine dinucleotide—reduced form, NADH; oxidized form NAD2; oxidized form FADFlavin adenine dinucleotide—reduced form, FADHATP) at 37 °C, pH 7.0. Its magnitude increases further from equilibrium when the Phosphate potential—Defined by the free energy of ATP hydrolysis (ΔGATP (kj/mol) the greater the phosphate potential and in healthy muscle cells at rest ranges from about −64 to −68 kj/mol at rest and decreases with intense exercise but not below −48 kj/mol where the energy required for calcium pumping by the sarcoplasmic reticulum can no longer be met resulting in fatigue [The more negative the ΔG fatigue .°C, pH 7.0: Redox potential—Determined from the reduction status of the pyridine pool the redox potential describes the energy contributed to the proton motive force from NADH oxidation at 37 REDOX, the greater the redox potential and this value ranges from roughly −210 to −230 kj/mol when near full oxidation or full reduction respectively. Note that up to three ATP are produced from the oxidation of one NADH molecule when optimally coupled.The more negative ΔG2Max), has also been a disputed co-morbidity of this disease [Metabolic diseases present with deficient handling of glucose and lipids and developing comorbidities that ultimately lead to premature death . The mos disease ,15,16,17 disease  and can  disease ) to 50-f disease  during e disease ,21,22. T disease ,24 has r disease ,26,27,2831PMRS). Following a 12-week exercise training regimen, MOP capacity was restored, and glucose tolerance improved. The close association of MOP capacity and glucose tolerance has resulted in a glut of hypotheses involving mitochondrial dysfunction in the development of type 2 diabetes.Skeletal muscle mitochondria oxidize carbohydrates to support ATP production and fuel contracting muscle and as a consequence glucose uptake is largely driven by mitochondrial ATP production ,3,29. ExSkeletal muscle mitochondrial dysfunction in type 2 diabetes was suggested by Kelley and colleagues in 2002 after reporting reduced electron transport chain activity measured from NADH:O2 oxidoreductase . This st2 to be used by the electron transport system (ETS) ultimately generating ATP , here oting ATP  (for det2 for the use at complex I (CI) or complex II (CII) to produce ubiquinol. During the cycle three NAD+ are reduced to NADH; one each by isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, and malate dehydrogenase and one FAD+ is utilized by succinate dehydrogenase to produce FADH2 and subsequent QH2. Oxidation of carbohydrates produces an extra NADH from the PDH reaction and fatty acid oxidation produces an additional NADH and FADH2 from the 3-hydroxyacyl-CoA dehydrogenase reaction and acyl-CoA dehydrogenase respectively. This results in a total of 4:1 NADH:QH2 produced per acetyl-CoA from pyruvate oxidization and 4:2 NADH:QH2 per acetyl-CoA from fatty acids. The TCA cycle resides within the mitochondrial matrix and produces reducing equivalents for the ETS. Although the TCA cycle is complex, for brevity it will be described only in the production of reducing equivalents to permit ATP production and evaluate mitochondrial function . Compose1 portion of the membrane bound F1FO ATPase to increase ATP consumption without affecting other aspects of metabolism. Glucose consumption through glycolysis directly increased with increased ATP consumption demonstrating the pull-through effect of ATP demand [Flux through the TCA cycle is regulated in part by the redox potential and phosphate potential to maintain the energetic status of the cell. This regulation is described using a series of reservoirs as metaphors for the potential energy differences by Meyer and Wiseman (2011)  and demoP demand . The rel2 produced from the TCA cycle are incorporated into the ETS by the donation of electrons at NADH dehydrogenase (CI) or succinate dehydrogenase (CII), respectively. Donated electrons pass down an electrochemical gradient through membrane soluble carriers to complex III (CIII) and complex IV (CIV). The energy provided from donated electrons fuels proton pumping across the mitochondrial inner membrane and develops a proton motive force  that is a function of the membrane potential (~170 mV) and proton concentration gradient (10-30 mV) (1FO ATP synthase) as originally described by Mitchell in his “chemiosmotic theory” [Reducing equivalents feed into the ETS to support chemiosmotic ATP production. NADH and FADH0-30 mV) . This Δp theory” . During  theory” ,40. ANT  theory” ,42,43,442 does not pump any protons but is used to produce QH2 [2 to CIII which pumps 2 protons. Finally, electrons are transferred via cytochrome c to CIV where two molecules of cytochrome c are oxidized to reduce 2 to water, and 4 protons are pumped across the inner membrane. Together, this results in a total of 10 protons pumped per NADH and 6 protons per FADH2. The F1FO ATP synthase utilizes this proton gradient by coupling the backflow of protons into mitochondrial matrix with the synthesis of ATP at the cost of 8/3 protons per ATP synthesized. The non-integer proton value originates from the fact that 8 c-subunit rings are associated with each ATP synthase molecule in mammals, and a complete 360° rotation of the F1 catalytic head produces 3 ATP molecules [2. Thus, the amount of ATP produced per O2 is dependent on which substrates are used to energize mitochondria. Oxidation of NADH and transfer of electrons to CI provides the energy to pump 4 protons across the inner membrane contributing to the proton motive force. In contrast, CII oxidation of FADHduce QH2 . The eleolecules . AccountProduction of ATP through the ETS, like TCA cycle regulation, is driven by ATP consumption via feedback regulation of ADP, Pi, and ATP. Other controllers and influences on respiration have been proposed and suggested to alter metabolic flux ,47,48, hMITO is routinely measured through biochemical assays of isolated tissue  and forMitochondria isolated from skeletal muscle via centrifugation  allows swithin the same muscle group it permits direct comparison of mitochondrial efficiency to produce ATP from ADP. RCR comparisons must be from the same type of tissue and using the same substrate since different substrates or tissues will change both measures of leak and the maximal ADP-stimulated state. For example, RCR values using pyruvate-based substrate are >20 in skeletal muscle, >17 in cardiac muscle, and >3–4 in liver while fatty-acid based substrates produce values of >8, >10, and >2–3 respectively [Mitochondrial function is quantitatively measured using high-resolution respirometry (Oroboros Instruments) to record changes in dissolved oxygen with a clark-type oxygen electrode. These changes in dissolved oxygen are direct indicators of mitochondrial oxygen consumption at cytochrome c oxidase. Mitochondrial function is determined by a series of metabolic challenges to quantify the leak state, maximal-ADP stimulated state, and respiratory control ratio  measuring of phosphate metabolites [31PMRS is the primary method discussed because of its quantitative advantage measuring metabolites inherent to mitochondrial respiration while NIRS quantification of oxygen dynamics is an indirect measure of mitochondrial function.Skeletal muscle permits the quantification of MOP capacity because ATP demand can be controlled and quantified in discreet steps through graded exercise intensities. Since ATP production is driven by ATP consumption, measuring these together is crucial to determine how well the mitochondria respond to step changes in demand . The study of skeletal muscle metabolism is powerful because ATP synthesis is driven by ATP demand and metabolic demand is difficult to quantify in other tissues in vivo. MOP capacity is routinely assayed by near-infrared spectroscopy (NIRS) by measuring hemoglobin and myoglobin desaturation ,56,57,58abolites ,61,62,63a of substrate and product [Using in vivo magnetic resonance MOP capacity is estimated by measuring the dynamics of the coupled metabolite phosphocreatine (PCr). Utilizing the creatine kinase equilibrium, changes in PCr directly reflect changes in ATP consumption/production ,29,64,65 product . Tight c product ,68,69. M product  where th product . This te product ,70,71. HAscribing PCr recovery to mitochondrial function is no longer valid if any of the assumptions in 31PMRS can quantitatively determine mitochondrial function from several other perspectives , ATPase rates from initial rate of PCr hydrolysis, glycolytic ATP production, and others  but with some reports of reduced respiration per organelle (MITO) [MOP capacity can be altered by changes in  (fMITO) ,87,88,89 (fMITO)  and exer (fMITO) . Obesity affects MOP capacity as demonstrated in several models of murine species and in human subjects as well. In a detailed study by Turner et al. (2007) increased mitochondrial volume in mice and rats subjected to elevated fat intake were reported. C5BL/6J mice were acquired at 8 weeks of age and subjected to a high-fat diet for 20 weeks. Mice were sacrificed at 5 and 20 weeks of age and assayed for markers of mitochondrial volume including citrate synthase  and β-hydroxyacyl CoA dehydrogenase . Feeding a high fat diet resulted in increased fat mass to obese levels and mitochondrial volume was elevated in quadriceps muscles by 20% in 5 weeks and 57% in 20 weeks. This result was also shown in the same study in Wistar rats subjected to 4 weeks of HFD, the chronically obese Zucker rat, and db/db mouse showing 14%, 36%, and 17% increased mitochondrial volume. Taken together, these observations show that increased fat composition leading to obesity can directly alter mitochondrial volume and by extension the MOP capacity across species . The inherent effect of obesity on MOP likely depends on the stage of its progression since many other studies of obesity report contradictory effects on MOP capacity . Mice suPhysical activity level directly influences skeletal muscle MOP capacity as shown by the late Dr. John Holloszy and colleagues in over 50 years of work studying exercise training ,96,97,98inactivity on health and development of disease pathology. Inactivity is strongly related to development of obesity and insulin resistance [More recently, studies have focused on the direct effect of sedentary behavior or physical sistance . In factsistance . The rolsistance ,107,108.sistance . Finallysistance .Sedentary lifestyle is directly related to a reduction in skeletal muscle MOP capacity. Booth and colleagues (1987) demonstrated 25% reduced MOP capacity after merely 7 days of sudden inactivity in rat quadricep muscle . Both hiThe direct role of inactivity on MOP capacity also translates to human subjects . Berg etObesity and inactivity each can independently influence skeletal muscle MOP capacity. Therefore, these variables must be controlled for especially in studies of “lifestyle diseases” including obesity, peripheral arterial disease, type 2 diabetes, or chronic obstructive pulmonary disorder. The role of further sequalae of obesity, inactivity, and metabolic syndrome that may alter apparent mitochondrial function including substrate and oxygen availability must also be weighed in the measure of MOP capacity and mitochondrial function  but for Mitochondrial function has been widely studied in human T2D, since the early works by Kelley and Shulman ,118, whoMitochondrial function in vitro is routinely measured in mitochondria isolated from skeletal muscle biopsies of individuals with type 2 diabetes. Several studies report reduced mitochondrial function ,123,124 In contrast, normal mitochondrial function in muscle from individuals with diabetes compared to BMI-matched controls has been shown ,127. HeyStudies in type 2 diabetes have also reported mixed results when determining skeletal muscle mitochondrial function in vivo. Many investigators have suggested mitochondrial dysfunction ,128,129 Scheurmann-Freestone and colleagues (2003) demonstrated slowed PCr recovery in individuals with type 2 diabetes and concluded that MOP capacity was reduced because of mitochondrial dysfunction in type 2 diabetes . In this1C. Despite pronounced type 2 diabetes, no differences were measured in PCr recovery after contracting at workloads where muscle acidity remained negligible. This work by De Feyter et al. suggests that in vivo dysfunction reported in type 2 diabetes is likely a result of age, obesity, and/or physical activity and not the disease itself [Subsequent studies also concluded mitochondrial dysfunction in type 2 diabetes from reduced PCr recovery time and controlled for obesity by BMI matching controls and diabetic subjects ,129. Howe itself .Contradictory evidence appears to leave the question of mitochondrial dysfunction in type 2 diabetes unanswered. However, the literature suggests that conflicting reports of mitochondrial function in type 2 diabetes are the result of subject variability and not the disease itself. To correct this shortcoming, studies that report changes in mitochondrial function in vivo should also have mitochondrial function evaluated in vitro in the same subject. Further, studies must expand upon work by De Feyter et al. performing on well-controlled subjects matching for physical activity, obesity, and abiding by the assumptions in O, determined from the length tension relationship at which the maximal force is obtained). LO is crucial in studies of muscle metabolism since any deviation can vastly influence the metabolic demand [Muscle metabolism has been extensively studied in rat models ,139,140 c demand . QuantifMany animal models of type 2 diabetes are used to study various organ systems and their link to the disease. For the purposes of brevity, this review focused on type 2 diabetes skeletal muscle mitochondrial function, and the studies discussed are confined to two of the most commonly used models including the Zucker Diabetic Fatty (ZDF) and Goto-Kakizaki (GK) rat models. For reference, The Zucker diabetic fatty (ZDF) rat is perhaps the most widely used rat model for the study of the impact of type 2 diabetes. ZDF rats were developed through selective breeding of obese Zucker rats (OZR) showing the greatest increase in the severity of insulin resistance and poor glycemic control with time . OZR manThe OZR develop metabolic disease along a very predictable time course spanning many weeks. It begins with rapid increases in body mass until they are overweight/obese and gradually worsening glycemic control culminating in the development of a moderate level of hypertension  and the 31PMRS [Skeletal muscle mitochondrial function quantified in vitro in the ZDF rat shows no difference in mitochondrial function. Mitochondria have been isolated from the skeletal muscles of ZDF rat tibialis anterior ,150,151 ZDF rat mitochondrial function in vivo studies have reported inconsistent results. Wessels et al.  described slowed skeletal muscle PCr recovery rates compared to age-matched lean controls and reported mitochondrial dysfunction in two separate studies ,151. MeaThe Goto-Kakizaki (GK) rat is the most widely used non-obese model of type 2 diabetes . The spoMitochondrial function in vitro in GK rat skeletal muscle shows findings are consistent with both human T2D and ZDF rat literature and shows no evidence of mitochondrial dysfunction. Skeletal muscle isolated mitochondria in the GK rat include data from both quadriceps muscles ,167 and 31PMRS in the GK rat with the qualitatively consistent conclusion that mitochondrial function is not impaired [Skeletal muscle mitochondrial function in vivo has been measured by impaired ,168,169.impaired ,73,79. T31PMRS was measured from the gastrocnemius muscle using a custom built NMR probe [31PMRS and are often not an integral part of the experimental protocols. Although no differences were seen, 3.3 Hz is well beyond the MOP capacity of the hindlimb for twitch stimulation  reported normal mitochondrial function in the GK rat with similar PCr contractile steady states and recovery time constants compared to Wistar controls . Rats weMR probe . PCr kin as 1 Hz ) and mak as 1 Hz ,73. To t as 1 Hz ,53,62,70In a separate study, PCr kinetics were measured in the GK rat and its Wistar control at several contractile intensities in the aerobic range   of ATP hydrolysis drive the mitochondrial respiration. ATP demand is described along the x-axis in en place .2 limited” zone ATP production would become oxygen dependent. This would present in vivo with increased PCr hydrolysis and slowed PCr recovery time constant but without any mitochondrial dysfunction.If oxygen supply is limited enough for a given ATPase rate to fall within the grey “Metabolic Phenotype” zone, ATP production would not be limited but would result in a reduced phosphorylation potential. This means that more ADP would be present and more PCr would be hydrolyzed within this zone as a result of the reduced oxygen availability. Thus, although ATP production is not limited, this condition will present with a reduction in ADP sensitivity and mimic the result observed from changes in mitochondrial fractional volume . FinallyApplication of this conceptual framework demonstrates why therapeutic techniques that increase the mitochondrial fractional volume shift the sensitivity to ADP by changing the oxygen consumed per mitochondria . In pati2 was chronically measured, while also systematically reducing either the blood flow or arterial oxygen content. Utilizing this technique, Stainsby et al. demonstrated that VO2 remains unaltered in contracting skeletal muscle until oxygen supply is limited by either reduction in blood flow or by reduction in oxygen content at the same blood flow rates. A similar experiment was designed utilizing the concept in 31PMRS was utilized to determine the critical point for ATP production prior to, during, and after inflatable cuff occlusion and measuring oxygen limitations by 1H measures of myoglobin desaturation [31PMRS and blood flow by doppler ultrasound. Blood flow was restricted in a stepwise fashion by cuff occlusion and the resulting changes in mitochondrial ATP production rates because of these reductions in blood flow were quantified. While these experiments were performed with another goal, these authors illustrated an experimental design that can be used to define the critical point in human subjects subjected to modest workloads.The critical point where aerobic respiration becomes limited by oxygen supply was demonstrated many years ago by Stainsby et al. in 1964 using a canine gastrocnemius-plantaris muscle in situ . In thisturation ,186. Worturation  measurin31PMRS [2). When breathing normal air (~21% O2), diabetics demonstrated reduced MOP capacity compared to controls and this reduction was alleviated when breathing 100% O2 [2 limited” zones respectively.Cree-Green and colleagues showed that oxygen could be limiting MOP capacity in type 2 diabetes patients utilizing 31PMRS . PCr dyn 100% O2 . This te 100% O2  and Rich 100% O2 ,191,192. 100% O2 ,190, 2)  100% O2 ,189. Thequantitatively determined if oxygen is in fact limiting ATP production. This must be performed by concurrent measurement of mitochondrial fractional volume, ATP production, ATP demand, and oxygen supply to respiring muscle. To illustrate this point we present an example of such an experiment in 31PMRS together with measures of blood flow using 1H-based phase contrast angiography. In Panels D-F, as contractile intensity increases toward the aerobic capacity of rat hindlimb  ATP demuenching . Other muenching ) are impReview of available data suggests that in type 2 diabetes any deficit in MOP observed in vivo is likely vascular in nature provided there is proper matching of control subjects with their diabetic counterparts regarding lean body mass and daily activity levels. Future experiments must be designed to quantitatively determine such limitations to guide intervention strategies. Once these techniques are clinically approved, their application will vastly improve the understanding of diseases presenting with exercise intolerance and impaired activity of daily living. Cogent understanding will improve pharmacological and therapeutic interventions toward the goal of improving quality of life in these patients.


At the same time, such a layer facilitates the possibility to switch domain wall helicity by an external electric field or even to completely change the characteristic structure of a ferroelectric Bloch wall passing through it. Thus, ferroelectric Bloch domain walls are shown to be ideal nanoscale objects with switchable properties. The reported results hint towards the possibility to exploit ferroelectric domain wall interaction with simple nanoscale devices.The phase-field simulations of ferroelectric Bloch domain walls in BaTiO In other words, a sufficiently thin paraelectric layer is effectively polarized by the neighboring ferroelectric material. However, little is known about robustness of the inner polarization, present within the nanoscale thickness of ferroelectric Bloch walls [Nanometer-scale mixtures of paraelectric and ferroelectric materials in disordered solid solutions or in very fine artificial crystalline superlattices often show qualitatively similar domain phenomena as the parent ferroelectric materials. For example, in the case of BaTiOterfaces –5. In gech walls –7. For e3–SrTiO3 crystalline superlattice, formed by thin SrTiO3 paraelectric layers of 0.5–3 nm thickness, separated by about 13 nm thick BaTiO3 ferroelectric slabs . Phase-field simulations for pure BaTiO3 single crystal and for the BaTiO3–SrTiO3 crystalline superlattice at stress-free mechanical conditions were performed using the phase-field simulation code ferrodo [Pt values, as in Pt values in the two domain states had the same magnitude and sign.The BaTiOthose of , except  ferrodo . The app ferrodo . The ini


KRAS mutations are common in colorectal cancer (CRC). In this setting, mutation status determination in circulating-free DNA from blood samples (liquid biopsy) has been shown to be a viable alternative to tissue testing. The objective of this study was to compare the sensitivity of two liquid biopsy methods for detecting KRAS mutations in plasma samples from metastatic CRC patients. Samples with a positive (KRAS-MUT+) result and a mutant allelic fraction (MAF) < 5% according to the OncoBEAM RAS CRC assay were pairly analyzed by the Idylla ctKRAS Mutation Test (n = 116). In a cohort of 71 patients with at least 1 year of follow-up, the progression-free survival (PFS) was determined according to MAF values. Idylla detected KRAS mutations in 81/116 OncoBEAM KRAS-MUT+ samples with MAF < 5% and in 48/79 samples with MAF < 1%. Concordance between OncoBEAM and Idylla significantly improved at higher MAF values. PFS rates at 6 and 12 months tended to be lower in patients with MAF levels between 1% and 5% than in those with levels <1%. OncoBEAM demonstrated greater sensitivity for plasma detection of KRAS mutations than Idylla. Importantly, our data identified a “gray zone” below 1% MAF where Idylla showed reduced KRAS mutation detection, highlighting the importance of an accurate method to provide the mutational status of CRC patients. RAS) gene family, including the kirsten RAS (KRAS) and neuroblastoma RAS (NRAS) proto-oncogenes, to exclude RAS mutation-positive metastatic colorectal cancer (mCRC) patients from receiving anti-epidermal growth factor receptor (EGFR) therapy2. This is because anti-EGFR agents do not provide meaningful survival benefits versus anti-angiogenic/chemotherapy regimens in mCRC patients whose tumors are not wild type (WT) with respect to RAS genes6. Accurate detection of RAS mutations in these patients is therefore of high clinical importance for therapy selection. Traditionally, formalin-fixed paraffin-embedded (FFPE) tumor samples have been used to determine the RAS mutation status of CRC patients in routine clinical practice. However, RAS status determination in circulating-free DNA from blood samples (known as liquid biopsy) has been shown to be a viable alternative to FFPE sample testing11. The use of liquid biopsy RAS mutation tests that lack the necessary sensitivity may lead to mischaracterization of mCRC patients as WT and in turn may diminish patient response to anti-EGFR therapy and lead to worse outcomes. Therefore, a direct comparison of available methods for RAS mutation status from liquid biopsy samples is warranted in order to evaluate the relative performance during routine clinical practice.International guidelines recommend testing for hotspot mutations in the rat sarcoma viral oncogene homolog  technologies inherently provide greater sensitivity than quantitative PCR (qPCR) techniques for somatic mutation detection13. The Idylla ctKRAS Mutation Test is a qPCR assay that has an analytical sensitivity of ≤1% for KRAS mutations in exons 2 and 3 and ≤5% for mutations in exon 414. The OncoBEAM RAS CRC15 is a dPCR assay that detects mutations in both KRAS and NRAS oncogenes with an analytical sensitivity determined to be <0.02% mutant allelic fraction (MAF)16. The main goal of the present study was to evaluate the sensitivity of these two liquid biopsy methods for detecting KRAS mutations in plasma samples from mCRC patients, both of which detect mutations in codons 12, 13, 59, 61, 117 and 146 of the KRAS gene. In addition, we examined the clinical sensitivity of Idylla and OncoBEAM plasma KRAS mutation detection at <1% MAF by comparing plasma mutational analysis obtained by both platforms to those obtained by standard-of-care (SOC) FFPE tumor RAS testing on paired primary tumor tissue specimens.Several different platforms are available to perform blood-based KRAS testing results on primary tumor specimens were only available for 43 KRAS-MUT+ patients with MAF values < 1% at the hospital centers performing OncoBEAM testing  and the majority of them (97.3%) had colorectal adenocarcinoma. The most frequent site of metastasis was the liver (74.6%) followed by the lung (33.8%). Among the 559 plasma samples tested with the OncoBEAM RAS CRC assay, 265 (47.4%) were KRAS-MUT+, of which 147 (55.5%) had MAF < 5%. The 1-ml plasma requirement for Idylla testing could only be met for 116 out of the 147 samples, so the comparative analysis included only these 116 patients that were analyzed by the two liquid biopsy methods. SOC FFPE KRAS mutations in 81 out of 116 OncoBEAM KRAS-MUT+ samples with MAF <5%, resulting in a PPA of 69.8%. Idylla detected KRAS mutations in 48 out of 79 OncoBEAM KRAS-MUT+ samples with MAF <1%, with a PPA of 60.7%. Concordance between OncoBEAM and Idylla significantly improved at higher MAF values.The proportions of mutated samples at 3 different OncoBEAM-determined MAF% value ranges are shown in Table PFS rates at 6 and 12 months were lower in patients with MAF levels between 1% and 5% than in those with levels <1%  of OncoBEAM vs SOC FFPE KRAS testing results was 31/43 or 72.1%, whereas concordance of Idylla with SOC FFPE KRAS results was 20/43 or 46.5% (p = 0.0275). Idylla ctKRAS testing showed WT results in 14 out of 31 patients that were called KRAS-MUT+ by both OncoBEAM and SOC. Eight out of these 14 discordant cases were seen in patients with liver metastases  and MAF values were <5%, in order to examine the clinical sensibility at lower MAF. Patients with mCRC have been shown to exhibit a range of MAF values across newly diagnosed patients with primary tumor intact and those presenting with recurrent disease18. In cases with MAF < 1% by OncoBEAM, plasma mutational analysis was also compared to tissue-based methods on paired primary tumor specimens in order to independently examine the clinical sensitivity of Idylla and OncoBEAM plasma KRAS mutation detection.The accurate detection of hotspot mutations in the KRAS mutation frequency of 47.4% as determined by the OncoBEAM kit is in agreement with previously published reports of KRAS mutations in mCRC patients19. Overall, Idylla detected mutations in 69.8% of OncoBEAM KRAS-MUT+ samples, with higher concordance between the two methods at higher MAF levels. OncoBEAM has been shown to detect mutations as low as 0.02% MAF16, whereas the reported Idylla KRAS detection sensitivity is ≤1% MAF14. This means that most of the samples with low MAF analyzed by the Idylla ctKRAS kit will result in false-negative KRAS WT results and patients would inappropriately receive anti-EGFR therapy. In this scenario, the use of Idylla may incorrectly select patients for anti-EGFR therapy, exposing patients to undue side effects, increased medical costs, and worse outcomes due to lack of demonstrated clinical benefit of anti-EGFR therapy in patients with RAS mutations. Accordingly, the testing with Idylla ctKRAS resulted in a 16.9% reduction in clinical sensitivity for KRAS mutation detection vs SOC FFPE and OncoBEAM plasma testing. Since 37.4% of the 559 mCRC patients in this study were KRAS-MUT+ <1% MAF using OncoBEAM, it is estimated that a 16.9% drop in clinical sensitivity by Idylla might result in false negative calls for 45 out of 559 patients in this study. Even in cases of liver metastases, supposed to have greater shedding properties due to high vascularization20, Idylla was not able to detect KRAS mutations in 8 of 22 patients with tissue mutated status.The KRAS WT status in 8–11.6% of patients22. When inter-tumor heterogeneity between primary tumors and metastases is examined there is frequent mutational discordance24, and tumor genomic evolution likely occurring between tissue and blood sampling can also contribute to discordant results. Thus, the use of liquid biopsy methods is a valuable alternative to tissue-based testing to identify patients eligible for anti-EGFR therapy in routine clinical practice, avoiding the sample bias associated with intra- and inter-tumor heterogeneity. In addition, serial circulating tumor DNA (ctDNA) analysis enables monitoring of genomic changes during therapy and profiling of the global status of genomic alterations across different sites of disease25.While OncoBEAM exhibited superior clinical sensitivity when compared to Idylla on replicate samples across MAF values, 12 out of 43 patients that were KRAS-MUT+ by OncoBEAM at <1% MAF were WT by SOC FFPE tissue testing. Our initial suspicion was that these low MAF samples might be false positives determined by OncoBEAM, but 9 out of 12 were also determined to be KRAS-MUT+ by Idylla. Although these data represent a small subset, it lends support to the conclusion that some of these patients may in fact be KRAS-MUT+ and were missed by SOC tissue testing either due to molecular heterogeneity or an insufficiency of the tissue mutation method. Testing of DNA from a single primary tumor tissue block may wrongly assign RAS mutation status is now vital component of routine standard of care workup for newly diagnosed mCRC patients, which includes hotspots regions of both KRAS and NRAS genes26. Since approximately 50% of the tested samples will return a WT KRAS result, these samples should also be tested for NRAS mutations. Therefore, it is valuable an extended RAS test to detect not only KRAS mutations but also NRAS mutations. The OncoBEAM RAS CRC kit evaluates both KRAS and NRAS mutations concurrently whereas Idylla testing for KRAS and NRAS is split into two different test runs to be performed sequentially. For both methods, the incorporation of the RAS mutational status in the initial histological report is now possible, enabling rapid initiation of targeted therapy and global assessment of tumor mutation status in newly diagnosed metastatic patients. An advantage of the BEAMing technology is its quantitative nature and high sensitivity. It has been shown that high RAS ctDNA MAF is associated with low survival27, so ctDNA levels as reflection of tumor load could provide valuable information to predict the disease evolution in RAS mutant patients prior to, during, or following treatment29.Testing for extended KRAS mutations (MAF ≥ 1%) had a shorter PFS compared with those carrying low abundant KRAS mutations (MAF < 1%)19. Moreover, rapid progressors in our study had significantly higher MAF than slower progressors. This information can be readily incorporated to inform the clinician of those patients that may be “high risk” and require more intense treatment and radiological follow-up. As ctDNA can reveal occult metastatic disease that is not evident on radiological imaging30, we are now able to identify CRC patients at high risk of metastatic relapse and/or local recurrence, which is consistent with studies showing that ctDNA can signal residual disease after surgical resection32. It is unlikely that a single time-point following surgery will identify all patients who are going to relapse, but longitudinal sampling may resolve this issue and improve the sensitivity30. Moreover, WT patients that do receive anti-EGFR therapy can be monitored during treatment to detect acquired resistance due to RAS mutation emergence33.This concept of MAF as a prognostic tool is consistent with our prior investigations and supported by the observation that patients with higher abundant KRAS mutations, while we are now at the point where the technology should detect all RAS mutations beyond KRAS mutations, which is not the case for this Idylla kit. Finally, since there were no wild-type samples there was no evaluation of specificity, out of the scope of the study.Certain limitations exist in our study. One is the retrospective nature of the study and the lack of clinical information in a substantial proportion of patients, including the treatment they received and its potential effect on MAF values. Second, the small number of available tissue samples hindered a consistent comparison of concordance between plasma-based and tissue-based methods. Third, our data were limited to 34, highly sensitive and specific detection methods are required for ctDNA to deliver a clinically feasible and accurate approach. In this study, OncoBEAM demonstrated greater sensitivity for plasma detection of KRAS mutations than Idylla. Importantly, our data identified a “gray zone” below 1% MAF where Idylla shows reduced KRAS mutation detection accuracy vs OncoBEAM and SOC FFPE tumor KRAS testing. These findings serve as a reminder that liquid biopsy assays with diminished sensitivity may lack the dynamic range to provide the accurate mutational status to properly guide individualized anti-EGFR treatment decisions and risk stratification that may benefit patient outcomes.Given that the frequency of mutant DNA alleles in cell-free DNA is as low as 0.01%This was a multicenter, retrospective study performed in 4 Spanish centers from April 2016 to November 2017. The study was approved by the Institutional Review Board at each hospital  and was conducted in accordance with the principles of the Declaration of Helsinki. Prior to the analysis, all patients signed informed consent accepting the use of their blood samples stored in the Biobank for research purposes. Data from adult patients with a diagnosis of mCRC were included in the analysis if KRAS mutation status was determined in plasma both by OncoBEAM and Idylla kits and OncoBEAM KRAS-MUT positive results were <5% MAF. Patients having surgery of primary tumor or metastasectomy or that received chemotherapy/biologic agents <2 months prior the blood draw were excluded.KRAS mutant allele fraction was determined according to the kit’s instructions for use in those samples positive for KRAS mutation (KRAS-MUT+). Idylla testing was performed following the instructions and positive percent agreement (PPA) of KRAS-MUT+ results obtained by OncoBEAM and Idylla was calculated for patient samples having KRAS mutations at <5%, <1%, and <0.1% MAF by OncoBEAM. Due to the retrospective nature of the study, patient outcomes were available for a subset of patients and we determined the progression free survival (PFS) according to MAF values (1–5% vs <1%) in a cohort of 71 patients with at least 1 year of follow-up.Blood was collected in either Streck BCT or EDTA tubes and processed into plasma using methods suitable for testing by both the OncoBEAM RAS CRC assay (3 mL) and the Idylla ctKRAS test (1 mL). Plasma samples were tested with OncoBEAM RAS CRC and Finally, the clinical sensitivity of Idylla and OncoBEAM was evaluated by comparing plasma mutational analysis obtained by both methods on replicate samples to those obtained by standard-of-care (SOC) FFPE tumor KRAS  testing on paired primary tumor specimens of KRAS-MUT+ patients with MAF values < 1% as determined by OncoBEAM.Categorical variables were summarized in numbers and percentages, continuous variables were presented as medians, minima and maxima. Comparison of detection thresholds was performed using the exact Mantel-Haenszel test. The PPA was also calculated. Paired proportions were compared using McNemar’s test. Relationships between MAF levels and clinical pathological characteristics were assessed using the Fisher exact test and the Student t-test. Median PFS was calculated by Kaplan-Meier estimation and compared with the log‐rank test. A p-value lower than 0.05 was considered statistically significant. Statistical analyses were performed using the statistical software SAS version 9.4.


Excessive or chronic stress can lead to a variety of diseases due to aberrant activation of the glucocorticoid receptor (GR), a ligand activated transcription factor. Pregnancy represents a particular window of sensitivity in which excessive stress can have adverse outcomes, particularly on the developing fetus. Here we show maternal hepatic stress hormone responsiveness is diminished via epigenetic silencing of the glucocorticoid receptor during pregnancy. Provocatively, reinstallation of GR to hepatocytes during pregnancy by adeno-associated viral transduction dysregulates genes involved in proliferation, resulting in impaired pregnancy-induced hepatomegaly. Disruption of the maternal hepatic adaptation to pregnancy results in in utero growth restriction (IUGR). These data demonstrate pregnancy antagonizes the liver-specific effects of stress hormone signaling in the maternal compartment to ultimately support the healthy development of embryos. Matthew Quinn et al showed diminished hepatic stress responses in pregnant mice due to epigenetic-mediated decreases in glucocorticoid receptor expression. This decrease is necessary for fetal development and highlights the importance of understanding the tissue-specific effects of glucocorticoid activation in models of maternal stress. The physiological actions elicited by glucocorticoids are mediated by binding to and activating the glucocorticoid receptor (GR), a ligand-activated transcription factor. While glucocorticoids are essential to return the body to homeostasis following either a physiological or psychological stress, excessive glucocorticoid production or chronic stress is linked to an array of diseases, particularly metabolic diseases and obesity6.Glucocorticoids are steroid hormones that are synthesized and secreted under coordinated actions of the hypothalamic–pituitary–adrenal axis that gets activated in response to a variety of stressful stimuli. Once in the circulation, glucocorticoids regulate a host of physiological processes ranging from immune regulation to altering systemic metabolism10. It is well established that the environment in which the fetus develops can have a long-lasting impact on the health of the offspring post parturition. This is known as the developmental origins of the health and disease hypothesis11. While a variety of factors contribute to this phenomenon, maternal stress has been identified as key in driving fetal programming eliciting poor health outcomes in the offspring ranging from neuropsychiatric disease17 to cardiovascular disease21 and metabolic syndrome28. The effects of maternal stress have been attributed to aberrant activation of the glucocorticoid receptor (GR)34. The physiological mechanisms in which mothers combat maternal stress to avoid these deleterious effects of pathogenic GR signaling during pregnancy have yet to be fully realized.The biological response to pregnancy is systemic with almost every organ of the body adapting, with the primary goal to support the developing fetus. The liver for example, a mitotically dormant organ in adults, reenters the cell cycle to elicit a pregnancy-induced growth response40. Furthermore, we have recently demonstrated that loss of reproductive axis activity through ovariectomy promotes glucocorticoid hypersensitivity and a Cushing-like syndrome resulting in the development of obesity, hyperglycemia, and hepatic steatosis41, extending this antagonistic relationship beyond the reproductive tract into metabolic tissues. These findings lay a potential physiological foundation for reproductive axis activation in females, limiting the stress response to avoid deleterious effects of stress during pregnancy.Previous studies have established an antagonistic relationship between female sex hormones and stress hormone responsiveness in a variety of reproductive tissues34. This has led to a lack of understanding of how the maternal compartment regulates the stress response in various tissues and how these tissues contribute to the maladaptive response to stress in utero. Here, we show that during normal pregnancy, hepatic stress hormone responsiveness is dramatically reduced in mice. The decreased glucocorticoid sensitivity seen during pregnancy is a product of reduced GR expression elicited by epigenetic silencing. Furthermore, we found that downregulation of maternal hepatic GR expression is essential to allow for pregnancy-induced liver growth. This was evidenced by the finding that GR reinstallation specifically to hepatocytes blunts pregnancy-induced hepatomegaly resulting in smaller livers. Last and unexpectedly, maternal hepatic GR reinstallation resulted in in utero growth restriction of the fetuses. Our findings have uncovered a role of GR silencing in the maternal liver to allow for the physiological hepatic response to pregnancy and thus appropriate fetal development. The data presented herein demonstrate how aberrant hepatic glucocorticoid signaling can facilitate intrauterine growth restriction (IUGR). Further investigation into how the stress hormone response is regulated in other tissue systems during pregnancy is essential in understanding how maternal stress interferes with the systemic adaptation to pregnancy.The effects of GR activation during pregnancy in a tissue-specific fashion have largely been overlooked, as models of maternal stress rely upon systemic administration of glucocorticoids41 (GSE99309) uncovered ~40% (335 genes) of the hepatic pregnancy-regulated genes overlaps with the stress hormone transcriptome in virgin females. Assessing the fold change elicited by pregnancy to the fold change elicited by stress hormone administration in virgin females revealed nearly 70% of commonly regulated genes to be altered in an opposite direction (black box)  Fig. . Furtherox) Fig. , suggestox) Fig. . Last, pox) Fig. , definin42. We hypothesized that pregnancy-induced hepatic glucocorticoid resistance is due to the reduction in receptor expression. To support this idea, we observed a decrease in both hepatic GR protein and mRNA in pregnant mice compared with virgin mice  transcripts in pregnant mice   under the human alpha-1 antitrypsin promoter as previously describedFP) Fig. . Transcrion Fig. . Our Nanion Fig. . The dision Fig. . Collect53. Therefore, we initially examined if activation of maternal hepatic GR promotes placental dysfunction. Maternal hepatic stress hormone signaling did not elicit overt gross placental changes, as evidenced by comparable placental weights at 14.5 dpc between AAV-GFP- and AAV-GR-injected mice  B2) Fig. , suggestB2) Fig. .Fig. 3HeThe increased nucleated red blood cells in the placenta coupled to increased fetal corticosterone levels prompted us to investigate the effects of maternal hepatic GR reinstallation on the fetuses in utero. Pregnant mice that have hepatic GR reinstalled display a delay in weight gain compared with pregnant controls Fig. . Of noteIn the current study, we aimed to determine the effects of pregnancy on maternal hepatic glucocorticoid signaling. First, we found that the pregnancy-regulated hepatic transcriptome shows a large overlap with the glucocorticoid-regulated transcriptome. Of the intersecting transcriptomes, a vast majority of the genes and biological pathways are regulated in the opposite direction. This led us to hypothesize that pregnancy leads to a state of hepatic glucocorticoid insensitivity. To this end, we uncovered that under normal physiological conditions, pregnancy leads to a decrease in hepatic GR expression, which ultimately promotes decreased glucocorticoid responsiveness. By restoring hepatic GR expression during pregnancy with the use of AAV-mediated viral transduction, we uncovered that one of the physiological consequences of decreased maternal hepatic GR signaling is to allow for pregnancy-induced hepatomegaly to ensue. This was evidenced by the fact that GR reinstallation to the liver during pregnancy led to dysregulation of a cohort of genes involved in cell-cycle regulation. The dysregulated gene expression profile ultimately resulted in impaired hepatic proliferation and decreased liver weights in pregnant mice that have hepatic GR reinstalled. Finally, we discovered that hepatic GR downregulation is essential for normal embryonic development. Embryos whose mothers received AAV-GR displayed altered placental expression of 11β-HSD2 and had higher fetal corticosterone levels compared with pregnant AAV-GFP controls. Furthermore, mothers receiving AAV encoding for GR had delayed weight gain during the course of gestation due to decreased embryo weights. In sum, we have uncovered a pathogenic role for hepatic GR signaling in disrupting the hepatic adaptation to pregnancy and ultimately promoting intrauterine fetal growth restriction.55. One way the mother compensates for the hyperglucocorticoidism observed during pregnancy is through the increased production of corticosteroid-binding globulin (CBG), a protein which binds circulating glucocorticoids rendering them biologically inactive56. While CBG increases at a comparable rate to glucocorticoids during pregnancy, high levels of sex hormones, such as progesterone, can displace glucocorticoid binding to CBG, rendering them biologically active in the circulation57. The physiological importance of active stress hormones during pregnancy is highlighted by a recent report by Whirledge et al. showing that uterine-specific GR knockout mice display impaired implantation and decidualization, which ultimately leads to a sub-fertile phenotype58. While the uterine effects of glucocorticoids are now appreciated, nothing was known about the effects of stress hormone signaling, specifically in the liver during pregnancy. We found that unlike the uterus, the liver requires silencing of GR for both the maternal adaptation to pregnancy as well as fetal development. We speculate that the physiological downregulation of hepatic GR during pregnancy is a compensatory mechanism to avoid pathogenic glucocorticoid signaling in the liver during the Cushing-like state observed throughout pregnancy. One reason for this is that the liver needs to undergo growth to support the metabolic demand of pregnancy. Due to the increased proliferation in the liver during pregnancy, liver regeneration is actually restored in rodents in response to pregnancy9. In rats undergoing partial hepatectomy, glucocorticoid administration inhibits the regenerative process59. By downregulating hepatic GR during gestation, pregnancy is able to elicit liver growth and enhance the regenerative process. Further studies aimed at evaluating the regenerative response in the liver during pregnancy in the context of partial hepatectomy following GR reinstallation are needed. Another pathogenic effect of excessive GR activation is the onset of cholestatic liver disease, a disorder of excessive bile acid production61. While not investigated in the current study, aberrant maternal hepatic glucocorticoid signaling could potentially underlie the idiopathic nature of cholestasis of pregnancy and warrants further investigation.Pregnancy is characterized by enhanced systemic production of glucocorticoids in both humans and rodents41. Given the sensitizing effects of FSH on hepatic GR activation, it is possible that the low levels of circulating FSH during pregnancy could account, at least in part, to the diminished stress hormone responsiveness in the maternal liver and warrant further investigation. As an alternative, methylation is an epigenetic means by which GR expression can be controlled. We show that hypomethylation of the NR3C1 CGI shore and CGI and subsequent enhanced YY1 co-repressor recruitment contribute to hepatic GR downregulation in gravid mice   can increase hepatic glucocorticoid sensitivity through enhancing ligand-dependent phosphorylation of GR, which ultimately promotes the development of fatty liver disease49. Furthermore, in a sheep model of maternal stress, glucocorticoid administration leads to cell death of the placenta50. Our results extend beyond the attributed role of stress hormones in decreasing placental function to promote intrauterine growth restriction and show that hepatic stress hormone signaling can also have detrimental effects in the onset of fetal growth restriction. Further studies focused on the long-lasting effects of our model of fetal growth restriction post parturition are essential, as in utero growth restriction has been shown to lead to diseases in the offspring extending into adulthood64.Maternal stress is a well-established factor promoting fetal growth restriction. The classical role of maternal stress that elicits intrauterine growth restriction is through the alteration of placental function. For example, excessive maternal glucocorticoids can impair trophoblast function, leading to decreased proliferation, migration, and invasion, ultimately culminating in pre-eclampsia-like symptoms in ratsDespite a long-standing relationship between maternal stress and detrimental fetal outcomes, the molecular mechanisms and tissue-specific effects elicited by stress during pregnancy have largely remained elusive. Our present study in conjunction with previous reports reveal a precarious role of stress hormone signaling in different tissues during pregnancy in the regulation of embryo development. Understanding the tissues that require GR activation versus tissues with which glucocorticoid signaling needs to be silenced will be instrumental in uncovering the physiological and pathophysiological role of stress hormones during development.NR3C1. Vector DNA was packaged into AAV9 particles by use of triple-plasmid transfection of HEK293 cells65. Plasmids were purified by polyethylene glycol precipitation followed by CsCl centrifugation66. The titers of the purified viral stocks were determined by DNA dot blots as viral genomes (vg) per milliliter.Recombinant hAAT promoter-driven mouse GRα or green fluorescent protein (GFP) AAV vectors were constructed utilizing standard cloning methods with the coding sequence of mouse 11vg in 25 μl via retro-orbital venous sinus injection. All animal experiments were performed in at least two independent cohorts of animals to ensure reproducibility. All animal experiments were approved and performed in accordance with the Institutional Animal Care and Use Committee at the National Institute of Environmental Health Sciences, NIH.Female C57BL/6J mice randomized for experiments and male C57BL/6J mice were used as breeders for experiments  and used between 12 and 14 weeks of age. All experiments of mice had ad libitum access to standard mouse chow and drinking water. Mice were exposed to a 12:12-h light/dark cycle. For pregnancy studies, male and female mice were set up for timed mating and the presence of a vaginal plug was marked at 0.5 dpc. Male mice were removed from the cage at 0.5 dpc and female mice were euthanized at either 14.5 dpc or PND1. Female mice not pregnant at 14.5 dpc were removed from the study. Glucocorticoid sensitivity was assessed in the livers of female mice at 14.5 dpc via injection of dexamethasone  . Four hours post I.P. injection, mice were killed and the livers were harvested. To reinstall GR during pregnancy, plugged mice were injected with 1 × 10® Mouse Exon 1.0ST Array . Briefly, 100 ng  of the total RNA was amplified and labeled with the Affymetrix WT Plus Reagent Kit. Five-and-a-half micrograms of amplified biotin–cDNAs were fragmented and hybridized to each array for 16 h at 45 °C in a rotating hybridization oven. Array slides were stained with streptavidin/PE utilizing a double antibody staining procedure, and washed with the Wash and Stain kit as per the manufacturer’s protocol FS450-0001. Slides were scanned in an Affymetrix Scanner 3000 and data were obtained using TAC software. The resulting data were processed using OmicSoft Array Studio . Differentially expressed genes were detected with Partek Genomic Suite software and considered significantly different if they had p < 0.05 via ANOVA and passed a Benjamini–Hochberg multiple test correction for false positives with a q < 0.05. Heatmaps were generated with either R software package (version 3.3.3) or Partek Genomic Suite software.RNA was harvested from the livers of female virgins and mice pregnant at 14.5 dpc and used for whole-transcriptome analysis via Affymetrix GeneChipNR3C1 RNA was measured with primer/probes spanning either intron 1  or the exon 2/intron 2 junction . The relative fluorescent signal of the gene of interest was normalized to PPIB using the 2−ΔΔCT method.One-hundred nanograms of the total RNA were reverse-transcribed and amplified according to the manufacturer’s instructions for the iScript One-Step RT-PCR kit for probes . Quantitative real-time PCR (qPCR) was performed with the Biorad CFX96 sequence detection system using commercially available primer/probe sets . Nascent Glucocorticoid receptor protein expression was detected in liver lysates homogenized in SDS-sample buffer (Biorad) containing β-mercaptoethanol . Thirty micrograms of protein were separated on 4–20% TGX gel (Biorad) and transferred onto a 0.2 μM nitrocellulose membrane (Biorad). Membranes were blocked with Licor blocking buffer  followed by primary antibody incubation with anti-monoclonal glucocorticoid receptor antibody (1:1000)  or anti-β-actin (1:5000) . Protein was detected with fluorescent secondary antibody detection  (Licor) and imaged on the Licor Odyssey (Licor). Densitometry was measured with the Odyssey software, and protein loading was normalized with β-actin. Uncropped images of western blots presented in the article can be found in Supplementary Figure 67. Briefly, 50 ng of the total RNA was hybridized at 65 °C for 20 h, and absolute RNA counts were measured with the nCounter as per the manufacturer’s protocol (Nanostring Technologies). Data were normalized to spiked-in positive and negative controls. Data were further normalized to GAPDH and reported as fold change over virgin (±SEM).To measure mRNA expression, we performed Nanostring analysis from livers of virgins and 14.5 dpc pregnant mice injected with either AAV-GFP or AAV-GR using the PanCancer Pathway codeset  as previously describedTwo observers blindly counted the total observed mitotic figures in 20-x fields of hematoxylin- and eosin-stained slides. Counts were made at a multi-head microscope, and a determination of mitosis was made only if there was concordance between the observers. A mitotic index was calculated by taking the average mitotic number in 20 fields.Proliferating hepatocytes were confirmed with immunofluorescent staining of Ki67. Briefly, 4 μM frozen liver sections from 14.5 dpc pregnant AAV-GFP- and AAV-GR-injected mice were incubated with anti-Ki67 (1:200) (Abcam) overnight at 4 °C. Ki67+ staining was detected with Alexa-Fluor 594 secondary antibody (1:400) . Nuclei were counterstained with DAPI, and Ki67+ nuclei were visualized with the Zeiss AxioD Observer Epifluorescent microscope . Ki67+ nuclei were quantified by counting the number of Ki67+ nuclei per field.Circulating maternal corticosterone from maternal blood samples was collected via submandibular bleed at ZT3 colorimetric corticosterone EIA according to the manufacturer’s instructions . Whole embryos were homogenized, and the supernatants were used to measure fetal corticosterone with a colorimetric corticosterone EIA kit (Arbor Assays).T test when comparing two groups . A normality test was performed to determine distribution, and when failing, a Mann–Whitney post hoc test was performed. When comparing three or more groups, a two-way ANOVA was performed, followed by a Sidak post hoc test for multiple comparisons. Statistical significance was defined as p < 0.05 unless otherwise stated. Data are mean ± SEM (Prism 7).Statistical significance was determined by using a two-sided Student’s Further information on experimental design is available in the Reporting SummarySupplementary Information