Zika virus (ZIKV) emerged suddenly in the Americas in 2015 and was associated with a widespread outbreak of microcephaly and other severe congenital abnormalities in infants born to mothers infected during pregnancy. Vertical transmission of ZIKV in humans was confirmed when viral RNA was detected in fetal and placental tissues, and this outcome has been recapitulated experimentally in animals. Unlike other flaviviruses, ZIKV is both arthropod- and sexually-transmitted, and has a broad tissue tropism in humans, including multiple tissues of the reproductive tract. The threats posed by ZIKV have prompted the development of multiple in vivo models to better understand the pathogenesis of ZIKV, particularly during pregnancy. Here, we review the progress on animal models of ZIKV infection during pregnancy. These studies have generated a foundation of insights into the biology of ZIKV, and provide a means for evaluating vaccines and therapeutics. Flaviviridae family. ZIKV was first isolated from a rhesus macaque in the Zika Forest of Uganda in 1947 [Zika virus (ZIKV) is a mosquito-transmitted, positive-sense RNA virus in the  in 1947  and subs in 1947 , the epi in 1947 . During  in 1947 .Thousands of infants in the Americas have been born with congenital ZIKV syndrome (CZS), which will impair their future neurodevelopmental function . CZS is A correlation has been seen between the trimester of exposure to ZIKV and fetal outcome. In one study in Brazil, 55% of first trimester infections resulted in fetal abnormalities compared to only 29% during the third trimester . In the In response to the ZIKV pandemic and the outbreak of congenital disease, animal models were developed to study ZIKV pathogenesis and evaluate countermeasures, including new vaccines and therapies. The ability to control the infection route and gestational age has allowed researchers to confirm the teratogenic effects of ZIKV infection and study both host and viral factors associated with vertical transmission and neuropathogenesis.Mice are the most commonly used animals to model viral infections in humans. Their low cost, short breeding times, large litters, and ease of handling facilitate their widespread use. They often recapitulate important aspects of human disease, including after viral and bacterial infections ,14,15. MIn the context of pregnancy, there are advantages and disadvantages for using mice to study human infection and disease. Specific breeding schemes with knockout mice allow for the study of the function of individual genes by restricting their expression to the maternal or fetal side of the placenta. However, mice have a much shorter gestation period compared to humans, only 20 days, and the structure of their placenta differs . Even soIfnar1−/−) or administered a blocking anti-type I IFN receptor antibody (MAR1-5A3) (Ifnar1−/− dams were crossed to WT sires and then inoculated with ZIKV. Subcutaneous infections with ZIKV (French Polynesia strain) in these models on embryo day (E)6.5 and harvested seven days later on E13.5 showed the vertical transmission of ZIKV to the placenta and fetal heads [Ifnar1−/− dams. Intravaginal inoculation of Ifnar1−/− dams mated to WT C57BL/6 sires at E4.5 and E8.5 also led to fetal resorption and viral replication in the placenta and fetus on E18.5 [Peripheral challenge of adult wild-type (WT) C57BL/6 mice with ZIKV resulted in little virus replication and no disease . One reaAR1-5A3) . For veral heads . Decreason E18.5 . Examinaon E18.5 ,23.Ifnlr1−/− dams crossed to WT C57BL/6 or Ifnlr1−/− sires. Ifnlr1−/− dams were treated with an anti-Ifnar1 antibody and then inoculated with ZIKV on E12. Whereas Ifnlr1−/− × Ifnlr1−/− matings resulted in an increase in viral infection in the placenta and fetal heads, Ifnlr1−/− dam × WT C57BL/6 sire matings had a similar viral burden to WT C57BL/6 mice. This data suggested that the feto-placental unit had to be fully deficient in type III IFN signaling [Ifnlr1+/− dam × Ifnlr1−/− sire cross) was not tested. Treatment with pegylated mouse IFN-λ2 at E12 but not E6 reduced the ZIKV burden in the placenta and fetal heads late in gestation. Subsequent studies showed that the protective effect of IFN-λ2 required a developed placenta, which occurs by E10. These studies established the role of type III IFN signaling in restricting ZIKV infection in the placenta in a gestational stage-dependent manner, and have been corroborated in human tissues [The effects of type III IFNs during ZIKV infection have been evaluated in pregnancy using ignaling  to be af tissues ,54.ZIKV infection in non-pregnant and pregnant immunocompetent C57BL/6, BALB/c, Swiss Webster, or CD-1 mice  resulted in no clinical signs of disease and little viral replication ,55, presStat2 gene with the human STAT2 ortholog (hSTAT2-KI mice) [Rag1−/− mice, which lack mature B and T cells [Fetal abnormalities in these WT mouse models were only seen when non-physiological routes of infection were used, including intrauterine, intravenous, or direct injection into the amniotic fluid or uterus. In an effort to create a peripheral challenge model with a more immunocompetent mouse, one group replaced the mouse KI mice) . This suKI mice) . For the T cells . Using aIfnar1−/− dams mated to WT C57BL/6 sires yield pups that are heterozygous for Ifnar1 expression. Despite having the ability to respond to type I IFN, these fetuses were not protected from ZIKV, and many were resorbed [Ifnar1−/− × Ifnar1−/− matings), paradoxically, greater survival was observed after intravaginal inoculation on E5.5 than with fetuses that had an intact type I IFN response [Ifnar1−/− × Ifnar1−/− than Ifnar1−/− × WT C57BL/6 matings, such that the viral load did not directly correlate with the outcome. Type I IFN signaling in the placenta was shown to instigate fetal demise and may serve as a way to select against the development of fetuses that have been exposed to and sustained significant viral infections or inflammation. The effect of intact type I IFN signaling on fetal resorption was observed when WT C57BL/6 dams mated with WT C57BL/6 sires were inoculated intravenously with a Puerto Rican strain of ZIKV at E9.5. These fetuses were resorbed at day E17.5, even in the absence of viral RNA in fetal heads [In the context of the studies with type I IFN deficiency, it was found that factors other than a viral burden contribute to the fetal outcome. Type I IFN signaling in the placenta was shown to contribute independently to fetal resorption . Ifnar1−resorbed ,23. Howeresponse . Remarkaal heads .Ifnar1−/− dams mated to WT C57BL/6 sires had fetuses with reduced head circumferences and crown rump length when infected with ZIKV at E9 but not E12 [More severe fetal abnormalities correlate with the trimester in which a pregnant human is infected with ZIKV . In a la not E12 . Viral b not E12 .Ifnar1−/− mice that were mated to WT sires. Viral RNA was isolated from the placentas and fetal heads, with gestational age being a determinant of increased viral burden. Type I IFN was found to be a determinant of fetal resorption after ZIKV infection. Type III IFN-λ responses protected the placenta from ZIKV, and dams treated with pegylated IFN-λ2 had improved pregnancy outcomes. An immunocompetent hSTAT2-KI model was recently developed and also showed vertical transmission after subcutaneous ZIKV infection. Overall, mice have been used successfully to model many aspects of ZIKV infection during pregnancy and provided a way for establishing correlates of protection for ZIKV countermeasures.Mouse models provided a rapid platform for confirming the teratogenic potential of ZIKV infection. Fetal abnormalities were observed in fetuses from ZIKV infected pregnant Ifnar1−/− A129 dams that were immunized with a different live attenuated ZIKV vaccine containing deletions in the capsid protein [The epidemic of CZS in the Americas has spurred an accelerated effort by academia, the government, and industry to develop vaccines capable of preventing the potentially devastating consequences of maternal ZIKV exposure for the developing fetus. In the first description of the efficacy of ZIKV immunization against challenge during pregnancy, WT C57BL/6 dams were immunized with either live-attenuated NS1-mutant (LAV) or lipid nanoparticle-encapsulated mRNA (mRNA-LNP) vaccines . Approxi protein .b) females were primed by prior ZIKV infection and mated to allogeneic, WT BALB/c (H-2d) males [+ T cells, to maintain a state of tolerance to the fetus. The dams were given no immunosuppression during priming by ZIKV, and exhibited no signs of illness during this primary infection as a result. However, even this abortive infection was sufficient to protect fetuses after subsequent anti-Ifnar1 antibody treatment and ZIKV challenge during pregnancy (at E10 or E11), as judged by the markedly decreased ZIKV RNA burden in feto-placental units three days after challenge. Consistent with the earlier reports, a sizeable subset of feto-placental units was still positive for ZIKV RNA, despite high titers of circulating neutralizing antibody.Subsequent reports of vaccine efficacy in pregnant mice have made modifications that may provide specific advantages. For example, WT C57BL/6 (H-2d) males . AllogenIfnar1−/−Ifngr1−/−) were utilized to evaluate a codon pair deoptimized LAV ZIKV vaccine [Ifnar1−/−Ifngr1−/− dams were challenged with ZIKV six days after mating to Ifnar1−/−Ifngr1−/− sires, which resulted in 100% of unvaccinated dams succumbing prior to delivery, whereas all immunized dams gave birth to healthy-appearing pups. However, no further virological, morphological, or behavioral assessment of the pups was reported.In addition to studying infection and disease in fetuses, some groups have followed pups from vaccinated dams through birth or challenged immediately after birth. ICR female mice were immunized with a recombinant E ectodomain protein (E90: 450 amino acids) vaccine and mated with ICR sires. Pregnant dams were challenged with ZIKV at E13.5 in the cerebral lateral ventricle of the fetal brain. Immunized fetuses inspected five days later were protected from infection and neuroprogenitor cell injury, and neonates from vaccinated dams were protected against microcephaly . The perIfnar1−/− dams were mated to WT sires, and ZIKV-117 was administered the day prior to ZIKV challenge. Although treatment with an isotype control mAb resulted in fetal demise and resorption, the vast majority of ZIKV-117-treated fetuses remained morphologically intact at E13. The authors also used the WT dams + anti-Ifnar1 mAb model to show that the passive transfer of ZIKV-117 as prophylaxis or post-exposure therapy resulted in markedly reduced placental and fetal viral burdens, although some tissues remained positive for ZIKV RNA. ZIKV-117 treatment also significantly improved placental and fetal dimensions to levels similar to uninfected animals. Introduction of the L234A/L235A “LALA” mutations, which abrogates Fc-FcγR interaction and prevents antibodies from mediating antibody-dependent enhancement, did not alter the efficacy of ZIKV-117 against placental or fetal infection, suggesting that antibody effector functions were not required for the therapeutic efficacy of ZIKV-117.Studies of human patients have suggested roles for humoral immunity in controlling natural ZIKV infection . Based oIfnar1−/− dams were mated to WT sires and challenged subcutaneously on E6 with a Brazilian ZIKV strain, followed by single-dose EDE1-B10 treatment on the day following infection. A nine-fold reduction in the rate of fetal demise was observed in mAb-treated mice, and this correlated with reductions in ZIKV-induced damage to the junctional zone of the placenta. Administration of EDE1-B10 mAb to WT + anti-Ifnar1 mAb-treated mice one day after maternal infection with a mouse adapted ZIKV (Dakar strain) resulted in a markedly reduced ZIKV burden in the placenta  and fetal head (4900-fold reduction), with a substantial fraction of placentas and fetal heads containing no detectable virus. Notably, a LALA-mutant of EDE1-B10 performed similarly in the WT + anti-Ifnar1 model. Treatment with EDE1-B10 at a later time point  in the WT + anti-Ifnar1 model also reduced ZIKV RNA in maternal and fetal tissues, but to a lesser degree, implying that antibody-based therapeutics may need to be administered within a certain time window after infection to convey maximal benefit. This also suggests that antibody-mediated immune clearance of established placental ZIKV infection may be subject to physiologic limitations.A similar scheme was utilized to assess the efficacy of the cross-reactive, envelope-dimer epitope (EDE) mAb EDE1-B10 when given to pregnant mice infected with various strains of ZIKV . Ifnar1−Ifnar1−/− dams mated to WT sires to test the consequences of antecedent DENV infection on secondary ZIKV infection during pregnancy [+ T cell depletion abrogated this protective effect of DENV immunity on the fetus. Anti-CD4+ T cell depletion also increased the fetal resorption rate, but only in a subset of animals. The central role of CD8+ T cells in mediating cross-immunity against ZIKV during pregnancy was recapitulated by CD8+ T cell depletions in the WT dams + anti-Ifnar1 mAb model and in CD8−/− dams. Thus, for both humoral and cell-mediated mechanisms, murine models have generated evidence for protective mechanisms that can be corroborated in non-human primate models and ultimately, in humans.In a separate report, Regla-Nava et al. utilized regnancy . FetusesMacaca) have been used most frequently for the study of ZIKV pathogenesis during pregnancy (Macaca nemestrina) and rhesus (Macaca mulatta) macaques with isolates of ZIKV from French Polynesia, Cambodia, Brazil, and Puerto Rico. Vertical transmission of ZIKV was reported, although fetal abnormalities and brain injury were relatively minor [The non-human primate (NHP) model of ZIKV infection in pregnancy shares greater similarities with humans than mice ,62. NHPsregnancy . The firly minor ,71,72. Hly minor .Callithrix jacchus) infected with ZIKV also had prolonged viremia and viruria [Most ZIKV-infected pregnant pigtail and rhesus macaque dams appeared healthy, which is analogous to the asymptomatic or mildly symptomatic infections of humans . Minor c viruria , which c viruria . The infThe paucity of clinical signs observed in infected NHP dams correlates with the observation that many ZIKV infections in humans are asymptomatic. Although most human studies in pregnant women have focused on symptomatic cases, the frequency of CZS in asymptomatic cases may be similar, making fetal demise and miscarriages from ZIKV infection more common than initially thought . Among aAlthough many of the initial NHP studies reported few maternal clinical signs, ZIKV crossed the placental barrier and reached the fetus, resulting in minor brain abnormalities . ZIKV RNInfection studies in pregnant NHPs have also shown that exposure to ZIKV at earlier gestational times results in more severe outcomes for the fetus. The percentage of fetal death in utero was higher in rhesus macaques inoculated in the first trimester with a Brazilian ZIKV isolate, whereas second and third trimester infections survived to term . PregnanCZS was observed in NHPs when fetuses from rhesus macaque dams were inoculated with ZIKV early during gestation (<7 weeks). Fetuses infected early in pregnancy showed a reduction in occipitofrontal diameter and head circumference at term compared to non-infected NHPs, but no gross microcephaly was observed. However, these fetuses showed evidence of microcalcifications, hemorrhage, and neuronal necrosis. Fetuses that were exposed to ZIKV earlier in pregnancy also developed more severe spinal cord, ocular, and neuromuscular pathology. In comparison, fetuses exposed to ZIKV at a later time point (>12 weeks) sustained fewer neuropathologic lesions .Fetal demise was also seen in NHPs infected with ZIKV. Pregnant common marmosets inoculated with a Brazilian ZIKV isolate transmitted the virus to their fetuses, which developed neuropathology and early demise . In a meThe placenta was a target of ZIKV infection in rhesus macaques with significant pathology observed, including chorioamnionitis and deciduitis that resulted in damage to the placental villi ,85,86. DThe NHP model shares many aspects of human ZIKV disease during pregnancy and is important because human efficacy trials for vaccines and therapeutics may be challenging due to the waning of cases in endemic regions. NHP vaccine studies during pregnancy also are important, as highlighted by one study that showed efficacy in mice against vertical transmission, but not in pregnant rhesus macaques .In the sole study to date of an antibody-based therapeutic against ZIKV infection in pregnant NHPs, a cocktail of three LALA mutant anti-ZIKV monoclonal antibodies  was given to pregnant rhesus macaques intravenously three days post infection, at the time of peak viremia . PregnanWhereas mice and NHPs are most commonly used to study ZIKV infection during pregnancy, chicken eggs and pigs also have been utilized to assess the link to fetal abnormalities. Embryonated chicken eggs were used to evaluate ZIKV infection in developing embryos. Eggs injected through the amniotic membrane into the inner member space with ZIKV at E2 or E5.5 resulted in malformed chick brains . High doDomestic pigs have been used to study human viral infections. Pigs have similar anatomy, genetics, and physiology to humans, although their placental structures differ . PregnanIn just a few years following the ZIKV outbreak in the Americas, animal models have been developed and used to study the detrimental effects of ZIKV on the developing fetus. These models have confirmed vertical transmission, the teratogenicity of ZIKV, and the ability to cause CZS. Animal models have also provided a platform for testing therapeutics and vaccines against ZIKV during pregnancy. Both NHPs and mice have shown vertical transmission after ZIKV infection, with viral RNA present in the placenta and fetal head. Fetal abnormalities that mimic CZS in humans and damage to neuroprogenitor cells have been observed in both models as well. ZIKV infection in mice and NHPs has confirmed the increased incidence of severe outcomes when infection occurs at early gestational stages. Overall, these models recapitulate many of the phenotypes seen during ZIKV infection of pregnant humans.Despite the many advances in the field, key questions remain that these models may help to answer: (a) what is the basis for persistent viremia during pregnancy in macaques and humans?; (b) what is the role and cell type specificity of type I IFN signaling on fetal resorption?; (c) what are the long term effects of asymptomatic versus symptomatic infection during pregnancy on the mother  and children ?; and (d) what is the significance of breakthrough infection of the fetus and placenta after vaccination? The marked waning of human infections over the past year is likely to make it challenging to perform human clinical trials and obtain new clinical specimens. Thus, small and large animal models of ZIKV infection during pregnancy will continue to have utility in addressing these and other questions, as the threat of ZIKV outbreaks and associated CZS is likely to remain in endemic regions for the foreseeable future.


Coefficients of variation of the collected leachate volumes for both lysimeter types were similar; 7) tillage did not markedly affect nitrate leaching losses; 8) tested leaching models could accurately predict leachate volumes and could be calibrated to match nitrate leaching losses in calibration years, but only one model (SOILN) accurately predicted nitrate leaching losses in the majority of validation treatment years. Apparent problems with tested models: there was difficulty estimating sizes of organic N pools and their transformation rates, and the models either did not include a macropore flow component or did not handle macropore flow well.Eighteen pan lysimeters were installed at a depth of 1.2 m in a Hagerstown silt loam soil in a corn field in central Pennsylvania in 1988. In 1995, wick lysimeters were also installed at 1.2 m depth in the same access pits. Treatments have included N fertilizer rates, use of manure, crop rotation , and tillage . The leachate data were used to evaluate a number of nitrate leaching models. Some of the highlights of the 11 years of results include the following: 1) growing corn without organic N inputs at the economic optimum N rate (EON) resulted in NO


Obstructive sleep apnea (OSA) is an independent risk factor for cardiovascular diseases (CVD) and vascular health. Peripheral blood-derived CD34+ progenitor cells have been used as biomarker for CVD risk and may play a similar role in OSA and CVD risk assessment. Although there are some controversial results in the literature, OSA patients may have a reduction in the number and function of CD34+ cells. The damages promoted by OSA in CD34+ cells may lead to an increase in endothelial oxidative stress and endothelial inflammation which may lead to a reduced endothelial repair capacity. In this study, we explored the effect of continuous positive airway pressure (CPAP) on peripheral blood-derived CD34+ cells and arterial stiffness  in OSA patients.Nine overweight and obese subjects without prediabetes or diabetes were recruited. Eight out of nine subjects had moderate to severe degree of OSA. CD34+ cells were isolated from peripheral blood. Number and function of these cells were monitored before and after 3 months of treatment with CPAP. No significant changes were observed in the number of CD34+ cells, CFU-Hill’s colony formation unit (CFU) count or migratory response to the chemotactic factor SDF-1a after CPAP use. However, CXCR4 mRNA expression significantly increased by 2.2-fold indicating that CPAP may have a positive effect on SDF1a receptor (CXCR4), thereby improving migration of CD34+ cells mediated by SDF1a after the 3 month period. Interestingly, in clinical arena our results showed a reduction of pulse wave velocity  following CPAP therapy.Our findings suggest that 3-month CPAP intervention does not show statistical significant increase in CD34+ cell number and function, in mostly moderate to severe OSA subjects; however, it did demonstrate a positive trend. CPAP therapy, did help improve arterial stiffness parameter.The online version of this article (10.1186/s13287-019-1251-8) contains supplementary material, which is available to authorized users. Obstructive sleep apnea (OSA) is a disorder that affects children and adults and is characterized by repetitive apneas, causing hypoxemia, and unintended arousals from sleep . The preAlthough there have been many different markers of inflammation used to look at endothelial dysfunction, cell-based biomarker such as hematopoietic progenitor cells CD34+ cells to assess endothelial function and treat cardiovascular diseases –12 has bNine adults with OSA (as defined by apnea-hypopnea index AHI ≥ 5) who had chosen continuous positive airway pressure (CPAP) for their OSA treatment were enrolled. Subjects were over 18 years of age and were diagnosed by an in-house polysomnography , we performed a venous blood draw to investigate patients’ CD34+ cell number, function, and gene expression (see below). Routine blood laboratory tests were also performed. All outcome measures were repeated at visit 2, 3 months after treatment with CPAP.The diagnostic PSG and CPAP titration PSG were conducting using a Respironics Alice 6 LDXN Sleep Diagnostic System  at the Center for Sleep Disorders at the Medical Faculty Associates at The George Washington University. A standard set of clinical measurements was acquired during the PSG, including electrocardiogram, electroencephalogram, oxyhemoglobin saturation, and respiratory airflow. Registered polysomnographic technologists manually scored the sleep studies and identified sleep stages, apneic and hypopneic events, and arousals using standard AASM criteria . HypopneArterial stiffness was assessed through pulse wave analysis (PWA) and pulse wave velocity (PWV) using the AtCor SphygmoCor CP system. PWA was obtained from the radial artery while the subjects were seated at rest. An average of two measurements was obtained per visit with an operator index above 80. PWA measures include augmentation index (AI), augmentation index adjusted for a heart rate of 75 (AI-75), augmentation pressure (AP), and both systolic and diastolic blood pressures measured both centrally and peripherally. PWV measures the velocity of the pulse as it moves from a proximal artery (carotid) to a distal artery . For the PWV test, the subject was lying supine on an exam table. An average of two tests was obtained per visit. Higher values of AP, AI, Ai-75, and PWV are correlated to higher levels of arterial stiffness.−ΔΔCt method considering the difference in cycle threshold between visit 2 and baseline (visit 1). The migratory capacity of CD34+ cells were evaluated using the CytoSelect 24-well Cell Migration Assay kit . Cells were suspended in serum-free media and seeded at 100,000 cells per insert. Migration of the cells through a 3-μm polycarbonate membrane to the wells containing serum-free media (control) and chemoattractant SDF-1α (10 or 100 ng/mL) was assessed after cells were kept overnight in a CO2 incubator at 37 °C. Migratory cells were dissociated from the membrane and subsequently lysed and quantified by fluorescence (480 nm/530 nm) using CyQuant GR dye . The fluorescence ratios between cells exposed to the chemotactic factor and cells exposed to chemoattractant-free media (control) along the visits were used to analyze the migratory capacity of the cells.Peripheral blood samples (approximately 60 ml) were drawn from patients and diluted in phosphate buffered saline (1:1). Mononuclear cells (MNCs) were then isolated from whole blood using a Ficoll density centrifugation method. MNCs were counted, and an aliquot was used for CFU-Hill colony formation assay following the manufacturer’s instructions . At day 5, colony forming units (CFU) were counted. To isolate EPCs (CD34+), MNCs were magnetically sorted through a column after cells were stained with CD34 microbeads antibody . An aliquot of CD34+ cells were then stained with trypan blue and counted using an Auto Cellometer Mini . CD34+ gene expression analysis was performed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). CD34+ total mRNA was extracted and purified using the RNeasy mini kit (Qiagen). mRNA was then converted into cDNA by using the high capacity cDNA reverse transcription kit (Applied Biosystems). Gene expressions of samples obtained at both visits were assessed by a CFX96 real-time qPCR system (Bio-Rad) using TaqMan Universal Master Mix II (Applied Biosystems) and inventoried probes. The gene expression analysis included endothelial function , cell chemotaxis , and endothelial lineage cell surface marker (PECAM1). The expression of individual gene was normalized to either housekeeping 18S or GAPDH and calculated by using the 2This is a pilot study for proof of concept. Our objective was to determine if 12 weeks of CPAP therapy is sufficient time to positively affect OSA parameters and parameters of CD34+ cells in moderate to severe middle-aged OSA subjects.p value from a paired Student t test. All p values are nominal, with no adjustments for multiple comparisons. Where effect sizes are reported, they are calculated as Cohen’s d statistics for paired observations.Baseline characteristics are described using means and standard deviations for quantitative variables and count and percentage for categorical variables. Tables and figures that compare quantitative variables between visits 1 and 2 show means and standard deviations for each visit and a The demographic characteristics of the subjects enrolled in this study according to their severity of sleep apnea as well as the CPAP usage are described in Table p < 0.05)  that was reduced by approximately 13%  of CXCR4 mRNA expression . Since arterial stiffness is an indicator of CVD risk [Table CVD risk , 20, a rn = 4) to severe (n = 4) degree of AHI score, which had on an average more than 4 h of CPAP usage per day. Our results showed a trend for an increase of the number of CD34+ cells from subjects using CPAP. Additionally, we also observed a trend for an improvement of the cell (CD34+) function such as migratory response to SDF1a as a chemotactic factor. Interestingly, Jelic et al [Endothelial dysfunction has been described as an early maker for atherosclerosis  number and function may be reduced in a population having a more severe condition of the apnea syndrome.Since SDF1-CXCR4 interaction plays a key role on EPCs, our hypothesis is that CPAP favors CD34+ cells homing, mobilization, and differentiation via upregulation of the receptor CXCR4 in addition to VEGFA and endothelial nitric oxide. Therefore, the use of CPAP can facilitate endothelium repair process to slow down the progress of endothelial dysfunction in OSA. In fact, it is already known that VEGF expression is increased in the plasma of OSA patients , 30. ThiArterial stiffness (AS) is a noninvasive marker used to evaluate hypertension, arterial stiffness and vascular health in general, and increased stiffness indicates poor vessel contractility. It is associated with an increased risk of cardiovascular complications such as myocardial infarction and cerebrovascular accident , 31. MorWe also noted a reduction (13%) of BUN with CPAP use; however, no change in creatinine was noted. It is likely that CPAP use improves renal function, but this finding needs to be verified with a larger cohort study.In our study, we demonstrated that CPAP has potential benefits in mostly moderate to severe OSA patients with 12 weeks of intervention but the improvement in the number and functionality of EPCs (CD34+) as well as in AS is limited according to the severity of the sleep apnea degree. Our results indicate that CPAP can potentially improve endothelial dysfunction as evidenced by CD34+ cell data and AS data. Further studies with larger cohorts are needed for a better understanding of the effects and mechanisms of action of CPAP on CD34+ cells. The increased gene expression of CXCR4 in CD34+ cells and a trend towards increase in mRNA expression for related genes that control homing-in property of endothelial progenitor cells such as VEGFA and NOS3 support the hypothesis that CPAP can improve the functionality of CD34+ cells but the severity of the sleep apnea should be considered. Our results suggest that CXCR4 gene upregulation of CD34+ cells is involved in the molecular mechanism underlying the improvement of vascular health in OSA.Additional file 1:Table S1. Blood biochemistry before and after CPAP treatment. (DOCX 18 kb)Inclusion and exclusion criteria.


Rising winter air temperature will reduce snow depth and duration over the next century in northern hardwood forests. Reductions in snow depth may affect soil bacteria and fungi directly, but also affect soil microbes indirectly through effects of snowpack loss on plant roots. We incubated root exclusion and root ingrowth cores across a winter climate-elevation gradient in a northern hardwood forest for 29 months to identify direct  and indirect  effects of winter snowpack decline on soil bacterial and fungal communities, as well as on potential nitrification and net N mineralization rates. Both winter snowpack decline and root exclusion increased bacterial richness and phylogenetic diversity. Variation in bacterial community composition was best explained by differences in winter snow depth or soil frost across elevation. Root ingrowth had a positive effect on the relative abundance of several bacterial taxonomic orders . Nominally saprotrophic  or mycorrhizal  fungal taxonomic orders were also affected by both root ingrowth and snow depth variation. However, when grouped together, the relative abundance of saprotrophic fungi, arbuscular mycorrhizal fungi, and ectomycorrhizal fungi were not affected by root ingrowth or snow depth, suggesting that traits in addition to trophic mode will mediate fungal community responses to snowpack decline in northern hardwood forests. Potential soil nitrification rates were positively related to ammonia-oxidizing bacteria and archaea abundance . Rates of N mineralization were positively and negatively correlated with ectomycorrhizal and saprotrophic fungi, respectively, and these relationships were mediated by root exclusion. The results from this study suggest that a declining winter snowpack and its effect on plant roots each have direct effects on the diversity and abundance of soil bacteria and fungal communities that interact to determine rates of soil N cycling in northern hardwood forests. Snowpack depth and duration are critical factors that determine winter soil temperature in mid-latitude temperate ecosystems . The timSpecific microbial functional groups strongly influence soil N cycling and the effect of winter snowpack loss on their abundance and distribution may be a significant constraint on forest ecosystem function . For exaUnderstanding the effect of declining winter snow depth on root-microbial interactions is also critical because the effect of snowpack reduction on plant roots is likely to lead to changes in microbial community composition and functioning . Winter At the Hubbard Brook Experimental Forest , low elevation sites experience shallower winter snow cover, colder winter soil temperatures, and greater soil frost depth and duration compared to high elevation sites . Soil N in situ for 29 months across an elevation gradient to determine the direct  and indirect  effects of winter snowpack on soil bacterial and fungal communities, as well as on potential N mineralization and nitrification rates. Bacterial community diversity was hypothesized to be positively related to winter snow depth and duration. Bacterial and fungal community composition were also expected to co-vary with snow depth across the elevation gradient. Root exclusion was hypothesized to result in enrichment of saprotrophic fungi and reductions in mycorrhizal fungi and these trends were expected to increase in magnitude with declining winter snow depth. Lastly, we expected that soil N mineralization rates would be positively associated with saprotrophic fungi and that soil nitrification rates would be positively related to the abundance of ammonia-oxidizing bacteria and archaea.To address these questions, root ingrowth and root exclusion soil cores were incubated The Hubbard Brook Experimental Forest is located in the White Mountain National Forest in central New Hampshire, United States . Elevation ranges from approximately 225 m to 1100 m. Maximum annual air temperature is 19°C, minimum annual air temperature is -9°C, and the mean annual temperature has increased by approximately 0.3°C per decade over the last 50 years . Annual i), a dense root-mat and decomposing organic material (Oe), and a nutrient rich humus layer (Oa) that extends 5 to 6.5 cm below the soil surface (Fagus grandifolia), sugar maple (Acer saccharum), eastern hemlock (Tsuga canadensis) and yellow birch . Red spruce (Picea rubens) and balsam fir  are also found above 740 m.The soils at Hubbard Brook are acidic (pH 3.9 to 4.5) with an organic layer consisting of leaf-litter (O surface . The forAcer saccharum (sugar maple) which maximized sugar maple basal area (25–33.2 m2 ha-1) and minimized the basal area of other common tree species at Hubbard Brook  at bi-weekly intervals during winter. Soil frost depth was measured using methylene blue dye-filled frost tubes . Snow den = 36 exclusion and 36 ingrowth cores).Three 1 m × 2 m plots, separated by at least 50 m, were established at all six sites  in May 2011. Root exclusion and root ingrowth cores were incubated in all 18 plots . The roo-3) was determined after drying for 48 h at 60°C. A subsample of each core was also dried at 60°C for 48 h to determine gravimetric water content. Potential nitrification and net N mineralization rates were determined on subsamples of these same soils in short-term lab incubations as described previously  or NO3- per unit dry soil (nitrification) between Day 1 and 14 and divided by the length of the incubation  and mineral horizon soil (5–15 cm below soil surface) were removed from each core and kept separate during lab processing. A subsample of each soil type from each core was immediately stored at -80°C for DNA extraction and subsequent microbial community analyses. Fine roots ( < 2 mm) were removed using forceps and root biomass  using the Powersoil DNA Extraction Kit  following the manufacturer’s instructions. Replicate soil DNA extracts were combined prior to PCR amplification for each soil core. We amplified both the hyper-variable V4 region of the 16S rRNA gene and the internal transcribed spacer region (ITS1) for identification of bacteria and archaea or fungi, respectively. Modified forward and reverse PCR primers for bacteria and archaea (518F-807) or (ITS1F-ITS2R) for fungi included the forward and reverse Illumina Nextera adapters. PCR amplification conditions were described in detail previously . PCR proReads were trimmed at both the 5’ and 3’ end to the distal sequence priming site using CutAdapt v1.9.1 . Addition = 999) using the using the R package ‘vegan’ (n = 999). Snow depth and duration or frost depth and duration were converted into a continuous single variable by calculating the integral of the relationship between time (days) and snow (cm) using the R package ‘pracma’  and phylogenetic diversity (Core Type P = 0.004) in the organic horizon  and mineral soil horizons (adjusted R2 = 0.40) whereas winter snow and soil frost were less important drivers of fungal community composition compared to standing root biomass  were affected by root exclusion in the organic horizon  and two  biomass .Snow depth and duration were positively related to the relative abundance of seven bacterial orders in the organic horizon  and fiveSnowpack depth and duration were positively related to Russalales abundance in the organic horizon  and to tMicrobial functional group abundance was related to potential soil N cycling rates in the organic soil horizon only . Root inA decline in winter snowpack depth and duration has increased the incidence of winter soil freezing in many mid-latitude ecosystems . In addi-2 yr-1) compared to the lower elevation and higher elevation sites used in this study  at our mid-elevation site (511 m) is higher  or mycorrhizal , but the lack of a widespread root ingrowth effect on fungal functional guilds suggests that other traits or trait complexes, in addition to carbon acquisition strategy, mediate fungal functional group distributions at our field site. The seasonal timing of our sampling also has some implications for the inferences we can draw about the impact of snow and soil frost on soil fungal communities. We sampled after plant senescence in October 2013, a period when tree C allocation belowground is likely to be at annual minima , potentiPlatanthera orbiculate and P. macrophylla) at Hubbard Brook may be experiencing a population bottleneck evidenced by a higher proportion of orchids at adult-compared to juvenile life-stages as well as low juvenile survivorship. Seed establishment and juvenile survivorship of orchids during early mycoheterotrophic life-stages are known to be highly dependent on close association with the fungal symbiont  that are known to colonize the roots of orchids . Cleavitsymbiont , and theThe results from our study provide novel evidence that observed gradients in soil N cycling with elevation in a northern forest are linked to a direct effect of winter climate on tree roots, as well as indirect, root-mediated effects on bacterial N cycle functional groups, such as ammonia-oxidizing bacteria or archaea . Consequently, future reductions in winter snowpack depth can be expected to reduce soil nitrification rates directly by reducing ammonia-oxidizing bacteria or archaea abundance. We also observed significant declines in net N mineralization rates as ectomycorrhizal abundance declined and saprotrophic fungi increased, trends that were driven by reductions in winter snow depth. These patterns may have ecosystem-level implications and consequences for tree N acquisition for species such as sugar maple, which preferentially acquire N in the form of ammonium . Thus, rBecause plant roots exert significant influence on both soil bacterial and fungal microbial community composition and structure, incorporating a root-microbial interaction perspective into winter climate change research will lead to a broader understanding of the effect of snowpack decline on northern hardwood forests. Here we provide new evidence that as winter air temperature continues to increase in coming decades, we should expect that plant roots will mediate the response of soil microbial communities to winter snow loss in northern forest ecosystems. Specifically, should winter snow cover continue to decline as projected, our results suggest that the capacity of these soils to mobilize N could be compromised due to the negative effect of soil frost on soil bacteria, archaea and fungi, and that these effects will be driven by both direct effects of snowpack and indirect effects mediated by frost damage to plant roots.PS, AF, and PT conceived of this contribution to the winter climate gradient study at Hubbard Brook that was originally developed and implemented by PG, TF, JD, JM, LC, MF, and PT. The data collection and analysis were performed by PS (primarily), PG, JD, and JM (soil microclimate), and PS and JB (bioinformatics). The manuscript was written primarily by PS and PT and all authors contributed to refining the text.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A Bland-Altman plot was created to compare both methods. For TMTV assessed by the SUV4 method a Cox regression was applied to determine its effect on time to progression, progression-free survival, and overall survival. Kaplan-Meier curves and corresponding hazard ratios were used to estimate the effect of TMTV alone or in combination with interim positron emission tomography response on patients’ survival. The data relate to the research article entitled “Dynamic risk assessment based on positron emission tomography scanning in diffuse large B-cell lymphoma: post-hoc analysis from the PETAL trial” [1].Total metabolic tumor volume (TMTV) was measured in 510 patients with DLBCL participating in the PETAL trial. The present data provide information about the prognostic impact of total metabolic tumor volume using the fixed standardized uptake value (SUV Cox regression models investigating the effect of interim positron emission tomography-derived (iPET) and International Prognostic Index-derived factors on time to progression (TTP), progression-free survival (PFS), and overall survival (OS) are shown in 4 method and good iPET response using the ΔSUVmax method formed a low risk group, while patients with either high TMTV and good iPET response or low TMTV and poor iPET response were defined as an intermediate risk group. Patients with high TMTV and poor iPET response were allocated to the high risk group with corresponding Kaplan-Meier curves within the risk groups of the dynamic prognostic model being displayed in We present supporting data belonging to the research article “Dynamic risk assessment based on positron emission tomography scanning in diffuse large B-cell lymphoma: post-hoc analysis from the PETAL trial” . Fig. 1 l) plot ,3). Lowe. Lowe41m2NCT00554164 and under EudraCT 2006-001641-33) is a multicenter randomized controlled study that was approved by the Federal Institute for Drugs and Medical Devices and the ethics committees of all participating sites [The PETAL trial , Eastern Cooperative Oncology Group performance status (≥2), Ann Arbor classification (stage III or IV), lactate dehydrogenase level , and extranodal manifestations (>1). The same model was re-run using backward elimination with α = 0.05 as threshold for removing an explanatory variable from the model.An identity plot of TMTV according to the SUV


The National Cancer Institute conducted the Biospecimen Pre-analytical Variables (BPV) study to determine the effects of formalin fixation and delay to fixation (DTF) on the analysis of nucleic acids. By performing whole transcriptome sequencing and small RNA profiling on matched snap-frozen and FFPE specimens exposed to different delays to fixation, this study aimed to determine acceptable delays to fixation and proper workflow for accurate and reliable Next-Generation Sequencing (NGS) analysis of FFPE specimens. In comparison to snap-freezing, formalin fixation changed the relative proportions of intronic/exonic/untranslated RNA captured by RNA-seq for most genes. The effects of DTF on NGS analysis were negligible. In 80% of specimens, a subset of RNAs was found to differ between snap-frozen and FFPE specimens in a consistent manner across tissue groups; this subset was unaffected in the remaining 20% of specimens. In contrast, miRNA expression was generally stable across various formalin fixation protocols, but displayed increased variability following a 12 h delay to fixation. Although traditionally NGS is conducted using high quality RNA such as that obtained from fresh or frozen specimens, FFPE specimens can also be analyzed by NGS and may be particularly desirable if existing morphological and protein expression data is available along with useful clinical data. Use of FFPE specimens may also allow for the study of archival specimens, as NGS has been shown to be successful in FFPE specimens after as many as 32 years of storage2. While RNA is affected by preanalytical handling and FFPE processing, which introduces differences in molecular data obtained with FFPE and frozen specimens, previous studies have shown that expression data from paired FFPE and frozen tissues by NGS can be strongly to very strongly correlated11. Notably, the expression of 1494 transcripts was found to differ between matched FFPE and frozen specimens12. Further, the concordance between FFPE and frozen specimens was affected by RNA quality12, which is known to be influenced by a number of preanalytical factors including post-mortem interval, delay to fixation (i.e. cold ischemia time), fixation duration, temperature, and storage conditions (Reviewed in13). Variation in these preanalytical factors is typical in the clinical laboratories where FFPE biospecimens are generated, but how these factors affect NGS results is unclear.As Next-Generation Sequencing (NGS) technologies have become more affordable, reliable and powerful, they have been increasingly used to determine clinical diagnosis, to guide treatment and predict prognostic outcome  and miRNA expression profiling that are attributable to formalin-fixation and DTF. This study provides guidance on best-practice methodologies for FFPE tissue handling and processing as well as analytical methods to enable the accurate and reliable detection of clinically relevant expression-related endpoints using NGS- based platforms.Although a number of studies have investigated whether nucleic acids extracted from FFPE tissues can be analyzed using NGS platforms, few studies have addressed the effects of formalin-fixation and delayed fixation on the reliability of results of whole transcriptome sequencing and small RNA profiling using case-matched frozen tissues. The National Cancer Institute’s (NCI) Biorepositories and Biospecimen Research Branch (BBRB) developed the Biospecimen Pre-Analytical Variables research program (BPV) to address this and other potential pre-analytical effects of variable FFPE practices. As part of this program, biospecimens collected using strict SOPs were divided shortly after collection and pieces were snap-frozen or formalin-fixed after four different delays to formalin fixation (DTF)Libraries obtained using Illumina’s TruSeq Total Gold RNA kit represented snap-frozen and FFPE specimens from all 30 tumors and included specimens subjected to a delay to fixation of 1, 2, 3, or 12 h  than matched snap-frozen samples. For a snap-frozen sample processed under the Total RNA protocol, approximately 50–60% of reads mapped to the transcriptome, and another 15–25% aligned to other intragenic regions including exon-intron chimeras Fig. . HoweverRNA from a large subset of the DTF samples 82%) exhibited differential expression in reference to the matched snap-frozen control, regardless of the duration of the DTF . As shown in Table We compared lists of genes identified as differentially expressed between tumor/tissue types in snap-frozen specimens with lists generated for 1 or 12 h DTF FFPE specimens. Differential expression was defined as having a greater than 1.5-fold difference in expression between tissue/tumor types and a t-test significance of P < 0.001  studyTo examine and quantify the effects of tissue type, preservation protocol, contributing medical institution, and patient as well as their primary interactions, we constructed linear models for individual genes. This linear model was more powerful than the group comparison test as it used all 148 specimens per gene whereas each group comparison test only used up to 20 specimens (assuming no outliers were removed). To facilitate interpretation of linear models, we restricted the set of genes modeled to the 18,217 genes that had normalized mean expression levels of at least 16  and determined the effect size by calculating the relative least squares estimate of each factor level. We created gene lists that identified differences in expression with an effect size greater than 1.5-fold and unadjusted p-values less than 0.001 between preservation groups using linear contrasts.Very few genes had significant interactions between factors in a first pass analysis (<0.1%) and, therefore, factor interaction terms were omitted. As summarized in Table The linear model also demonstrated another interesting facet. Genes that were found to be differentially expressed in the simple group comparison between tissues under one preservation condition but not another Table  were alsWhen we investigated the high concordance in differential expression further, we observed a strong positive correlative relationship (Pearson r = 0.97) between the relative density of material in coding and UTR regions (using measures such as FPKM) of snap-frozen and FFPE material Fig. , but theAll 150 samples yielded RNA of sufficient quality and thus were utilized for miRNA analysis  was negatively correlated with DTF time with the typical upper quartile value decreasing by approximately 75 counts (or 15%) for 12 h DTF samples when compared to snap-frozen controls. This decline in counts is most likely due to the increased presence of degradation products of other RNAs that are within the selected RNA nucleotide size range (<34 bases). Quality review of the adapter-clipped library sequences showed a notable increase in the proportion of library material that was greater than 25 bases in length for 12 hour DTF samples when compared with other DTF time points.DTF had a more general impact on the quality of miRNA data. miRNA detection levels were consistently negatively correlated with DTF time across all three tissue types , with only one exception. However, there was a tendency to detect fewer differentially expressed miRNA in specimens with a 12 h DTF. This coincided with the lower detection level associated with 12 h DTF. Regardless, similar final results can be expected from differential analysis experiments using tissues subjected to a delay-to-fixation of up to a 12 h.17 even in specimens with comparable exome expression data to a snap-frozen counterpart. Thus, gene expression is more difficult to quantify without enrichment for exonic material using FFPE tissue in comparison to snap-frozen specimens at the gene level and especially the isoform level. Consistent with qRT-PCR results in these specimens18 DTF had little effect on measured RNA expression, but there was an increase in the abundance of small RNA fragments (<34 bases), likely due to RNA degradation, and a noticeable increase in the variability of miRNA expression measurements for a subset of miRNAs.We found that differential analysis of miRNA and RNA from FFPE specimens exposed to various delays-to-fixation by NGS can yield reliable and reproducible results so long as comparative analysis is carefully and consistently performed and specimens are limited to those derived from similar protocols (preservation and processing). In particular, when directly comparing miRNA or mRNA expression in formalin-fixed samples vs. snap-frozen samples, differences due to preservation method alone can be anticipated. These differences are due to a large set of RNA (in the thousands) and a smaller number of miRNA whose expression measurements are altered in a generally consistent manner by formalin-fixation across tissue types. As reported previously, formalin-fixation also led to a large increase in off-target reads associated with intronic regions14; thus, any observable difference cannot be attributed to variations in specimen processing nor can they be attributed to between-patient variability as effects were observed for some but not all specimens from the same tumor. Importantly, the FFPE specimens that lacked the induced formalin-associated upregulated expression also had the same higher percentage of intronic reads observed in all FFPE specimens (relative to snap-frozen controls), indicating this phenomenon is not due to a labeling error. Analysis of the genes upregulated in the majority of FFPE specimens revealed enrichment of genes with roles in nucleosome and chromatin assembly, as well as histone core genes.Although systemic differences in levels of several thousand transcripts were observed between most formalin-fixed specimens and snap-frozen matched specimens, such effects were not observed in 20% (24 of 120) of DTF specimens  in FFPE samples relative to their matched snap-frozen counterparts and this was not observed. The increase in intronic reads could also be due to formalin interference with proteins that perform splicing, allowing transcription of a large amount of immature RNA to go unspliced, but formalin-fixed specimens had comparable splice junction rates  to that of their matched snap-frozen counterparts. A remaining conjecture is that the formalin fixation process increases levels of proteins bound to intronic regions and thus prevents the intronic regions of these genes from being digested quickly.The cause behind the higher percentage of intronic reads in FFPE specimens versus snap-frozen specimens is unclear. Previously, this has been attributed to differences in library construction methods14. Consistent with these results, DTF had little to no effect on RNA-Seq results. However, DTF of 12 h increased the variability in miRNA-Seq data and as previously reported resulted in decreased DV200 in the kidney specimens14. This lack of effect on RNA-Seq results is interesting as it may allow for more flexibility in terms of time between surgical excision and fixation. However, the shortest DTF explored in this study was 1 h; therefore, we cannot rule out the possibility that particularly labile mRNA may be affected by delays shorter than 1 h. Previous studies using microarrays have reported significant differences in gene expression when snap-freezing was delayed by 15 min rather than 0 min19, 20 min versus 5 min20, 30 min rather than 5 min21 or 45 min versus 10 min22, and when RNAlater preservation was delayed by 30 min23, but other microarray studies found no significant effects of 40 min24 and 5 min versus 2 h25 cold ischemia. Further, an increase in the number of short RNA fragments was noted after a DTF of 12 h and is consistent with the decreased DV200 in these kidney specimens after a delay of 12 h14. This is presumably due to degradation of RNAs and consistent with findings of reduced RIN with ischemia of 45 min26 or 90 min versus 10 min27. The increase in short RNA fragments results in a species-dependent decrease in miRNA counts and increase in miRNA count variability. Consequently, while RNA-seq may be possible in specimens subjected to a 12 h DTF, use of these specimens for miRNA-seq would result in less power in the detection of small changes in expression despite only limited observed effects on RNA quality metrics14 unless sequencing depth was increased to compensate.We previously reported the results of nucleic acid quality assays on the biospecimens from the NCI BPV study, finding limited effects of DTF on RNA quality metricsWe conclude that FFPE specimens preserved using a consistent protocol are likely suitable for gene expression and miRNA profiling. However, it may be necessary to adopt library preparation protocols that enrich for exonic material and ensure that delays to fixation are limited to 3 hours or less. Primary factors affecting the analysis of RNA isolated from FFPE specimens include a very consistent increase in off-target library material and increases in expression of specific genes. One way to compensate for the increase in off-target reads is enriching for target exonic material during library preparation or by increasing sequencing depth 2-fold relative to snap-frozen specimens. Similarly, while less impacted by FFPE than whole transcriptome sequencing, miRNA sequencing may require a 10–20% increase in standard read depth for FFPE specimens subjected to a delay to fixation of 3–12 h to compensate for the increase in off-target material observed with longer DTF. It is important to note, that despite adherence to strict protocols and detailed specimen annotation, the effects of formalin-fixation on the measured expression of select genes and isoforms (3–4 k in number) in 80% of specimens remains problematic, rendering their absolute and even relative quantification unreliable until more is known about the associated mechanism.14. Each tumor was divided into six samples one of which was snap-frozen in a prechilled cryosette in liquid nitrogen vapor soon (<1 hr) after resection and stored in liquid nitrogen. The remaining samples were subjected to 1, 2, 3 or 12 h of cold ischemia in a humidified chamber at room temperature prior to fixation in 10% neutral buffered formalin (NBF) for 12 hours and paraffin-embedding. FFPE blocks were stored for 3 to 18 months prior to extraction.The effect of DTF on RNA and miRNA-Seq results were investigated using specimens from 30 tumors including 10 renal clear cell carcinomas (Kidney), 10 serous ovarian carcinomas (Ovary), and 10 colon adenocarcinomas (Colon) collected at 4 medical centers using Institutional Review Board (IRB)-approved protocol was used by each medical site for the collection of human biospecimens for research purposes in accordance with the Helsinki Declaration of 1975, as revised in 1983 . Informed written consent was obtained from each patient. Further details regarding consent, acquisition and processing are detailed in a previous publication14 and for relevant specimens is available in Supplemental Table All nucleic acid extractions were performed by the Van Andel Research Institute (VARI). RNA was extracted from snap-frozen and FFPE specimens using QIAsymphony kits. Briefly three freshly cut 10 µm thick FFPE sections were placed in deparaffinization solution at 56 °C, then digested with proteinase K at 56 °C for 15 min before loading into the QIAsymphony sample drawer for RNA extraction. Similarly, snap-frozen specimens were disrupted using the TissueLyser II homogenizer and supernatants were loaded into the QIAsymphony sample drawer. Specimen quality was assessed as part of a previous reportRNA samples were converted into cDNA libraries using the Illumina TruSeq Stranded Total RNA sample preparation kit (Illumina \# RS-122-2303) and sequenced using a HiSeq 2500. Briefly, Total RNA samples were concentration normalized, and ribosomal RNA (rRNA) was removed using biotinylated probes that selectively bind rRNA species. This process preserved mRNA and other non-coding RNA species including long intergenic noncoding RNA (lincRNA), small nuclear RNA (snRNA) and small nucleolar RNA (snoRNAs). The resulting rRNA-depleted RNA was fragmented using heat in the presence of divalent cations. Fragmented RNA was converted into double-stranded cDNA, with dUTP utilized in place of dTTP in the second strand master mix. A single ‘A’ base was added to the cDNA along with forked adaptors that included index, or barcode, sequences that were attached via ligation. The resulting molecules were amplified via polymerase chain reaction (PCR). During PCR the polymerase stalled when a dUTP base was encountered in the template. Since only the second strand included the dUTP base, the first strand was the only viable template, thereby preserving the strand information. Final libraries were quantified, normalized and pooled. Pooled libraries were bound to the surface of a flow cell and each bound template molecule was clonally amplified up to 1000-fold to create individual clusters. Four fluorescently labeled nucleotides were then flowed over the surface of the flow cell and incorporated into each nucleic acid chain. Each nucleotide label acts as a terminator for polymerization, thereby ensuring that a single base was added to each nascent chain during each cycle. Fluorescence was measured for each cluster during each cycle to identify the base that was added to each cluster. The dye was then enzymatically removed to allow incorporation of the next nucleotide during the next cycle.Multiplexed FASTQs were created using Illumina’s CASAVA 1.8.2 software. Reads were demultiplexed using fastq-multx and quality-trimmed using fastq-mcf [ver. EA-Utils 1.04.773], which removed low quality bases from the ends of reads, low quality reads, and residual Illumina adapters. We used EA Genomics v9 RNA pipeline for RNA-Seq quantitation. Briefly, the human transcriptome is defined by University of California Santa Cruz (UCSC), but with the addition of the Ensembl lincRNAs. We implemented STAR v2.4 as an aligner specifically designed for RNA-Seq. Post-alignment, the resulting bam file was fed into the RNA-Seq quantification software RSEM, version 1.2.14. For this study, we only utilized gene-level results that were log(count +1) based with normalization via upper quartile methods of generally detected genes. Hg19 was used as the reference genome.Post-quantification, we further characterized reads into those which aligned to UTRs, and those which aligned to intragenic regions. In both cases we used the SAMPLE.star.bam, coupled with custom region definitions. Using these definition files, we parsed the alignments found in the SAMPLE.star.bam, and counted the number of alignments to each region of interest. We counted reads per UTR per gene. In a similar manner we characterized intragenic reads as those that fall between the 5′ and 3′ end of a gene, but not within known exons or the UTRs. For this analysis, we defined the intragenic regions separately and performed a special alignment and quantification using RSEM to determine intragenic counts per gene.Protocol for laboratory processing: Total RNA samples were converted into indexed cDNA sequencing libraries using Illumina’s TruSeq Small RNA sample preparation kit (Illumina \# RS-200-0012). Starting with ~200 ng of total RNA, a single stranded adenylated DNA adapter was added to the 3′ hydroxyl group using T4 RNA Ligase 2 deletion mutant. The T4 RNA ligase 2 deletion mutant prevents ligation of the adapter to the 5′ end due to the absence of ATP. A 5′ adapter was then added to the 5′ phosphate using T4 RNA Ligase in the presence of ATP. Following adapter ligation, single stranded cDNA was created by a reverse transcription reaction. The cDNA was then PCR amplified using a common sequencing primer and an indexed primer that is unique to each sample. The cDNA libraries were analyzed for quality and fragment size ranges using the Agilent 2200 Tapestation . Libraries were then size-selected, retaining fragments of between roughly 125–160 bp, using BluePippin , resulting in a mean library size about approximately 135 bp. The final libraries were then quantified by qPCR , and normalized to 2 nM in preparation for sequencing on a HiSeq 2500.Processing pipeline for miRNA-Seq data: Multiplexed FASTQs were created using Illumina’s CASAVA 1.8.2 software. Reads were demultiplexed using fastq-multx and quality-trimmed using fastq-mcf [ver. EA-Utils 1.04.773], which removed low quality bases from the ends of reads, low quality reads, and residual Illumina adapters. Removal of this residual adapter is particularly important for miRNA analysis, as miRNAs are shorter than the overall read length, and therefore the adapter sequence should be present on nearly every sequencing read. Post-clipping, it was ensured that the average read length of the remaining sequence was in line with typical small RNA sizes. After clipping, a basic alignment of miRNAs to those found in the mirBase database, allowing for 1 base mismatch, was performed. The reads associated with each miRNA were counted and normalized using upper quartile methods.In general, principal component analysis used all samples including outliers and was performed using SAS v9.4 PROC PRINCOMP (no further options). The analysis was conducted on log-transformed signal, centered by gene.Hierarchical clustering in general used correlation as a measure of similarity and centroid-based linkage.28 with the same fold change threshold and a (multiple testing) corrected or adjusted p-value of 0.01. The linear model for the RNA-Seq analysis utilized 148 of the original 150 samples (specimens 4-4 and 51-4 omitted). The linear model was performed using SAS v9.4 PROC MIXED with subject as a random effect and terms for fixed effects of tissue type, collection site, and preservation protocol. Only effects with unadjusted p < 0.001 were kept for meta-analysis across genes.Simple differential analysis of groups using the RNA-Seq data was performed using two methods. In one method, the well-known t-statistic with a mildly stringent unadjusted p-value of 0.001 was combined with a fold change threshold of 1.5 to generate comparator lists. In a comparator method, we used the RNA-Seq differential method DESeq2We used Levene’s test (two-sided) for homogeneity of variance (SAS) when examining variation in miRNA expression by protocol.Supplementary Data 1


CCR5 gene, an immune system gene with both general and specific functions. This has been interpreted as allowing flexibility and diversity of gene expression in response to varying disease loads. A broad expression repertoire is useful to humans—the only globally distributed primate—due to our unique adaptive pattern that increased pathogen exposure and disease loads . The main objective of the study was to determine if the previously observed human pattern of increased variation extended to other members of our genus, Homo. The data for this study are mined from the published genomes of extinct hominins , an ancient human (Ust’-Ishim), and modern humans (1000 Genomes). An average of 15 polymorphisms per individual were found in human populations . There were 94 polymorphisms identified across extinct Homo  with 41 previously observed in modern humans and 53 novel polymorphisms . Neither the frequency nor distribution of polymorphisms across gene regions exhibit significant differences within the genus Homo. Thus, humans are not unique with regards to the increased frequency of regulatory polymorphisms and the evolution of variation patterns across CCR5 gene appears to have originated within the genus. A broader evolutionary perspective on regulatory flexibility may be that it provided an advantage during the transition to confrontational foraging (and later hunting) that altered human-environment interaction as well as during migration to Eurasia and encounters with novel pathogens.Humans have a comparatively higher rate of more polymorphisms in regulatory regions of the primate  CCR5 (a G protein-coupled receptor) is best known for its adaptive immune system role in binding the M-tropic human immunodeficiency virus (HIV) and creating a gateway to host cell infection [CCR5 genes present high levels of gene conversion with the chromosomally adjacent CCR2 [CCR5 gene structure, open reading frame (ORF), and amino acid identity are evolutionary highly conserved [cis-regulatory region polymorphisms in comparison to 36 non-human primate species of apes, Old World Monkeys, and New World Monkeys [CCR5Δ32 [Chemokine receptors facilitate communication between cells and the environment , 2 and mnfection –12. In sent CCR2 –18. Primonserved –21 and ionserved . There ionserved . New Woronserved . Further Monkeys . Humans [CCR5Δ32 , 25, tha[CCR5Δ32 –29) asso[CCR5Δ32 –42.CCR5 is 6,065 bases long with an ORF of 1,056 bases that codes for a protein with 352 residues. Two common transcripts (B with three exons and the more stable A with four) likely resulted from non-coding upstream polymorphisms in two separate gene promotors  upstream of Exon 1 and the downstream promoter (PD) upstream of Exon 3 [Located on Chromosome 3 (3p21), human f Exon 3 , 20. Thef Exon 3 .CCR5 particularly interesting from a broader evolutionary perspective. Homo has one of the broadest adaptive ranges of any species [Homo. For example, there is strong evidence for increased disease risk via genetic load in extinct Homo and past human populations [CCR5 has been well studied due to its role in HIV infection  but no work has explored variation within the genus Homo more broadly.The plasticity in regulation of gene expression via alternate transcripts and increased polymorphisms –22 makes species  and huma species , 20 may  species –46 that ulations  and archulations –52. CCR5CCR5 was conducted prior to the generation of deep coverage, high quality paleogenomes for extinct hominins, such as Neandertal species and the newer Denisova species. While paleogenomic sample sizes are not robust to make statements on selection or add to a discussion of other evolutionary forces acting on variation, they provide an evolutionary dimension to understanding the patterns of variation characterizing our genus and insights into possible adaptations to new environments, subsistence regimes, and pathogens [Homo and examine if the pattern of humans having a significantly higher number of cis-regulatory region polymorphisms  [The plethora of research on the evolution athogens . Plus, tathogens and Neandathogens  and the athogens . Understathogens  or high-athogens  versus cathogens . Thus, tMonkeys)  is speciCCR5 was examined in humans and extinct hominins to address the questions: are there shared patterns of variation across the genus for polymorphism frequency and is the distribution of polymorphisms across the gene suggestive of a common evolutionary trajectory? Based on previous studies on human-nonhuman primate gene structure and variation and the finding that human polymorphisms allow flexible CCR5 gene expression [Homo. Both expectations were met.Variation in pression , 20, theHomo data are: Denisova 3 [Modern human data is from the 1000 Genomes Project , which cnisova 3 , Denisovnisova 3 , Vindijanisova 3 , Altai Nnisova 3 , El Sidrnisova 3 , Mezmaisnisova 3 , and an nisova 3 —see Tablnisova 3 , 66, 67.CCR5 gene (NM_000579 and NM_001100168) were downloaded from the National Center for Biotechnology Information (NCBI). The modern human variation data (CCR5 and cis-acting elements) were downloaded from 1000 Genomes via ftp as variant call format (VCF) files (http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/). All data were downloaded to and analyzed using the University of Alaska Research Computing Systems. All files were aligned to the human genome GRCh37/hg19. VCF files for six extinct hominin species and one extinct human (Ust’-Ishim) were downloaded from the Max Planck Institute Leipzig. Ancient DNA often contains C-to-T deaminations at the end of reads [https://reich.hms.harvard.edu/datasets) [The human reference sequences for two common transcript variants for the of reads . Despiteof reads , 70–75; atasets)  confirm U and PD), ORF, and CCR5. The target area for PU was the most inclusive range (-1976 to +33) which avoided overlap with PD and because little difference was noted between putative PU regions studied by Mummidi et al [D was the most productive range (+119 to +828). Significant difference in distribution of polymorphisms across gene structure for all samples was tested using Monte Carlo methods for the exact test.Distribution of polymorphisms was guided by the structure provided by Mummidi et al.  and incle range -76 to +33CCR5 [Homo).Previous research has examined gene structure , gene vaCCR5  is speciHomo samples , an average of 13 per individual included in analysis (Tables Homo (n = 41) have been previously observed in modern humans , they only occur in PU  from opportunistic non-confrontational scavenging to confrontational scavenging and, later, top predatory behaviors; this alteration to hominin-environment interaction brought hominins into greater and regular contact with animal carcasses –84. Neanelection , 76, whiCCR5, particularly CCR5Δ32 which has a more recent origin [Homo identified in this study suggests a potential evolutionary adaptation. A key event distinguishing members of the genus Homo from the last common ancestor with Australopithecus was the shift to confrontational scavenging and, later, hunting; this alteration to human-environment interaction added a new point of disease contact as evidenced by modern data showing hunting bushmeat  alters  did too –94. GiveS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


When primitive vertebrates evolved from ancestral members of the animal kingdom and acquired complex locomotive and neurological toolsets, a constant supply of energy became necessary for their continued survival. To help fulfill this need, the endocannabinoid (eCB) system transformed drastically with the addition of the cannabinoid-1 receptor (CB1R) to its gene repertoire. This established an eCB/CB1R signaling mechanism responsible for governing the whole organism's energy balance, with its activation triggering a shift toward energy intake and storage in the brain and the peripheral organs . Although this function was of primal importance for humans during their pre-historic existence as hunter-gatherers, it became expendable following the successive lifestyle shifts of the Agricultural and Industrial Revolutions. Modernization of the world has further increased food availability and decreased energy expenditure, thus shifting the eCB/CB1R system into a state of hyperactive deregulated signaling that contributes to the 21st century metabolic disease pandemic. Studies from the literature supporting this perspective come from a variety of disciplines, including biochemistry, human medicine, evolutionary/comparative biology, anthropology, and developmental biology. Consideration of both biological and cultural evolution justifies the design of improved pharmacological treatments for obesity and Type 2 diabetes (T2D) that focus on peripheral CB1R antagonism. Blockade of peripheral CB1Rs, which universally promote energy conservation across the vertebrate lineage, represents an evolutionary medicine strategy for clinical management of present-day metabolic disorders. The gravity of this crisis becomes readily apparent when examining the Diabetes Atlas released by the International Diabetes Federation (IDF). According to the 2015 Atlas, 415 million adults worldwide (1 in 11) have diabetes and nearly half of that population is presently undiagnosed , eyes (retinopathy), peripheral nerves (neuropathy), heart (cardiomyopathy), and blood vessels (atherosclerosis) , 4. HypeIn addition to hyperglycemia-induced vascular complications, the devastation of the T2D pandemic stems from its inseparable connection with obesity. A meta-analysis of human clinical studies detected that obesity was associated with a 90-fold increase in diabetes risk and concluded that weight loss could be the “most important therapeutic task” for diabetics . A longiWith the obesity-T2D symbiosis in mind, it seems therapeutically counterproductive that many of the available anti-diabetes medications  lead to weight gain rather than loss . As for In this paper, we will propose that dysregulated hyperactivity of CB1Rs and their parent endocannabinoid (eCB) signaling system at the molecular level drives excess energy intake and storage among individuals and the modern metabolic disease pandemic across global society. Based on evidence from vertebrate biological evolution over millions of years as well as human cultural transitions over thousands, we seek to characterize the peripheral eCB/CB1R system's functions as “vestigial” for fully developed adult humans in a modern setting of plentiful energy supply  obtainable with minimal physical effort. By extending this evolutionary line of thinking to therapeutics, we propose a clear rationale for the usage of peripheral CB1R blockers in the clinic. Owing to their downstream  effects on key metabolic circuits of the central nervous system (CNS), we will establish the superiority of this pharmacological approach over the central CB1R antagonist Rimonabant and current therapeutics targeting “conventional” neurotransmitters. Finally, we discuss potential new therapeutic strategies as well as currently existing ones focused on limiting peripheral CB1R activation. Our central hypothesis is that human cultural evolution has fundamentally shifted the peripheral endocannabinoid system from an energy thermostat, optimized by natural selection, to a pathologically dysregulated agent of the modern metabolic disease pandemic. Drug design efforts aimed at blocking the peripheral component of modernization-induced CB1R overactivity will pave the way for safe and effective defense against the societal infiltration of obesity and T2D Cannabis plants (marijuana). Early indications of the eCB system's metabolic integration derived from marijuana's traditional medical usage in ancient civilizations  as an appetite inducer, although it possessed several other purposes such as pain relief. These observations of antiquity were replicated by Western medicine in “clinical trials” during the 1800s  in the 1990s  current activation  . DSI protivation .In agreement with the well-known hunger stimulation capacity of marijuana, CB1Rs exist in regions and circuits of the brain responsible for regulating food intake and reward . A landmark study published less than a month after the Dies Mirabilis revealed the involvement of central eCBs in the promotion of feeding behavior. It found that leptin, an adipocyte secretory factor capable of crossing the blood-brain barrier (BBB), directly opposes the actions of CB1R-mediated feeding behavior by reducing hypothalamic eCB levels . The rodSince endogenous leptin and exogenous SR141716A both induce appetite suppression, the 2001 study by Di Marzo et al. encouraged utilization of this pre-clinical candidate compound as a therapeutic for patients with metabolic diseases. As a result, SR141716A gained formal approval 5 years later within the European Union and entered the market as the anti-obesity drug Rimonabant. Early clinical trial data from across the continent generated optimism for its therapeutic potential. It delivered on its expected benefits of weight loss and reduced hyperglycemia , and as The gold standard of this class, identified as JD5037, epitomized the clinical objective of these “next generation” peripheral CB1R blockers. Despite not binding to brain CB1Rs as Rimonabant did, JD5037 administered to rodents with diet-induced obesity (DIO) was equally effective with respect to weight loss and other metabolic benefits . These eThe medical importance of CB1R's broad distribution and influence over every aspect of energy homeostasis and metabolism, from the brain's hunger signals to nutrient utilization in the periphery, is highlighted by a 2015 review article  which deHydra vulgaris, a basal animal with a “nerve net” as its primitive nervous system, modulates feeding behavior at a physiologically-relevant concentration. Furthermore, Rimonabant specifically blocks AEA's stimulation of Hydra mouth closure  which lived around 1 billion years ago , and undreceptor  into CB1receptor , higher receptor , 66, enereceptor , and lowreceptor . Based oCiona intestinalis (sea squirt)  , 71. In r hunger , just asr hunger . In thisr hunger , reflectr hunger , 76. StaMammals utilize a unique form of temperature regulation not possessed by “cold-blooded” vertebrates such as frogs and fish. Known as brown adipose tissue (BAT) thermogenesis, it allows their bodies to generate internal heat from energy stored in BAT deposits. Interestingly, conventional white adipose tissue, which stores energy as opposed to liberating it, can differentiate into BAT , althougAnother category of mammals with extensive need for energy reserves are hibernating mammals, which are not confined to a specific lineage but rather spread throughout the phylogeny. These organisms extensively utilize BAT when they are not actively acquiring energy during the winter months, a physiological state which would require lowered levels of eCBs within adipose tissue. BAT thermogenesis suppression serves as the final component within the “peripheral → central → peripheral” metabolic circuit of eCB/CB1R-mediated energy conservation, shared among mammals regardless of hibernation capacity and exhibited under conditions of excess energy intake see . In contThroughout the natural history of vertebrates, the coordinated central-peripheral crosstalk of the eCB/CB1R system conserved energy for “wild” organisms living in environments with unpredictable food availability. When humanity rapidly  civilized and successively developed agricultural and industrial technologies in order to secure a more stable and energy-dense food supply, the original selection pressures for the eCB system's evolution in vertebrates became obsolete. Despite a lack of empirical evidence regarding CB1R function in cartilaginous fish , the liver's role as the only fat deposit available to sharks  for fuelPerhaps the most convincing evidence for the tremendous influence of liver CB1Rs on overall metabolic dysfunction comes from mice with CB1Rs specifically deleted from the hepatocytes, but preserved in all other locations. Upon receiving high-fat diet, these conditional knockout mice did not develop numerous metabolic consequences associated with the DIO mouse model  despite gaining a typical amount of weight . StudiesThe constant “feedforward” loops of eCB/CB1R activity within individual organs can aggregate and form a systemic cycle that, if left unbroken, leads to progression of metabolic pathologies. The traditional “diet and exercise” remedy serves as one such circuit breaker by reducing overall eCB/CB1R system tone. In clinical intervention studies of obese and/or T2D patients, both caloric restriction and increased physical activity  have produced marked improvements in cardiometabolic risk in tandem with decreased circulating eCB concentrations –103. TreZooming into the eCB/CB1R system of adipose tissue reveals a controversial picture , which cThe theme of individual eCB system polymorphisms motivated a recent study of an ethnically and metabolically diverse Brazilian population. A polymorphism impairing expression of fatty acid amide hydrolase (FAAH), the enzyme responsible for degrading AEA, correlated with severity of both insulin resistance and obesity. This relationship with metabolic dysfunction depended on whether the polymorphism was homozygous or heterozygous, given that homozygotes displayed higher plasma AEA levels . Individin vitro experiments with human skeletal muscle cells  cleared JD5037 to begin Phase I clinical trials as a therapeutic for the most severe version of non-alcoholic fatty liver disease (NAFLD). NAFLD affects 75–80% of obese individuals  and possAlthough the door remains open for a peripheral CB1R blocker approved for a more broad clinical population, the results of the JD5037 clinical trials will certainly shape the future of the compound class just as Rimonabant did before it. A previously underappreciated aspect of central eCB signaling, which was overlooked and disregarded by Rimonabant's manufacturer before advancing the drug to clinical trials , comes fMany brain circuits and regions  serve as integrative hubs for both physiological energy balance and psychological stress response . Thus, tin vitro study of differentiated neurons, pharmacological activation of CB1Rs was able to counteract the high glucose-induced impairment of neurite outgrowth , safety concerns that have lingered ever since the cancellation of Rimonabant must be addressed. The most pressing issue stems from the unacceptable psychiatric side effects, which have seemingly been resolved by the non-brain penetrant design of JD5037. Whereas acute treatment of mice with Rimonabant produces noticeable anxiety in a behavioral assay known as the elevated plus maze (EPM), treatment with JD5037 does not alter EPM performance relative to vehicle-treated controls . Additioutgrowth . The higdiabetes . These fdiabetes , 135. WiAs was observed in the JD5037 pre-clinical studies, blockade of peripheral CB1Rs indirectly leads to restoration of impaired LepR and MC4R signaling in the hypothalamus, indicating that a drug unable to enter the brain can still affect key brain circuits involved in metabolism. This homeostatic restoration of the CB1R-LepR-MC4R axis at both the central and peripheral levels bears remarkable similarity to the molecular changes induced by a bariatric surgical procedure known as a sleeve gastrectomy, which essentially shrinks the stomach. The procedure performed on rats resulted in significant downregulation of CB1R in the stomach  and the hypothalamus  at day 90 of the post-surgery period, with reversal of leptin resistance and a dramatic increase in hypothalamic MC4Rs accompanying these CB1R expression changes . The bloCB1Rs of the digestive tract, including tongue, stomach, and intestines, have not been previously discussed in this work, but are reviewed extensively elsewhere , 137. InDue to the ubiquity of CB1Rs in the periphery, there exists an intriguing possibility of having a CB1R antagonist compound “library” able to tackle a variety of metabolic ailments. A chief priority for this agenda is the design of a liver-specific blocker, given the evolutionary conservation of hepatic CB1R function and dysfunction. A possible approach for delivering targeted effects to the liver utilizes prodrugs that only liver enzymes can metabolize to active form . ExploitConscious changes to the diet aimed at reducing endogenous eCB levels can also produce therapeutic benefits. One of the most vital and easily implementable dietary modifications would consist of improving O3:O6 ratio via an omega-3 supplement such as krill oil. When provided to an obese and overweight cohort over the course of 24 weeks, it simultaneously reduced eCBs and triglycerides in circulation . Since fThe recently emerging field of eCB/CB1R developmental biology reveals a particular plasticity of this system in response to maternal energy status . This phenomenon warrants consideration when selecting the ideal metabolic interventions for certain individuals. Experimental evidence from rodents demonstrates that either maternal food deprivation  or obesiIn summary, this paper postulates that the rapid transitions from hunter-gatherer to agriculturalist and finally industrialist society has led to the modern crisis of metabolic disease via hyperactivity of the CB1Rs originally intended to promote survival. Peripheral CB1R blockade represents a novel “evolutionary medicine” strategy for eliminating a root cause of the epidemic. We have integrated evidence from across many scientific disciplines to suggest that in obese adult humans, peripheral eCB system overactivity represents an evolutionary “mismatch” accessible to pharmacological agents (and modifiable by various lifestyle interventions) due to its focused role in energy homeostasis. By contrast, we have utilized the same evolutionary logic to demonstrate that current approaches cannot safely target central CB1Rs due to their pleiotropic functions in stress and mood regulation that emerged during vertebrate evolution.in vivo screening option due to their eCB/CB1R system's similarity to its human counterpart. The development of CB1R blockers with a neutral antagonist profile  could provide distinct safety advantages over inverse agonists such as JD5037 while still preserving efficacy , a peripheral CB1R blocker able to fulfill this role will prove instrumentally useful as a “preventative medicine” approach to reducing the global metabolic disease burden. efficacy . In suppefficacy . This exefficacy . Disruptefficacy . Thus, pThe phrase “leave no stone unturned” should apply to all future basic and clinical research on the eCB/CB1R system. This paper has certainly attempted a comprehensive incorporation of new findings and ideas into the established concept of CB1R hyperactivity driving metabolic disease. However, it has only scratched the surface of the complexity involved in this pathological relationship. One of the most basic unanswered questions in all of cannabinoid biology remains mostly unanswered, especially with respect to non-neuronal signaling : Why areIP conceived and wrote the manuscript. AV, WM, FL, and AD critically reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.