Panicum hallii to experimental salinity treatments. Coastal genotypes demonstrated less growth reduction and superior ion homeostasis compared to the inland genotypes in response to saline conditions, supporting a hypothesis of local adaptation. We identified several QTL associated with the plasticity of belowground biomass, leaf sodium and potassium content, and their ratio which underscores the genetic variation present in this species for salinity responses. Genome-wide transcriptome analysis in leaf and root tissue revealed tissue specific overexpression of genes including several cation transporters in the coastal genotype. These transporters mediate sodium ion compartmentalization and potassium ion retention and thus suggests that maintenance of ionic homeostasis of the coastal genotypes might be due to the regulation of these ion transporters. These findings contribute to our understanding of the genetics and molecular mechanisms of salinity adaptation in natural populations, and widens the scope for genetic manipulation of these candidate genes to design plants more resilient to climate change.Soil salinity can negatively impact plants growth, development and fitness. Natural plant populations restricted to coastal environments may evolve in response to saline habitats and therefore provide insights into the process of salinity adaptation. We investigated the growth and physiological responses of coastal and inland populations of  Plants experience a host of complex environmental stresses such as soil salinity, drought, and high temperature in their natural habitats that can impact their growth, physiology, and ultimately their fitness. Plant populations may over time evolve adaptations that maintain fitness in the face of stressful local environmental conditions . Coastal+ accumulation by long distance signaling. In Arabidopsis, the hyperosmolarity-gated calcium channel (OSCA1) was identified as a putative hyperosmotic stress sensor that triggers cytosolic Ca2+ which subsequently induces stomatal closure  and coastal ecotypes   . Regulat species . HoweverP. hallii obtained from collections across Texas and New Mexico   until leaf relative water content, aboveground biomass, and belowground biomass were measured on 37th DAS  which is equivalent to ~130 mM NaCl solution and often considered moderate salinity stress for many species  . To test species .P. hallii [one representative genotype of the inland hallii ecotype (HAL2) and one representative genotype of the coastal filipes ecotype (FIL2)] with 12 replicates in each genotype x treatment combination . These plants were established in a split-plot design. Blocks were randomly assigned to one level of treatment and genotypes were nested in each block. Plant growth condition and treatment application were carried out in the same manner described for the population study except this time we applied a different concentration of NaCl solution progressively . Based on our results from Experiment 1, a moderately higher level of salinity stress was chosen to test for the response at the genotype level. On 37th DAS aboveground fresh biomass, belowground fresh biomass, the ratio of fresh biomass (aboveground/belowground), leaf relative water content, leaf water potential, leaf sodium content (Na+), leaf potassium content (K+), and the ratio (Na+/K+) were measured. Leaf water potential was measured by using a Scholander-type pressure chamber . Detailed protocols for measuring leaf osmotic potential, leaf relative water content, and ion contents were provided in the Detailed physiological and growth response of plants to the salinity treatment were tested on two genotypes of P. hallii, we implemented a QTL mapping study using recombinant inbred lines (RILs) from a cross between P. hallii var. FIL2 and P. hallii var. HAL2. This mapping population consists of 380 RILs developed by single seed descent (th DAS and aboveground biomass (AGB), belowground biomass (BGB), the ratio of aboveground to belowground biomass (RB), leaf relative water content (RWC), Na+, K+, and the ratio (Na+/K+) were measured . A detailed description of the linkage map construction for this RIL mapping population can be found on Dryad (https://doi.org/10.5061/dryad.73n5tb2w8). In this RIL mapping experiment all the traits except Na+, K+ and Na+/K+ were measured in both control and salinity treatment conditions. As such, this experimental design can be considered a two factorial design and our aim was to identify the main effect of inland versus coastal alleles (G) on phenotypic variation and the interactive effect of coastal/inland allele with treatment (GxT) on phenotypic variation. This analysis strategy relied on a reaction norm perspective. Here, the QTL mapping analysis was split into a portion based on the simple additive effect of QTL averaged across the control and salinity treatment by taking the average of replicates from the two treatment levels. This analysis will detect QTL that are robust to the environments and that have constitutive effects. To study the QTL that show QTL x salinity treatment effects, the difference among the replicates across the treatment was also studied (A genetic linkage map composed of 901 markers evenly distributed across the nine  studied . Prior t studied . The “sc studied  package Constitutive: Ymean (mean of control and stress for each RIL) = µ + QTL + cohort (if applicable) + QTL x cohort (if applicable) + errorQTLResponsive: YDiff (Difference from Stress to Control) = µ + QTL + cohort (if applicable) + QTL x cohort (if applicable) + errorQTL+, K+ and Na+/K+):The following QTL model was implemented for the traits which were measure only on salinity treated plants (NaIonic: YTreat (Trait measure in treatment condition) = µ + QTL + errorQTLTo detect potential epistatic interactions the “scantwo” function was implemented. Permutation (n=1000) for each trait was performed with stratification by cohort to obtain the null distribution for main and epistatic interaction effects. The stepwise QTL function was used to complete a forward-backward search by adding/dropping QTL effects and their epistatic effect. The threshold value for Type I error was set as 0.1 for all the traits and QTL intervals were calculated with a threshold of 1.5 LOD drop from the peak. Estimated QTL effects were obtained by the “qtlStats” function from the qtlTools package  with theth DAS around 10 AM to study global gene expression. 3′ TAGSeq libraries were constructed as described by P. hallii var. hallii (HAL2) reference genome (v2.0), filtered for mapping quality, and expression counts were generated using the v2.1  gene models of the mentioned reference genome. Raw reads can be found in NCBI Bioproject PRJNA853054. Detailed method for library construction and count matrix generation can be found in To characterize the transcriptome response to salinity a RNA-sequencing strategy was implemented. To do so, ~8 replicates of each genotype (HAL2 or FIL2) x treatment combination from the mature plant experiment described in section 2.1 were randomly selected for further characterization. The first emerging leaf and the total root systems of plants were sampled on 37https://github.com/tahia/NatVariation_salinity_adaptation_Phallii.All code and scripts for the analyses and plotting can be found in P. hallii spans from the saline rich coastal habitats along the Gulf Coast of Texas to the arid Chihuahuan desert in the southwest. The edaphic conditions vary considerably across this distribution, and exhibit a strong soil sodium deposition gradient  were selected and a salinity stress was applied (Detailed in -05) compared to control, while the coastal genotype demonstrated no significantly detectable reduction. Similarly, Na+ and Na+/K+ showed significant GxT and the coastal genotype exhibited significantly lower Na+  and Na+/K+  under salinity treatment compared to the inland genotype  mapping was carried out using the RIL mapping population from HAL2 and FIL2 parents. In congruence to our earlier findings (see section 3.2), belowground biomass, Na2) for traits on the mean, difference and ion homeostasis related traits  ranged from 0-21%, 0-5% and 24-36% respectively  had positive effect from coastal alleles; however, the fourth QTL (qK-5@77) had the largest and positive effect from the inland allele. In addition, an epistatic interaction between qK-1@10 and qK-9@17.2 was detected with RILs that possess either or both coastal alleles at these loci maintaining higher leaf potassium than RILs with both inland alleles  the inland alleles increased Na+ while for the other two (qNa-3@40 and qNa-5@77.5) the coastal alleles elevated the trait. Noticeably, all three QTL detected for the ratio of leaf sodium to potassium content  overlap with the intervals of detected QTL for Na+ and K+ individually resulting in three ionic QTL clusters. Since Na+ and K+ demonstrated a weak negative correlation (Pearson correlation coefficient = -0.29) in this mapping population, these QTL clusters imply a plausible association of these loci with ionic balance. Altogether, several genetic loci were identified that were associated with the response difference of belowground biomass and the ionic homeostasis related traits during salinity stress. Generally, the coastal genotype contributed alleles towards a more resilient response. However, the detection of several QTL with inland alleles as positive contributors for ionic homeostasis suggests that the inland genotype may also possess genetic regulators of ionic homeostasis.In this QTL analysis framework, the overall mean and difference of genotypic values between treatment levels for a given trait were defined as either ‘constitutive’ or ‘responsive” respectively for our measures phenotypes  in a given QTL interval was compared to random genomic background using a permutation test  during salinity response, the enrichment of specific Gene Ontology (GO) terms in a given QTL interval was tested (Method section 2.4). GO terms such as response to oxidative stress, antioxidant activity and anatomical structure development were significantly enriched  for qBGB-3@15.8. This result suggests that the presence of potential candidate genes associated with stress responses may contribute to the maintenance of belowground growth in saline conditions. Intervals for qNa-5@77.5 and qNa/K-5@77.5 were found to be enriched with response to oxidative stress and antioxidant activity while intervals for qK-9@17.2, qNa-9@17.2, and qNa/K-9@17.2 were enriched with oxidoreductase, transferase and phosphorelay sensor kinase activity. Since these QTL resided in two major QTL clusters it was not surprising that these intervals would be enriched with similar GO terms. Apart for these two treatment QTL clusters, the qNa-3@40 interval was enriched with cell redox homeostasis and chemical homeostasis terms. In the candidate gene lists for the majority of ion content QTL, we noticed the presence of three e family , genes i pathway  and the er genes . The fre+ from root to leaf tissue in the coastal genotype.To study transcriptional reprogramming during salinity stress, the first fully expanded leaf and the total root systems from experiment 2 were sampled , Treatment (T) and Genotype x Treatment interaction (GxT). Detected differentially expressed genes (DEGs) were categorized in four groups based on their fixed effects; DEG that had significant i) solitary Genotype effect (G), ii) solitary Treatment effect (T), iii) both Genotype and Treatment additive effects (G+T) and iv) an interaction of Genotype x Treatment (GxT). In leaf tissue 5072, 2408, 3893, and 2448 genes were identified for G, T, G+T and GxT categories respectively , 5,775 genes showed significant variation only for treatment (T category), 2,818 genes displayed both genotype and treatment specific additive effects (G+T category) and 1,105 genes demonstrated significant genotype specific response to salinity (GxT category)  like antiporters is considered as a key mechanism to avoid the toxic effects of Na+ in the cytosol both in root and leaf tissues  in the P. hallii var HAL2 reference genome, two were differentially expressed in this experiment. PhHAL.3G288900 was upregulated in leaf tissue for the inland genotype under salinity stress, whereas the coastal genotype had higher expression in root tissue constitutively. The other NHX2 detected in this study (PhHAL.2G474100) was upregulated in leaf tissue for both genotypes during salinity treatment. This compartmentalization of Na+ by NHX is driven by the proton-motive force generated by the vacuolar H+-ATPase (V-H+-ATPase) and the plastic response of V-H+-ATPase has been reported during salinity stress (+/H+ exchanger (SOS1) (+ into phloem recirculation and shuttle Na+ from shoot tissue to maintain ion homeostasis. SOS1 (PhHAL.3G490000) and HKT1 (PhHAL.4G025000) were upregulated in leaf tissue for both the genotypes under salinity stress and are perhaps involved in Na+ recirculation from leaf tissue to phloem. Overall, NHX2 was upregulated in a genotype and tissue specific manner whereas SOS1 and HKT1 demonstrated leaf specific upregulation irrespective of genotype.Compartmentalization of Na tissues . Among ty stress . A vacuor (SOS1)  and highr (SOS1)  play an + homeostasis by driving K+ uptake from the soil (KT transporter (PhHAL.2G242200) was upregulated during salinity in the coastal genotype while a HAK (PhHAL2G.001500) transporter was upregulated during salinity stress irrespective of genotype. However, the upregulation of several KT (PhHAL.2G242200) and KUP  transporters in leaf tissue in the inland genotype suggest their possible involvement in maintaining cytoplasmic K+ levels when external Na+ inhibits K+ uptake and cellular Na+ replaces K+ (+ uptake (KAT1 (PhHAL.1G101800) was upregulated in the coastal genotype during salinity treatment and could be involved in K+ uptake in leaf tissue.KT/HAK/KUP family transporters help to maintain Kthe soil . In rootlaces K+ . Inward + uptake . KAT1 (P+ channels (KCO) activated by membrane depolarization due to high Na+ influx can release K+ from the vacuolar pool to cytosol or mediate K+ exclusion across the plasma membrane  in coastal genotypes compared to inland genotypes. This implies that the coastal population might have evolved efficient Na+ exclusion and K+ retention as a mechanism to maintain ion homeostasis.Under a model of local adaptation, we hypothesized that coastal genotypes would outperform inland genotypes in the presence of salinity treatment. We detected aboveground and belowground growth and biomass stability in response to salinity for plants collected from coastal habitats compared to inland habitats which support our hypothesis. This is consistent with previous report that the salt tolerant genotypes of Proso millet, another terparts . Studiests (+/K+  and someenotypes . Our phy2 ~24 -36%) implies the presence of substantial genetic variation in this population in response to salinity. We detected several QTL for Na+, K+, and Na+/K+. The majority of these QTL effects were driven by the coastal alleles contributing superior ion homeostasis . Moreover, we noticed enrichment of a priori salinity tolerant candidate genes such as the HKT gene family  was upregulated in leaf tissue for the inland genotype under salinity but it was constitutively overexpressed in root tissue for the coastal genotype irrespective of treatment conditions. The constitutive expression pattern of NHX in root tissue suggests that the coastal genotype might be primed for grown in a saline habitat. In addition, KT (PhHAL.2G242200) was upregulated in the coastal genotype relative to the inland genotype during salinity treatment in root tissue. Another K+ channel (KAT1) (PhHAL.1G101800) was upregulated in the coastal genotype compared to the inland genotype during salinity treatment in leaf tissue. Conversely, two outward rectifying K+ channels (KCO) (PhHAL.9G082300 and PhHAL.9G082500) were upregulated during salinity treatment in the inland genotype in leaf tissue. It is likely that the coastal population may have evolved to strictly regulate the expression of various ion transporters to retain potassium and regulate cytosolic ion homeostasis. Moreover, these two KCO family genes were detected as candidate genes in one QTL cluster  for which the coastal allele contributed to superior leaf ion homeostasis. Among these two KCO family genes, PhHAL.9G082500 had elevated non-synonymous to synonymous substitution rate (dN/dS = 1.52) and all four nonsynonymous codon substitution were derived in the coastal genotype  implying that this gene might be under positive selection in the coastal lineage and could contribute to efficient K+ retention in these putatively adapted genotypes.Overexpression of various cation transporters have been reported to confer salinity tolerance. For example, overexpression of  species . SeveralP. hallii-exposed populations. However, it is also reasonable to infer that some of the molecular mechanisms maintaining ion homeostasis could be the result of crosstalk between shared pathways for salinity and water stress  and Integrative Biology Research Grant for graduate students, UT Austin. This research was also supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science Program grant DE-SC0021126 to TJ.We thank the lab members of the Juenger Lab for helping with phenotypic data collection and Shane Merrell for his support while conducting experiment in UT greenhouse facilities. We also thank Robert Heckman and Zeba I. Seraj for their constructive comments on this 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


There is strong evidence showing that sedentary behaviour (SB) increase the risk to develop several chronic diseases and to premature death . A dose response relation is observed with a more marked risk when people spend more than 7 hours/day in sitting position . The study INCA 3 on the lifestyle habits of French population indicated that 40% of people between 18 and 79 years had a high risk for health conditions with more than 7 hours of daily SB . The economic consequences of this risk have never been evaluated. The aim of this study was to estimate the economic burden of SB-related diseases in France.From meta-analysis or large cohorts based on individual SB time, we identified relative risk (RR) to develop cardiovascular disease (CVD), colon cancer, breast cancer and all-causes premature death after co-variables adjustments including physical activity. From RR and prevalence of SB time in France, a population attributable fraction approach was used to estimate the yearly number of cases for each disease. Data from the national health insurance were used to calculate the annual average costs per case for each disease. Then, disease-specific and total health-care costs attributable to prolonged SB time were calculated. Indirect costs for private sector and households were calculated in a second stage.In France, 66 528 premature deaths/year appear related to a daily SB time ≥ 8,6 hours Each year prolonged SB cost 559 millions € for the national health insurance, including 359 millions € for CVD (≥ 10 hours of daily SB), 170 millions € for breast cancer (≥ 6 hours of daily SB), and 31 millions € for colon cancer (≥ 5 hours of TV/day).These preliminary results showed that many deaths could be avoided by reducing prolonged SB prevalence in France. Moreover, direct health-care costs attributable to SB related diseases represent a high economic burden for the French health system. To address this issue, strong responses should be implemented to tackle SB, complementary to physical activity promotion.


While upstream neural afferents of GnRH neurons, including kisspeptin neurons in the rostral hypothalamus, are proposed as critical loci of E2 feedback action, the underlying mechanisms governing the shift between E2 negative and positive feedback are still poorly understood. Indeed, the precise cell targets, neural signaling factors and receptors, hormonal pathways, and molecular mechanisms by which ovarian-derived E2 indirectly stimulates GnRH surge secretion remain incompletely known. In many species, there is also a circadian component to the LH surge, restricting its occurrence to specific times of day, but how the circadian clock interacts with endocrine signals to ultimately time LH surge generation also remains a major gap in knowledge. Here, we focus on classic and recent data from rodent models and discuss the consensus knowledge of the neural players, including kisspeptin, the suprachiasmatic nucleus, and glia, as well as endocrine players, including estradiol and progesterone, in the complex regulation and generation of E2-induced LH surges in females.A fundamental principle in reproductive neuroendocrinology is sex steroid feedback: steroid hormones secreted by the gonads circulate back to the brain to regulate the neural circuits governing the reproductive neuroendocrine axis. These regulatory feedback loops ultimately act to modulate gonadotropin-releasing hormone (GnRH) secretion, thereby affecting gonadotropin secretion from the anterior pituitary. In females, rising estradiol (E A fundamental tenet of hypothalamic-pituitary-gonadal (HPG) axis regulation is sex steroid feedback: the ability of gonadal steroid hormones  to circulate back into the brain and regulate the neural circuits, including gonadotropin-releasing hormone (GnRH) neurons, that govern the HPG neuroendocrine axis. Although feedback loops were proposed decades ago, the detailed mechanisms by which gonadal sex steroids act in the brain to inhibit (“negative feedback”) or stimulate (“positive feedback”) GnRH secretion still remain poorly understood, in part because GnRH cells themselves lack the requisite sex steroid receptors for steroid feedback. Thus, other “upstream” brain cells communicating with GnRH neurons are posited to serve as loci of sex steroid feedback action. Though advances were made in recent years with the discovery of the neuropeptide kisspeptin, the precise brain cells, neural signaling factors and receptors, and physiological and molecular mechanisms by which ovarian-derived estrogen acts in the brain to stimulate GnRH release (“estrogen positive feedback”) still remain major gaps in knowledge. In the present review, we summarize essential background on neuroendocrine mechanisms of estrogen positive feedback, highlight recent advances on this topic, and discuss some critical gaps in knowledge that need addressing to better understand how the LH surge is both generated and modulated. Given other recent in-depth reviews on this and related topics , we will2) and testosterone (T)]. Besides regulating reproductive physiology and behavior, circulating E2 and T also provide feedback loops to the brain to modulate GnRH secretion. During most of the female cycle, lower levels of ovarian E2 provide negative feedback on pulsatile GnRH release, keeping it within a proper homeostatic range (2 levels at the end of the follicular phase (proestrus in rodents) paradoxically “switch” from being inhibitory to stimulatory, providing positive feedback activation of GnRH cells. This E2 positive feedback induces a massive increase in GnRH secretion , in turn driving the synthesis and secretion of gonadal sex steroids  that mediate both sex steroid positive and negative feedback . Thus, s rodents . More re rodents .Kiss1 gene, directly stimulates GnRH neurons. Humans and rodents lacking either kisspeptin or its receptor, KISS1R, are completely infertile, with low LH and gonadal sex steroids due to diminished GnRH secretion  in a region-specific manner: for both sexes, E2 or T increases Kiss1 mRNA levels in the RP3V whereas these sex steroids reduceKiss1 mRNA levels in the ARC  while another more anterior population is in the RP3V . The RP3eminence , 2021. T process . Indeed, the ARC . Convers the ARC ,2009. Bos of ERα , and we s of ERα , have shignaling .2 regulates the Kiss1 gene differently in the two hypothalamic Kiss1-expressing populations has ultimately proven to be a crucial discovery in understanding sex steroid feedback loops. Indeed, this finding led to the proposal that differential effects of E2 on Kiss1 mRNA in the ARC and RP3V reflect different roles of the two kisspeptin populations  pulses.The seemingly simple finding that Eulations , with ARlsewhere  and willlsewhere , and dirnerator” , may be KISS neurons in driving GnRH pulses and mediating steroid feedback, it is now widely believed that RP3VKISS neurons comprise the neural conduit mediating E2 positive feedback on GnRH neurons in female rodents  at the time of the LH surge in proestrus or OVX + E2-treated females  by regulating multiple intrinsic currents in these kisspeptin cells  display an LH surge which occurs solely around the time of lights off and which repeats daily at the same time as long as E2 is elevated  showed circadian increases in both R3PV Kiss1 mRNA levels and RP3VKISS neuron activation  12], with lower levels earlier in the subjective morning or later in the subjective night. Importantly, in these females, the observed circadian increases in RP3V Kiss1 levels and RP3VKISS neuron activation occurred synchronously with the circadian onset and duration of the LH surge  and diestrus females (low E2) do not display an LH surge, though the underlying reason for the E2-dependence of RP3V kisspeptin neuron circadian changes is still not entirely known. As noted below, the vasopressin receptor, V1a, is E2-sensitive  suggest that VIP is an important communicator to extra-SCN brain areas because these knockout mice display disrupted or altered circadian rhythms, and sometimes arrhythmicity  may link the SCN to GnRH neurons . A propoVIP→GnRH circuit is important for the LH surge: (1) in vitro VIP treatment can directly stimulate GnRH neuron electrical activity in female mouse brain slices (Vip mRNA levels in the SCN delays and reduces (but does not abolish) the in vivo LH surge and lowers GnRH neuron activation . Still, these limited data suggest VIP may facilitate some aspect of GnRH surge timing, though more compelling direct evidence is still needed, especially in vivo. Indeed, while VIP activates GnRH neuron electrical activity in vitro, several studies report that central VIP injections in young adult female rats surprisingly inhibit the LH surge and GnRH neurons tivation . This wativation , though tivation , though deficits , though  neurons , via mecnjection , complicly VIPR2 , making diestrus ; what thKISS neurons . However, another study in SCN-lesioned OVX + E2 rats  used reverse microdialysis to increase extracellular AVP levels specifically in the POA/RP3V area which then led to LH surge-like secretion  intermediary neurons. In addition to possible polysynaptic effects, extra-synaptic mechanisms are also possible, but this needs to be tested. While intriguing, this hypothesis awaits confirmatory findings and additional studies are needed to identify the location and phenotype of any possible intermediary neurons. In addition, several studies previously suggested that SCN-regulated RFRP-3 neurons in the DMN may provide inhibitory regulation on LH surge generation by acting on GnRH or kisspeptin neurons  mRNA were reported in the RP3V region of adult female rats (KISS neurons to determine if these neurons contain a circadian oscillator that helps time downstream activation of GnRH secretion (KISS neurons were shown to express PER1 with an E2-sensitive daily rhythm (in vitro Per2-luciferase expression in RP3V brain slices (lacking the SCN) and demonstrated a circadian rhythm, though a limitation was that the Per2 expression in this case was not specific to kisspeptin neurons and could be in any number of the many heterogeneous cells in the RP3V region. Still, it is important evidence that circadian rhythms can persist in the RP3V without the input of the SCN; future studies can ascertain if this SCN-independent rhythm occurs specifically in kisspeptin cells.The circadian nature of the GnRH and LH surge, the demonstrated circadian pattern of RP3Vale rats , that stecretion . Most RPy rhythm . Howevery rhythm . The samBmal gene from just kisspeptin neurons using Cre/lox technology. Both studies found that conditional loss of Bmal in kisspeptin neurons does not impact fertility  and its receptor (PR) are also critical contributors to the LH surge mechanism. Classic studies showed that P4 treatment is able to amplify the magnitude, and in some cases advance the timing, of the LH surge induced by E2  to prevent PR expression in just that area. Unlike control rats, female rats infused with PR antisense oligos near the RP3V did not exhibit any LH surges, suggesting that P4 acts somewhere in that region to influence surge generation (KISS neurons highly express PR (2 transgenic mice with selective KO of PR in just kisspeptin cells (termed “KissPRKO” mice) did not show LH surges or proper RP3VKISS neuron activation, as measured by cfos coexpression  selectively in RP3VKISS neurons of KissPRKO females, while leaving PR deleted from all other kisspeptin populations  from ERα-expressing astroglia harvested from the POA region  LH and FSH section. Because LH and FSH are both required for ovarian E2 synthesis and secretion, the ARCKISS population is therefore indirectly required for preovulatory LH surges in so much as it is required for stimulating the tonic gonadotropin secretion that activates the ovaries. However, whether ARCKISS neurons play a more direct role in the GnRH surge mechanism within the brain, separate form stimulating GnRH pulses, is less certain. Indeed, there is some contradictory evidence on this possibility. The three most compelling pieces of data arguing against a critical role of ARCKISS in the neural GnRH surge process include are that (1) elevated E2inhibitsKiss1 (and Tac2 and Pdyn) gene expression in the ARC  long-term fiber photometry recording of in vivo ARCKISS neuron activation (which correlates strongly with occurrence of LH pulses) over the course of the female mouse estrous cycle) demonstrates no increase in activation during the afternoon or evening of proestrus when the LH surge occurs  RP3VKISS neurons , this does not implicate the ARCKISS population in the normal endogenous GnRH surge mechanism. Moreover, if ARCKISS neurons provide amplification of the surge, ablation of such neurons would be predicted to reduce the surge amplitude, but the rat studies reported an enhanced surge magnitude in the absence of ARCKISS neurons  also show activation in these species and may also be involved (Overall, while the current data suggesting that ARCinvolved .KISS neurons are more prevalent in males than females, but show moderately increased Kiss1 levels in the presence of E2 and on proestrus versus diestrus (KISS neurons play a role in HPG axis regulation specifically during the LH surge remains unknown. A few studies in mice have experimentally activated MeAKISS neurons via optogenetics or chemogenetics but reported only minor increases in LH secretion (KISS neurons are not major players in the E2-induced LH surge mechanism. It remains possible MeAKISS may play a modulatory role in pheromone-induced LH surges induced by conspecific exposure or in aspects of socio-sexual behavior (Finally, we first described the presence of a small estrogen-sensitive kisspeptin population in the medial amygdala region of rodents . MeAKISSdiestrus , 2018. Wecretion ; notablybehavior , though The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The initial introduction of utilizing double helix structural oligonucleotides known as SNP typing with excellent specificity (STexS) in a standard PCR greatly improved the detection of single nucleotide polymorphisms (SNP) by enhancing amplification rates of primer-matching strands and interrupting mismatched strands by constant instability of kinetics regarding alignment attaching and detaching. The model was beneficial overall in detecting SNP variants consisting of large amounts of wildtype strands such as EGFR mutation genotyping for early detection of non-small cell lung cancer. While the STexS PCR is advantageous in detecting SNPs and biomarkers, limitations were yet observed. Despite the ability to detect variants 10 times more effective than a typical amplification-refractory mutation system PCR, it could only perform optimally in DNA concentrations around 101 ~ 105. To further enhance STexS specificity to perform detecting viral-RNA variants such as the infamous SARS-CoV-2, a novel improvement of the regular TaqMan Probe using Com-probes to inhibit high copy wild targets and amplify low copy mutant targets. By introducing the novel STexS II, omicron variants of SARS-CoV-2 were able to be successfully detected in high concentrations of normal genes. Currently, the dominant concept in locating SNP is through PCR. Amplification of highly specific primers matching to certain mutated genomic regions results in time-efficient and cost-effective results that are relatively reliable3. To further increase specificity, various implements are added to the PCR process. As the basis of PCR is separated into three steps; denaturation, annealing, and extension, improvements were observed in several aspects4. Supplements such as probes are designed to give fluorescent signals during annealing, enabling comparative quantitative analysis that led to qPCR5. Other approaches focused on customizing primer sequences to obtain highly specific matches and increasing the annealing temperature to block potential dimers6. The recently introduced SNP typing with excellent specificity (STexS) model also focuses on the annealing process. By implementing double helix structured oligonucleotides within the primer, small portions of matched primers were successfully discriminated from the abundant mismatched templates7. All the methods mentioned above eventually benefit the overall PCR process to detect variants.Detection of genetic alterations such as SNPs is crucial in tackling various genetic disorders and cancers. As research revealed genetic disorders and various cancers can be triggered by a single nucleotide mutation, the importance of distinguishing such diseases through methods of genotyping continues8. To make matters worse, mutated variants of SARS-CoV-2 continue to derail the overall effort to effectively diagnose and suppress9. The Coronaviridae is an RNA virus replicating by reverse transcription followed by DNA replication and transcription. The extended replication process enables the virus to have more alterations of the original template which then can form characteristics not seen in previous infections9. This gradually led to distinct variants known as Alpha, Beta, Gamma, Delta, and Omicron. While the other four subtypes have been relatively subdued through various types of vaccines, the Omicron variant continues to spread among the population. Reports indicate SARS-CoV-2 can be detected via RT-PCR 2 to 3 days prior to symptoms being observed10. Further reports show the virus to have a high transmission rate within 1 ~ 2 week after the initial exposure11. To cause the SARS-CoV-2 to further diminish, effective detection methods that can discriminate infected strands within yet inflicted strands are essential.While the STexS platform was able to perform outstandingly in a genomic environment, limitations were soon to be seen when implemented in a viral circumstance. Among the varieties of the virus, SARS-CoV-2 has been the dominant threat to public health. The ability to disseminate worldwide and inflict high casualties during a short period is concerningTo achieve such goals, this study focused on developing the previous STexS method to effectively detect and amplify mutated regions of SARS-CoV-2 while simultaneously diminishing unnecessary strands by inhibiting signal emission to increase specificity. As a result, we introduce an enhanced method utilizing a novel probe mixture named Combination and Competition probes (Com probes).1 to 108 copies. The typical ARMS PCR was able to detect variants within the 101–103 range. STexS was able to improve the detection range to 101–105. Despite the enhanced specificity, viral variants could be seen in far concentrated amounts that exceed the current detection limit. To overcome the challenge and increase specificity, methods such as TaqMan and minor groove binder (MGB) included TaqMan probes were introduced for distinct purposes12. While the original Taqman probe focused on normalizing and quantifying relatively small concentrations around 101–105, MGB included TaqMan probes that tend to perform optimally in high concentrations around 104–108. To detect variants of RNA-based viruses such as SARS-CoV-2, the spectrum must be broadened that envelops small and high concentrations13. As a result, the concept of STexS II was based on novel TaqMan probe enhancements  to be above the lowest annealing temperature to rule out unnecessary dimersase Fig. . Further14. This condenses the PCR workflow to quantify several targets simultaneously. The multiplex method of probes was possible as probes were designed to react independently with no interference within each group15. While this is the main approach for using multiplex probes, the STexS II Com probes were implemented to interfere with and block nonspecific signals, gradually increasing the gap between mutated and nonspecific signals. Each Com probe was designed to attach wildtype regions that parallel to the mutated sequence (Table 2) and high (108) concentrations  and the Korean National Culture Collection for Pathogens (NCCP). Both institutes are government affiliations that confirm integrity and regulations.Each primer was designed and manufactured to match the omicron variant mutation location Q954H and T547K. Probes were designed and manufactured to match above mentioned primers. Probe elongation was performed by adding one base to the 3' end. A Series of probes elongation was tested for optimal performance and Tm adjustments. Com probes were designed to not emit any fluorescent color only to act as a competitive combination in binding nonspecific probes. The sequence of Com probes was designed to be one base different from the main probe.Each qPCR was performed consisting of forward and reverse primers matching the Q954H and T547K region SARS-CoV-2. For quantitative measures, the main hydrolyzable fluorescent probes were attached with a FAM or JOE fluorescence in the 5' end for Fluorescence Resonance Energy Transfer (FRET) and a BHQ1 quencher in the 3' end. Com probes were added an additional phosphate in the 3' end to inhibit DNA polymerases to attach and amplify. The buffer solution of each PCR was calculated to total 20 μL using Enzynomics™ solution (cat no. RT 431M). The PCR was performed within 45 ~ 50 cycles after a 10 min reaction in 95 °C, with each cycle consisting of 10 ~ 15 s in 95 °C and 10 ~ 15 s in 60 °C in the CFX9600 Real-Time System (Biorad™). Omicron variant standard samples were performed with the same PCR platform.RNA samples of omicron variants of SARS-CoV-2 were obtained publicly through the National Culture Collection for Pathogens (NCCP). Three sample collections NCCP43326, NCCP43410, and NCCP43408 were stored for further STexS II trials.Supplementary Information 1.Supplementary Information 2.Supplementary Legends.


Silene uniflora. We found that the general pattern of reversion is driven by the absence of a widespread stress response in zinc-adapted plants compared with zinc-sensitive plants. We show that ancestral plasticity that moves expression closer to the optimum value in the new environment influences the evolution of gene expression among genes that are likely to be involved in adaptation and increases the chance that genes are recruited repeatedly during adaptation. However, despite convergence in gene expression levels between independently adapted lineages, ancestral plasticity does not influence how similar expression values of adaptive genes become. Surprisingly, we also observed that ancestral plasticity that increases fitness often becomes genetically determined and fixed, that is, genetically assimilated. These results emphasize the important role of ancestral plasticity in parallel adaptation.Phenotypic plasticity in ancestral populations is hypothesized to facilitate adaptation, but evidence is piecemeal and often contradictory. Further, whether ancestral plasticity increases the probability of parallel adaptive changes has not been explored. The most general finding is that ancestral responses to a new environment are reversed following adaptation (known as reversion). We investigated the contribution of ancestral plasticity to adaptive evolution of gene expression in two independently evolved lineages of zinc-tolerant  Silene uniflora.Adaptation to new environments often involves changes in gene expression. This study shows a role of ancestral gene expression plasticity in heavy metal adaptation of two independent lineages of  Whether repeated adaptation to the same environment results in similar changes at the molecular level is key to understanding this balance6, as well as the predictability of future responses to environmental change7. Adaptation to novel environments often involves gene expression changes, but previous studies have found varying degrees of parallelism during repeated adaptation11. These changes occur at various levels, including in the overlap of shared differentially expressed genes, fold changes of these genes or final expression levels12. Understanding the mechanisms that influence the extent of parallelism is an important step in predicting evolutionary responses to new environmental challenges13.The contributions of determinism and contingency in shaping evolution are hotly debated16. In addition to generally preserving the genetic variability of a colonizing population17, plastic responses to new environments could provide the basis for adaptation by moving the trait values in some individuals closer to the new local optimum18. Beneficial plasticity of this kind could be retained in locally adapted populations or genetically assimilated and canalized into constitutive expression differences19. Alternatively, ancestral plasticity that takes expression levels further away from the new optimum is potentially maladaptive and could hinder adaptation to the novel environment21.Phenotypic plasticity in ancestral populations  is suspected to play a role in facilitating adaptation to new environments24, but the relationship between plasticity and evolutionary parallelism has received limited attention25. Other properties of gene expression in ancestral populations, such as ancestral expression level or tissue expression location, are associated with increased co-option and potentially parallelism27. If phenotypic plasticity substantially facilitates the repurposing of traits during adaptation28, then beneficial plasticity may result in greater parallelism than when plasticity is maladaptive.Current evidence suggests a variety of possible impacts of ancestral plasticity on adaptation31 . However, there are examples of ancestral plasticity in particular genes or traits facilitating subsequent adaptation35. Most expression studies on the topic examine transcriptome-wide patterns in ancestrally plastic genes, rarely considering whether genes involved in evolutionary adaptation to the new environment are more likely to have possessed beneficial ancestral plasticity, when compared with the whole transcriptome37. Transcriptome-wide assessments include changes that may not directly contribute to adaptation (in the evolutionary sense), such as those stemming from general stress responses. As a result, estimates of the contribution of ancestral plasticity to adaptation may be distorted in whole transcriptome analysis.Previous studies have generally found that most ancestral plasticity across transcriptomes is reversed in derived populations, that is, it takes expression values further from the new optimumSilene uniflora from contaminated metal mines and local zinc-sensitive coastal populations38. In this species, ancestral coastal populations have repeatedly colonized contaminated mine soils throughout Great Britain and Ireland over the past 250 years39, producing locally adapted populations that can grow at high concentrations of zinc40. As a result, expression differences between closely related mine-coast pairs should resemble the expression differences between the current mine populations and their coastal ancestors. Common changes across replicates are likely to represent adaptive changes rather than drift6. Extant coastal populations also provide an approximation of the ancestral plastic response to zinc. This system provides an ideal opportunity to investigate the role of ancestral plasticity in adaptation across multiple evolutionary replicates.Here we investigate the relationship between ancestral plasticity, adaptation and parallelism using independently evolved lineages of zinc-tolerant S. uniflora largely exclude zinc from their shoots, preferentially accumulating zinc in their roots40. We quantified gene expression in the roots of two independently derived, zinc-tolerant populations from geographically distant, derelict mines  and their nearest and most closely related zinc-sensitive coastal populations  was dominated by a general and widespread stress response. In total, 51.1% of the transcriptome  was differentially expressed in both sensitive populations between treatments, with an overwhelming majority being shared across populations  in the contaminated soils40. Given that this phenotype has evolved in parallel due to strong selection, we also expected a component of the transcription profiles to show parallel changes in tolerant populations. In the control treatment, principal component analysis (PCA) of transcriptome-wide gene expression levels separated populations by zinc tolerance  on PC1 and by geographic origin  on PC2 .PC2 Fig. . Within-PC2 Fig. . In thes11 and 400 genes represents a greater overlap than expected by chance . The degree of similarity in gene expression levels between populations can be quantified by comparing the absolute per-gene log2-transformed shrunken fold changes , but expression was also highly similar in tolerant populations (CEC|FC|T1-T2 = 0.12), despite substantial expression divergence and genome-wide genetic differentiation from the nearest coastal populations . In other words, for the 400 CEC genes, parallel evolution in mine populations produced expression similarity comparable to that observed between sensitive populations, which is the product of shared ancestry, gene flow, drift and selection.In total, 2,119 and 2,884 genes were differentially expressed in control conditions between T1 and S1, and T2 and S2, respectively, of which 532 were shared . This level of parallelism is high compared with other systems, such as repeated adaptation to elevation43. This difference may be due to the strength and specificity of selection that metal toxicity imposes, rather than more multifarious selection along elevation gradients. These shared genes had highly correlated expression shifts , which may also explain this difference. Overall, these results suggest that highly parallel patterns of differential gene expression across evolutionary replicates can be acquired very early in adaptation and over very short timescales. This is true for both the identity of the genes and the magnitude of expression shifts. Papadopulos et al.38 identified both shared and non-shared genetic changes across mine-adapted populations and concluded that there may be a highly polygenic basis to adaptation. These evolved expression shifts could be caused by the same or different underlying genetic variants. The responsible variants may be cis- or trans-acting, may have arisen via gene duplications, and may either directly affect gene expression or target a few upstream regulators—we are unable to assess this from transcriptomic data alone. Regardless of the nature of the genetic changes that have occurred, they have produced remarkably similar gene expression across independent mine colonization events. Previous experimental evolution studies in Drosophila, Tribolium and Ipomoea have demonstrated the evolution of gene expression plasticity in response to heterogeneous environments within 22–130 generations51. We demonstrate that this can also occur in wild plant populations in comparable timeframes and is repeatable between independent colonizations of a novel habitat.There were three times as many genes with constitutive differences between the sensitive ecotype and the tolerant ecotype (CEC genes) as genes with DP. In the literature, there is considerable variability across taxa in the ratios of constitutive to plastic differences associated with local adaptationA. thaliana Zinc Transporter 1ZIP1, which encodes a protein that mediates the uptake of zinc from the rhizosphere52, Heavy Metal Atpase 2  and Metal Tolerance Protein 1 . These are upregulated in zinc hyperaccumulators such as Arabidopsis halleri57 and when overexpressed confer increased metal accumulation and tolerance59. The function of these genes is consistent with increased zinc accumulation in the roots of zinc-tolerant S. uniflora populations40. DP genes were enriched for 248 GO terms, including 7 associated with metal tolerance . Overexpression of GSTs results in enhanced zinc and cadmium tolerance62. These GSTs were also differentially expressed between conditions in susceptible populations, further hinting at a role of ancestral plasticity in adaptation. These results indicate that genes that have been repeatedly recruited for a role in zinc tolerance across multiple species41 have also undergone repeated gene expression changes in zinc-tolerant populations over a few hundred generations.Examining sets of shared genes with expression patterns consistent with a role in adaptation sheds light on the mechanisms underlying zinc tolerance. CEC genes were enriched for 222 GO terms, including terms associated with metal tolerance , in a new environment (Lp) and in adapted populations in the new environment31  with the subsequent change in expression between the ancestral population and an adapted population in the new environment22 . The relationship between PC and EC  can be characterized in three ways: (1) ‘reinforcement’, where the initial PC and subsequent EC both move expression in the same direction towards the new optimum  ‘ovmum Fig. ; and (3)mum Fig. . During 36.We evaluated the degree of reversion, reinforcement and overshooting in our transcriptome dataset. To avoid spurious assignment to these categories resulting from very small expression changes, only genes showing substantial changes in PC and EC  in susceptible plants between conditions (PC); and (2) between mine and coast plants in zinc (EC); see 11, but they account for only 1.8% of the transcriptome. DP and CEC genes are unlikely to include all genes that are involved in adaptation to zinc contamination—some may only be important in a single population or not detected under the framework applied here. Nevertheless, the DP and CEC sets are likely to be enriched for genes involved in adaptation, making them informative as to whether adaptive genes have different responses to ancestral plasticity versus the largely non-adaptive background transcriptomic response.The majority of genes displaying substantial PC and EC across the transcriptome undergo high stress responses in sensitive plants in zinc and remain at unstressed levels in tolerant populations in the zinc treatment. As such, most of the transcriptome is not directly involved in adaptation. Examining the evolutionary response to ancestral plasticity across the predominantly non-adaptive transcriptome provides an indication of the probability that an ancestral plastic response moves expression closer to the new optimum in the zinc-contaminated environment. The large number of subsequent evolutionary reversions indicates that this probability is low Fig. . WhetherP = 2.48 × 10−7; binomial two-sided test) suggests that DP genes may carry a fitness cost for being expressed at an inappropriate/inaccurate level for a given concentration of zinc and have fine-tuned the ancestral level of plasticity.To understand whether ancestral plasticity facilitates adaptive evolution, we considered the proportion of genes undergoing reversion, reinforcement and overshooting in the DP and CEC gene sets. Among DP genes with substantial PC and EC , 79.6% underwent reversion, 3.5% reinforcement and 16.8% overshooting Fig. . The hig19. Among CEC genes showing substantial PC and EC , only 68.4% show signs of reversion, with 28.0% undergoing reinforcement and 3.6% overshooting  or transcriptome-wide . We conducted parametric bootstrapping as recommended in ref. 29 to reduce bias stemming from the presence of Lp in calculations of PC and EC . Ancestral plasticity may facilitate the evolution of differences by moving expression closer to the new optimum, which could then lead to constitutive adaptive changesing Fig. . This is EC Fig.  generall65. Of the 400 CEC genes, 310 are not zinc-responsive in either tolerant population but display substantial PC; these have been repeatedly canalized. Other definitions of genetic assimilation only include cases where the derived trait value is similar to the ancestral value in the new environment66. Of the 310 canalized genes, 114 do not display substantial EC, that is, the ancestral response was close to the new optimum. These included HMA2 and ZAT (see Zinc tolerance pathways section). For an additional 69 of these canalized CEC genes, the ancestral response took expression closer to the new optimum . Altogether 183 genes have undergone genetic assimilation , emphasizing the importance of ancestral plasticity during rapid adaptation to new environments.Here we define genetic assimilation as: when a trait with an environmentally induced response that increases fitness becomes genetically determined and canalized Lp/Lo) and evolutionary change in control conditions . However, the common denominator of Lo in both variables would tend to produce a positive correlation67, potentially making these results unreliable. Most constitutive differences have been found to have evolutionary changes in the opposite direction to ancestral plasticity (reversion and overshooting were not distinguished)12, but whether there was an increase compared with the transcriptome-wide pattern was not assessed. Here, we demonstrated that although most ancestral plasticity is maladaptive, ancestral plasticity that can move expression closer to the new optimum contributes to adaptation.Other studies have looked for a role for ancestral plasticity in producing constitutive expression differences by establishing a positive correlation between ancestral plasticity , genes differentially expressed in the control in only one population pair were more likely to show reversion . Genes lacking ancestral plasticity can rapidly evolve plastic responses with comparable expression convergence to ancestrally plastic genes. In summary, ancestral plasticity facilitates the repeated recruitment of genes but does not necessarily lead to greater convergence in expression levels during adaptation.In addition to affecting gene recruitment, ancestral plasticity may also affect the degree of expression convergence in repeatedly recruited genes. Comparisons of expression levels for DP or CEC genes that had ancestral plasticity versus those without returned no significant differences  and the solution adjusted to 600 µM ZnSO4 solution in the remaining three tanks (zinc treatment). Eight days later, roots from each individual cutting were flash frozen in liquid nitrogen and stored at −80 °C. For each individual within a treatment, roots of one cutting per tank  were pooled, homogenized and RNA extracted using a Qiagen RNeasy plant mini kit , 8.4% fragmented, 16.6% missing. After filtering  across all samples were removed. Variance-stabilizing transformed counts for 27,970 genes across all conditions were calculated and used in downstream analysis. This transformation reduces the dependence of the variance on mean expression values, making it more suitable for visualizing between-sample differences74. Principal components analysis of these counts for (1) all genes in control conditions  allons Fig.  and (3) ons Fig.  were calP = 0.05). Differentially expressed genes between T1 and S1, and T2 and S2 were identified in (1) control and (2) zinc treatments separately using contrasts  were identified using DESeq2’s in-built models with a single combined factor for population + condition  for a given set of genes. The sign of the log2 fold change depends on the order of comparisons being made ; the absolute value must be taken to meaningfully summarize the difference in expression levels . The median was used to summarize the values of |FC| as their distribution is highly skewed. Pairwise Wilcoxon signed-rank tests with Benjamini–Hochberg correction were used to detect significant differences in the distributions of |FC| between different pairs of population/treatment groups.Quantification of fold changes of genes between populations and/or treatments used empirical Bayes shrinkage, calculated with the lfcShrink function in DESeq2Lo, mean expression value across S1 and S2 in control; Lp, mean expression value across S1 and S2 in zinc; La, mean expression value across T1 and T2 in zinc. These were used to calculate the initial plastic change (PC = Lp−Lo) and subsequent evolutionary change (EC = La−Lp) for each gene, as in ref. 12. Only genes with substantial plastic and evolutionary change were assigned as undergoing reversion, reinforcement or plasticity . Genes were defined as having substantial (1) PC if they were differentially expressed between conditions in susceptible populations, combining data across S1 and S2  = S and  = T) and (2) EC if they were differentially expressed between tolerant and susceptible populations in zinc, combining data across both population pairs . Data across ecotypes were combined to gain maximum power to detect small shifts in expression; alternative approaches outlined in 36: (1) Reinforcement, if EC×PC > 0; (2) Overshooting, if EC×PC < 0 and |EC | < 0.5×|PC|; or (3) Reversion, if EC×PC < 0 and |EC| > 0.5×|PC|. Significant differences in the relative proportions of these categories between sets of genes  were assessed using a two-tailed binomial test. Parametric bootstrapping of gene assignment to these categories following ref. 29 was implemented in R and repeated 100 times per gene  using SNPhylo78 v20180901 , which also showed differential expression between treatments in S1 and S2, table outlining enriched GO terms.Supplementary Data 4For genes displaying the DP expression pattern (see main text for definition), but which did not show differential expression between treatments in S1 and S2, table outlining enriched GO terms.Supplementary Data 5For genes displaying the CEC expression pattern (see main text for definition), table outlining enriched GO terms.Supplementary Data 6For genes displaying the DP expression pattern (see main text for definition), table outlining enriched GO terms.Supplementary Data 7Data for each individual sequenced.Supplementary Data 8Phylip format file of sites used to construct the phylogenetic tree in Fig. 1a.


Testosterone deficiency is reportedly correlated with an elevation of cholesterol in plasma, but the mechanism remains unclear. Our objective was to investigate the effects of testosterone deficiency on cholesterol metabolism and the corresponding molecular changes in vivo and in vitro.  SD rats were randomized into three groups: sham-operated (SHAM), subtotal orchiectomized (SO), and orchiectomized (ORX) and fed for 8 weeks. HepG2 cells were cultured with medium containing testosterone with the final concentrations of 0, 10, 30, and 300 nM. Method of isotope tracing and fluorescence labelling was adopted to investigate cholesterol metabolism. Several key molecules of cholesterol metabolism were also analyzed.  SO and ORX rats displayed dysfunctional liver uptake of cholesterol. HepG2 cells incubated with testosterone of lower and excessive level exhibited reduced capacity of cholesterol uptake. Further investigation revealed that lack of testosterone induced increased proprotein convertase subtilisin/kexin type 9 (PCSK9) and decreased low-density lipoprotein receptor (LDLR) both in vivo and in vitro. Moreover, the androgen receptor (AR) antagonist flutamide mimicked the effects of testosterone deficiency on PCSK9 and LDLR indicating the role of AR as a mediator in triggering attenuating liver cholesterol uptake in which testosterone instead of dihydrotestosterone (DHT) is the major functional form of androgen.  Testosterone deficiency attenuated cholesterol liver uptake mediated by the PCSK9-LDLR pathway, in which AR and testosterone without transforming to DHT play important roles. Risk of cardiovascular diseases (CVD) in men is about three times higher than that in women , explanaCholesterol homeostasis is maintained by the balance among several metabolic pathways including the de novo synthesis, intestinal absorption of cholesterol, and the liver uptake. The body gains cholesterol endogenously from the de novo synthesis rate, limited by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Cholesterol is also gained exogenously through diet which is absorbed in the intestine where cholesterol is transcellularly transported by Niemann–Pick C1-like 1 (NPC1L1) and secreted back to the lumen by ATP-binding cassette subfamily G 5/8 (ABCG5/8) dimer. In contrast, cholesterol in plasma is removed from circulation mainly by the liver uptake which is essentially mediated by LDLR-induced endocytosis. PCSK9 escorts LDLR to lysosome for ubiquitination-related degradation .Statins which have been proven efficacious by data of high quality for many years and recommended as the first-line therapy for CVD risk attenuating de novo synthesis of cholesterol. Subsequently, PCSK9 inhibitors and ezetimibe emerged against the backdrop of increasingly reported adverse effects of statins and the plight of refractory hypercholesteremia as alternative medications aimed at facilitating cholesterol liver uptake and reducing intestinal absorption, respectively . Each ofThe goal of this research is to identify the specific pathway of cholesterol metabolism affected by testosterone deficiency and explore the molecular mechanisms involved. To achieve this, we adopted the methods of isotope tracing and fluorescence and identified attenuated cholesterol liver uptake as a pathogenic factor of hypercholesteremia in the state of testosterone deficiency both in animals and the cell line. We further revealed that PCSK9 upregulation and concomitant LDLR reduction are responsible for the impaired liver clearance of LDL in which testosterone acts as the functional form mediated by AR. These results provide new insights into the molecular basis of hypercholesteremia in testosterone deficient patients upon which precise treatment could be accomplished.Male Sprague Dawley rats (8-week of age) were obtained from Shanghai Laboratory Animal Center in China. They were housed in pathogen-free facilities with a reversed 12 : 12-h light-dark cycle. The temperature was kept at 22 ± 2°C. Rats had access to a normal chow diet and water ad libitum. Body weights were measured weekly. All procedures were conducted in accordance with the guidelines of Animal Research Committee of Shanghai Ninth's People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine and Institutional Animal Care and Use Committee (IACUC).n = 6), SO (n = 6), and ORX (n = 6). After anaesthetization and regular disinfection, orchiectomy was carried out as previously described  cholesterol per ml plasma/percentage of intravenous dose 3H cholesterol per ml plasma)∗100.Cholesterol absorption was also measured by the plasma dual isotope ratio method as previously described . In brieμCi/ml 14C-acetate sodium . 24 hours later, cells were harvested, and the total lipids were extracted using 10%KOH/95% ethanol (v:v 1 : 10). Then, total cholesterol was isolated from total lipids using a solvent mixture of petroleum ether/diethyl ether/acetate acid (v:v:v 80 : 30 : 1). Radioactivity was determined by liquid scintillation counting . The results are presented as follows: [14C] in extracted cholesterol (μCi)/dry cell weight (mg).Cholesterol biosynthesis experiment was applied as previously described , 16. Celμg/mL of BODIPY-labeled LDL  for 2 h at 37°C. Then, cell slides were then incubated with DAPI  for 5 min. Images were acquired using an Olympus microscope. Fluorescence density was analyzed using ImageJ software.Cholesterol liver uptake in cell was carried out according to the manufacturer's guide HepG2 cells were incubated with 15 Cell and liver protein samples were extracted and prepared as previously described . ProteinTotal RNA was extracted from tissue samples using TRIzol reagent. RNA concentrations were measured using Nanodrop . cDNA was synthesized using a PrimeScript TMRT Master Mix  according to manufacturer's instructions. qPCR was performed with LightCycler 96  using the Hieff qPCR SYBR Green Master Mix  according to the guidelines of the kits. The detailed qRT-PCR protocol has been described previously. Beta-actin served as the housekeeping gene. The primer sequences used are listed in It has been substantiated that patients with hypercholesteremia suffer from higher cardiovascular risk. To examine the effects of testosterone deficiency on cholesterol homeostasis and cardiovascular diseases, we used testosterone-deficient SD rats having undergone orchiectomy and subtotal orchiectomy, respectively. As expected, testosterone level six weeks after the operation in the rats of both groups markedly reduced compared with the sham-operated (SHAM) group .Although the food intake was similar among three groups , testostCholesterol homeostasis depends on exogenous absorption in intestine, uptake and de novo synthesis of the liver, and abnormality in either of which can lead to dyslipidemia in clinical cases.μCi 3H-cholesterol, we observed no difference of cholesterol intestinal absorption among SHAM, SO, and ORX rats  on various key regulators in cholesterol homeostasis.To investigate the molecular changes resulting in liver uptake inadequacy in the testosterone-deficient state, key mediators involved in the liver uptake were studied. Both SO and ORX rats demonstrated lower LDLR protein content in liver than that in the SHAM rats . IntriguμM AR antagonist flutamide and displayed PCSK9 elevation  and mediated by histone 1 nuclear factor P (H1NFP) [α (HNF4α) [The body gains cholesterol either endogenously from de novo synthesis or exogenously from intestinal absorption and mainly clears cholesterol by liver uptake . Our stu (H1NFP)  or newly (HNF4α) . FurtherExcessive testosterone has deleterious effects on the cardiovascular system. Our data showed that excessively high testosterone attenuates cholesterol uptake of hepatocytes in which PCSK9-LDLR alteration similar to testosterone deficiency was observed. It is presumably associated with accompanied hyperhomocysteinemia in patients with hyperandrogenemia . In factα reductase. Testosterone and DHT are both ligands to activate AR followed by the cascade in the downstream. In this study, we found that AR antagonist flutamide can abolish the effect of testosterone while 5-α reductase cannot, which indicates the crucial role of AR in testosterone-induced modulation of liver cholesterol uptake. Testosterone activates AR either located on the membrane which triggers nongenomic, rapid cascade, or on the nuclear which mediates the genomic effect by regulating transcription factors [Testosterone is transformed into DHT which is catalyzed by 5 factors . Besides factors . Elabora factors . Further factors . EGF has factors . TestostStatins have been strongly substantiated to ameliorate or prevent atherosclerotic cardiovascular diseases (ASCVD) by lowering LDLc levels and universally considered as the first choice of lipid lowering . HoweverIn conclusion, we have provided the first evidence to the fact that attenuated cholesterol liver uptake caused by the PCSK9-LDLR pathway is responsible for hypercholesteremia in the state of testosterone deficiency, in which AR and testosterone without transformation to DHT are required. These findings have pharmacological and clinical implications for tailored therapy of hypercholesteremia in aging men.


This review aims to discuss the most recent evidence identifying the presence of distinct white adipocyte subpopulations in white adipose tissue (WAT) and how these may be altered with increasing adiposity and/or metabolic disease. We conceptualize how changes in adipocyte subpopulations may contribute to alterations in WAT function and the development of metabolic diseases such as type 2 diabetes mellitus (T2DM), nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD).Studies utilizing novel analytical approaches support the existence of distinct white adipocyte subpopulations in both human and murine WAT. Adipocyte subtypes are potentially functionally distinct and may have different roles in WAT function and obesity-associated metabolic diseases.The exploration of white adipocyte heterogeneity using novel analytical technologies, has unveiled a new layer of complexity in the study of WAT biology. Interrogation of potential functional differences between adipocyte subpopulations and their role in the function of different WAT depots is now needed. Through understanding the mechanisms regulating white adipocyte subtype development and potential pathophysiological consequences of changes in the presence of adipocyte subpopulations, studies could provide novel therapeutic targets for the treatment of T2DM, NAFLD, and CVD. In addition to serving as a storage depot for energy substrates, white adipose tissue (WAT) is an endocrine organ that contributes significantly to whole-body energy metabolism and homeostasis. It is the excessive accumulation of WAT that defines obesity, and WAT dysfunction is thought to link obesity to a host of metabolic complications. At a population level, obesity is an independent risk factor for multiple metabolic diseases including type 2 diabetes mellitus (T2DM) , nonalcoOne approach that has been used to explore and identify changes in WAT function is via the exploration of whole-tissue gene expression profiles using RNA sequencing (RNAseq), which enables researchers to identify the presence and quantity of RNA in biological samples. Advances in RNAseq technologies over the last decade have facilitated the exploration of gene expression profiles at the level of single cells (scRNAseq). Such advances have consequently permitted the identification and further exploration of WAT SVF cell populations in both physiological and obesity-associated pathophysiological settings . However▪▪]. In order to overcome this, rather than sequencing a whole cell, RNA within nuclei isolated from WAT can be sequenced via single-nuclei RNAseq (snRNAseq) consequently enabling the transcriptomic profiling of single adipocytes. The technical differences, advantages, and limitations of single-cell and snRNAseq for the study of WAT biology have been recently described in detail by Yang Loureiro et al.. The first subclass referred to as AdipoLEP (because of a high expression of the leptin-producing gene) was found to have an enriched expression for genes encoding proteins involved in cell–cell matricellular interactions (TNS1 and SPTBN1) and modifiers of leptin signalling/secretion (PTPN11 and DDR2) [15▪▪]. In contrast, the second subclass referred to as AdipoPLIN  had an enriched expression of genes involved in glucose and lipid metabolism as well as adiponectin secretion [15▪▪]. Through implementing novel approaches that enabled the authors to identify the location of each adipocyte subclass within WAT samples, subclasses AdipoPLIN and AdipoLEP were found to colocalize whilst having a reciprocal expression pattern for genes involved in triglyceride (TAG) biosynthesis and hydrolysis (highly expressed in AdipoPLIN and underrepresented in AdipoLEP). Thus, it is possible that anatomically related subpopulations of adipocytes may have functionally different roles in lipid handling within the same WAT depot [15▪▪]. This study also identified a third less abundant adipocyte subclass (AdipoSAA) characterized by a distinct expression of retinol-binding proteins. The authors speculate that AdipoSAA may have a particular role in the modulation of WAT inflammation [15▪▪]. Additional studies are warranted to explore the role of adipocyte subtypes in the regulation of tissue inflammation in the context of obesity-associated WAT dysfunction.Historically, unilocular adipocytes were considered to be uniform in both form and function; however, we now appreciate that this somewhat simplistic view is likely inaccurate. Multiple snRNAseq studies have identified adipocyte subpopulations within murine and human WAT depots 12▪▪,▪▪,15▪▪. et al.[▪▪] detected seven distinct adipocyte subpopulations within paired human abdominal SAT and VAT biopsies and noted strong depot-specific associations of adipocyte subtypes. Compared with VAT, the presence of adipocyte subpopulations in SAT that were associated with a higher expression of genes involved in TAG biosynthesis, fatty acid desaturation, and lipogenesis [▪▪]. Whilst yet to be explored, it is possible that such depot-specific differences in adipocyte subtypes are a contributing factor to the established depot-specific differences in lipid handling and metabolism. Interestingly, whilst some similarities in the types of adipocyte subpopulations in mouse models were observed, there was an absence of depot-specific subtype enrichment as seen in humans [▪▪]. Whilst other SVF cell types may have a good cross-species concordance, the authors suggested that adipocytes in mice do not appear to map clearly to human adipocyte subpopulations [▪▪]. Thus, caution should be exercised when attempting to extrapolate findings from studies exploring adipocyte subtypes in murine models to humans, at least until the clinical relevance of findings from preclinical models has also been confirmed in human tissues/cells.Using snRNAseq, Emont et al.▪▪ detectogenesis ▪▪. Whilsn humans ▪▪. Whilsulations ▪▪. Thus,A recent study has demonstrated that the quality of adipocyte transcriptomic profiling may be improved by using full-length snRNAseq, which results in an enhanced gene coverage that may permit more accurate profiling and identification of adipocyte subtypes within WAT ▪. This wLEP (AdipoLEP) was positively associated with BMI, whilst those with a high expression of adiponectin and genes involved in glucose and lipid metabolism (AdipoPLIN) were inversely associated with BMI [▪▪]. This study also demonstrated that short-term exposure to insulin (following a hyperinsulinemic–euglycemic clamp) induced a robust transcriptional response specifically in AdipoPLIN, which also correlated with overall insulin sensitivity [▪▪]. These findings could suggest that the capacity of WAT to respond to insulin is determined by the presence and function of a specific adipocyte subtype rather than the overall capacity of the depot to respond to insulin per se[▪▪]. Furthermore, these findings may indicate that shifts in adipocyte subpopulations with increasing adiposity may modify the secretion of adipokines, which are known to be associated with metabolic diseases, such as T2DM, NAFLD, and CVD . Specifically, only one adipocyte subtype (hAd7) in SAT was found to be associated with the presence of T2DM whilst only representing a small proportion (1%) of total adipocytes [▪▪]. Despite the potential function of hAd7 being unclear, the presence of this subtype and its gene markers were found to be inversely associated with HOMA-IR, suggesting that changes in the presence of this subtype are linked to the development of T2DM and systemic insulin resistance [▪▪]. Adipocytes were also found to be most strongly associated with BMI-adjusted waist-to-hip ratio (a measure of WAT distribution) supporting the notion that the adipocyte subtypes are likely to be depot-specific. Although not statistically significant, changes in human SAT adipocytes were also found to be associated with high-density lipoprotein (HDL) concentrations [▪▪]. Similarly, the proportion of so-called ‘basal’ SAT adipocytes (hAd1) and changes in low-density lipoprotein (LDL) concentrations had a near-significant association and the expression of selective genes from this subtype  were positively associated with LDL concentrations in humans [▪▪]. Such evidence could indicate that changes in the proportion and function of specific adipocyte subpopulations are associated with the development of a proatherogenic lipid profile; however, this is yet to be explored.In WAT, adipocytes were found to be the cell type most likely to mediate the association between cellular changes in SAT with the presence of T2DM adjusted for BMI ▪▪. Speciipocytes ▪▪. Despisistance ▪▪. Adipotrations ▪▪. Similn humans ▪▪. Such Despite the feasibility of using snRNAseq to interrogate adipocyte heterogeneity in WAT, it is important to consider that we do not yet understand the roles of different cell subpopulations on whole tissue function or on whole-body metabolic health. Exploring SVF WAT progenitor cell populations using scRNAseq, recent work has suggested that clustering in transcriptomic profiles was largely driven by the anatomical location of specific WAT depots (i.e. SAT vs. VAT) . Convers▪▪], it potentially adds an additional layer of complexity to our understanding. Mechanistic studies exploring the transition of adipocyte subtypes will be crucial over the next few years. Our ability to understand the characteristics and functionality of WAT depots at a single-cell level  is governed by the existence and availability of appropriate methodology. This area is in its infancy and much work is needed to enable us to understand if, how, where and when changes in adipocyte subpopulations contribute to functional changes in a given WAT depot. Technologies, such as ‘Live-seq’ may eventually facilitate the temporal transcriptomic recording of single adipocytes [Whilst sparse, current evidence from both murine models and humans suggests that shifts in specific adipocyte subpopulations within WAT are more closely associated with changes in BMI and obesity-associated metabolic dysfunction. Figure 15ipocytes . StudiesNone.J.B., J.K.S., and C.D.B. are supported by the National Institute for Health and Care Research through the NIHR Southampton Biomedical Research Centre (Grant Number IS-BRC-20004). This research was funded in part by the Wellcome Trust (Grant number 206453/Z/17/Z to J.K.S.).There are no conflicts of interest.


Mobile health approaches in community based participatory projects keep the promise to encourage sustainable behavior in citizens by engaging communities in data collection and connecting their individual level behavior to larger level community health. This review evaluates the effectiveness of the implementation of citizen science for health promotion of non-communicable diseases in urban community-based participatory research projects.A scoping review was conducted using PRISMA-ScR. Pubmed and Web of Science were searched for citizen science studies. We included research 1.) conducted in an urban setting, 2.) related to environmental sustainability, 3.) focused on non-communicable diseases, and 4.) applied citizen science methodology.32 community-based participatory research projects were identified using the following data collection technologies: mobile applications (n = 7), photovoice (n = 6), Stanford Healthy Neighborhood Discovery Tool (n = 6), monitoring (n = 6), mixed-methods (n = 4) and sensors (n = 3). The Stanford Healthy Neighborhood Discovery Tool was most effective at delivering real time data collection, exploring new communication and dissemination opportunities for remote and marginalized communities, and at offering a flexible and cost-effective approach to identify health promotion interventions. Studies that implemented photovoice tools and mobile applications had challenges with regard to recruitment and retention of participants as well as privacy concerns.Mobile health technologies in community-based participatory research projects may be a promising way to uncover unknown local risk factors, raise awareness and identifying targeted policy solutions to promote healthy and sustainable environments in urban spheres. The effectiveness of mobile health applications for health promotion of non-communicable diseases may vary between community-based participatory studies by data collection method.• Mobile health technologies in communities may be a promising way to promote healthy and sustainable environments by raising awareness and offering targeted policy solutions in urban spheres.• The effectiveness of mobile health applications for health promotion of non-communicable diseases may vary between community-based participatory studies by data collection method.


Objective The aim of this study was to investigate the effect of immediate dentin sealing (IDS) technique using universal adhesive under simulated pulp pressure on microtensile bond strength (μTBS) of indirect resin composite restorations and dentin permeability.Materials and Methods Fifty extracted caries-free human third molars were used for specimens' preparation. Each molar's occlusal table was abraded flat and their roots were separated under continuous water cooling. Forty specimens were used for microtensile bond strength test (μTBST) evaluation. The μTBST specimens were randomly assigned to two groups according to the dentin sealing time; Immediate dentin sealing (IDS) and delayed dentin sealing (DDS). Each group was further subdivided into two subgroups according to the adhesive system used for dentin sealing: iBOND self-etch adhesive and GLUMA Bond Universal. All specimens were exposed to simulated pulp pressure for 1 week then restored using computer-aided design/computer-aided manufacturing (CAD/CAM) resin composite blocks. The μTBS was evaluated for all tested subgroups after 24 hours and 6 months of water storage. The remaining 10 teeth were used for the preparation of dentin discs for dentin permeability evaluation. They were divided into two groups according to type of self-etch adhesive used. Fluid filtration rate was evaluated after etching, with smear layer and after adhesive application. Results obtained were statistically analyzed using Shapiro–Wilk test and Weibull analysis.Results Statistically significant difference was recorded between µTBS mean values of both IDS and DDS techniques at 24 hours and after 6 months of water storage. GLUMA Bond Universal adhesive had significantly higher bond strength compared with iBond at both IDS and DDS techniques, but both adhesives showed a significant reduction in the Weibull characteristic strength after 6 months of water storage. Significant reduction in dentin permeability was recorded by both adhesives without any significant difference between them.Conclusions The IDS technique using universal adhesive in self-etch mode is an effective strategy for improving the final bond strength of CAD/CAM resin composite restorations and reducing dentin permeability. Indirect dental restorations have witnessed a massive increase in the use of computer-aided design and manufacturing (CAD/CAM) techniques over the last decade, likely due to impressive developments in intraoral imaging and manufacturing technologies.Generally, adhesive–dentin interface is considered as the weak link of any bonded indirect restoration; therefore, microleakage and postoperative sensitivity remain a concern.Consequently, contemporary efforts have been performed to mitigate postoperative sensitivity through dentin sealing using available adhesive systems.in vitrostudies have assessed the bond strength of universal adhesives, there is little data on how to achieve state-of-the-art dentin sealing of CAD/CAM resin composite restorations using self-etch mode of universal adhesive systems, which is becoming more common in clinical practice due to its reduced chair time and ease of use. It is also worth noting that, in vital teeth with positive pulpal pressure, dentinal fluid transudation through polymerized adhesive layers may prevent near-perfect dentin sealing while using simplified adhesive systems. In light of these considerations, the twofold purpose of this study was to evaluate the effect of IDS technique using self-etch mode of universal adhesive system on microtensile bond strength (μTBS) of indirect resin composite restorations and dentin permeability in presence of simulated pulpal pressureAlthough someTherefore, the null hypotheses tested in the current study were: (1) There is no difference in bond strength of IDS and DDS techniques either after 24 hours or 6 months of water storage, (2) There is no difference in bond strength exists when self-etch mode of universal adhesive and a self-etch adhesive used for IDS and DDS when tested after 24 hours and 6 months of water storage, and (3) there is no difference in the ability of both tested adhesive systems to reduce dentin permeability.in vitrostudy was approved by the Research Ethics Committee (REC), Faculty of Dentistry, Suez Canal University . All tested materials and their description, composition, batch number, and manufacturer's information are displayed inThiswww.randomizer.org): 40 for the microtensile bond strength test (µTBST) and 10 specimens for evaluation of dentin permeability.After sample size calculation, 50 human extracted crack and caries-free human mandibular third molars were extracted from patients aged between 18 and 25 years for the current study. All soft tissue remnants were removed, then teeth were stored in distilled water containing 0.2% thymol at room temperature for not more than 3 months at 4°C till testing. Each molar's occlusal table was abraded flat 1 mm behind the DEJ to reveal dentin surface with 600-grit SiC paper  under running water to create a standardized smear layer. Roots of each tooth were then separated by a diamond saw  under continuous water cooling. Roots separation was achieved at a direction parallel to the occlusal surface 2 mm below the cementoenamel junction and pulp tissues were removed from the exposed pulp chambers. All specimens were checked for possible cracks at X20 magnification stereomicroscope . Then, the flat dentin surfaces were then rinsed with water for 2 minute and blot dried. Specimens were randomly distributed through a research random assignment tool (n = 20) according to the dentin sealing time: IDS and DDS. Each group was further subdivided into two subgroups (n = 10) according to the adhesive system used for dentin sealing; iBOND self-etch adhesive (A1) and GLUMA Bond Universal (A2).The µTBST specimens were randomly divided into two groups . The LED guide tip diameter was 14 mm and was kept at zero distance with the specimen surface on curing. Prevention of an oxygen inhibited layer formation was achieved through coating of the adhesive layer with a layer of glycerin gel and light cured for 10 seconds. Meanwhile specimens of IDS A2 subgroup were immediately sealed using GLUMA Bond Universal. The adhesive was applied according to the manufacturer's instructions to the dentin surface with an applicator brush and rubbed for 20 seconds. The entire dentin surface was dried sufficiently by blowing mild air for more than 5 seconds, then the adhesive layer was isolated with glycerin gel and further light cured for 10 seconds. The specimens were temporarily restored with provisional resin discs that were fabricated through light curing of temporary restorative material  for 40 seconds in a silicone mold. A chemically set temporality cement  was used for cementing the provisional discs.Dentin surfaces of this group specimens were sealed immediately after preparation. Specimens of IDS A1 subgroup were immediately sealed using iBOND self-etch adhesive according to the manufacturer's instructions. The adhesive was agitated for 20 seconds with a brush and the dentin surface was carefully air-dried for 5 seconds with a flow of oil-free air to evaporate the solvent and water from the bonding layer. Then, adhesive was light cured for 10 seconds at a light irradiance of 1200 mW/cmAll temporally restored specimens were subjected to simulated pulpal pressure after the luting phase.After preparation of DDS group specimens, they were temporarily restored using provisional resin discs and then stored for 7 days under simulated pulp pressure as described in IDS group. Following that delay, the provisional discs were removed and dentin surfaces cleaned. Dentin surfaces were then sealed according to their assigned subgroups following the manufacture instructions as in IDS group .Permanent restoration of all specimens was performed through milling of cylindrical nanohybrid resin composite blocks  with a diameter of 12 mm and height of 4 mm using a milling machine . The fitting surface of each block had been abraded under water cooling with 600-grit SiC paper followed by airborne-particle abrasion with 50 um aluminum oxide particles for 10 seconds to create a flat surface with standardized roughness for proper cementation. After rinsing with running water, the fitting surface of each block was air-dried and primed using a silane coupling agent for 60 seconds, then air dried before final cementation. The blocks were seated after gentle dispensing of self-adhesive resin cement on the prepared specimens. Using an especially fabricated cementation unit, a static load (1 kg for 5 minutes) was applied during block cementation.n = 5) was measured after 24 hours and the other half was measured after 6 months of water storage in distilled water. Then, each specimen was serially sectioned perpendicular to the adhesive tooth interface into beams with a cross-sectional bonded area of ~1 mm2using a diamond saw . At a cross-head speed of 0.5 mm/min, tensile load of 500 N was applied till beam failure. Ten beams were selected for measuring μTBS of each specimen and recorded in mega pascal (MPa) by a computer software connected to the testing machine . Maximum tensile load was divided by the specimen cross-sectional area to obtain results in units of stress (MPa). Then, mean values were calculated for each tooth at each testing time.Each specimen was mounted in acrylic resin block using cylindrical Teflon mold of 15 mm diameter and 40 mm height. Half of each subgroup specimens  from the prepared specimens (one disc each). Each disc was of 1 mm thickness that was carefully examined under a stereo microscope after sectioning to ensure being free of coronal enamel or pulpal tissue.All discs were etched with phosphoric acid etching gel  for 15 seconds to remove the smear layer prior to thorough rinsing with deionized water and slight air-drying. Air bubble displacement inside a graduated glass tube was determined in the beginning of the procedure and after 10 minutes of switching the pressure pump. Any specimen that showed any signs of dentinal blockage or leakage was excluded immediately from the experiment and replaced to end up with 10 specimens as planned. Fluid filtration (Q) was measured from the linear air bubble displacement to the nearest 0.01 mm according to the following equationQ = displacement x cross sectional area of the pipette:Permeability was expressed in terms of fluid filtration (Jv) where: Jv = Q/AT−2min−1,Jv = fluid filtration rate in ml cmQ = fluid flow in mL,2A = dentinal surface area in cmT = time in minutes./Baseline fluid filtration ratex100.Baseline fluid filtration represents the maximum fluid flow of each specimen and was randomly assigned to a value of 100% permeability. Then, the fluid flow rate was measured after smear layer creation using 600-grit sand paper discs under water irrigation and after tested adhesive application. Dentin sealing percentage of each specimen was obtained using the following equation, with each specimen serving as its own control: Permeability reduction percentage = Difference between fluid filtration rate at the baseline and after treatment protocolt-test was used to compare between the tested groups.μTBS and permeability reduction percentage showed a parametric distribution when checked using Shapiro–Wilk test. Bond strength data were analyzed using Weibull analysis. Weibull parameters were calculated using maximum likelihood estimation, and 95% confidence intervals were calculated with Monte Carlo simulations. The different groups were compared at the characteristic strength (63.2% probability of failure). IndependentIDS technique significantly increased the μTBS compared with the DDS for both tested adhesives after 24 hours and 6 months of water storage. GLUMA Bond Universal adhesive showed significantly higher μTBS values compared with iBOND self-etch adhesive after 24 hours and 6 months of water storage at both IDS and DDS techniques. Both materials suffered a significant reduction in the Weibull characteristic strength after 6 months of water storage in comparison to the 24 hours values .p = 0.05 (α=0.05), a significant decrease in the permeability reduction percentage resulted after smear layer creation in comparison to the base line values. Both tested adhesives showed significant reduction in dentin permeability percentage in comparison to the smear layer mean values. Meanwhile, insignificant difference was recorded in the permeability reduction percentage between both tested adhesives  in their ingredients, both of which have been shown to interact chemically with hydroxyapatite.Unfortunately, after 6 months of water aging, both tested adhesives showed reduction in bond strength values. It is conceivable that their chemical interaction, which has been shown to deteriorate by aging, could be the reason for their reduced values.Regarding dentin permeability evaluation, assessment of hydraulic conductance has been documented to be an appropriate method for evaluating dentinal tubule occlusion.Under the limitations of the present study, it can be concluded that the IDS technique using self-etch mode of the universal adhesive is an effective strategy for improving the final bond strength of CAD/CAM resin composite restorations and reducing post-cementation sensitivity.