Hybridization between humans and Neanderthals has resulted in a low level of Neanderthal ancestry scattered across the genomes of many modern-day humans. After hybridization, on average, selection appears to have removed Neanderthal alleles from the human population. Quantifying the strength and causes of this selection against Neanderthal ancestry is key to understanding our relationship to Neanderthals and, more broadly, how populations remain distinct after secondary contact. Here, we develop a novel method for estimating the genome-wide average strength of selection and the density of selected sites using estimates of Neanderthal allele frequency along the genomes of modern-day humans. We confirm that East Asians had somewhat higher initial levels of Neanderthal ancestry than Europeans even after accounting for selection. We find that the bulk of purifying selection against Neanderthal ancestry is best understood as acting on many weakly deleterious alleles. We propose that the majority of these alleles were effectively neutral—and segregating at high frequency—in Neanderthals, but became selected against after entering human populations of much larger effective size. While individually of small effect, these alleles potentially imposed a heavy genetic load on the early-generation human–Neanderthal hybrids. This work suggests that differences in effective population size may play a far more important role in shaping levels of introgression than previously thought. A small percentage of Neanderthal DNA is present in the genomes of many contemporary human populations due to hybridization tens of thousands of years ago. Much of this Neanderthal DNA appears to be deleterious in humans, and natural selection is acting to remove it. One hypothesis is that the underlying alleles were not deleterious in Neanderthals, but rather represent genetic incompatibilities that became deleterious only once they were introduced to the human population. If so, reproductive barriers must have evolved rapidly between Neanderthals and humans after their split. Here, we show that observed patterns of Neanderthal ancestry in modern humans can be explained simply as a consequence of the difference in effective population size between Neanderthals and humans. Specifically, we find that on average, selection against individual Neanderthal alleles is very weak. This is consistent with the idea that Neanderthals over time accumulated many weakly deleterious alleles that in their small population were effectively neutral. However, after introgressing into larger human populations, those alleles became exposed to purifying selection. Thus, rather than being the result of hybrid incompatibilities, differences between human and Neanderthal effective population sizes appear to have played a key role in shaping our present-day shared ancestry. The recent sequencing of ancient genomic DNA has greatly expanded our knowledge of the relationship to our closest evolutionary cousins, the Neanderthals –5. NeandWhile some introgressed archaic alleles appear to have been adaptive in anatomically modern human (AMH) populations –14, on aIt is less clear why the bulk of Neanderthal alleles would be selected against. Were early-generation hybrids between humans and Neanderthals selected against due to intrinsic genetic incompatibilities? Or was this selection mostly ecological or cultural in nature? If reproductive barriers had already begun to evolve between Neanderthals and AMH, then these two hominids may have been on their way to becoming separate species before they met again , 20, 21.We set out to estimate the average strength of selection against Neanderthal alleles in AMH. Due to the relatively short divergence time of Neanderthals and AMH, we still share much of our genetic variation with Neanderthals. However, we can recognize alleles of Neanderthal ancestry in humans by aggregating information along the genome using statistical methods , 13. Herμ. Building on a long-standing theory on genetic barriers to gene flow [ℓ in the genome, we can express the present-day expected frequency of Neanderthal alleles in our admixture model in terms of the initial frequency p0, as well as a function gℓ of the recombination rates r between ℓ and the neighboring exonic sites under selection, and the parameters s, t, and μ  of modern humans are expected to carry the Neanderthal allele. The function gℓ decreases with tighter linkage to potentially deleterious sites, larger selection coefficient (s), longer time since admixture (t), and higher density of deleterious exonic sites (μ). If a neutral Neanderthal allele is initially completely unassociated with deleterious alleles, pℓ,t would on average be equal to p0. Our model explicitly accounts only for deleterious alleles that are physically linked to a neutral allele. However, in practice, neutral Neanderthal alleles will initially be associated (i.e. in linkage disequilibrium) not only with some linked, but also with potentially many unlinked deleterious alleles. This is because F1 hybrids inherited half of their genome from Neanderthal parents, which leads to a statistical association even among unlinked Neanderthal-derived alleles. Therefore, p0 should be thought of as an effective initial admixture proportion in the sense that it implicitly absorbs the effect of these physically unlinked, but statistically associated deleterious Neanderthal alleles. Technically this is because the effect of unlinked loci (assuming multiplicative fitness) can be factored into a constant multiplier of gℓ, and so can be accomodated into the model by rescaling p0 . Therefore, we average over the uncertainty in the locations of these alleles . We assuene flow –27, at end μ see , S2 Textnd 36 of ). In prap0, s, and μ), we minimised the residual sum of squared deviations (RSS) between observed frequencies of Neanderthal alleles [p0, s, and μ for autosomal variation in Neanderthal ancestry in the EUR and ASN populations.To estimate the parameters of our model . We obtain similar estimates if we assume that the Neanderthal ancestry in humans has reached its equilibrium frequency or if we account for the effect of multiple selected sites . These estimates correspond to less than 1 in 10,000 exonic base pairs harboring a deleterious Neanderthal allele, on average. As a result, our estimates of the average selection coefficient against an exonic base pair (the compound parameter (μs) are very low, on the order of 10−8 in both samples , but the 95% bootstrap CI overlap (p0) are mildly confounded with estimates of the strength of selection per exonic base (μs). That is, somewhat similar values of the expected present-day Neanderthal allele frequency can be inferred by simultaneously reducing p0 and μs  against deleterious Neanderthal alleles are very low, on the order of the reciprocal of the effective population size of humans. This raises the intriguing possibility that our results are detecting differences in the efficacy of selection between AMH and Neanderthals. Levels of genetic diversity within Neanderthals are consistent with a very low long-term effective population size compared to AMH, i.e. a higher rate of genetic drift [s fall in a region of parameter space for which simulations suggest that Neanderthals have a strong excess of population-specific fixed deleterious alleles, compared to humans , different from the autosomes, which allows us to detect a sex bias in the direction of matings between AHM and Neanderthals. Although our formulae can easily incorporate sex-specific selection coefficients, we keep a single selection coefficient (sX) to reduce the number of parameters. Therefore, sX reflects the average reduction in relative fitness of deleterious Neanderthal alleles across heterozygous females and hemizygous males.We finally turn to the X chromosome, where observed levels of Neanderthal ancestry are strongly reduced compared to autosomes , 13. ThipX0,, μX, and sX using our modified model to [μX and sX, see μXsX, i.e. the average selection coefficient against an exonic base pair on the X. In μXsX for the X, along with analogous estimates of μs for autosomal chromosomes. For the X chromosome, there is also strong confounding between pX0, and μXsX, to a much greater extent than on the autosomes (note the larger spread of the X point clouds). Due to this confounding, our marginal confidence intervals for μXsX and pX0, overlap with their autosomal counterparts  exclusively to sex-biased hybridization, our result would imply that matings between Neanderthal males and human females were about three times more common than the opposite pairing . Our approach represents one of the first attempts to estimate the strength of genome-wide selection against introgression between populations. The method we use is inspired by previous efforts to infer the strength of background selection and selective sweeps from their footprint on linked neutral variation on a genomic scale –39. We hμ) and average strength (s) of purifying selection. Therefore, our findings of weak selection are conservative in the sense that the true strength of selection per base pair may be even weaker. We argue that the bulk of selection against Neanderthal ancestry in humans may be best understood as being due to the accumulation of alleles that were effectively neutral in the Neanderthal population, which was of relatively small effective size. However, these alleles started to be purged, by weak purifying selection, after introgressing into the human population, due to its larger effective population size.Here, we have shown that observed patterns of Neanderthal ancestry in modern human populations are consistent with genome-wide purifying selection against many weakly deleterious alleles. For simplicity, we allowed selection to act only on exonic sites. It is therefore likely that the effects of nearby functional non-coding regions are subsumed in our estimates of the density  still segregate for deleterious alleles introduced into Eurasian populations via interbreeding with Neanderthals. However, given that the initial frequency of the admixture was very low, we predict that a typical EUR or ASN individual today only carries roughly a hundred of these weak-effect alleles, which may have some impact on genetic load within these populations.As our results imply, selection against deleterious Neanderthal alleles was very weak on average, such that, after tens of thousands of years since their introduction, these alleles will have only decreased in frequency by 56% on average. Thus, roughly seven thousand loci (≈ −4)7000 ≈ 94% compared to modern humans, who lack all but roughly one hundred of these deleterious alleles. This would obviously imply a substantial reduction in fitness, which might even have been increased by a small number of deleterious mutations of larger effect that we have failed to capture. This potentially substantial genetic load has strong implications for the interpretation of our estimate of the effective initial admixture proportion (p0), and, more broadly, for our understanding of those early hybrids and the Neanderthal population. We now discuss these topics in turn.Although selection against each deleterious Neanderthal allele is weak, the early-generation human–Neanderthal hybrids might have suffered a substantial genetic load due to the sheer number of such alleles. The cumulative contribution to fitness of many weakly deleterious alleles strongly depends on the form of fitness interaction among them, but we can still make some educated guesses (the caveats of which we discuss below). If, for instance, the interaction was multiplicative, then an average F1 individual would have experienced a reduction in fitness of 1 −  do not strongly change when we include multiple deleterious sites or consider large windows surrounding each focal neutral site (up to 10 cM) in our inference procedure .Strictly, under our model, the estimate of  hybrids . These u hybrids  have recengtsson ). In praIf the predicted drop in hybrid fitness is due to the accumulation of many weakly deleterious alleles in Neanderthals, as supported by our simulations, it also suggests that Neanderthals may have had a very substantial genetic load (more than 94% reduction in fitness) compared to AMH Drosophila yakuba and D. santomea [Xiphophorus sister species [Whether or not the many weakly deleterious alleles in Neanderthals were a cause, or a consequence, of the low Neanderthal effective population size, they have had a profound effect on patterning levels of Neanderthal introgression in our genomes. More generally, our results suggest that differences in effective population size and nearly neutral dynamics may be an important determinant of levels of introgression across species and along the genome. Species coming into secondary contact often have different demographic histories  and so We have here considered the case of introgression from a small population  into a larger population (humans), where selection acts genome-wide against deleterious alleles introgressing. However, from the perspective of a small population with segregating or fixed deleterious alleles, introgression from a population lacking these alleles can be favoured . This coS1 and N1 be the introgressed  alleles at the selected and linked neutral autosomal locus, respectively, and S2 and N2 the corresponding resident (human) alleles. The recombination rate between the two loci is r. We assume that allele S1 is deleterious in humans, such that the viability of a heterozygote human is w(S1S2) = 1 − s, while the viability of an S2S2 homozygote is w(S2S2) = 1. We ignore homozygous carriers of allele S1, because they are expected to be very rare, and omitting them does not affect our results substantially  is a function of the recombination rate r between the neutral and the selected site, the selection coefficient s, and the time t in generations since admixture  region of the X chromosome . As abovf is equivalent to the gene flow factor , and that the selected site is located in the middle of that exon. Lastly, the effects of selection at linked sites will be small if their genetic distance from the neutral site is large compared to the strength of selection (s). In practice, we may therefore limit the computation of ℓ, but also explored larger windows of size 10 cM to show that our results are not strongly affected by this choice. Taken together, these assumptions greatly simplify our computations and allow us to calculate the expected present-day frequency of the Neanderthal allele at each SNP along the genome.Lastly, we introduce a parameter ℓ, and denote the total number of exons in this window by ith nearest exon to the focal locus ℓ be li base pairs. The probability that the ith exon contains the nearest selected site is then i − 1 exons closer to ℓ than exon i. Conditional on the ith exon containing the selected site, the frequency pt of N1 at locus ℓ and time t is computed according to r replaced by ri, the recombination rate between ℓ and the center of exon i. Then, we can write the expected frequency of the neutral Neanderthal allele at site ℓ surrounded by Specifically, consider a genomic window of size 1 cM centered around the focal neutral site f as given in Eqs The last product term accounts for the case where none of the website. We use estimates from Sankararaman et al. [pn. Although pn is also an estimate, we generally refer to it as the observed frequency, in contrast to our predicted/expected frequency pt. Sankararaman et al. [pn for samples originating from Europe (EUR) and East Asia (ASN)  SNPs for which [gℓ is calculated according to s and μ. We did not perform a grid search for p0. Rather, for each combination of s and μ, we analytically determined the value of p0 that minimizes the RSS asgℓ is given in nl considered autosomal (X-linked) SNPs. For details, we refer to Our inference method relies on minimizing the residual sum of squared differences (RSS) between E[or which  providednl. We then resampled these segments (with replacement) to create a bootstrap chromosome of the same length as the original chromosome. Once all appropriate bootstrap chromosomes were created , we obtained for each bootstrap sample the combination of p0, μ, and s that minimises the RSS according to Eqs We obtained confidence intervals by calculating 2.5 and 97.5 percentiles from 1000 bootstrapped genomes. We created these chromosome by chromosome as follows. For a given chromosome, for each non-overlapping segment of length 5 cM, and for each of 676 parameter combinations, we first calculated the denominator and the numerator of s and μ, suggesting that our findings are reasonably robust to our choices.In To test whether selection against alleles introgressed from Neanderthals can be explained by the differences in ancient demography, we simulated the frequency trajectories of deleterious alleles in the Neanderthal and human populations, between the time of the Neanderthal–human split and the time of admixture . We assuFor each simulation run, we recorded the frequency of the deleterious allele in Neanderthals and humans immediately prior to admixture. Our simulations show that the majority of deleterious alleles that are still segregating at the end of the simulation are fixed differences . This maHarris and Nielsen  independS1 TextHere, we describe several models of a single pulse of admixture between Neanderthal and modern humans, and derive approximations for the present-day frequency of a neutral introgressed Neanderthal allele linked to one or multiple sites under purifying selection in humans. We then demonstrate the accuracy of these approximations by comparing them to numerically iterated recursion equations and individual-based simulations. Lastly, we consider models of single and multiple waves of continuous introgression and show that one cannot distinguish between these models and a single-pulse admixture model using the present-day frequency of introgressed alleles as the only source of information.(PDF)Click here for additional data file.S2 Textμ that, at any given exonic base pair, a deleterious Neanderthal allele is segregating in the modern human population. We then discuss the details of our inference procedure and expand on our results.Here, we introduce the last model parameter, the average probability (PDF)Click here for additional data file.S3 Texts) and the exonic density of deleterious sites (μ) that we estimated Click here for additional data file.S1 Figt = 2000 (red) and the equilibrium given in Eq. (8) of s = 0.0001, and y0 = 0 for all lines, and p0 = 0.04 (dotted), 0.034 (dashed) and 0.03 (full line).Lines represent Eq. (6) of (EPS)Click here for additional data file.S2 Figt = 2000 (red) and the equilibrium given in Eq. (8) of s = 0.0004, and y0 = 0 for all lines, and p0 = 0.04 (dotted), 0.034 (dashed) and 0.03 (full line).Lines represent Eq. (6) of (EPS)Click here for additional data file.S3 Figt = 2000 (red) and the equilibrium from Eq. (13) of sf = sm = 0.0001, and yX,0 = 0 for all lines, and p0 = 0.04 (dotted), 0.034 (dashed) and 0.03 (full line).Lines represent Eq. (12) of (EPS)Click here for additional data file.S4 Figt = 2000 (red) and the equilibrium from Eq. (13) of sf = sm = 0.0004, and yX,0 = 0 for all lines, and p0 = 0.04 (dotted), 0.034 (dashed) and 0.03 (full line).Lines represent Eq. (12) of (EPS)Click here for additional data file.S5 Figr (recombination rate) and s (selection coefficient). Values of r range from 1 × 10−5 (red circle border) to 1 ×10−2 (black border), s ranges from 1 × 10−5 (yellow circle area) to 4 × 10−4 (light blue area). For each parameter combination, the mean frequency of N1 after t = 2000 generations was calculated across 1000 independent runs. Grey lines represent approximate 95% confidence intervals for simulation results (mean ±1.96 × standard error), and a black line with slope 1 is shown for reference.The figure shows 676 circles representing different combinations of (EPS)Click here for additional data file.S6 FigpIJ∞, from Eq. (15) of t = 2000 generations and until the equilibrium is reached, respectively. A: The neutral locus is flanked by one locus under selection on each side, and recursions followed Eq. (17) of a = 0.0002 and b = 0.0004, respectively. The initial frequency of N1 is p0 = 0.04.Curves show (EPS)Click here for additional data file.S7 FigpIJX,∞, from Eq. (21) of t = 2000 generations and until the equilibrium is reached, respectively. A, B: The neutral locus is flanked by one locus under selection on each side. C, D: The neutral locus is flanked by two loci under selection on one side. A, C: Selection coefficients against introgressed deleterious mutations at locus af = 0.0001 (am = 0.0003) and bf = 0.0002 (bm = 0.0006), respectively. B, D: Selection coefficients are identical in the two sexes; af = am = 0.0001 and bf = bm = 0.0002. In all panels, the initial frequency of N1 is pX,0 = 0.04.Curves show (EPS)Click here for additional data file.S8 Figr = 10−4, s = 5 × 10−4, x0 = 0.04, and y0 = 0.001. The duration of admixture in the single-wave model is τ = 500. Additional parameters for the dual-wave model are τ1 = 75, τ2 = 1075, τ3 = 1500. The solid black line represents a single-pulse model without change of time.By changing time in the single-pulse model (dashed and dotted black lines) as described in (EPS)Click here for additional data file.S9 Figp0, making this a profile RSS surface. Regions shaded in orange represent parameter values of higher RSS.Each value of the RSS is minimized over (EPS)Click here for additional data file.S10 Figp0, making this a profile RSS surface. Regions shaded in orange represent parameter values of higher RSS. Black circles show bootstrap results of 1000 block bootstrap reestimates, with darker circles corresponding to more common bootstrap estimates.Each value of the RSS is minimized over (EPS)Click here for additional data file.S11 Figp0, making this a profile RSS surface. Regions shaded in orange represent parameter values of higher RSS.Each value of the RSS is minimized over (EPS)Click here for additional data file.S12 Figp0, making this a profile RSS surface. Regions shaded in orange represent parameter values of higher RSS. Black circles show bootstrap results of 1000 block bootstrap reestimates, with darker circles corresponding to more common bootstrap estimates.Each value of the RSS is minimized over (EPS)Click here for additional data file.S13 Figp0, making this a profile RSS surface. Regions shaded in orange represent parameter values of higher RSS. Black circles show bootstrap results of 1000 block bootstrap reestimates, with darker circles corresponding to more common bootstrap estimates.Each value of the RSS is minimized over (EPS)Click here for additional data file.S14 Figt = 2000 generations after Neanderthals split from the EUR (grey) and ASN (pink) populations. Dots and horizontal lines show the value of p0 that minimizes the RSS and the respective 95% block-bootstrap confidence intervals. Each value of the RSS is evaluated at the values of the selection coefficient (s) and exonic density of selection (μ) given in Table A in Results are shown for a model where only the nearest-neighboring exonic site under selection is considered, and for (EPS)Click here for additional data file.S15 FigGenomic regions with low exonic density (low exonic density rank) contain higher average Neanderthal allele frequency in both in Europeans (grey circle) and Asians (pink circle), a pattern recreated in our model. Dashed lines represent the 95% block bootstrap confidence intervals. The length of segments used to create the bins is 2 cM.(EPS)Click here for additional data file.S16 FigGenomic regions with low exonic density (low exonic density rank) contain higher average Neanderthal allele frequency in both in Europeans (grey circle) and Asians (pink circle), a pattern recreated in our model. Dashed lines represent the 95% block bootstrap confidence intervals. The length of segments used to create the bins is 1.5 cM.(EPS)Click here for additional data file.S17 FigGenomic regions with low exonic density (low exonic density rank) contain higher average Neanderthal allele frequency in both in Europeans (grey circle) and Asians (pink circle), a pattern recreated in our model. Dashed lines represent the 95% block bootstrap confidence intervals. The length of segments used to create the bins is 0.5 cM. There are 9 bins, rather than 10 bins, in this figure because there are many 0.5 cM bins with zero exonic sites. Therefore, we collapsed our results together into a smaller number of bins.(EPS)Click here for additional data file.S18 Figp0, which makes this a profile RSS surface. Regions shaded in orange represent parameter values of higher RSS.This surface is constructed using windows of 10 cM, but otherwise analogous to (EPS)Click here for additional data file.S19 Figp0, which makes this a profile RSS surface. Regions shaded in orange represent parameter values of higher RSS.This surface is constructed using windows of 10 cM. Each value of the RSS is minimized over (EPS)Click here for additional data file.S20 Figp0, which makes this a profile RSS surface. Regions shaded in orange represent parameter values of higher RSS. The window size 1 cM.This surface is constructed using the fraction of EUR and ASN alleles at each site with confident Neanderthal calls . Each value of the RSS is minimized over (EPS)Click here for additional data file.S21 Figr (recombination rate) and s (selection coefficient). Values of r range from 1 × 10−5 (red circle border) to 1 × 10−2 (black border), s ranges from 1 × 10−5 (yellow circle area) to 4 × 10−4 (light blue area). For each parameter combination, the mean and variance of the frequency of N1 after t = 2000 generations was calculated across 1000 independent runs.The figure shows 676 circles representing different combinations of (EPS)Click here for additional data file.S22 Figp0, which makes this a profile RSS surface. The window size 1 cM.Each value of the RSS is minimized over (EPS)Click here for additional data file.S23 Figs for ASN (the larger of the two CI) that we inferred. Note that monomorphic sites are not shown, but are included in the denominator of the fraction of sites. In contrast to Nn = 500 and u = 10−8.(A) A two-dimensional histogram of the difference in allele frequency between the Neanderthal and human population, and the deleterious selection coefficient over all simulated sites. (B) The fraction of sites in the simulations where there is a human- or Neanderthal-specific fixed difference, binned by selection coefficient. Dotted lines indicate the nearly-neutral selection coefficient (i.e. the inverse of the effective population size) for Neanderthal (right) and Human (left) populations. Solid lines show the 95% CI of (EPS)Click here for additional data file.S24 FigN2 = 1000.Details are as in (EPS)Click here for additional data file.S25 FigN2 = 2000.Details are as in (EPS)Click here for additional data file.S26 FigTb = 10 generations prior to admixture with Neanderthals. The long-term effective size of the human population prior to the bottleneck was set to Nh = 14400, and the effective size during the bottleneck to 1861 (see In contrast to (EPS)Click here for additional data file.S27 FigTb = 100 generations.Details are as in (EPS)Click here for additional data file.S28 FigTb = 1000 generations.Details are as in (EPS)Click here for additional data file.


PIK3CA and TP53, two genes commonly deregulated in the late stage, had an effect on the maintenance of the CD26+ cell population. When CD26− tumor daughter cells were sorted and cultured, the emergence of tumor spheres containing CD26+ cells occurred. These findings shed light to the origin of colorectal cancer stem cells with metastatic properties, which has an implication on conventional treatments by surgery or adjuvant chemotherapy for tumor debulking.Colorectal cancer results from genetic aberrations which accumulate over a long period of time, with malignant and metastatic properties acquired at a relatively late stage. A subpopulation of CD26+ colorectal cancer stem cells are known to be implicated in metastasis. We quantified CD26+ cancer cells in 11 primary tumor samples by flow cytometry, and showed that tumors having confirmed or suspected metastases harbored a relatively high CD26+ level in these samples. We hypothesized that this subpopulation of cancer stem cells arises in the late stage of carcinogenesis from the bulk of tumor daughter cells which are CD26−. The manipulation of  Patients with colorectal cancer may suffer from grave prognostic implications when metastatic disease develops. The understanding of cancer metastasis has evolved with the cancer stem cell model, which proposes that cancer is maintained and initiated by a rare population of tumor cells having unique self-renewal properties . RecentlThe prevailing model of colorectal cancer suggests that carcinogenic and metastatic properties are acquired in a stepwise process, with accumulations of new genetic aberrations . Given tTP53 and PIK3CA. Mutations in both these genes are considered late events and occur in a substantial proportion of colorectal cancer, with approximately 70% of sporadic tumors harboring TP53 mutations [PIK3CA mutations [We focused on genes which likely play a role in the later stage of carcinogenesis, such as utations  and arouutations .PIK3CA and TP53 on CD26+ tumor cell maintenance. We hypothesized the possibility that these CSC with metastatic properties may arise from non-stem tumor cells. Finally, we discussed how our findings may apply to the current models of colorectal carcinogenesis.The current study focused on the possible origin and mechanism of maintenance of CD26+ colorectal cancer cells, which were shown to be CSC with metastatic properties. We quantified CD26+ in tumor samples by flow cytometry and correlated the findings with clinical data, in order to investigate if a high CD26+ level correlates with a later stage of cancer. We evaluated the functional roles of To investigate the notion that CD26+ cells appear in the later stage of colorectal carcinogenesis, we identified the percentage of CD26+ cells within clinical specimens from various tumor stages by flow cytometry. Our group has previously demonstrated that the number of primary tumors with liver metastases harboring >1% of CD26+ CSC was greater than that of primary tumors without metastases . In thisp = 0.061, p = 0.13, data not shown). Univariate analyses were performed to investigate the prognostic implication of age, gender, metastases, tumor size, degree of differentiation, and CD26+ population percentage . The site of metastases was also not found to be related to CD26+ level  . As was the case for SW480, the low baseline level of CD26+ restricted the possible effect of PIK3CA inhibitors and thus may have impacted the statistical significance. We hypothesized that CD26+ cells emerged from non-stem colon cancer cells under the influence of genes which characterize the late stage of colorectal carcinogenesis. We investigated whether an inhibition of , n = 3) . Experim, n = 3) . For SW4TP53 could influence the percentage of CD26+ cells found in the resultant cultures, TP53 was knocked down within the SW480 cell line using a targeting small interfering RNA (siRNA). The maximal knockdown as evidenced by Western blot was 48 h after transfection    B,C. To i, n = 6) D.Because the cancer stem cell population in a cancer is by definition small, the effect exerted on this intricate cell population by the knockdown of a tumor suppressor gene could be minute. When another cancer cell line, SW620, was tested, the change in CD26+ cell population was about 3.88 ± 3.42-fold . The origin of cancer stem cells is still unsettled in the research community. Possibilities include tumor daughter cells, transit amplifying cells, or tissue stem cells ,10. The Our group previously demonstrated that a CD26+ tumor cell subpopulation in primary tissue has stem-like properties in that these cells can initiate sphere formation and tumor formation in serial dilution implantation experiments . This stTP53 is a tumor suppressor gene, whose mutation is known to be a late event and is implicated in around 70% of colorectal cancer cases [PIK3CA is an oncogene found to be mutated in around 25% of tumors and is also a late event [TP53 enlarges the CD26+ population, while inhibition of PIK3CA has the opposite effect. We further hypothesized that this CD26+ cell population had stem-like properties, and could arise in a colorectal tumor with a predominant population of CD26− daughter cells at the late stage. To this end, we used fluorescent-activated cell sorting to select CD133−/CD26− cells for culture using colorectal cancer cell lines HCT116 and HT29. After a period of 7 to 14 days, sphere formation was detected in CD133−/CD26− cell cultures from both cell lines, indicating the formation of cells with stem cell-like properties . Cells wPIK3CA and TP53, we observed that the CD26+ population with stem-like properties can be initiated or maintained. This finding does not favor the mechanism of non-genetic phenotype plasticity, although this possibility is not completely excluded. Also, the increase in CD26+ CSC subpopulations can be due to either an increase in cell formation, an increase in cell survival, or a decrease in cell differentiation. Each mechanism likely contributes to some extent to our observation. As CD26− and CD26+ cells may interchange in the cell culture due to some inherent genetic changes, each population should not be viewed in isolation but in a dynamic equilibrium. Taken together, the emergence of the CD26 cell surface marker allows for two possible interpretations; either the cells has acquired stem-like properties during the cell culture, or the change in the cell surface marker is merely an effect of non-genetic phenotype plasticity in response to environmental stress . HoweverVarious research groups have shown that differentiated cells may spontaneously convert to stem cells ,14. ProvWe understand the limitations of this study include several aspects. While the CD26+ level is expected to be high in tumors with confirmed or suspected metastases, it is also important from the clinical point of view to quantify CD26+ levels in patients with early stages of cancer in the hope of identifying those who may be more prone to developing metastatic disease. It remains unknown whether a high CD26+ level in early-stage tumors represents the possibility of undetected micrometastases or truly increased risks of future metastasis, or whether this is merely an incidental finding. The limitation in obtaining viable cell samples from clinical specimens for flow cytometry, which is particularly true for the inherently contaminated colorectum, complicated the analyses. Future investigations correlating the CD26+ level of the primary tumor with quantities of circulating tumor cells or cell-free nucleic acid may be useful. Limitations were also identified for the in vitro studies. First, cancer cell lines may have acquired many more genetic aberrations with passages compared to cancer cells in vivo. Second, the percentage of CD133+ and CD26+ cells were found to be much greater in cancer cell lines than in tissue obtained from patients. Third, the in vitro model is at its best a simulation of its in vivo counterpart, where the tumor microenvironment may contribute to malignant cell growth, maintenance, and metastasis ,19. We dThe theory of cancer stem cells has casted doubt on the current treatment for cancer, because chemotherapy at its best only debulks the tumor, while many of the CSC, critical to tumor maintenance but more resistant to chemotherapy ,21,22, a2 humidity. For sphere formation, cancer cell obtained from fluorescent-activated cell sorting (FACS) were cultured on ultra-low attachment plates , and also incubated under the aforementioned conditions.HT29, SW480, SW48, SW620 and HCT116 (obtained from American Type Culture Collection (ATCC), Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)  and supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1% Penicillin-Streptomycin (Life Technologies), and placed in an incubator with a temperature of 37 °C, under 5% COCells were harvested from culture, washed with phosphate buffered saline (PBS), and incubated with the appropriate staining antibodies in binding buffer for 15 min at room temperature in the dark. After being washed with PBS twice, the cells were analyzed on a Cytomics FC500  or BD FACSCalibur ). Cell sorting was performed with a MoFlo XDP Cell Sorter (Beckman Coulter). In all cytometry analyses and cell sorting experiments, an appropriate isotype control was used, and the positive population was defined where the fluorescent signal intensity exceeded that of the 99th percentile of the isotype control. The data acquired were analyzed using FlowJo . Antibodies used include CD133-PE  and CD26−PE/Cy5 (Miltenyi Biotech). PIK3CA inhibitor  was used as a culture medium. Cells were harvested after 48 h for downstream analyses.One hundred and fifty thousand cells per well were seeded in an adherent six-well culture plate (Corning Inc.) under the culture conditions described above. On the subsequent day, fresh antibiotics-free DMEM with 10 µM p53  at 40 nM was delivered to the cells using Lipofectamine 2000 , according to the manufacturer’s instruction. Nonsense scrambled oligo  was used as a control. Cells were harvested after 48 h post-transfection for analyses. For Western blot analyses, ice-cold radioimmunoprecipitation assay buffer (RIPA) buffer  containing phenoylmethylsulfonyl fluoride (1 mmol/L) and protease inhibitor was used for cell lysis. The lysate was centrifuged at 12,000× g for 15 min to obtain the supernatant. The harvested protein was suspended in sodium dodecyl sulfate buffer and resolved by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. Transfer to polyvinylidene difluoride (PVDF) membranes  was carried out. The antibody against TP53 was purchased from Cell Signaling Technology . The membranes were developed after probing with horseradish peroxidase-conjugated secondary antibodies. Small interfering RNA (siRNA) targeting Fresh tumor resection specimens were obtained with informed consent from 11 colorectal cancer patients who underwent surgical resection at the Department of Surgery, Queen Mary Hospital, University of Hong Kong, approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB). The specimens were immediately minced on ice, suspended in DMEM/F12 medium , and dissociated with collagenase (Invitrogen) and hyaluronidase . Enzymatically disaggregated suspensions were filtered and washed three times with PBS. Red blood cells were removed by Histopaque-1077 . The resulting single tumor cells were used for flow cytometry analyses.p-Values < 0.05 were considered statistically significant. Analyses were performed with the SPSS 20 statistics software .Continuous variables were expressed as means ± SD. Treatment groups were compared with the independent or paired sample t test where appropriate. Comparisons involving variables with non-normal distribution were performed by the Mann-Whitney U test. Comparisons of nominal variables were performed via Chi-square test or Fisher’s exact test where appropriate. Knowledge of the timing and cell type where cancer stem cells with metastatic properties may arise is critical for effective treatment, although this probably varies with different cancers. In colorectal cancer, disease develops over a long period of time by stepwise acquisitions of mutations which are relatively well-defined. Detectable metastases usually occur as the terminal event. This study supports the possibility that emergence of CD26+ colorectal cancer stem cells with metastatic properties can be under the influence of genes that are usually mutated late in the disease course.


We show that the orientation of cassette arrays relative to replication influences attC site folding and cassette excision by placing the recombinogenic strands of attC sites on either the leading or lagging strand template. We also demonstrate that stability of attC sites and their propensity to form recombinogenic structures also regulate cassette excision. We observe that cassette excision dynamics driven by these factors differ between MIs and SCIs. Cassettes with high excision rates are more commonly found on MIs, which favors their dissemination relative to SCIs. This is especially true for SCIs carried in the Vibrio genus, where maintenance of large cassette arrays and vertical transmission are crucial to serve as a reservoir of adaptive functions. These results expand the repertoire of known processes regulating integron recombination that were previously established and demonstrate that, in terms of cassette dynamics, a subtle trade-off between evolvability and genetic capacitance has been established in bacteria.Integrons ensure a rapid and “on demand” response to environmental stresses driving bacterial adaptation. They are able to capture, store, and reorder functional gene cassettes due to site-specific recombination catalyzed by their integrase. Integrons can be either sedentary and chromosomally located or mobile when they are associated with transposons and plasmids. They are respectively called sedentary chromosomal integrons (SCIs) and mobile integrons (MIs). MIs are key players in the dissemination of antibiotic resistance genes. Here, we used  in silico and in vivo analyses, we show that cassette excision is highly regulated by replication and by the intrinsic properties of cassette recombination sites. We also demonstrated differences in the dynamics of cassette recombination between mobile and sedentary chromosomal integrons (MIs and SCIs). For MIs, a high cassette recombination rate is favored and timed to conditions when generating diversity (upon which selection can act) allows for a rapid response to environmental conditions and stresses. In contrast, for SCIs, cassette excisions are less frequent, limiting cassette loss and ensuring a large pool of cassettes. We therefore confirm a role of SCIs as reservoirs of adaptive functions and demonstrate that the remarkable adaptive success of integron recombination system is due to its intricate regulation.The integron system confers upon bacteria a rapid adaptation capability in changing environments. Specifically, integrons are involved in the continuous emergence of bacteria resistant to almost all antibiotic treatments. The international situation is critical, and in 2050, the annual number of deaths caused by multiresistant bacteria could reach 10 million, exceeding the incidence of deaths related to cancer. It is crucial to increase our understanding of antibiotic resistance dissemination and therefore integron recombination dynamics to find new approaches to cope with the worldwide problem of multiresistance. Here, we studied the dynamics of recombination and dissemination of gene encoding cassettes carried on integrons. By combining  Antibiotics are essential to the success of modern medicine, but their efficacy has been impeded by the emergence of multiresistant bacteria. In 1989, integrons were identified as systems responsible for the dissemination of resistance genes among Gram-negative bacterial pathogens , primari4–intI), a site-specific tyrosine recombinase which catalyzes cassette rearrangements; (ii) a primary recombination site for the insertion of cassettes, attI; and (iii) a promoter, Pc, driving the expression of proximal cassettes in the array (attC). Cassette arrays represent a low-cost repository of valuable functions for the cell and most likely reflect a history of adaptive events. The number of cassettes in the array can be very large in SCIs (more than 200), while it rarely exceeds eight in MIs  a gene encoding the integron integrase  form, attC sites are recombined in a single-stranded (ss) folded form (–attC site recombines about 103 times more frequently than the top strand (attC sites does not rely on the nature of their primary sequence but rather on the structure of their folded single-stranded DNA (ssDNA), as they share only 3 conserved nucleotides at the cleavage site , the unpaired central spacer (UCS), and the variable terminal structure (VTS), which ensure strand selectivity and high levels of attC recombination (–attC site length (from 57 to 141 bp) is mostly due to differences in the VTS loop (attC site of the aadA7 gene (attCaadA7 site) to complex branched secondary structures in larger sites such as Vibrio cholerae repeat (VCR) sites .–15.attI p strand . The prep strand , 17. In age site . Folded bination –18. The VTS loop  19), wh, whattI erae SCI ). Due tolication .attC sites leads to the excision of a cassette from the array. Recombination between the attC site of the excised cassette and the attI site allows for the reinsertion of the cassette in the beginning of the array, placing it downstream of the Pc promoter where it is more highly expressed. Hence, cycles of excision and insertion shuffle cassettes in the array and change their expression patterns , 23attC patterns  are probpatterns . Stress patterns , 27. Thepatterns .attC sites, the regulation of cassette excision is dependent on attC site folding, meaning that bacteria must regulate it subtly. This is particularly important because the presence of stable and long hairpin structures can also be detrimental for the maintenance of bacterial genomes (attC site integrity ensured by the single-stranded DNA binding (SSB) protein which hampers folding of attC sites in the absence of the integrase  and by the propensity to form cruciform structures due to supercoiling . However, if the rate is too high and the balance between cassette excision and insertion is shifted toward excision, then cassettes could be rapidly lost, decreasing the probability of their vertical transmission. Since cassette excision directly depends on simultaneous folding of consecutive  genomes . We haventegrase ; on the rcoiling .in silico analyses and in vivo experiments to study the parameters that play important roles in maintaining an adequate level of cassette excision: orientation of cassette arrays relative to replication, CDS lengths, and attC site properties. Finally, we discuss the results obtained for MIs and SCIs in terms of integron evolutionary biology.In order to gain a better understanding of cassette excision dynamics in integrons, we performed both attC site (attCbs) is located on the lagging strand template in which large regions of ssDNA are available , its folding is favored, increasing the frequency of attC × attI recombination . In the data set of SCI-containing genomes, Vibrio genomes are overrepresented, reflecting the general bias toward human pathogen species. We therefore repeated this analysis after exclusion of Vibrio genomes and found that this difference remained significant  as a function of attCbs orientation relative to replication excluding Vibrio strains. Dots correspond to the number of attC sites for each of the analyzed 34 MCIs and 9 SCIs. Tests of the differences between data sets were performed using the Wilcoxon rank sum test . MCI, mobile chromosomal integron; SCI, sedentary chromosomal integron; bs, bottom strand; Lag st temp, lagging strand template; Lead st temp, leading strand template. Download FIG S1, EPS file, 0.5 MB.Distribution of the number of Copyright © 2017 Loot et al.2017Loot et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the attC sites is promoted, e.g., when the distance between both sites was smaller than the length of ssDNA found between two Okazaki fragments or smaller than the size of supercoiled plectonemes, cassette excision is likely to be favored. Therefore, we decided to analyze the cassette length. We used two independent proxies for the length of cassettes to test this hypothesis: CDS length and the distance between identified attC sites. On the one hand, CDS length underestimates cassette length but is highly correlated with it, since most cassettes have one single CDS and small adjacent regions. On the other hand, the distance between attC sites provides an exact measure of cassette length but is affected by inaccuracies in the detection of attC sites (a missed attC can lead to the doubling of a cassette length). We performed these analyses for the 393 integrons identified by the IntegronFinder program  except in Vibrio strains. (B) Violin plots showing the distribution of mean CDS lengths per replicon and per integron for replicons, MIs, and SCIs (excluding ARGs or not excluding ARGs). (C) Violin plots showing the distribution of mean lengths between two consecutive attC sites per integron for MIs and SCIs. (D) Violin plots showing the distribution of pairwise differences in mean CDS lengths between a replicon and its integron for MIs and SCIs. Download FIG S2, EPS file, 1 MB.Cassette and CDS length analysis of the 393 integron-containing strains identified by IntegronFinder and their replicons. Tests of the differences between data sets were performed using the Wilcoxon rank sum test. Differences are significant . We extr trends .10.1128/mBio.02296-16.3FIG S3 Vibrio strains and their SCIs. The horizontal green bar indicates the position of the SCI. bps, base pairs; CDS, coding sequence. (A) CDS lengths as a function of their positions in chromosome 1 (Chr1) of Vibrio vulnificus CMCP6 strain. (B) CDS lengths as a function of their positions in chromosome 2 (Chr2) of Vibrio fischerii MJ11 strain. (C) CDS lengths as a function of their positions in chromosome 1 (Chr1) of Vibrio parahaemolyticus RIMD 2210633 strain. (D) CDS lengths as a function of their positions in chromosome 2 (Chr2) of Aliivibrio salmonicida LFI1238 strain. (E) CDS lengths as a function of their positions in chromosome 1 (Chr1) of Vibrio alginolyticus ATCC 17749 strain. (F) CDS lengths as a function of their positions in the whole genome of Vibrio rotiferanus DAT722 strain. Download FIG S3, PDF file, 0.6 MB.CDS length analysis of Copyright © 2017 Loot et al.2017Loot et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the attC sites involved in the reaction . These analyses were performed on 185 attC sites from MIs and 1,744 attC sites from SCIs , whereas SCIs contain fewer such sites (only 52%). Moreover, contrary to MIs, SCIs contain sites with an extremely low pfold value . These low-pfold attC sites are mostly found in Vibrio SCIs. When attC sites exclusively found in Vibrio strains were excluded from the data set, we found no significant difference among the pfold values of MI and SCI sites  Proportion of attC sites as a function of attCbs pfold values for MIs and SCIs, excluding Vibrio strains or not excluding Vibrio strains. bs, bottom strand. (B) Proportion of attC sites as a function of the length of the attC site for MIs and SCIs, excluding Vibrio strains or not excluding Vibrio strains. bps, base pairs. Download FIG S4, EPS file, 1.3 MB.Supplementary data on Copyright © 2017 Loot et al.2017Loot et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the attC site length comparison between MIs and SCIs showed that the former have smaller VTSs, which can be as short as 3 nucleotides. The attC sites of SCIs often have longer VTSs. The length of MI attC sites is relatively heterogeneous, ranging from 56 to 141 bp, with a majority of small attC sites (<100 bp)   and S4B.d 129 bp  and S4B.and SCIs  and S4B.tC sites .10.1128/mBio.02296-16.5FIG S5 attC site properties. For each data set, the slope of the regression, as well as the corresponding coefficient of determination R2 and P value are indicated. MI, mobile integron; SCI, sedentary chromosomal integron; bps, base pairs; bs, bottom strand. (A) attCbs pfold values as a function of attC site lengths for MIs and SCIs, excluding Vibrio strains or not excluding Vibrio strains. (B) Difference in ΔG between the bottom and top strands (ΔGbs − ΔGts) of attC sites as a function of their length for MIs and SCIs, excluding Vibrio strains or not excluding Vibrio strains. Download FIG S5, JPG file, 1 MB.Supplementary data on Copyright © 2017 Loot et al.2017Loot et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the attC sites from the INTEGRALL database (G of the folded bottom strands (ΔGbs) is on average 2.12 kcal/mol lower than the ΔG of the folded top strands (ΔGts), suggesting that folded bottom strands are more stable . This value was not significantly different from the one observed for MI attC sites . SurpritC sites . We obsetC sites . This coce in ΔG .attC sites are enriched in purines, especially in guanines, which contribute to the difference in folded strand stability   . However strands . Additioin silico analyses, we performed in vivo excision tests for several synthetic cassettes using the previously described excision assay  (the reticulating agent of peptidoglycan in Escherichia coli). Comparison of the number of clones growing with and without DAP yields a recombination frequency for a given reaction. Corresponding strains without integrase were used as controls to assess the rate of false-positive events potentially due to replication slippage.In order to better understand the biological significance of our on assay   between two attC sites (E. coli lacZ DNA fragments)  oritemplate . For theagments) . Finally10.1128/mBio.02296-16.6FIG S6 attC sites used in synthetic cassettes. Download FIG S6, PDF file, 0.2 MB.Supplementary data on Copyright © 2017 Loot et al.2017Loot et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the attCaadA7-attCereA2 and attCaadA7-VCR126 cassettes. First, we tested that the expression of dapA from the Plac promoter . Second, we tested for sequence-specific effects by replacing selected lacZ DNA fragments by hubP fragments from V. cholerae  and attCaadA7-VCR126 (B and D). Experiments were performed in the presence (+) or absence (−) of the IntI1 integrase. Synthetic cassette lengths and orientation of attCbs relative to the replication are indicated. (A and B) Experiments were performed in the presence (+) and absence (−) of IPTG. (C and D) V. cholerae hubP DNA fragments were used instead of E. coli lacZ DNA fragments. bs, bottom strand; Lag st temp and Lead st temp, lagging and leading strand template, respectively; bps, base pairs; nd, not detected events. Download FIG S7, EPS file, 1.1 MB.Recombination frequencies for different synthetic cassettes in control experiments. Cassette excision assay was performed for several synthetic cassette combinations: Copyright © 2017 Loot et al.2017Loot et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (a) attCaadA7-attCereA2synthetic cassettes. The first set of synthetic cassettes was flanked by attCaadA7 and attCereA2 MI sites ..(a) attC10.1128/mBio.02296-16.8FIG S8 attCaadA7 (gray) and attCereA2 (orange) are shown in red. (A) Sequences of attCaadA7, attCereA2, and the slippage-resulting attC site. The sequences of the top strands are shown. (B) Proposed model for the slippage events. The model proposed is that attC site folding permits a slipped mispairing mechanism during replication, resulting in a cassette excision event, which reconstitutes a functional dapA gene independently of integrase expression. This scheme represents the attCbs carried on the leading strand template, but in cases where the attCbs is carried on the lagging strand template, the slippage mechanism is the same. bs, bottom strand; ts, top strand; Lag st temp, lagging strand template; Lead st temp, leading strand template. Download FIG S8, EPS file, 1 MB.False-positive recombination events due to slippage. Direct repeats of Copyright © 2017 Loot et al.2017Loot et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (b) attCaadA7-VCR126synthetic cassettes. In order to assess the importance of the attC site pfold value on cassette excision, we tested the excision of cassettes flanked on one side by the previously used attCaadA7 site and on the other side by the VCR126 site. VCR126 has a very low pfold value  that is significantly lower than that of attCereA2  VCR-VCR synthetic cassettes. We also tested two sets of cassettes flanked on both sides by VCR sites. First, we combined VCR126* and VCR126** sites. The most stable structures of these sites are non-recombinogenic , but this effect was less pronounced for VCR16*-VCR64 cassettes (6 × 10−6  and S6),and 0.24  and S6).assettes  and H. AOur study aimed to understand the rules that govern cassette array dynamics in SCIs and MIs and to determine the cause of shorter CDS lengths in cassettes compared to the rest of the genome.in silico analyses reveal that attCbs in SCIs are predominantly carried on the leading strand template and that these SCI arrays are significantly larger, up to 200 cassettes in SCIs of Vibrio spp. Our in vivo results show that such orientation relative to replication limits cassette excision and thus stabilizes large cassette arrays. This may explain why this orientation was much less frequent among MCIs, for which a higher cassette mobility might be favored over the preservation of a larger reservoir of genetic functions that will stay accessible through horizontal gene transfer. Interestingly, the five SCIs identified in inverse orientation were found exclusively in Xanthomonas species and their number of cassettes did not exceed 22. Comparison of SCIs in these two closely related genera is particularly interesting, since according to phylogenetic analyses, the acquisition of integron systems in both Vibrio and Xanthomonas occurred independently and thus can be regarded as two single ancestral events , at least in one orientation. However, this was not the case  of this characteristic, in particular whether the reduced length of CDSs in cassettes reflected a constraint in maximal distance between two consecutive the case . When bothe case . Also, wtemplate . Taken tattC sites in our synthetic cassettes, we tested the impact of their properties on cassette excision. When cassettes were flanked by two attC sites with high pfold values, cassette length did not influence the recombination rate when the bottom strands were carried on the lagging strand templates. Because of their high pfold values, simultaneous folding of the two attC sites could occur in three possible ways. (i) Both sites fold from ssDNA during the passage of the replication fork (the “ss pathway”). (ii) Both sites are extruded from dsDNA (the “ds pathway”). (iii) One site is folded from ssDNA, and the other is extruded from dsDNA. The third pathway could explain the very high efficiency of recombination that we obtained for +3,000-bp cassettes when bottom strands were carried on the lagging strand template. On the other hand, the difficulties of recombining +3,000-bp cassettes when both high-pfold attCbs were carried on the leading strand template, and therefore must have been extruded from dsDNA, might be explained by topological constraints such as the presence of independent topological domains in bacterial chromosomes . This eecombine .attC site pfolds, and more generally attC site biophysical properties, control cassette excision dynamics. Moreover, the in silico analyses demonstrated that MIs mostly contain small attC sites with high pfold values and folded bottom strands which are more stable than folded top strands. This ultimately favors their recombination by the integrase , unlike their MI counterparts . Using the IntegronFinder program (https://github.com/gem-pasteur/Integron_Finder), we analyzed 2,484 bacterial genomes, including 2,626 replicons labeled as chromosomes and 2,006 replicons labeled as plasmids. IntegronFinder ensures an automatic and accurate identification of integrons, cassette arrays, and attC sites.The sequences and annotations of complete genomes were downloaded from NCBI RefSeq  Accession numbers of the genomes and positions and lengths of integrases. (B) Bacterial strains. (C) Plasmids. (D) Primers and synthetic fragments (sequences are given in the 5′ → 3′ direction). Download Copyright © 2017 Loot et al.2017Loot et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the attC site comparison, we used the attC sites published and classified in reference attC sites and 1,744 different sedentary CI attC sites). We reran IntegronFinder with the clustering parameters of 15 kb (-dt 15000 instead of 4 kb by default) for six known SCIs that IntegronFinder could not properly aggregate because of this threshold. The sequences of attC sites included the full 7-bp-long R box .For MI and SCI G values of the minimal free energy (MFE) structure. In order to obtain the pfold (probability of folding a recombinogenic structure) values, folding predictions were performed with and without constraints. pfold was calculated as the Boltzmann probability of the constrained (recombinogenic) structure in the ensemble: Eu is the Gibbs free energy of the unconstrained  ensemble, Ec is the Gibbs free energy of the constrained (recombinogenic) ensemble, R is the gas constant, and T is the absolute temperature  was introduced by transformation into the MG1655ΔdapA derivative strains containing insertions (in the attB lambda site) of plasmids carrying a dapA gene interrupted by the synthetic cassettes , DAP, and glucose, strains were cultivated for 6 h in the presence of the appropriate antibiotic (Carb), DAP, l-arabinose (Ara), and IPTG to allow intI1 expression (Pbad) and dapA expression (Plac promoter), respectively. Then, cultures were plated on agar containing either LB with IPTG and Sp or LB with DAP, IPTG, and Sp. Recombination activity was calculated as the ratio of the number of cells growing in the absence of DAP over the total number of cells. For each reaction, we confirmed cassette excision by performing PCRs with SW23begin/DapA-R primers generating products of 715, 763, 784, and 832 bp for attCaadA7-attCereA2, attCaadA7-VCR126, VCR126*-VCR126**, and VCR16*-VCR64 cassettes, respectively.The pBAD::assettes . These s


The progressive character of tooth formation records aspects of mammalian life history, diet, seasonal behavior and climate. Tooth mineralization occurs in two stages: secretion and maturation, which overlap to some degree. Despite decades of study, the spatial and temporal pattern of elemental incorporation during enamel mineralization remains poorly characterized. Here we use synchrotron X-ray microtomography and Markov Chain Monte Carlo sampling to estimate mineralization patterns from an ontogenetic series of sheep molars . We adopt a Bayesian approach that posits a general pattern of maturation estimated from individual- and population-level mineral density variation over time. This approach converts static images of mineral density into a dynamic model of mineralization, and demonstrates that enamel secretion and maturation waves advance at nonlinear rates with distinct geometries. While enamel secretion is ordered, maturation geometry varies within a population and appears to be driven by diffusive processes. Our model yields concrete expectations for the integration of physiological and environmental signals, which is of particular significance for paleoseasonality research. This study also provides an avenue for characterizing mineralization patterns in other taxa. Our synchrotron imaging data and model are available for application to multiple disciplines, including health, material science, and paleontological research. Teeth form incrementally, creating microscopic features that have been a subject of study since they were first observed by van Leeuwenhoek in the 17th century . Tooth fEnamel formation begins when enamel-forming cells (ameloblasts) secrete a matrix of proteins and enzymes that control the formation of amorphous calcium phosphate (ACP) and its transformation into ordered hydroxyaparite (HAp) crystallites ,13,23,31in vitro and has been difficult to study in vivo. For this reason the spatial and temporal pattern of enamel mineralization remains contested and largely unknown [The majority of mineral is added during maturation, when ameloblast cells complete enamel secretion and rest on the surface of the tooth ,14,36. N unknown ,15–19,34Two principle models have been proposed to explain the geometry and timing of maturation during mineralization. The first model was proposed by Shoichi Suga based on radiographs of primates and ungulates, and it describes a series of four waves of increasing mineralization moving to and from the EDJ through time ,16,18,34Here we address longstanding uncertainty in the timing and geometry of mineralization by constructing an empirical model describing ungulate molar mineralization over time. Our model is built from quantitative synchrotron X-ray microtomography (μCT) and Markov Chain Monte Carlo (MCMC) methods using the molars of sheep that died between the ages of 0–1.5 years old. We use sheep because of their established use as a model organism, and their enamel thickness and formation time broadly reflect patterns observed in large mammals, particularly herbivores used in paleoclimate reconstruction. Our method allows us to construct the first HAp-based dynamic picture of mineralization, including secretion and maturation phases, from discrete density measurements made from different individuals. This method differs from previous efforts to characterize mineralization in that it quantitatively reconstructs the timing, magnitude, and variation of mineralization over time in a population of animals.We dissected 45 first molars (M1s) and 18 second molars (M2s) from the mandibles of 45 Dorset sheep that died of natural causes between birth and 540 days of age . The aniThe teeth were scanned on beamline ID17 of the European Synchrotron Radiation Facility  in two batches with an isotropic voxel size of 46μm. A second scan was conducted on a subset of teeth at higher resolution (13μm). We used synchrotron imaging because monochromatic X-ray beams with single energy values can be used to quantify hydroxyapatite densities in mineralizing teeth . In contHAp density values (ρ) from attenuation coefficients were calculated in grams of HAp per cubic centimeter from pixel grey values (Px) using the equations ρ = 6.9 x 10-5Px + 1.54 and ρ = 2.8 x 10-4Px + 1.49 for first and second sample scans, respectively ,41. To qa, slope s and offset o, we used this function to re-assign size-modeled ages tm to each section as a function of its extension length el, according to the equationerf-1 is the inverse error function, and elmax is the maximum length of the tooth when mature. Fitting of a, o and s for extension, maturation onset and completion was conducted using the NLopt module for python, implementing the Multi-Level Single-Linkage (MLSL) and Constrained Optimization BY Linear Approximations (COBYLA) algorithms for global and local optimization, respectively [In order to model tooth growth over time, we used the EDJ length (degree of secretory-cell extension at death) and the age-at-death of each animal to calculate age as a sigmoid error function of enamel extension. When estimating extension curves, Gaussian rather than exponential or logistic functions were chosen based upon indications that highest extension rates are observed shortly after initiation , 42,43. ectively ,45.3 midway from the EDJ to the enamel surface. This is because mineralization data demonstrate that while trajectories are variable, all locations are either entering maturation phase with steep increases in mineral density (maturation onset), or leaving it with declines in mineral addition rate (maturation completion), at approximately 40% and 85% densities. To further validate our M1 and M2 extension modeling experimentally, we raised a single sheep for this study at the Harvard Concord Field Station. The animal received four 8mg/kg subcutaneous calcein injections, and was sacrificed by 180mg/kg IV sodium pentobarbital injection. Through polarized light and fluorescence microscopy, synchrotron imaging and dissection we determined M1 and M2 initiation times to be 49 days prior to and 84 days after birth, respectively. These values were employed for fitting extension and maturation curves  in developing enamel:ρd and ρm are the measured and modeled pixel HAp densities over all time intervals t, ρtd and ρtm are the measured and modeled density for a particular time t, and σt is the estimated density error for each measurement. In these calculations we used a flat prior. Our model assumes that for a given pixel location density, measurements from each tooth are independent, and measurement error or natural biological variation amounts to 5% of all measurable HAp densities. In order to explore the posterior distribution of ρm, we employed Metropolis-Hastings with four walkers and 150,000 samples per pixel, of which we stored 100 for our model of tooth mineralization. The final model includes over 12,000 pixel locations, with density estimates calculated for 280 days per pixel (interpolating across measurements from each of 45 samples), and 100 density estimates per pixel-day (336 million density estimates).To estimate mineral density increases for each location throughout the tooth crown we used an MCMC technique to sample from the Gaussian of likely mineralization histories given our observations for each coordinate in all teeth  rapidly extend in size around birth, with extension rates slowing between 100–150 days . SeveralBy standardizing M1 shapes and assembling mineralization trajectories  into a dThis mineralization model is accessible in an HDF5 data format that can be adapted to a variety of purposes .Suga  proposedThe consistency of this pattern is striking given the independent calculation of over 12,000 mineralization trajectories that each contribute to the model  Files. OThe implications for reconstructing body and environmental chemistry are best understood by comparing the predicted timing of secretory and maturational mineral deposition of the current and earlier models . In all This method can be used to model the mineralization of human teeth, for which many aspects of mineral incorporation remain unknown. An empirical model of mineral density changes can provide a framework for interpreting the etiology of enamel defects that are typically caused by mineralization disruption . These h13C values in an experimental animal that reflect both secretory and maturation geometries [An empirical model of molar mineralization will be especially valuable for evaluating the relationship between stable light isotopes and the seasonal dietary, behavioral and environmental factors that produced them. In archaeology and paleontology, repeated samples of tooth carbon stable isotopes have been used to infer seasonal grazing upon wet or arid adapted plants, or foraging of terrestrial or marine resources ,50–52. Oometries ,30.Mineralization also informs elemental and isotopic sampling from archaeological and fossil teeth, which can become powerful tools for understanding the behaviors and diets of past organisms ,25. For The method outlined here, combining an ontogenetic series of synchrotron-based density measurements into an MCMC modeling framework, can be used to reconstruct biomineralization processes in other complex structures and taxa. This is important since formation processes remain elusive for many biomineralized structures that combine organic and inorganic components . These sMus teeth, possibly facilitated by a variety of physiological processes [Our mineralization model relies upon density estimates and cannot directly assess mineral phase transitions that may also impact biomineralization, isotopic and trace element analyses. These transitions, including that from ACP to HAp, are now thought to be integral to biomineralization processes and have been observed in a several taxa –24,64,65rocesses ,47,48,66rocesses ,54.Despite the heterogeneity of maturation among individual teeth, synchrotron μCT data from many individuals and tooth locations results in an overall dynamic model that is highly consistent in secretory and maturation geometry with respect to the EDJ, and in the magnitude of density increases. The dynamic model of mineralization presented here shows that sheep molars mineralize in secretion and maturation waves with distinct spatial orientations that are separated temporally until the end of mineralization. This is consistent the view that mineralization is an ordered process, and possibly conserved among taxa . DensityS1 TextThis file contains a list of definitions for terms commonly employed by this manuscript. An associated reference list is provided.(PDF)Click here for additional data file.S2 TextThis text provides additional detail on synchrotron mineral density measurement, flattening and standardizing virtual enamel sections, estimating initiation and extension, calcein labeling, model conversion between different tooth types, and MCMC methods for estimating mineral density increase over time. An associated reference list is provided.(PDF)Click here for additional data file.S1 DatasetVirtual buccal enamel sections extracted from synchrotron scanned tooth volumes at 45μm resolution. Brighter pixels indicate dense, highly mineralized enamel, and darker pixels indicate less dense, poorly mineralized enamel. In file name, day of animal death is indicated first, followed by scan batch (1 or 2), followed by animal ID number. 13μm resolution scans (“hi-res”) not used to construct the model are also included.(ZIP)Click here for additional data file.S1 FileH5PY file holding the mineralization model results derived from synchrotron scans and MCMC sampling. File is best opened by HDFView software. The file contains four data sets: age_mask, ages, locations and pct_min_samples. Age_mask data records where each of the 45 scanned M1s (x-axis) contains or does not contain mineral density data for each of 12,000 pixels locations (y-axis). Age_mask data is binary: the presence of data is recorded by a 1, and absence by a 0. Ages data records the age in days of each of the 45 animals. Locations data records the standardized X and Y tooth coordinates (first and second columns of the x-axis) for each of 12,000 pixels. Pct_min_samples data records model estimated mineral density for all locations over time. This dataset contains three axes, with 100 mineral density % samples (x-axis columns) for each of 12,000 pixels (y-axis rows), over all 45 specimen ages (z-axis pages).(H5)Click here for additional data file.S2 FileAnimation showing mineral density increase described by the mineralization model. Dark blue colors indicate no mineral addition, while warmer colors  indicate higher rates of mineral deposition. Mineral density increase has been Gaussian blurred for the purposes of visualization, with the standard deviation of the Gaussian set to one pixel in both x and y axes, and 8 days in the z (time) axis.(MP4)Click here for additional data file.S3 FileAnimation showing 3D density data collected from synchrotron imaging of a single first molar. Red colors indicate dense, highly mineralized enamel, and blue colors indicate less dense, poorly mineralized enamel. Visualization produced using VGStudio MAX 3.0 software.(AVI)Click here for additional data file.


Purpose: Due to the antimicrobial property, menthol have significant potential for food preservation and foodstuff shelf life improvement. Nevertheless, menthol instability, insolubility, and rapid crystallization in aqueous media make it unsuitable for used in food products. This work was aimed to prepare menthol-loaded nanostructured lipid carriers (NLCs) to enhance its antimicrobial activity.Methods: Morphology, particle size and size distribution, encapsulation efficiency percent (EE%), and physical stability of the optimized formulation, prepared by hot melt homogenization method, were characterized by scanning electron microscopy, particle size analyzing, gas chromatography, and X-ray diffraction (XRD) methods. Minimum inhibitory concentration and minimum bactericidal concentration of menthol-loaded NLCs were evaluated and compared with conventional menthol emulsion against various Gram-positive  and Gram-negative bacteria (Escherichia coli), as well as one fungus .Results: Menthol-loaded NLCs were spherically shaped nanosized (115.6 nm) particles with narrow size distribution (PDI = 0.2), suitable menthol EE% (98.73%), and appropriate physical stability after 90 days of storage period. XRD results indicated that menthol was in the amorphous form in the nanoparticles matrix. Antibacterial assay results revealed that the menthol-loaded NLCs exhibited significantly higher in vitro antimicrobial property than conventional menthol emulsion. The results also indicated that menthol-loaded NLCs had better effect on fungi than bacteria, and furthermore, antibacterial efficiency on Gram-positive bacteria was higher than Gram-negative bacteria.Conclusion: In conclusion, NLCs could be a promising carrier for improvement of antimicrobial activity and preservation efficacy of essential oils in foodstuffs. Due to the aromatic property of essential oils, they have been widely used in cosmetic industries for the production of soaps, perfumes, and toiletries.2 The large bioactivity of essential oils has been confirmed by several studies, including antibacterial, antiviral, anti-inflammatory, antifungal, antimutagenic, anticarcinogenic, and antioxidant activities. This group of oils may provide the natural antimicrobials that food industry requires leading to reduced need for synthetic preservative excipients.4 The main constituents of essential oils have unsaturated carbon chains, which is well-known that are susceptibility to oxidation mediated by light or heat. The high volatility of essential oils also limits their free use without a vehicle. Furthermore, the low aqueous solubility limits the possible application of essential oils in aqueous based foodstuffs such as beverages. All of these factors limit the application of essential oils as candidates for preserving food solutions.5 Menthol is a monocyclic monoterpene alcohol naturally obtained from peppermint or other mint oils. It is widely used as a flavoring agent for toothpaste, hygiene products, chewing gum, etc. Menthol is generally available in the form of crystals or granules with a melting point at 41-43 °C. Although, previous researches have shown that menthol has antibacterial and antifungal activity, its high volatility, instability, insolubility, and rapid crystallization in aqueous mediums are the crucial problems concerning its applications and shelf life.6 The microencapsulation method is an appropriate technique to solve above mentioned problems. In this perspective, encapsulation procedures provide an effective protection of naturally compounds against chemical reactions and undesirable interactions with other components in food. Furthermore, it improves solubility, diminish migration, and preserve the bioactive compounds stability during food processing and storage.7 In food engineering, protection of bioactive compounds such as vitamins, antioxidants, proteins, and lipids could be achieved using nano encapsulation technique to produce functional foods with enhanced functionality and stability.8 As lipid based nanoparticles are composed by lipids, they have the ability of interaction with several bacterial and fungal cell types.10 Therefore, in the present study menthol-loaded nanostructured lipid carriers (NLCs) were prepared to improve the solubility, stability, and antimicrobial efficacy of menthol for potential application as preservative in food industry.Increased concern of consumers on the various side effects of synthetic preservatives and development of antibiotic resistant strains have attracted great attention to the use of natural compounds with antimicrobial activity in food and pharmaceutical industries.® 80, Mueller Hinton Agar, Mueller Hinton Broth and Nutrient dextrose agar were supplied from Merck Chemicals Co. (Germany). Poloxamer® 407 was obtained from Sigma-Aldrich Company (USA). Glyceryl Palmtostearate (Precirol ATO-5®) was kindly donated from Gattefossé Company (France). Miglyol® 812 was prepared from Sasol Company (Germany). All other used chemicals and reagents were of analytical grade.Menthol, Tween11 Briefly, 200 mg menthol was dissolved in 200 mg Miglyol® and added to 1.8 g melted Precirol®at about 70 °C. Subsequently, Poloxamer® (1.5 g) was dissolved in water and added drop-wise into the oil phase under stirring at 20000 rpm by a homogenizer . Finally, after 15 min, keeping the temperature at 70 °C and the same stirring rate allowed the hot formulation to cool down at room temperature. NLC structures are usually composed of solid lipid (e.g. Precirol®) and liquid lipid (e.g. Miglyol®). To have an equivalent comparison between NLC and emulsion in their antimicrobial activity evaluation, a similar liquid lipid was chosen for the preparation of NLC and emulsion. Emulsion was also prepared by gradual water addition into the mixture of menthol (200 mg), Miglyol® (2 g), and Tween® 80 (200 mg) under stirring with homogenizer (5000 rpm). Each formulation was prepared and characterized in triplicate.Menthol-loaded NLCs were prepared by hot melt homogenization method as described previously.®, and Poloxamer® and optimized NLCs formulation.Particle size distribution of prepared NLCs was analyzed using dynamic light scattering system (DLS) and reported as intensity-weighted average (z average) and polydispersity index (PDI). Zeta potential of prepared nanoparticles was also analyzed by the same system . The morphology of prepared nanoparticles was obtained using scanning electron microscope (SEM)  operating at 15 kV. The specimens were mounted on a metal stub with double-sided adhesive tape and coated under vacuum with gold (100-150 A°) in an argon atmosphere prior to observation using a direct current sputtering technique  at a scan rate of 0.05 °/s to assess the crystalline structures of menthol, Precirol® Ultra-15 with MWCO of 100 kDa  tube. The formulation was added to the upper chamber of the Amicon® tube, and then the tube was centrifuged  at 5000 rpm for 5 minutes. The clear solution in the bottom of Amicon® tube was used for the determination of unloaded menthol. The EE% and LC% of menthol-loaded NLCs formulations were calculated according to the following equations:The EE% and LE% values were expressed as the percentage of encapsulated menthol to the added menthol or to the used lipid, respectively. EE% was determined by the first separation of the un-entrapped menthol by centrifugation method using Amicon is the amount of initial menthol used, and W(Free Menthol) is the amount of free menthol detected in the lower chamber of Amicon® tube after centrifugation of the NLCs formulation (extracted by ether in triplicate). Accordingly, in equation (2), W (Entrapped Menthol) is the amount of loaded menthol, and W  is the amount of used lipid in the preparation process. The rinsed formulation was used for the further experiments.where, in equation (1), WSamples (15 mL) of each formulation were stored in plastic tubes in the dark place at room temperature (25 ± 1 °C), and the physical stability of the menthol-loaded NLCs was evaluated in terms of the mean particle size of NLCs after 90 days.The concentration of menthol was determined using gas chromatography method . The DB5 capillary column  was used to separate and quantify the sample compounds. The carrier gas (Helium 99.999%) flow rate was 2.0 mL/min. The temperatures were as follows: injector = 250 °C, detector = 250 °C, column = 50-250 °C (4 °C/min) and injected volume = 1 μL. Data were collected and processed by a computer equipped with Chromcard software . The reference standard of the menthol was accurately weighed and dissolved in diethyl ether to produce the stock standard solution and was subsequently diluted to a series of appropriate concentration for construction of calibration curves and determination of the limit of quantification (LOQ). The internal standard solution was added in each concentration solution. Calibration curves were constructed with six different concentrations by plotting the peak area ration of analyte to internal standard versus analyte concentration. Calibration curves showed good linearity (0.9966) over a relatively wide concentration range 3.0-7.5 mg/100 mL, and LOQ was calculated as 0.134 mg/mL.Escherichia coli ATCC 25922), two Gram-positive bacteria (Staphylococcus aureus ATCC 25923 and Bacillus cereus ATCC 11778), and fungi . All the microorganisms were provided by the microbiological laboratory, Drug Applied Research Center, Tabriz University of Medical Science, Tabriz, Iran. After activating, the cultures of bacteria were maintained in their appropriate agar media at 4 °C and used as stock cultures. A single colony from the stock plate was transferred into Mueller Hinton Broth and incubated over night at 37 °C. After incubation, the cells were harvested by centrifugation at 3000 rpm for 15 min, washed twice, and re-suspended in saline solution to provide an optical density equal to 0.5 McFarland standard turbidity  /mL of bacterial and 108 CFU/mL of fungi).The antimicrobial activity of the menthol essential oil formulations was tested against four food-related microorganisms including one Gram-negative bacterium  was considered as MIC. There were different control groups: a) media, b) media + menthol free NLC and emulsion, c) media + menthol-loaded NLC and emulsion (without addition of bacteria), d) media + menthol free NLC and emulsion with addition of bacteria, and e) media + bacteria. Turbidity of wells was measured spectrophotometrically  at the wave length of 620 nm. Subsequently, to determine the MBC, samples (5 μL) from tubes in which no growth was observed were cultured in plate (containing Mueller Hinton agar medium) and incubated for 24 h at 37 °C. To determine the MIC value for C. albicans, 16 dilution series from 2496 to 4.87 µg/mL of menthol were prepared, and 50 μL of fungi suspension was added and incubated at 37 °C for 24 h, and the MIC value was determined as the lowest concentration of essential oil inhibiting visible growth of fungi on the agar plate when there was visible growth on the control plates.13 Each experiment was performed in triplicate. All procedures were performed under sterile conditions. In each test, microorganism strain in Muller Hinton broth (with blank NLCs or emulsion) and Muller Hinton Broth alone were used as positive and negative growth controls, respectively.The MIC values were assessed using the broth microdilution method by sterile 96-well microtitre plates.All experiments were repeated in triplicate, and the data were expressed as a mean value ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) with multiple comparisons between deposition data using a Tukey honest signiﬁcant difference (HSD) test using SPSS software . A P-value <0.05 was considered statistically signiﬁcant.14A PDI value of 0.1 to 0.25 indicates a narrow size distribution whereas a PDI value greater than 0.5 indicates a broad distribution.15 Particle size and PDI value of prepared emulsions were 1489 nm and 0.556, respectively. The particle size and size distribution of prepared menthol-loaded NLCs were analyzed, and the size distribution profile of optimal formulation is represented in 14 Optimum formulation had z-average value of 115.6 nm and PDI value of 0.243. Lower particle size of nanoparticles resulted in higher clearness of the prepared nano suspension considered as a critical advantage for the drinking stuffs such as beverages. On the other hand, one of the strategies to address stability issues is the particle size; lower particles reduce the sedimentation rate, and therefore, the particles can stay suspended longer in nano suspension formulations. Furthermore, a narrow particle size distribution can minimize the saturation solubility difference and drug concentration gradients within the medium and thus inhibit the occurrence of Ostwald ripening.16 SEM images of nanoparticles showed spherical particles which verified the size analysis data of NLCs , which is a critical advantage for a nanoparticular formulation and guarantees the stability of nanoparticles in distribution mediums such as drinking stuffs.Particle size and size distribution are the key parameters of colloidal systems and have significant effects on the final nanoparticles’ behavior including dissolution, bioavailability, content uniformity, and stability. Dynamic light scattering (DLS) is widely used to determine the size and size distribution of small particles suspended in liquid medium. of NLCs b. Partic® as surfactant in the NLCs formulation. Furthermore, polymorphic and crystallization form of Precirol® in NLCs formulation is another reason for higher EE% value of NLCs.19 EE and LC percentage values for emulsion system were achieved 91.4% and 9.1 %, respectively. Representative chromatograms of menthol standard solution and menthol-loaded NLCs formulation after injection into GC column are shown in EE% of optimized NLCs formulation was found 98.73% ± 0.5 with LC% of 9.8% ± 0.05. High EE% was predictable due to the high lipophilicity of menthol and high concentration of Poloxamer® and Poloxamer® in NLCs was much weaker than that of bulk lipid. It indicates that Precirol® in menthol-loaded NLCs was partially recrystallized and formed the less-ordered crystals.The physical state of the nanoparticles was analyzed by X-ray diffraction method . The dif19 In the present study, the absorbance of microtiter plate before and after the incubation of bacteria, with different concentrations of menthol-loaded NLCs and emulsion, was assessed spectrophotometrically. After incubation and increasing the number of bacteria it is possible to find out the MIC values of menthol-loaded NLCs and emulsion. As shown in Plant’s essential oils and extracts have been used for many thousands of years in food preservation, pharmaceuticals, alternative medicine, and natural therapies. It is necessary to scientifically investigate those plants used in traditional medicine to improve the quality of healthcare.B. cereus in comparison with other microorganisms. Correspondingly, both NLCs and emulsion formulations exhibited the highest MIC values in the case of E. coli, suggesting that menthol possesses higher antibacterial efficiency against the Gram-positive bacteria , this mechanism appears to be dependent on the lipid composition and net surface charge of the bacterial membranes. Furthermore, the essential oil might cross the cell membranes, penetrate the interior of the cell, and interact with intracellular sites critical for antibacterial activity. The outer layer of the Gram-negative outer membrane is composed primarily of lipopolysaccharide molecules and forms hydrophilic permeability barrier providing protection against the effects of highly hydrophobic materials. This may explain the low sensitivity of Escherichia coli to the cytotoxic effect of the lipophilic monoterpenes such as menthol.23 NLC formulations also showed promising antifungal activity against candida  who explored the mechanism of action of three monoterpenes, speculated that the antimicrobial effect of monoterpenes such as (+)-menthol, thymol and linalyl acetate, is partially due to the disruption of the lipid fraction of the plasma membrane, causing in changed permeability and leakage of intracellular materials.29 Emulsions are homogeneous thermodynamically stable transparent dispersions of two immiscible liquids stabilized by an interfacial film of surfactants. However, one major drawback of emulsions is that formation requires high surfactant concentration, which causes toxicity when used in pharmaceutical applications. On the other hand, NLCs could be prepared by a simple and industrially scaling up homogenization method. It has been demonstrated that NLCs increase the retention of a drug in the particle, hence, enhancing the release time and reducing the amount of drug required for the therapeutic action. This proficiency could be employed in controlling essential oils release to improve efficacy and reduce the amount and toxicity of used essential oils. Besides, controlling the release of drug molecule, nanocarriers also protect essential oils against possible thermal or photo degradation, oxidation, or evaporation which guarantees increased stability, flavor, and function, consequently prolonging the final product shelf life. Considering these features, these systems can actually represent an interesting approach for overcoming essential oils restrictions. Nanocarriers could also protect essential oils against possible thermal- or photo-degradation, oxidation or evaporation, assure increased stability, flavor, and function, and consequently extend the final product shelf life.30Antimicrobial effect of menthol may be due to the alteration of plasma membrane permeability and leakage of intracellular materials. candida . The sucMenthol was successfully loaded into NLCs in the amorphous structure in the ratio of 1:10 Menthol:lipid by dissolving in the oil phase. Menthol-loaded NLCs were around 100 nm with narrow size distribution (PDI 0.2). The antimicrobial efficiency of the encapsulated menthol was tested on four different microorganisms, demonstrating that MIC and MBC values in the case of NLCs were lower than the menthol emulsion. It can be concluded that essential oils could be used as antibacterial supplement and food preserving agents; however, further investigations are required on the potential antibacterial applicability of this nanostructure in other applications such as topical and oral uses especially against the drug-resistant microorganisms.This paper was financially supported by Tabriz University of Medical Sciences.Not applicable.The authors declare no conflict of interests.