ESR1 and APOE gene polymorphisms and the risk of ONFH.Osteonecrosis of the femoral head (ONFH) is a disease with a high disability rate, and genetic factors are closely related to its pathogenesis. This study aimed to investigate the possible correlation between ESR1 gene and two genetic variants (rs7259620 A < G and rs769446 C < T) of the APOE gene with the risk of ONFH was investigated. Correlations between gene polymorphisms and ONFH risk were assessed using logistic regression analysis, with calculation of odds ratios (ORs) and 95% confidence intervals (CIs).In this case–control study, the potential association between three genetic variants  of the ESR1 gene exhibited a correlation with a decreased risk of ONFH under the homozygous , dominant , and additive  models. The stratification analysis revealed that rs9322332 was linked to a lower risk of ONFH in subgroups characterized by individuals aged over 51 years and non-smokers. Nevertheless, there were no notable correlations found between ESR1 rs2982573 and rs10872678, as well as APOE rs7259620 and rs769446, with the risk of ONFH.The overall analysis demonstrated that rs9322332 in the ESR1-rs9322332 is closely linked to a decreased risk of ONFH, thereby enhancing our understanding of the relationship between gene polymorphisms and ONFH.The online version contains supplementary material available at 10.1186/s13018-023-04447-4. Osteonecrosis of the femoral head (ONFH), also referred to as ischemic necrosis of the femoral head, is a frequently seen and challenging condition in orthopedics. It is characterized by the interruption or impairment of blood supply to the femoral head, leading to the death and insufficient repair of bone cells and bone marrow cells. Subsequently, this causes structural changes in the femoral head and its eventual collapse, resulting in joint pain and dysfunction . AccordiESR1) gene encodes the ERα, which is localized within the bone and plays a crucial role in regulating bone metabolism [ESR1, thus playing a physiological role, while estrogen deficiency may cause changes in bone microstructure, resulting in increased cortical bone vascular aperture, increased bone trabecular separation and decreased bone trabecular number, thus leading to the occurrence of ONFH [ESR1 polymorphisms on the quality of the femoral head in patients with Turner syndrome, revealing that ESR1 rs2234693 is potentially linked to decreased bone mineral density (BMD) in the femoral neck and total hip regions [ESR1 gene polymorphisms and the risk of ONFH is scarce.Estrogen receptor (ER) is a group of nuclear receptor superfamily members that function as transcription factors in the nucleus, consisting of two types: ERα and ERβ . The Esttabolism . Relevan of ONFH –13. Curr regions . HoweverAPOE), located on chromosome 19, plays a crucial role in plasma lipid metabolism by facilitating the hepatocyte-mediated uptake and removal of chylomicrons, very low-density lipoproteins (VLDL), and high-density lipoproteins (HDL) lipoproteins [APOE-deficient individuals may exhibit severe hyperlipidemia, which can potentially influence the microcirculation of the femoral head and contribute to the development of ONFH [APOE gene polymorphisms and the risk of developing steroid-induced ONFH. Some studies have suggested a possible association between the rs7412 C/T and rs429358 T/C loci and an elevated risk of SONFH [APOE gene polymorphisms and ONFH.Apolipoprotein E  and APOE gene (rs7259620 A < G and rs769446 C < T) polymorphisms on the susceptibility to ONFH. The findings of this study are anticipated to shed light on potential biomarkers for the diagnosis and treatment of ONFH.Given that This study included a sample of 505 individuals diagnosed with ONFH and 512 healthy controls, all of whom were obtained from the Affiliated Hospital of Weifang Medical University and the Second Affiliated Hospital of Inner Mongolia Medical University. The inclusion criteria for ONFH cases were as follows: (1) Patients experiencing pain in the hip joint, buttock, or groin area, accompanied by pain in the knee joint and restricted internal and external rotation of the hip joint; (2) Diagnosis of ONFH confirmed by X-ray, computed tomography (CT), and magnetic resonance imaging (MRI); (3) No history of direct trauma, osteoarthritis, ankylosing spondylitis, hip joint-related diseases , cardiovascular and cerebrovascular diseases, metabolic disorders, or bone metastasis. The group of healthy controls consisted of individuals who met the following inclusion criteria: (1) No pain in the hip joint, buttock, or groin area; (2) No evidence of lesions on imaging examinations; (3) No chronic alcohol use or steroid use; (4) No history of direct trauma, osteoarthritis, ankylosing spondylitis, cardiovascular and cerebrovascular diseases, metabolic disorders, or bone metastasis. Demographic characteristics and clinical data of the study participants were collected through questionnaires and a review of patient medical records. Additionally, this study was approved by the ethics committee of the Affiliated Hospital of Weifang Medical University and the ethics committee of the Second Affiliated Hospital of Inner Mongolia Medical University, and conducted in compliance with the principles outlined in the Declaration of Helsinki. Informed consent was obtained from all participants before the commencement of the experiment.ESR1 gene, as well as two SNPs (rs7259620 and rs769446) in the APOE gene, were selected for genotyping. These SNPs were selected based on the following process: (1) Data on ESR1 and APOE gene polymorphisms were obtained from the 1000 Genomes Project database; (2) Screening criteria were implemented, necessitating a minor allele frequency (MAF) above 5% and Hardy–Weinberg equilibrium (HWE) exceeding 0.01; (3) The SNPs to be studied were identified through a combination of primer design and an extensive literature search. After a 12-h fasting period, 5 mL of peripheral venous blood was collected from the study participants using a vacuum blood collection tube containing EDTA-K2 anticoagulant, and mixed upside down and used in subsequent experiments. Genomic DNA extraction was performed using a genomic DNA isolation kit (GoldMag Biotechnology) following the manufacturer’s instructions. The concentration of the DNA was measured using a Nanodrop 2000 spectrophotometer . SNP genotyping was carried out utilizing the Agena MassARRAY platform , and the data analysis was performed using the Agena Typer 4.0 software. Additional file Three SNPs  in the t-test for continuous variables and χ2 test for categorical variables. HWE in controls was calculated using the χ2 test to further explain the good representativeness of the study population. Logistic regression analysis with odds ratios (ORs) and corresponding 95% confidence intervals (CIs) was used to assess the association between ESR1 and APOE gene polymorphisms and the risk of ONFH. Multifactor dimensionality reduction (MDR) analysis was employed to explore SNP-SNP interactions. The reliability of significant findings was assessed using false positive report probability (FPRP) analysis. Statistical significance was set at a p value of less than 0.05. The flowchart of this study is shown in Fig. The characteristics of the study participants were analyzed using the p = 0.004) factor was observed between the cases and controls, whereas there were no significant differences in terms of age (p = 0.242) and gender (p = 0.282) distribution between the two groups. The detailed characteristics of the study participants are shown in Table This case–control study encompassed a total of 1,017 participants, with 505 cases of ONFH  and 512 healthy controls . The average age of ONFH patients was 51.65 ± 14.43, whereas the average age of healthy controls was 50.59 ± 14.39. It is noteworthy that a statistically significant difference in smoking .The basic information and MAF of SNPs for ESR1 and APOE gene polymorphisms and the risk of ONFH using different genetic models, including co-dominant, dominant, recessive, and additive models. The findings of the overall analysis examining the association of ESR1 and APOE gene polymorphisms with ONFH risk are summarized in Fig. ESR1-rs9322332 and a decreased risk of ONFH in the overall analysis, especially in homozygous , as well as in dominant  and additive  models. However, this study found no association between other SNPs of ESR1 and APOE genes and ONFH risk.This study investigated the correlation between ESR1 and APOE gene polymorphisms with ONFH risk, we performed stratified analyses based on age , females , and non-smokers . However, this study did not observe any associations between several other SNPs  and ONFH when analyzed using different genetic models.To further investigate the correlation of ge Table , gender ge Table , smokingge Table , and cliESR1 and APOE gene polymorphisms (Table p = 0.009). Furthermore, the optimal multi-locus prediction model was a combination of five loci , demonstrating a CVC of 10/10 and a testing balanced accuracy of 0.551 (p < 0.001). Additionally, the interaction between each locus was demonstrated in the dendrogram , dominant , and additive  models. The stratified analysis revealed that this polymorphism has a protective effect against ONFH in non-smoker and aged over 51 years old. These results emphasized the significance of ESR1-rs9322332 in the pathogenesis and advancement of ONFH and suggest its potential as a novel biomarker for ONFH treatment.In the present study, we investigated the potential link between ESR1 rs2982573, rs10872678, and rs9322332, currently, there is limited research available. Liu et al. conducted a study showing that individuals in the Taiwanese population carrying the TC + CC genotypes of ESR1 rs2982573 had a lower likelihood of developing osteoporosis when consuming at least three cups of coffee per week [ESR1 rs2982573 and the risk of ONFH. This discrepancy could be attributed to differences in the geographical locations of the study participants, as our study focused on individuals from inland China. For rs9322332, Andrew May et al. discovered that carrying the C allele of ESR1-rs9322332 was associated with a decrease in bone mineral content among black South African children [ESR1-rs9322332 and a lowered risk of ONFH, indicating a potential association between the rs9322332 mutation’s impact on bone mineral content and the reduced risk of ONFH. However, there are no studies on the association between rs10872678 and ONFH or other diseases.Regarding the per week . Howeverchildren . InteresAPOE rs7259620 and rs769446, there has been a significant amount of research conducted thus far. Cai et al. investigated the correlation between APOE gene polymorphisms, diet, and dyslipidemia in the Yao minority area, and found no significant association between rs7259620 and dyslipidemia [APOE gene variations on the risk of dyslipidemia in diabetes, and no statistical differences were observed in rs449647 variants among T2D patients with and without dyslipidemia [APOE gene and the risk of ONFH, but no significant association was found between them. We speculate that such results may be related to the study participants’ region of residence, race, type of disease, and a variety of other factors.Regarding the ipidemia . Park etipidemia . Furtheripidemia , 24. In ipidemia –29. In oESR1 and APOE gene polymorphisms on the risk of developing ONFH. Our findings indicated that the ESR1-rs9322332 polymorphism was linked to a decreased risk of ONFH among individuals aged over 51 years, suggesting an age-dependent effect of ESR1-rs9322332 on ONFH risk. We also found a significant correlation between ESR1-rs9322332 polymorphism and a reduction in ONFH risk among female participants. However, considering that the HWE p value < 0.05 in the female population , 3.89 (95% CI 1.22–12.4), and 4.26 (95% CI 1.32–13.7) times higher, respectively, compared to non-smokers [The onset of ONFH is affected by many factors, including age, gender, and smoking status. ONFH is a disabling condition that primarily affects young to middle-aged individuals. In China, the mean age at diagnosis is 50.40 ± 13.71 years, with a predominant number of patients falling within the age range of 41 to 60 years old . AdditioESR1 rs9322332 and the risk of ONFH, and validate our findings through in vivo animal experiments.Certain limitations exist in this study that should be acknowledged. Firstly, the participants were exclusively recruited from a single hospital, possibly leading to selection bias. Secondly, functional experiments were not performed in this study. In future studies, we will expand the sample size to further investigate the association between ESR1-rs9322332 and a lower incidence of ONFH, particularly among individuals over the age of 51 and non-smokers. However, further validation with a larger sample size is necessary. In summary, this study provides valuable insights into the role of ESR1 gene polymorphisms in the prevention and diagnosis of ONFH.This study has established a strong link between Additional file 1. Table S1. The primers of the selected SNPs. Table S2. Association between ESR1 and APOE polymorphisms and ONFH risk stratified by clinical staging. Table S3. P values obtained through Hardy-Weinberg equilibrium under overall and subgroup stratification.


Although many data sets are discrete and heavy tailed , not many discrete heavy tailed distributions are available in the literature. In this paper, we discuss thirteen known discrete heavy tailed distributions, propose nine new discrete heavy tailed distributions and give expressions for their probability mass functions, cumulative distribution functions, hazard rate functions, reversed hazard rate functions, means, variances, moment generating functions, entropies and quantile functions. Tail behaviour and a measure of asymmetry are used to compare the known and new discrete heavy tailed distributions. The better fits of the discrete heavy tailed distributions over their continuous counterparts as assessed by probability plots are illustrated using three data sets. Finally, a simulated study is performed to assess the finite sample performance of the maximum likelihood estimators used in the data application section. If data are continuous and heavy tailed then the data should be modeled by continuous heavy tailed distributions. By continuous heavy tailed distributions, we mean distributions that have mostly polynomial tails. If data are discrete and heavy tailed then the data should be modeled by discrete heavy tailed distributions. By discrete heavy tailed distributions, we mean discrete versions of continuous heavy tailed distributions.In reality, many data sets  are discrete in nature and have heavy tails. Three examples are given later in Section 5 , 2. HoweY be a non-negative continuous random variable having a heavy tail with cumulative distribution function (CDF) specified by FY(⋅). We define a random variable X say having a discrete heavy tailed distribution as one specified by the probability mass function (PMF)x = 0, 1, …. The corresponding CDF, hazard rate function (HRF), reversed hazard rate function (RHRF), mean, variance, moment generating function (MGF), entropy and quantile function (QF) arex = 0, 1, …. Expressions for mean, variance, MGF and entropy are given mostly as infinite sums. We suppose that these infinite sums converge at least for some of the parameter values; that is, we supposeThe aim of this paper is to review known discrete heavy tailed distributions, propose several new discrete heavy tailed distributions, list their properties and illustrate data applications showing superiority of discrete heavy tailed distributions. Let The technique used for constructing the new discrete distributions has been used by  to constSeveral of the new discrete heavy tailed distributions are highly flexible in modeling insurance data sets. One of the new discrete heavy tailed distributions has the most flexible tail behaviour, see The contents of the paper are organized as follows. Known discrete heavy tailed distributions are listed in Section 2 and new discrete heavy tailed distributions are listed in Section 3. Comparison of the known and new discrete heavy tailed distributions in terms of tail behaviour and a measure of asymmetry is given in Section 4. The better fits of the discrete versions over continuous counterparts are illustrated using three insurance data sets, see Section 5. The data sets are listed in Appendices A, B and C. The method of maximum likelihood is used for estimating parameters of the discrete and continuous heavy tailed distributions. Finite sample performance of the method is assessed in Section 6 via a simulation study. Some conclusions and future work are noted in Section 7.In this section, we list thirteen known discrete heavy tailed distributions. Their applications can be read from the cited papers and references therein.X has this distribution due to .t, discrete Fréchet, discrete generalized Pareto, discrete inverse exponential, discrete inverse gamma, discrete inverse paralogistic, discrete inverse transformed gamma, discrete Lévy and discrete paralogistic distributions all have power tail behaviours. Of these the discrete inverse Rayleigh, discrete inverse exponential and discrete Lévy distributions are not so flexible because their power exponents are fixed constants. The discrete extended Weibull, discrete Rayleigh, discrete Weibull, discrete Weibull geometric, exponentiated discrete Rayleigh and exponentiated discrete Weibull distributions all have exponential type tails. Of these the discrete Rayleigh and exponentiated discrete Rayleigh distributions are not so flexible because the power exponents inside the exponentials are fixed constants.We see that the discrete Burr type III, discrete inverse Rayleigh, discrete inverse Weibull, discrete log-logistic, discrete lognormal, discrete Student’s The discrete additive Weibull geometric and discrete reduced modified Weibull distributions have tails that are products of two exponential type terms. The discrete Burr and discrete Pareto distributions have exponential type tails with the exponent taking a logarithmic form. The discrete modified Weibull distribution has a double exponential type tail. The discrete log Cauchy distribution has a logarithmic tail. The discrete log gamma distribution has a power tail multiplied by a power of a logarithm. The discrete transformed gamma distribution has a power tail multiplied by an exponential type tail.A. The discrete inverse exponential and discrete inverse paralogistic distributions allow for 0 ≤ A ≤ 1 only. The discrete Pareto distribution allows for 0.317 ≤ A ≤ 1 only. The discrete Rayleigh distribution allows for A = 0.075908 only. The discrete Lévy distribution allows for 1/2 ≤ A ≤ 1 only. Hence, the discrete Rayleigh distribution is the least flexible in terms of asymmetry. The discrete Lévy distribution is the second least flexible. The discrete Pareto distribution is the third least flexible. The discrete inverse exponential and discrete inverse paralogistic distributions are the fourth least flexible. The remaining discrete distributions are fully flexible in terms of asymmetry.All but the discrete Pareto, discrete Rayleigh, discrete inverse exponential, discrete inverse paralogistic and discrete Lévy distributions can take the full range of possible values of In Sections 5.1 to 5.3, we illustrate three applications involving insurance data. In each of these sections, we show that at least four of the discrete distributions in Sections 2 and 3 provided better fits than their continuous counterparts. Many of the other discrete distributions in Sections 2 and 3 provided better fits too. The method of maximum likelihood was used to obtain estimates of the distributions. We maximized the likelihood function directly by using the function m, 2022) .The first data set is from  and are The second data set is from  and are https://search.r-project.org/CRAN/refmans/ExamPAData/html/travel_insurance.html The data values are given in Appendix C in The third data are travel insurance data in Canadian dollars. The data were obtained from p-values were 0.21, 0.23 and 0.38.All three data sets are positively skewed and have heavy tails. We tested heavytailedness of the three data sets using ’s test bWe fitted six of the discrete distributions in Sections 2 and 3 and their continuous counterparts. The probability plots of the fits are shown in The fits of the continuous versions do not appear reasonable for any of the six distributions. But the fits of the discrete versions appear reasonable for all six distributions. Their plotted points are much closer to the diagonal lines. We fitted six of the discrete distributions in Sections 2 and 3 and their continuous counterparts. The probability plots of the fits are shown in Again the fits of the continuous versions do not appear reasonable for any of the six distributions. The fits of the discrete versions appear reasonable for all six distributions excluding the discrete inverse gamma distribution. We fitted four of the discrete distributions in Section 3 and their continuous counterparts. The probability plots of the fits are shown in Yet again the fits of the continuous versions do not appear reasonable for any of the four distributions. The fits of the discrete versions appear reasonable for all four distributions. In this section, we assess the finite sample performance of the maximum likelihood estimators of discrete heavy tailed distributions in terms of biases and mean squared errors. The following simulation study was used.b = 1;choose the discrete Weibull distribution, the particular case of the exponentiated discrete Weibull distribution in Section 2.13 for a = 2 and q = 0.5;set initial values as n from the discrete Weibull distribution by using the QF in Section 2.13 for b = 1;simulate a random sample of size a and q for the sample in step c);compute the maximum likelihood estimates of i = 1, 2, …, 1000;repeat steps c) and d) a thousand times, giving the estimates compute the biases of the estimators ascompute the mean squared errors of the estimators asn = 10, 11, …, 100.repeat steps c)–g) for n = 10, 11, …, 100 are shown in Plots of the biases and mean squared errors versus n; b) the biases for n ≥ 80; f) the mean squared errors generally decrease to zero with increasing n; g) the mean squared errors for n ≥ 80.We can observe the following from the figure: a) the biases generally decrease in magnitude to zero with increasing n, the mean squared errors always decreased to zero with increasing n, the biases always appeared small enough for all n ≥ 80, and the mean squared errors always appeared small enough for all n ≥ 80.The observations noted are for particular parameter values and for a particular discrete heavy tailed distribution. We have not presented results for other choices in order to save space and avoid repetitive discussion. But the same observations held for a wide range of parameter values and for all of the discrete heavy tailed distributions in Sections 2-3. In particular, the biases always decreased to zero with increasing We have studied discrete heavy tailed distributions, taken as discrete versions of continuous heavy tailed distributions. We have reviewed thirteen known discrete heavy tailed distributions and introduced nine new discrete heavy tailed distributions. We have given expressions for the PMF, CDF, HRF, RHRF, mean, variance, MGF, entropy and QF for each of the discrete heavy tailed distributions.We have compared the known and new discrete heavy tailed distributions by tabulating their tail behavior and a measure of asymmetry. The tail behavior was either a polynomial or a polynomial multiplied by a logarithmic term or a polynomial multiplied by an exponential term for the new discrete heavy tailed distributions. All but three of the new distributions allowed the measure of asymmetry to take the full range of possible values.We have shown that the discrete heavy tailed distributions can provide better fits than continuous versions to three real data sets on insurance payments. The better fits were assessed in terms of probability plots. The observed and expected probabilities were closer to each other when the discrete heavy tailed distributions were fitted.We have assessed the finite sample performance of the maximum likelihood estimators of the discrete heavy tailed distributions by a simulation study. The study showed the biases and the mean squared errors of the estimators appeared small enough for all sample sizes greater than or equal to 80. Two of the three data sets (see Sections 5.1-5.2) have sample sizes less than 80. Hence, the conclusions in Sections 5.1-5.2 should be treated conservatively.t distributions, multivariate t distributions, bivariate skew t distributions, multivariate skew t distributions, bivariate generalized hyperbolic distributions, multivariate generalized hyperbolic distributions, mixtures of these distributions, and others. Further discrete heavy tailed distributions can be constructed using known mechanisms for generating continuous heavy tailed distributions, see, for example, [Future work are to study discrete heavy tailed distributions for bivariate, multivariate, matrix variate and complex variate data. These distributions could be based on bivariate example, . MoreoveS1 File(TXT)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.


Nanoparticle-assisted nuclear magnetic resonance (NMR)chemosensingexploits monolayer-protected nanoparticles as supramolecular hoststo detect small molecules in complex mixtures via nuclear Overhausereffect experiments with detection limits down to the micromolar range.Still, the structure–sensitivity relationships at the basisof such detection limits are little understood. In this work, we integrateNMR spectroscopy and atomistic molecular dynamics simulations to examinethe covariates that affect the sensitivity of different NMR chemosensingexperiments . Our results show that the intensityof the observed signals correlates with the number and duration ofthe spin–spin interactions between the analytes and the nanoparticlesand/or between the analytes and the nanoparticles’ solvationmolecules. In turn, these parameters depend on the location and dynamicsof each analyte inside the monolayer. This insight will eventuallyfacilitate the tailoring of experimental and computational setupsto the analyte’s chemistry, making NMR chemosensing an evenmore effective technique in practical use. As such, NMR spectroscopyrepresents an ideal technique for analyzing mixtures. However, multispeciescrowding and overlapping of different resonance frequencies typicallyhamper the direct detection of single species in mixtures. To addressthis issue, we proposed a “nanoparticle-assisted NMR chemosensing”approach for the NMR identification of target analytes in complexmixtures.1 This method capitalizes on thereduced tumbling rates of nanoparticles and on the supramolecularhosting abilities of ligand shell-protected gold nanoparticles (AuNPs)to promote the selective magnetization/saturation transfer to theanalytes and the subsequent removal of the signals from the otherspecies.7 The ligands constituting the AuNP’s coating form a micelle-likepseudophase that can incorporate hydrophobic guests in water .9 Functional groups inserted intothe coating ligands can provide additional or alternative interactionswith the guests.10 The residual conformationalmobility of the coating ligands can even promote the formation oftransient and adaptable binding pockets with a cavitand-like structurein the monolayer.13AuNPs are particularly suited for this applicationbecause theyare excellent platforms for designing macromolecular hosts, as confirmedby various proposed applications.14 The modest detection limit ofNOE pumping was subsequently improved by shifting to saturation transferdifference (STD) experiments. In this approach, the spin populationsof the analytes interacting with the AuNPs are indirectly alteredthrough sustained irradiation at a limited portion of the monolayer’sresonance frequencies, providing a more efficient magnetization transfer.17Several protocols can be used for nanoparticle-assistedNMR chemosensing.In early nuclear Overhauser effect (NOE) pumping experiments, magnetizationwas transferred from the AuNP to the analytes via a transient NOEafter the signals from the fast-diffusing species  were dephased by a diffusion filter.18 and the modification uni-WASTY.19 InHPwSTD, the water molecules in long-lived association with the AuNP act as additional reservoirsof saturation . This NOE is generated in the fast motion regime and results in a negative polarization of the signals stemming from theunbound analytes and in the consequent reduction of the intensityof the signals produced by those analytes that are interacting withthe AuNPs in water STD (wSTD) and waterLOGSY experiments.More recently, we proposed a high-power water-mediated saturationtransfer difference (HPwSTD) experimentturation 1A. HPwST20 the role of the specific interactions among the AuNPs, analytes,and solvent molecules remains poorly understood.18 On the one hand, higher affinities for an analyte should resultin lower detection limits.17 However, increasingan analyte’s bound fraction leads to a severe signal broadeningthat restricts this approach’s applicability.18 On the other hand, variables such as the location of theanalyte in the monolayer, its mobility, and its orientation couldaffect the saturation transfer efficiency and hence the sensitivityof the AuNP toward different analytes. The relevance of these parametersin relation to the detection protocol is also still unknown. Here,we report an integrated experimental and computational study investigatingthe chemical parameters that control the sensitivity of NMR protocolsduring biomarker detection.Despite the potential of the STD-based protocolsto significantlydecrease the detection limit,1-AuNP, and one of two analyteswith a similar structure, namely, serotonin (Ser) and dopamine . Ser 1-AuNP was fixed at 0.93 μM and those of the analytes were 0.5 and 1.4 mM forSer and Dop, respectively. These conditions were selected to ensurethat the same amount of each analyte  was bound to 1-AuNP. Note that the number of bound analytes per AuNP is constantfor both samples, but the molar fractions of bound and unbound analytesare different (vide infra). We determined the raw 1H NMR spectrum for each sample  that does notinteract significantly with 1-AuNP under the adoptedexperimental conditions. Indeed, no signals of Phe were detected in the STD or HPwSTD spectra , confirming that the Ser and Dop signals observed in the STD spectrawere due to only the AuNP–analyte interactions.When the STD experiments were performed on thesesamples 2C,D,theanalytes 2E,F. ExpT1 values of theanalyte protons are markedly different, STD experiments may not providean accurate image of analyte–target interactions.21 On this basis, we measured the exchange-averaged T1 values in the presence of 1-AuNPand compared them for Ser and Dop. We found that the two analytesrelaxed similarly. In particular, the aromatic  and aliphatic  protons provided similar T1 valuesacross Ser and Dop (Table S2), even ifthey are quite different from those of other protons within each molecule.Reassured by the absence of relevant relaxation differences, we determinedthe saturation transfer efficiency, ηSTD% (or ηwSTD%), of each proton type as ηSTD% = 100× (InHoff – InHon)/InHoff, where InHoff and InHon are the integrated signal intensities for proton Hn in the off- and on-resonance spectra, respectively.22It isknown in the context of epitope mapping that STD responsesdepend on the longitudinal relaxation times of the ligand protons.In particular, when the STD%values between the two samples is still inadequate. In an STD experiment,the measured ηSTD% values are proportional to themolar fraction of the bound analyte18 [asimilar relation holds in the case of HPwSTD (see section S5 for details)]. In our experiments, the 1-AuNP/Ser sample contains a lower unbound molar fraction of analytethan does the 1-AuNP/Dop sample, and consequently, themeasured ηSTD% would be smaller even in the caseof equal efficiency of saturation transfer. Indeed, because the concentrationof the bound analyte is set to be equal, the ηSTD% measured in our samples is dependent on the total analyte concentration.We hence introduced a concentration-normalized saturation transferefficiency, calculated as ηSTDN% = ηSTD% × R, where R is the ratio between the concentration of the selected analyte andthe concentration of the analyte used in the smaller amount. In ourcase, the concentration of Ser is 0.5 mM and the concentration ofDop is 1.4 mM, so R = 1 for Ser and R = 2.8 for Dop.18However, we noted that notwithstandingthe controls and treatmentsdescribed above, a direct comparison of the ηSTDN% that arelarger than those of Dop in the STD experiments. In particular,the average ηSTDN% values for all of the Ser and Dop protonswere 5% and 3%, respectively , because the presenceof the NOE from bulk water reduces the saturation transfer efficiency(see section S5 for details). For thisreason, the ηwSTDN% values measured were corrected for thebulk water contribution before normalization as ηwSTDN% = (ηwSTD – ηwSTD0%)R, whereηwSTD0% was measured in the absence of 1-AuNP. The signalsobtained with wSTD are more intense than those in standard STD experimentsand less intense than those in HPwSTD experiments. The ηwSTD0 % values forSer and Dop are 14% and 16%, respectively, with a small preferencefor Dop over Ser.The normalizeddata reveal that Ser has average values of ηectively 2I. Interectively 2J. As alectively 2G,H,K, wOverall, the results presented above confirmedthe different sensitivitiesof STD, wSTD, and HPwSTD experiments. They also revealed that eventhough AuNPs bind the same number of analyte molecules, the net responseof the different NMR experiments depends on the analyte’s identityand the chemical equilibrium between its bound and unbound states.STD correctly detects Ser, while the signals for Dop are barely abovethe signal-to-noise ratio under the conditions employed. Instead,wSTD detects the two analytes with a similar sensitivity and a weakpreference for Dop. Lastly, HPwSTD features a sensitivity much largerthan that of STD, and the preference for Dop is substantially enhanced.1-AuNP in explicit water to equilibrateits structure. This simulation shows that the ligands extend in waterto 2.5 nm from the center of mass (COM). Water molecules enter differentregions of the coating monolayer at different rates [radial waterexchange rate (see section S1)]. At short distancesfrom the gold center (<1.5 nm), water molecules are rarely exchanged,indicating a stable and tightly packed solvation shell. As the distanceincreases, the exchange of water molecules accelerates following asingle-exponential function with a rate λ of 1.6 nm–1 until it reaches the nominal value of 58.2 nm–2 ns–1 in the bulk solvent. The hydrodynamic radiusof 1-AuNP, i.e., the distance at which the exchange ratereaches 55.1 nm–2 ns–1 , is located at 3.0 nm [confirmed also by DOSY (see section S3)].To obtain molecular information about these different behaviors,we used a computational approach to analyze the specific AuNP–analyteinteractions. We first performed a 100 ns molecular dynamics (MD)simulation of 1-AuNP in the presence of 10 moleculesof Ser, Dop, or Phe. The average distribution of the ligands, as describedby the radial distribution function (RDF), is nearly identical forthe three analytes . All analytemolecules, including Phe, were found inside or in the vicinity ofthe monolayer. This result stems from the very high concentrationof analytes and AuNP attained in silico, which favorsfull binding of the analytes to 1-AuNP in the case ofboth high (Ser and Dop) and low (Phe) affinity. Still, this setupreproduces well the interaction conditions of the nanoparticles inthe experiments (except for the negative control Phe). In fact, thenumber of Ser and Dop molecules included in the monolayer at any timepoint of the simulations (10 per nanoparticle) was similar to thatof the NMR experiments (15 per nanoparticle) as calculated from theaffinity constant. The full-binding conditions allowed us to obtaininformation about the bound states of the two analytes, which arethe ones experiencing saturation transfer.To study how the analytes interact with the AuNP’smonolayerand the surrounding water molecules, we performed three 1 μssimulations of 1-AuNP’s monolayer. We also identified that Phe, the nonbinding analyte,formed significantly fewer π-stacking interactions with 1-AuNP than its cationic counterparts (Ser and Dop). Nevertheless,Dop featured more π-stacking interactions than did Ser, suggestingthat this form of interaction is not the driving factor behind STDsignals.A deeper analysis of the π-stacking interactions revealedthat the aromatic moieties of analytes and ligands stack mainly ina “parallel displaced” geometry (–1 (section S1), which corresponded to expected lifetimes (λ–1) of 1.49, 0.96, and 0.64 ns for Ser, Dop, and Phe,respectively.We further characterized the specific AuNP–analyteinteractionsby studying the contacts responsible for the NOEs in the differentNMR protocols. We computed the number of proton–proton contacts(<0.4 nm) between the analytes and the ligands, grouping all ofthe chemically equivalent protons 1A,B. ThiNote that these computations include only contactslasting longerthan 0.5 ns  to ensure preciseestimates of the contact times .The robustness of this threshold was assessed by calculating and plottingthe total contact time as a function of the minimum contact time threshold. 1-AuNP, followed by Dop and Phe. The cumulativeresidence times between the monolayer and the analyte (number of contacts× expected lifetime) are 48.7, 19.2, and 10.8 μs for Ser,Dop, and Phe, respectively. Accordingly, the cumulative residencetime of Ser is 2.5 times larger than for Dop. This figure matchesquite well the experimental results, where ηSTDN% values ofSer were 1.7 times larger than those of Dop.Hence, Ser is the analyte making the most and longest contactswith Interestingly,the trends in the total number of contacts and thecontact’s duration also correlate well with the analytes’affinities 4A, sugge1Ser/H2Ser and H1Dop form the most contacts with thealkyl chain of the ligands (positions 1–5) compared to theother aromatic hydrogens. These positions likely identify the mosthydrophobic portion of the two analytes that penetrates more deeplyinto the monolayer. These contact patterns are consistent with theexperimental results, which showed larger values of ηSTDN% for H1Ser/H2Ser andH1Dop than for the rest of the signals .27The contact analysis discussed above can explainthe results fromwSTD. It suggests a similar ability of the nanoparticle to transfersaturation to the two analytes, with a weak preference for Dop whenthe most persistent contacts are considered. This result matches wellwith the experimental data, where ηpatterns 5C and thSTDN% values for Ser and Dop significantly increase from4% in STD to 15% in wSTD and 57% in HPwSTD. These figures measurethe relative effectiveness of the different protocols and the respectivesaturation sources. The larger ηwSTDN% values obtained withwSTD compared to the ηSTDN% values from STD confirmthat the slowly tumbling water molecules are a larger and more effectivesource of magnetization than the monolayer’s ligands, as confirmedby calculations that indicate that the number of contacts with thesolvent molecules is 2-fold larger than that of the contacts withthe monolayers. Calculations also indicate that the contribution frommonolayer spins favors Ser while the contribution of solvation waterslightly favors Dop, and both of these suggestions are confirmed byexperiments.In theend, HPwSTD experiments can be examined on the basis ofthe results discussed so far, even if dissecting the individual contributionsis not trivial, and only a qualitative analysis is possible. The firstrelevant information, provided by the experiments, is that the averageηSTDN% values measured with these experiments compared to those measuredwith STD and wSTD. Also, because (i) both saturation transfer mechanismsare enhanced in HPwSTD, (ii) solvation water molecules are a moreeffective saturation source, and (iii) Dop is more susceptible togaining saturation from the solvent than Ser, the sensitivity forDop is expected to be greater than that for Ser. Nicely, this expectationalso agrees with the experimental results.In the HPwSTD experiments, contacts with the nanoparticle’smonolayer and the solvation water molecules both saturate the analytes,and the negative contribution of bulk water is minimized. Strongersignals are expected, as confirmed by the larger η19 The advantageof the HPwSTD protocol hence rests in its generality, because it canexploit all of the possible saturation reservoirs without knowingthe binding mode of the analyte in advance. Our data confirm thatclosely associated water molecules are more efficient as a saturationsource than the monolayer’s spins. In addition to a sufficientaffinity for the analyte, the ideal nanoparticle host should ensuregood exposure of the bound analyte to solvation molecules. Accordingly,we recently showed how HPwSTD is effective even in the case of silicananoparticles, where no monolayer contribution was possible.20Experimental andcomputational results showed that Ser and Doplocate themselves in the monolayer of our anionic nanoparticle toreceive saturation, albeit to different extents, from the nanoreceptorand the solvating water molecules. This behavior differs from whatwe recently found in a typical protein–substrate system, wherethe saturation was transferred to the analytes primarily through theprotein’s spins.In this work, we compared the sensitivitiesof different saturationtransfer NMR protocols for the nanoparticle-assisted detection oforganic analytes. Experimental and computational results indicatethat nanoparticle/analyte systems can behave differently by selectingthe macromolecular receptor, the solvating water, or both as the mainsource of saturation to be transferred to the analytes. This choicedepends on the binding site’s structure and the analyte’sbinding pose. In this regard, MD simulations can provide precise informationabout the docking of the analytes to the monolayer and the specifichost–guest interactions. In addition, MD contact analysis provedto be a reliable method for predicting the affinity of nanoparticlesfor analytes and hence explaining the sensitivity of STD experiments.These results can assist researchers in designing chemosensing experimentsand virtual screening protocols attuned to the chemistry of analytesand nanoparticles of interest.


Sapindus mukorossi Gaertn) is a popular woody plant in Vietnam, often used as a cleaning product due to its ability to wash, foam and emulsify due to high saponin content. In this study, the performance of fermentation by two microbial strains, namely Saccharomyces cerevisiae active dry yeast (ADY) and Levilactobacillus brevis lactic acid bacteria (LB) along with the addition of pomelo peel (flavedo) was evaluated during 15 days in terms of sugar removal, antioxidant and antibacterial activities, foaming power, volatile composition, and sensory acceptability. The results showed that the soluble solid content of original extracts experienced a significant decrease from 14.5% to a stable range of 9.4–11.0% until day 15 for all fermented samples, which correlated with a reduction by approximately 60% in reducing sugars (from 12.52 g L−1 to 4.77–6.56 g L−1). In addition, the saponin content of fermented extracts was in the range of 118.2–145.0 mg L−1 while antioxidant activities were extremely reduced after 15 days of fermentation. Increases in pomelo peel imparted fermented extracts with greater antibacterial activity against Staphylococcus aureus ATCC 6538, Proteus mirabilis ATCC 25933, and Candida albicans ATCC 10231, and LB had higher activity than ADY overall. Regarding the volatile profiles, two main compounds in the original extracts, including trilaurin (75.02%) and 1-dodecanoyl-3-myristoyl glycerol (24.85%), were completely removed and replaced by new alkanes, alkenes, alcohols, esters, and organic acids, and particularly d-limonene (86.34–95.31%) upon pomelo addition. Additionally, the foaming ability and stability of fermented extracts were also enhanced and there was clear distinction between fermented and unfermented samples using principal component analysis based on sensory liking data which showed consumers' preference towards fermented samples with a high percentage of pomelo peel.Soapberry ( The microbial fermentation by yeast and lactic acid bacteria fortified with pomelo peel waste was performed to purify and improve smell of soapberry extract. Sapindus mukorossi Gaertn) belongs to the family Sapindaceae, commonly known as soapnut, and is a common wild plant in tropical and subtropical climates from Japan to India.1 The fruit pericarp contains a high level of saponins (10.1–11.5%) with potential applications as detergents, surfactants, and antibacterial agents in functional cosmetics such as shampoo and cleansers.2–4 Saponins are secondary metabolites with a structural characteristic consisting of an aglycone part (triterpene or steroid) linked to mono/disaccharides, typically glucose, arabinose, rhamnose, xylose and galactose via ester linkages.2,3,5 The majority of plants can only produce one of two types of saponins, namely triterpenoid (dicotyledons) or steroid (monocotyledons).5 The applicability of soapberry extracts is limited due to impurities such as sugars (10%), proteins, oils and mucilages native to fruit along with unpleasant odors.4 Many studies have been done to purify soapberry extracts such as solvent extraction, use of macroporous resins, ultrafiltration, and foam separation; however, these methods have disadvantages in terms of equipment cost, environmental concerns, and purification efficiency.6,7Soapberry  and lactic acid bacteria Levibactobacillus brevis (LB). In addition, the valorization of pomelo peel wastes was conducted to improve the unpleasant smell, stickiness, antibacterial ability and foaming power, thereby making the extract easy to apply in other cosmetic products. The fermentation broth was analyzed for changes of physicochemical parameters , antioxidant activities , microbiological qualities (microorganism density and minimum inhibitory concentration), volatile composition, foaming ability and sensory acceptability. To the best of our knowledge, this is the first study into how microbial fermentation and the addition of pomelo peel waste affect the volatile profiles of soapberry extract. This combination was promising as a practical method for application at the household level.However, there has been little information on the application of other microorganisms into the purification of soapberry extracts and alteration of volatile composition; for example, yeast fermentation has been used in few studies to purify soapberry extract.Levilactobacillus brevis UCCLB521 was isolated from kombucha tea at the Microbiology Laboratory (Nguyen Tat Thanh University).Soapberry fruits  were collected in Kbang  in February, 2022 and convectively sun-dried for 24 h to reach the moisture content of 11.29% before storage in PE plastic bags at room temperature, in a dry place. Green pomelo (Tan Trieu cultivar) was grown at Bien Hoa . Thermosacc® Dry commercial yeast preparation was supplied by Lallemand Inc. (France) while lactic acid bacteria Escherichia coli ATCC 8739 and Proteus mirabilis ATCC 25933), two Gram-positive bacteria (Staphylococcus aureus ATCC 6538 and Bacillus cereus ATCC 11778), and one yeast strain  were kept frozen in Mueller–Hinton broth (MHB) medium containing 15% v/v glycerol.Pathogenic microorganisms, including two Gram-negative bacteria . Mueller–Hinton agar (MHA), Mueller–Hinton broth (MHB), De Man, Rogosa and Sharpe (MRS) agar, and yeast extract were purchased from Hi-Media Laboratory . Other chemicals were of analytical grade.−1) for 2 h. After extraction, the mixture was cloth filtered and centrifuged at 1220g for 10 min using a PLC-05 centrifuge . Subsequently, the clear extracts were pasteurized at 80 °C for 2 min and then rapidly cooled on an ice bath to room temperature. The green pomelo peel (flavedo) was peeled off the white part , and ground before the addition of water (mass ratio of 1 : 1) and pasteurization using the same procedure described above. Pomelo peel was added to original soapberry extract (SBE) at two levels of 7.5% (7.5% PP) and 15% (15% PP) prior to inoculation of activated yeast (ADY) and Levilactobacillus brevis (LB) at 10% inoculum size. The fermentation was conducted under static conditions at room temperature for 15 days and analyzed at day 1, 3, 5, 10, and 15 for the content of total saponins, total phenolics, antioxidant activities and some other physicochemical parameters  in comparison with control samples (0% PP). In addition, fermented soapberry extracts after 15 days fermentation were evaluated for foaming ability and foam stability, antibacterial activity, volatile composition, and sensory attribute.The dried soapberry was ground and sifted through a 40-mesh sieve, followed by extraction with distilled water at a material : solvent ratio of 1 : 5  and 5 mL sulfuric acid (78% w/v diluted in water) and incubated for 15 min. The absorbance of the sample was measured at 560 nm using a spectrophotometer and total saponin content in the extract was calculated based on a calibration curve using Quillaja saponin standard and expressed in mg Quillaja saponin equivalent per liter of extracts (mg QSE per L).The vanillin–sulfuric acid method was used to quantify the total saponin content as described in the literature.16 In addition, based on the initial and post-fermentation glucose content, the substrate consumption rate  was calculated.pH and total soluble solid (°Brix) were measured using the HI 2211-02 pH meter  and the Master-53M hand-held refractometer , respectively. Reducing sugar content expressed as g glucose per liter was spectrophotometrically determined based on the chromophore from the reaction of reducing sugar and DNS reagent at boiling conditions.17 The antioxidant activity of DPPH was calculated against the Trolox calibration curve and expressed in mg Trolox equivalent per liter of extracts (mg TE per L).Antioxidant activity was evaluated through DPPH free radical scavenging capacity based on the purple color change of DPPH solution (0.6 mM) measured at 515 nm upon reaction with antioxidants.18 The ABTS cationic radical scavenging activity was calculated against the Trolox calibration curve and expressed in mg Trolox equivalent per liter of extracts (mg TE per L).ABTS free radical scavenging activity was carried out based on the discoloration of ABTS (7.4 mM) solution measured at 734 nm upon reaction with the antioxidant.et al.19 based on the chromophores formed between the working reagents  with antioxidants. Ferric reducing antioxidant activity was calculated against the Trolox calibration curve and expressed in mg Trolox equivalent per liter of extracts (mg TE per L).Ferric reducing antioxidant power (FRAP) was determined according to Arriola −1, glucose 20.0 g L−1, peptone 5.0 g L−1, and agar 20.0 g L−1) and MRS agar plates, respectively. The agar plates were then incubated for 48 h at 30 °C and colonies were counted to determine the bacterial density expressed in log number of colony-forming units per milliliter of extract (log CFU mL−1).To determine the density of lactic acid bacteria and yeast, 0.1 mL of diluents was poured on YPDA  were serially diluted (dilution factor of 2) using 0.9% NaCl solution in a 96-well plate before adding MHB medium (50 μL) and microbial cultures (50 μL) at a concentration of 108 CFU mL−1. After aerobic incubation , the MIC was verified as the concentration (μL of extract per mL) with no turbidity as indicative of microbial growth.MIC (minimum inhibitory concentration) is the minimum concentration to inhibit pathogenic microorganisms.n-hexane, followed by centrifugation at 5000 rpm for 10 min. The supernatant layer was used for analysis on a GC-MS system  with a silica fused capillary 30 m long column  with particle diameter of 0.25 mm and a film thickness of 0.25 μm using helium as carrier gas. The temperature program was set as follows: the oven temperature was set to 50 °C for 1 min, then increased by 30 °C min−1 to 80 °C and further increased by 10 °C min−1 to 230 °C, hold for 2 minutes and then increase 25 °C min−1 to 280 °C, hold for 8 min. Other fixed parameters were flow rate of 1 mL min−1, injection volume of 2 μL, scanning wavelength range of 1–3345 m/z, ionization voltage of 70 eV, and ionization source temperature of 250 °C.The volatile composition and contents were analyzed by gas chromatography coupled mass spectrometry (GC-MS). Briefly, sample aliquot (2 mL) was shaken with 2 mL of The volatile compounds were identified by comparing their mass spectra with the mass spectral library of National Institute of Standards and Technology (NIST)  and by matching their calculated retention indices with the retention indices found in the library. Subsequently, semiquantification was performed to determine their relative concentrations in percent using the peak area of the internal standard .21 Briefly, 5 mL of the test solution was transferred into a 2 × 18 cm cylindrical test tube before vigorously shaking at a shaking amplitude of 5 cm and a frequency of 3 Hz. Foam height (cm) was recorded at 0, 5 and 10 min to evaluate foam strength over time.The foaming ability and foam stability were determined according to the tube shaking method.22The overall acceptability of seven soapberry samples was evaluated by sensory panelists of 30 participants using a five-point hedonic scale (from 1 = ‘extremely dislike’ to 5 = ‘extremely like’) based on sensory attributes, namely smell and stickiness. All participants were students at our institution between the ages of 18 and 25, with women comprising 63%. The participants were not subject to the training session but the repeated contact with original soapberry extracts for one month before the actual sensory evaluation. In addition, the panelists were asked to sign the consent form following ASTM E3314-21 – Standard Guide for Protection of Respondents and Informed Consent for Sensory Evaluation Studies and IFST Guidelines for Ethical and Professional Practices for the Sensory Analysis of Foods. Seven samples (20 mL each) were prepared in covered plastic cups coded with three digits and refrigerated at 5 °C before being randomly presented to panelists using a Latin square design.post hoc Tukey test, were performed at 5% significance level using R version 4.1.2.23 Triplicates were used for all tests and measurements. Principal Component Analysis (PCA) based on sensory data was also computed using the FactoMineR package.24,25All statistical techniques, including normality test (Shapiro–Wilk's test), homoscedasticity of variances (Levene's test), one-way ANOVA, and Saccharomyces cerevisiae and lactic acid bacteria Levibactobacillus brevis are presented in et al.6 who presented the decrease in total soluble solid from 18.29% to 15.30% after yeast fermentation. It is noticeable that for ADY, although the sugar depletion was slower at the first three days in the samples fortified with pomelo peel  for ADY and 8.00–8.50 (log CFU mL−1) for LB. Agricultural wastes such as fruit peels have been utilized as carbon source for microbial fermentation by lactic acid bacteria in the study of Parra-Matadamas et al.26 who concluded that microbial growth was improved upon addition of 1% pomelo peel into fermentation medium, as illustrated by the increase in microbial density from 7.96 log CFU mL−1 to 9.02 log CFU mL−1.The changes of soluble solid content (°Brix) and reducing sugar of soapberry extract with and without the addition of pomelo peel during fermentation with commercial yeast elo peel , these velo peel . On the QS > 0.09, 28 and the production of various metabolites, such as carbonic gas, ethanol and other by-products.29 However, there is a difference in the relationship of sugar and °Brix values in soapberry extracts; in which, the high °Brix level of soapberry extracts (14.5%) in this study was not mainly due to sugar. Although sugar decreased sharply but °Brix decreased insignificantly because soluble solid content is inclusive of saponin which are not consumed by microorganisms. Heng et al.6 also concluded that the fermentation by yeast increased the saponin purity by 75.5%.On the other hand, the sugar content of LB samples showed no differences for the entire process, except for day 1, as illustrated by the fast sugar consumption , the impregnation pre-treatment of malic acid resulted in a slight decrease of total saponin content. Another explanation is the adsorption of saponin and other compounds into non-viable lactic acid bacteria and yeast cells depositing at the bottom of fermentation broth.32,33 In addition, the increase in saponin content at the remaining days of fermentation could be explained by the diffusion of native saponins present in pomelo peel after long maceration in soapberry extract.27Saponin is a natural surfactant that has many applications in the cosmetic and pharmaceutical fields.34 Therefore, the changes of phenolic content and antioxidant activity of fermented soapberry extract were evaluated during 15 days of fermentation and are presented in 27 and the fermentation caused chemical modification of compounds by precipitation, protein binding or adsorption by microorganisms.35,36Natural preparations rich in physiologically active substances are increasingly being used in cosmetic products for their antioxidant, anti-inflammatory, anti-aging, antibacterial, and photoprotective effects.27 It is believed that the active compounds such as antioxidants are present at higher concentration in citrus peel than juice.37 During fermentation, microorganisms release enzymes, such as esterases, reductases and decarboxylases that alter the integrity of the cell membranes and hydrolyze the ester bonds of phenol polymers and glycosides.38 In addition, due to the use of two-phase fermentation, the fluctuation in the antioxidant contents and activities are likely the result of complex relationship of bioconversion by microbial metabolism, antioxidant diffusion from pomelo peel, and their adsorption into pomelo peel.It is also concluded that the slight increase in phenolics during the first five days can be explained by the alterations as a result of plant cell wall disruption, allowing the phenolic compounds to diffuse out of the pomelo peel.Staphylococcus aureus ATCC 6538. Bacterial growth can be stifled by lactic acid and other fermentation byproducts, particularly bacteriocins produced by lactic acid bacteria.39 Furthermore, the essential oils derived from pomelo flavedo also contributes to the increased antibacterial activity of soapberry extracts, as illustrated by the higher activity upon increasing the percentage of pomelo peel in the fermentation medium.MIC for pathogenic organisms including bacteria and fungi were performed on the pre- and post-fermentation soapberry extracts with the addition of pomelo peel and are presented in 40 Foaming ability is one of the important properties of saponins,5 so determining the foaming ability of saponins present in extracts before and after fermentation is also very important. The foaming ability and foam stability over time of the fermented soapberry extract were shown in et al.,3 which indicated that fermentation not only improved saponin purity but also significantly promoted foam stability. Wei et al.7 also concluded that fermented soapberry extracts showed increase in detergent activity by 11.3% for dirty cloths and 20% for protein and sebum with double antibacterial activity against Trichophyton rubrum ATCC 294 and Candida albicans ATCC 10231. The increase in foaming capacity may be due to fermentation leading to some bioconversion such as partial hydrolysis of saponins to the corresponding aglycone (sapogenins), which is more active than saponins.5 In addition, the removal of components such as proteins, carbohydrates and other components resulting from fermentation increased surface tension, thereby increasing the foaming power.3,7tSaponin is a natural surfactant that has hydrophilic sugar molecules and a hydrophobic sapogenin moiety.41 In case of fermented samples with the addition of pomelo peel, d-limonene appeared to dominate (86.34–95.31%) and their volatile composition was mainly alkanes, alkenes, alcohols, esters, and organic acids. The predominance of d-limonene in fermented samples was associated with the occurrence of this substance in native pomelo peel,42 which plays an important role in improving the aroma of fermented extracts upon the addition of pomelo peel.The volatile composition of soapberry extracts fermented by ADY and LB were compared with non-fermented soapberry extracts (SBE) and presented in 8 Proteolysis is one of the major biochemical processes in flavor development that occurs during fermentation. Two main pathways are involved in the conversion of free amino acids to volatile compounds, including the reduction catalyzed by lyase and the pathway initiated by aminotransferases.43 In addition, lipolysis and fatty acid oxidation in fermented foods are the main sources of flavor compounds, which can be produced by lipase present in relatively high concentrations in Lactococcus and Lactobacillus species.44It is known that fermentation can alter the organoleptic properties of foods and other fermented products.d-limonene, and the lower stickiness resulting from the depletion of sugar content. Furthermore, PCA technique was also applied to discriminate the seven samples and the PCA results presented in The overall likings of seven soapberry extracts based on five-point hedonic scale are shown in d-limonene to the fermented extracts. This is also reflected in the high sensory acceptability of fermented samples and the clear clustering between fermented and unfermented ones. In addition, the foaming ability and antibacterial activity of the extract were also significantly enhanced compared to original soapberry extract.Fermentation of soapberry extract by yeast and bacteria improved saponin purity by removing impurities, mainly sugars, in the original extract. In particular, the combination of fermentation and pomelo flavedo has completely eliminated the unpleasant smell of soapberry extracts and imparted a pleasant scent, mainly Quoc-Duy Nguyen: conceptualization; data curation; investigation; methodology; visualization; writing – original draft; writing – review & editing. Quoc-Duy La: investigation; data curation; writing – original draft. Nhu-Ngoc Nguyen: visualization; data curation; writing – original draft. Thi-Ngoc-Lan Nguyen: conceptualization, data curation.There are no conflicts to declare.RA-013-D3RA01858J-s001


Saccharomyces cerevisiae in six laboratory environments and found that the global-epistasis scaling is nearly invariant across environments. Instead, the environment tunes one global parameter, the background fitness at which most mutations switch sign. As a consequence, the distribution of mutational effects is remarkably predictable across genotypes and environments. Our results suggest that the effective dimensionality of genotype-to-phenotype maps across environments is surprisingly low.Predicting how new mutations alter phenotypes is difficult because mutational effects vary across genotypes and environments. Recently discovered global epistasis, where the fitness effects of mutations scale with the fitness of the background genotype, can improve predictions, but how the environment modulates this scaling is unknown. We measured the fitness effects of ~100 insertion mutations in 42 strains of  The effects of mutations on microbial growth rate follow a pattern of global epistasis that is invariant across environments. Adaptive evolution can lead to profound changes in the phenotypes and behaviors of biological systems, sometimes with adverse and sometimes with beneficial consequences for human health, agriculture and industry –5. HowevMuch of prior empirical work measured the effects of individual mutations on fitness-related phenotypes and characterized how these effects vary across genetic backgrounds and environments , 12–21. Several recent studies have shown that many mutations tend to make the phenotype of an organism in which they occur less extreme , 37–46, Saccharomyces cerevisiae in six conditions (see Methods). All background genotypes are segregants from a cross between two strains of yeast  and differ from each other by ~2×104 SNPs throughout the genome (− h−1 (Methods).Probing whether global epistasis models can capture G×G, G×E and G×G×E interactions at both microscopic and macroscopic levels hinges on measuring the effects of many mutations across multiple genetic backgrounds and environments. To this end, we measured how ~100 quasi-random barcoded insertion mutations constructed in our previous study  affect ge genome . Our enve genome –52, in ae genome . pH 3.2 −1 to increasing it by Δλ = 0.13 h−1, with the median effect Δλ ≈ 0 h−1. We validated a subset of these estimates with an independent low-throughput competition assay  of our mutations are beneficial in at least one strain and condition, and, of those, 98% (86/88) are also deleterious in at least one strain and condition. Even within the same environment, between 33% (31/94) and 65% (61/94) of all mutations change sign across background strains, and between 10% and 39% of mutations change sign across environments in the same strain . Thus, tA recent global epistasis model suggests that the effects of most mutations on GR should linearly decline with the GR of the background genotype . One strTo probe the microscopic global epistasis model quantitatively, we modeled the effect ncratic” , 39.F-test, P < 0.05 after Benjamini-Hochberg correction), and explained on average 46%  of variance in the effects of mutations across background strains and environments (Methods). When tested individually, 38% (205/545) of global epistasis slopes , P < 0.05 after Benjamini-Hochberg correction), with 98% (200/205) of them being negative  of pairwise comparisons , S4, S5,ronments , S7.Taken together, the near-invariance of global-epistasis slopes across environments and the linear relationship between slopes and intercepts indicate that the microscopic G×G, G×E, and G×G×E interactions for most of our mutations are largely captured by a simplified version of To understand the implications of The behavior of higher moments of the DFE is less obvious. We find that the DFE variance is predicted to depend on To understand this prediction intuitively, consider an idealized case where the effects of all mutations vary according to the generalized global epistasis equation with  to zero . Then, apositive .Finally, the generalized global epistasis equation predicts that the skewness of the DFE ought to decline monotonically with To test these predictions, we compared the empirical DFEs across environments. We find that pairs of strains with matched adjusted GRs have significantly more similar DFEs than pairs of strains with the same absolute GR in different environments or the same strain in different environments  and S9, The fact that all our predictions hold indicates that the linear generalized global epistasis equation with uncorrelated noise terms quantitatively captures the variation in the DFE shape across genotypes and environments. However, if the environment truly modulates only one effective parameter, the pivot GR, then we should be able to predict DFE shapes in any environment once its pivot GR is known. To test this prediction, we turned to our previous work where we measured DFEs of 163 yeast strains (a superset of the 42 strains used in this study) in a rich YPD environment –H.These results show that microscopic global epistasis imposes simple, predictable, and general constraints on the G×G, G×E and G×G×E interactions at the macroscopic level. Specifically, the external environment controls a single effective parameter, the pivot growth rate ive mean .To the extent that our observations hold beyond the specific set of strains, mutations and environments investigated in this study, they have a number of important implications. In genetics, the generalized global epistasis model can be incorporated into QTL analyses to improve predictions of the phenotypic effects of mutations. In evolutionary biology, our results point to the existence of a universal class of distributions of fitness effects of mutations, which could explain why evolutionary dynamics of fitness are so similar and predictable across systems , 53–55. Supplement 1Supplement 2Supplement 3


Euwallacea fornicatus and its fungal symbiont, Fusarium euwallaceae, into Israel, the United States and South Africa has had a devastating impact on many economically and ornamentally important tree species. Currently, there is no effective control measure in place to control this beetle pest and its symbiont. As a first step towards controlling this beetle in South Africa, this study investigated the efficacy of commercialized entomopathogenic products against the beetle. Results showed that although effective under lab conditions, currently recommended field application methods of these products to the outer bark of trees have limited effects on beetle survival and reproduction.The accidental introduction of the ambrosia beetle Euwallacea fornicatus, was first reported in South Africa in 2018. The beetle has now spread to eight provinces of the country and has had a devastating impact on both native and non-native tree species. This is especially true for trees located in urban and peri-urban environments. Recent predictions are that the South African E. fornicatus invasion will cost an estimated ZAR 275 billion (approx. USD 16 billion) if it continues to spread uncontrollably, justifying an urgent need for its effective management in the country. One option is biological control, which is preferred over the use of chemicals due to its lower environmental impact. We tested two broad-spectrum fungal entomopathogenic agents, Eco-Bb® and Bio-Insek, which are commercially available in South Africa, for efficacy against E. fornicatus. Initial laboratory assays yielded promising results. However, beetle infestation trials using treated pieces of woody castor bean stems showed little effect on beetle survival and reproduction.The invasive ambrosia beetle,  Euwallacea fornicatus , commonly known as the polyphagous shot hole borer (PSHB), is a small ambrosia beetle (Coleoptera: Curculionidae: Scolytinae) residing in the Xyleborini [Fusarium euwallaceae [leborini . Despiteallaceae ,4. The aallaceae .F. euwallaceae, is killing many old and well-established trees. If left unchecked, it is estimated that PSHB will result in economic losses of approximately ZAR 275 billion (approx. USD 16 billion) in the next 10 years, much of this necessitated by the responsible removal of some 65 million infested trees in urban environments [The first official report of PSHB in South Africa was published in 2018, although there is evidence that the beetle has been in the country since at least 2012 ,6. It haThe unique lifestyle of ambrosia beetles deep within trees makes these insects notoriously difficult to control ,13,14. IPlatanus ×acerifolia) sawdust, 20 g agar, 10 g sucrose, 5 g casein, 5 g corn starch, 5 g yeast, 1 g Wesson’s salt mix, 0.35 g streptomycin, 2.5 mL wheat germ oil, 5 mL 96% ethanol and 500 mL ddH2O. Dry ingredients were mixed first before adding the wet ingredients and autoclaving for 25 min at 121 °C. Approximately 20 mL of diet was then poured into 50 mL polyethylene centrifuge tubes. The tubes were loosely capped and allowed to dry in a flow hood for 7–14 days, allowing all condensation to evaporate. The tubes were sealed and maintained at 4 °C until use.Beetles were reared on a sawdust-based artificial diet as described by Cooperband et al. . IngrediQuercus sp., Rietondale, Pretoria, South Africa, 2021). Logs were maintained in emergence chambers, and the emerging beetles were collected. Foundress beetles were surface disinfested in 70% ethanol for 10 s and allowed to air dry before being transferred to individual rearing tubes. Each rearing tube was capped with a ventilated lid and maintained in a plastic container in the laboratory. Following the establishment of the beetle colonies, the adult female progeny was transferred to fresh diet tubes every 8–10 weeks to maintain the population.Initial foundress beetles for rearing were collected from naturally infested oak logs )) were excluded due to their limited host range. A fourth product, BroadBand ® , was further excluded due to it only being available for purchase at commercial scale and not quantities suitable for laboratory testing. The two remaining products, Bio-Insek  and Eco-Bb ®, were selected for screening against PSHB , Lecatech nst PSHB  based onTwo laboratory assays were performed to assess the efficacy of these agents against the PSHB: (1) direct application of entomopathogen fungal suspension to beetles, and (2) application of entomopathogen fungal suspension to filter paper on which the beetles were maintained. Assays were incubated for 10 days in darkness at 23 °C without providing food.2O with 0.2 g Eco-Bb® or 0.4 g Bio-Insek spore powder, respectively, scaled down from the instructions given on the product packaging. Suspensions were mixed thoroughly by vortexing for 1 min. From these suspensions, 1 mL was pipetted to evenly cover the surface of individual 35 mm Petri dishes. The beetles were then individually exposed to one of the treatments  for 1 min by allowing them to walk in the suspension [In the first assay, 8-week-old female beetles were excavated from their rearing tubes. Female beetles were selected to be used in the assays as they are the dispersing sex and would be most likely to encounter the biological control agents. Fecundity of the female beetles was not determined prior to use in the assays. Each trial replicate consisted of eight beetles that were exposed to one of the entomopathogenic agents or a control treatment of sterile distilled water, and three replicates were performed per agent (n = 24 beetles per treatment). A suspension was prepared for each of the entomopathogenic agents  by mixinspension . Each of®, 3× Bio-Insek, and 3× control) and eight beetles were added per Petri dish (n = 24 beetles per treatment). Beetles were inspected daily, and mortality was recorded.In the second assay, spore suspensions of the entomopathogenic agents or sterile water to serve as a control were prepared as described previously, and 1 mL was used to impregnate 55 mm (ø) filter paper disks. Nine 65 mm Petri dishes were lined with the impregnated filter paper discs (3× Eco-BbRicinus cummunis), a common reproductive host of the beetle in South Africa. Collected stems were sectioned into 40 pieces (12.1 cm × 3.3 cm (average length by diameter)) and were used within 24 h of collection. Both the Eco-Bb® and Bio-Insek products were tested at two concentrations in the infestation assay. These included (1) the recommended dose as indicated by the manufacturer, and (2) 10 times (10×) the recommended concentration . The suspension was spread evenly over the surface of the agar plate using a sterilized spreader, and plates were incubated at 23 °C in the dark for 24 h. After 24 h, germinating spores were counted within a 1 cm2 block on the agar surface. Spores were considered viable if a germ tube longer than the spores’ width had emerged. Five replicates were prepared for each suspension for each assay.To confirm viability of the spores contained within the product packaging throughout the time of the study, a viability assay was performed for each assay. After spore suspensions were prepared, 100 µL of suspension was pipetted onto the surface of a clean malt extract agar plate . Initially, Shapiro–Wilk and Levene’s tests were performed to determine if the data were normally distributed and homogenous. Two of the three parameters  passed these tests and were found to be normally distributed and homogenous, whereas one parameter  did not pass these tests. Where the parameters were normally distributed and homogenous, both a one-way ANOVA and a Kruskal–Wallis rank sum test [For both laboratory assays, a Kaplan–Meier estimator  was usedsum test  with an sum test  were per® and Bio-Insek both resulted in a reduced median survival time (MST) of PSHB beetles compared to the control treatments . Of the 24 initially introduced beetles, 19 beetles (79%) were recovered from the control treatment replicates, 21 beetles (88%) were recovered from the recommended dose Eco-Bb® treatment replicates and 20 beetles (83%) were recovered for each of the three remaining treatments, i.e., Bio-Insek at recommended and 10× doses and the 10× dose of Eco-Bb®. The presence of the entomopathogenic fungi had no significant effect on the boring activities of the beetles, and similar numbers were found to have had infested the castor bean stems in all trial replicates . The application of the entomopathogens to the castor bean stems also had little effect on beetle mortality, with similar numbers of living and dead beetles recovered from the different trials .For each of the four respective fungal treatments and control treatment, comparable numbers of beetles were recovered/harvested from the castor bean stems  were observed for beetles recovered from the infestation assay. Mycosis matching that of the inoculated fungal agents was observed for only four of the beetles recovered from the infested castor bean pieces. Two of the beetles were recovered from different Bio-Insek applications, one from a recommended dose replicate and one from a 10× dose replicate, each showing signs of c agents . This su®, showed promise as preventative agents of PSHB. These products were able to reduce the survival of the beetles in both direct and indirect application assays compared to control treatments.Laboratory assays investigating the efficacy of two entomopathogenic products that are commercially available in South Africa, Bio-Insek and Eco-BbThe MST values of the indirect application assays were lower in both products compared to the direct application assays. This is similar to results observed by Davis et al. 2018 , who alsDespite initial success in the laboratory assays using the two fungal products, wood boring assays using woody castor bean stems treated with both the recommended dose of the products and 10 times this amount, had no significant effects on the survival of the beetles. There were no statistically significant differences between treatments for the beetles that infested the treated castor bean stems or the number of live beetles recovered. Signs of beetle reproduction were also seen in trial replicates, except those of the castor bean stems treated using the recommended dose of Bio-Insek. However, the recovery of living beetles from the material treated with both the recommended and 10× doses of Bio-Insek, and the presence of eggs and larvae seen in the 10× treated material, suggests that the beetles would be able to reproduce in this material after a longer incubation period.In vitro application of fungal entomopathogens against infestation by bark and ambrosia beetles using application methods such as dipping ,18,27, dBiological control has been used against invasive pests for more than a century. This is largely due to the lowered environmental risk offered and the potential for self-sustainability of pest management without a need for continued re-application. In this study, we tested two entomopathogenic agents commercially available in South Africa for their efficacy against the invasive ambrosia beetle PSHB. Despite promising initial results, infestation assays to determine their usefulness as preventative agents under more natural conditions showed little effect on beetle survival. To use entomopathogenic agents as a management strategy for PSHB, further work on the identification of more specific fungal strains adapted to the PSHB’s environment would be required, which could reduce the number of spores required to establish infection. Additionally, other modes of application should be considered, i.e., using spore dispersal traps, which could obtain higher rates of beetle exposure during the limited window of opportunity of a dispersing female. However, if these agents are to be used as more than just a preventative measure against the PSHB or other invasive ambrosia beetles, a method to actively deliver the agents into already established gallery systems will also be required.


There was no significant difference between lung squamous cell carcinoma and lung adenocarcinoma in the polymorphism of ACE1 rs4646994 (p > 0.05). We also found that the ACE1 rs4646994 DD genotype frequency was inversely correlated with the risk of EGFR mutation in lung adenocarcinoma patients. Conclusions: Our study indicated that ACE1 rs4646994 polymorphism increases the risk of lung cancer in patients with pulmonary nodules from China.Background: Currently, many detection methods have high sensitivity to the diagnosis of lung cancer. However, some postoperative patients with pulmonary nodules are eventually diagnosed as having benign nodules. The ideal evaluation of an individual with a pulmonary nodule would expedite therapy for a malignant nodule and minimize testing for those with a benign nodule. Methods: This case–control study is designed to explore the relationship between ACE1 rs4646994 polymorphism and the risk of lung cancer in patients with pulmonary nodules, for which 400 individuals with lung cancer and benign pulmonary nodules were included. A DNA extraction kit was used to extract DNA from peripheral blood. The relationship between ACE1 rs4646994 and the risk of lung cancer in patients with pulmonary nodules was determined by the chi-square test, logistic regression analysis and cross analysis. Results: The results showed that after adjusting for age and gender confounding factors, the risk of lung cancer in patients with pulmonary nodules carrying the DD genotype was more than three times that of the I carriers (II + ID) genotype (OR = 3.035, 95% CI, 1.252–7.356,  Lung cancer is a primary cancer type with high incidence and mortality, and the number of new cases of lung cancer in China has continued to rise . The oveA pulmonary nodule is an abnormal area in the lung that is less than 3 cm in size, and most pulmonary nodules are benign (not caused by cancer) . The risAngiotensin-converting enzyme (ACE) encodes an enzyme involved in blood pressure regulation and electrolyte balance that converts angiotensin I into a physiologically active peptide angiotensin II, and degrades bradykinin . ACE plaThis pilot study was approved by the Research Ethics Committees of the Shanghai Jiao Tong University, under protocol KS1407, in the city of Shanghai, China in 2014. All participants signed their informed consent.For our study, IRB approval and a waiver of written informed consent were obtained. Inclusion criteria are as follows: (1) Patients must be greater than or equal to 18 years old, regardless of gender. (2) Patients have positive pulmonary nodules found by conventional dose CT or low-dose spiral CT (LDCT): (a) non-calcified nodules with a diameter of 5–20 mm; and (b) all three imaging types of pulmonary nodules can be included in the group, namely solid nodules, partial solid nodules (mixed ground glass density nodules) and pure ground glass density nodules. (3) Patients are prepared to receive surgical treatment of pulmonary nodules. (4) Patients have newly diagnosed pulmonary nodules who received baseline CT within 60 days before enrollment. (5) Patients must be able to fully understand the informed consent, agree to participate in the study and sign the informed consent. The exclusion criteria are as follows: (1) pregnant or lactating women; (2) patients received any diagnostic puncture treatment before enrolled the group, such as percutaneous lung biopsy, bronchoscopy biopsy or surgery; (3) receive any blood transfusion within 30 days before enrollment; (4) patients with any malignant tumor (except for non-melanoma skin cancer) diagnosed by pathology 2 years before enrollment; and (5) unable to understand or sign informed consent. Finally, subjects with lung cancer (n = 300) were included as the case group and matched with subjects with benign nodules (n = 100) as the control group. Both groups were investigated for clinical demographic data, including age, gender, and histological type.®-MGB probe assays were used to genotype the polymorphisms in 384-well plates on LightCycler® 480 system . The primers and probes of TaqMan® assays were designed using Primer Express Oligo Design software v3.0 . PCR reactions were performed in a 6 TaqManp < 0.05 was considered for all statistical analyses. The associations between different genotypes and lung cancer risk in patients with pulmonary nodule were estimated with an individual odds ratio (OR) and 95% confidence interval (CI).All data were analyzed using SPSS 20.0 statistical software . The p > 0.05). Most cases of lung cancer were LUAD, followed by SCLC and SCC. Among the 400 patients with pulmonary nodules included in the study, a total of 146 patients were tested for EGFR gene mutation. There was no significant difference in EGFR gene mutation between lung cancer patients and benign lung disease patients (p = 1.000). It is noteworthy that EGFR mutations exist in patients with benign nodules  was higher than that in the control group, suggesting that the DD genotype is closely associated with the risk of lung cancer. Compared with the I carrier (II + ID) genotype, the DD genotype significantly increased the onset risk of lung cancer . In the female subgroup (p = 0.041). Compared with the I carrier (II + ID) genotype, DD genotype . The results shown in p = 0.030).The distribution of the ACE1 rs4646994 genotype and allele in the case and control groups is shown in subgroup , patientp = 0.032). Compared with the I genotype (II + ID) carriers, patients with the DD genotype are about three times more likely to develop LUAD than patients with benign nodules . The comparison of ACE1 rs4646994 between the SCC group and control group manifested no statistical significance (p > 0.05). In addition, there is no significant difference between lung squamous cell carcinoma and lung adenocarcinoma in the polymorphism of ACE1 rs4646994 (p > 0.05). As shown in p > 0.05). p > 0.05).The distribution of genotypic frequencies of ACE1 rs4646994 in the SCC, LUAD, and control group is listed in p = 0.044) is lower than that in patients carrying genotype II. The DD genotype decreases the onset risk EGFR mutation when compared with the I carrier (II + ID) genotype . No evidence shown there was relationship between ACE1 rs4646994 gene polymorphism and EGFR mutation in male (p > 0.05) and female (p > 0.05) lung adenocarcinoma patients (p > 0.05).As shown in patients . As showp = 0.031). The results of a previous meta-analysis did not show any significant association between ACE1 rs4646994 polymorphism and lung cancer risk [p = 0.492). More interestingly, we found that patients with pulmonary nodules carrying the DD genotype had an increased risk of adenocarcinoma and a lower probability of EGFR gene mutation .Angiotensin-converting enzyme (ACE) gene polymorphism is one of the most studied genetic systems in recent years, including cardiovascular, metabolic, immune, cancer, aging, neurodegenerative diseases and mental illness ,23,24,25cer risk . The incp = 0.041). No statistically significant association between ACE1 gene polymorphism and lung cancer risk was found in male patients with pulmonary nodules . However, it is certain that in both male and female subgroups, we observed that the DD genotype of ACE1 was positively correlated with lung cancer susceptibility in patients with pulmonary nodules. Male patients with pulmonary nodules carrying the DD genotype of ACE1 rs4646994 were more strongly associated with lung adenocarcinoma than lung squamous cell carcinoma . Due to the limited number of samples, there was no lung squamous cell carcinoma in female patients diagnosed with lung cancer. There was no significant correlation between the ACE1 polymorphism and EGFR mutation after gender stratification. However, we observed that patients with the DD genotype of ACE1 rs4646994 were less likely to have EGFR mutations in male and female subgroups of lung adenocarcinoma patients, which was consistent with the results of no gender stratification analysis. It is noteworthy that in this study, we observed that patients with benign nodules also had EGFR mutations, suggesting that patients with benign nodules should also be screened for EGFR mutations.In the gender stratification analysis, we found that DD genotype carriers of ACE1 rs4646994 had a significantly increased risk of lung cancer in female patients with pulmonary nodules . It was reported that advanced age is a risk factor for lung cancer and pulmonary nodule [In patients with pulmonary nodules under 45 years old, no statistical association was found between ACE1 rs4646994 and lung cancer risk, while in older patients (>45), the risk of lung cancer was significantly increased in patients with the DD genotype (DD vs. ID + II: OR = 3.423, 95% CI, 1.284–9.126, y nodule ,28. In tAs far as we know, this is the first study exploring the association between ACE1 rs4646994 polymorphism and lung cancer risk in patients with pulmonary nodules. This study is also one of the few that has investigated the association between gene polymorphism and the risk of SCC and LUAD. However, there are some limitations in our research. It is difficult to avoid selection bias and information bias in the whole research process. Although the number of samples included in this study meets the experimental requirements, the number of respondents in the stratified analysis is not sufficient. In order to further understand the role of the ACE1 gene in the development of lung cancer and determine the complex relationship between ACE1 polymorphism and gene–environment interaction, a larger sample size and various expression studies are needed. In addition, as with all association studies, it is certainly necessary to reproduce our findings in independent studies and with different ethnic populations.In conclusion, our study indicates that the DD genotype of ACE1 rs4646994 may contribute to an increased risk of lung cancer in patients with pulmonary nodules. Furthermore, the possibility of EGFR mutation in lung adenocarcinoma patients with the ACE1 rs4646994 DD genotype was lower than that of II or ID genotype carriers.