In an age of expanding international consortium research, there is a growing need to disentangle technical site effects from sample characteristics of interest. Numerous statistical and machine learning methods have been developed to control for, model, or attenuate site effects – yet to date, no comprehensive review has discussed the benefits and drawbacks of each for different use cases. Here, we provide an overview of the different existing statistical and machine learning methods developed to remove unwanted site effects from independently collected neuroimaging samples. We focus on linear mixed effect models, the ComBat technique and its variants, adjustments based on image quality metrics, normative modeling, and deep learning approaches such as generative adversarial networks. For each method, we outline the statistical foundation and summarize strengths and weaknesses, including their assumptions and conditions of use. We provide information on software availability and comment on the ease of use and the applicability of these methods to different types of data. We discuss validation and comparative reports, mention caveats and provide guidance on when to use each method, depending on context and specific research questions.Site differences, or systematic differences in feature distributions across multiple data-acquisition sites, are a known source of heterogeneity that may adversely affect large-scale meta- and mega-analyses of independently collected neuroimaging data. They influence nearly all multi-site imaging modalities and biomarkers, and methods to compensate for them can improve reliability and generalizability in the analysis of genetics, omics, and clinical data. The origins of statistical site effects are complex and involve both  Multi-site data analysis is now the typical practice in consortium projects such as the Enhancing NeuroImaging Genetics through Meta-Analysis (ENIGMA) consortium , the Cohstatistical power by increasing the total number of observations. This improves the ability to detect small but likely true effects. Another goal is to boost the generalizability and hence the scientific value of the results by directing the same hypothesis to independent cohorts. Similarly, multivariate pattern analysis (MVPA) needs large, heterogeneous and representative data to effectively detect subgroups (“biotyping”) and to develop classification tools through supervised machine learning that generalize well to unseen data  site without impacting the model's capacity to estimate other effects that co-vary with the site. Insufficient correction for site effects can leave serious confounds in the data that obscure interpretations, impair the generalizability, replication and reproducibility and hinder multivariate approaches such as MVPA and machine learning (ML). In turn, overcorrection of site effects may interfere with the estimation of effects of interest and the effects of confounders or other nuisance variables that are needed for a valid model  .http://enigma.ini.usc.edu/protocols/imaging-protocols). In addition, data quality control (QC) procedures that often cannot be fully automated but require a qualitative assessment by a reader, can be standardized , which is the primary topic of this article, the MR imaging platform, its field strength, properties of the main magnet, gradient coils and receiving coils and the sequence specification, such as voxel size, are all examples of major sources of different raw signal properties that propagate into the derived features , 16. Sitdardized . Furtherdardized .Sample characteristics are the other major source of site effects, as “site” and “sample/cohort” are often indistinguishable. Sample characteristics comprise the population from which participants are sampled, or the criteria by which healthy controls are defined, including tolerance thresholds for current or past illness. In clinical samples, diagnostic criteria, their operationalization and potentially ill-defined context variables, as well as inclusion and exclusion criteria have an influence. Cohort characteristics are thus the most disputable source of site related variability that must be handled carefully because they may carry true variability of the effect of interest. We give an overview over potential sources of site difference in across subjects. Fully automated analyses such as by the FreeSurfer segmentation pipeline (http://surfer.nmr.mgh.harvard.edu) lead to regional cortical thickness, surface area, cortical volume, or gyrification indices, and many of these features are also available at the vertex level. Voxel-based analyses generate measures in stereotactic atlas space with increasingly precise intersubject coregistration  as much as possible, but have made adjustments for consistency within this document and the flow of the text. Lastly, the difference between standardized and unstandardized values is often marked by selecting different parameters . For readability and the convenience of the reader, we abstain from this differentiation and note at this point that all values in the following need to be considered as standardized.For the convenience of the reader, we aim to provide a consistent selection of parameter names and the notation of equations and mathematical symbols. We follow general notation practices, such as denoting the mean of x by as One very simple harmonization technique is adjusting the observed values by a simple estimate of the residuals from a multiple regression analysis that includes site and a set of covariates as predictors. The even simpler approach—of just regressing out site effects without considering a covariate system—is not recommended , 38.This approach is based on the additive modeling of site effects and covariate effects:f being the feature index, s being the site index, αf the grand mean of the feature across sites, X represents the covariate matrix, βf the coefficients associated with X and γsf the site effect parameters. The residual terms ϵisf are assumed to have a mean of 0. Using ordinary least squares (OLS), for each feature f, the estimators with f are considered in the way that during the OLS procedure the covariate effects “compete” with the site effects and attenuate their correlation with the dependent data. However, the approach has several disadvantages: first, multicollinearity between site and covariates may lead to unstable estimations of the regression coefficients and to both undercorrection or overcorrection of site and covariate effects. Second, different scaling behaviors of the sites  are not modeled. Particularly when aiming to combine independently collected data, we advise against using this procedure, as the variance retained in Xisβf is strongly dependent on the covariate effects and the degree of collinearity between covariates and sites. Nygaard et al.  offset from the grand mean per site per feature, and δsf, a (multiplicative) effect affecting the dispersion around the mean, thus changing the variance of the error term to δsf ϵisf.The non-site effect related components include: αmean only in which the estimates for mean s and featurefare estimated from the sample , and in an empirical Bayes (EB) mode, in which those best estimators  adjustment, a procedure in which these additive effects  and multiplicative effects  are removed per feature per site by subtracting the additive effects and dividing by the multiplicative site effects. To do this, ComBat can be estimated in two ways: s sample mean for feature f is the mean of all individuals in this sample for this feature: F) and the corresponding variance s sample means γsf from The hyperparameters s and feature f) we can deduce that the mean s of those F, is Inverse Γ distribution with the mean In addition, assuming that Estimating the parametric batch effect adjustmentssf and We place prior distributions over γsf and For both parameters we aim to find the posterior conditional distributions for γApplying Bayes' theorem, it can be shown that the expected values for As we can see from the estimates for lgorithm .The rationale behind the EB step and creating this prior is its robustness toward outliers, particularly in smaller samples, by borrowing information from all features for the variance estimate. As the EB adjustment diverges from the prior with increasing sample size, this results in comparable results (to the L/S adjustment) for medium sample sizes, but results in strong adjustments for small sample sizes, protecting the site effect mean and variance estimates from the influence of outliers .Based on the estimates of of f and site s. Beyond this, yet depending on the implementation, ComBat allows the user to either align data from all sites to the (grand) mean and (pooled) variance of all sites or to that of a reference site . Second, sites with less data are more heavily regularized than those with more data, leading to unbalanced adjustments in case of large sample size differences between sites.isf is modeled as an empirical average of the variance from all data. This forces all sites to have the same (homoscedastic) noise term ϵisf and leads to a loss of the biological meaning that is included in heteroscedasticity (e.g as described in the example above). Instead, the modeling of the noise term in ComBat leads to ϵisf becoming sample size dependent: The ϵisf in a data set with two sites with n1 = 10000 samples and n2 = 100 will be different from the ϵisf of two sites with n1 = 500 and n2 = 500. The limitation to a unified, average noise term within the ComBat framework also prevents the approach to cater for the differences in precision that result from site sample differences as described in the second implication. To conclude, Combat is not able to model heteroscedastic distributions and large sample differences between sites, and should not be used in those cases.Both implications are a direct result from the modeling of modeling noise in ComBat: within the ComBat framework, the variance term ϵX and a site , problems occur: First, the algorithm may force the sample to align with a generally assumed linear age trajectory, i.e., strongly increasing the values of this site. Second, this shift may be particularly strong if no non-linear age effects are modeled over the life-span . In this respect, Pomponio et al.  are not preserved and variability of the imaging phenotypes explained by this (unmodelled) covariate will be removed from the total variance. Further, ComBat assumes that the site effects o et al.  recommenAs a practical disadvantage, different implementations of ComBat with different functionality exist in parallel: for example, the version of Fortin et al.  cannot pf(x):ComBat-GAM  allows for modeling of non-linear covariate effects by placing one covariate or a set of covariates within a function f(Xisf) is a smooth function over covariates x1, x2, x3, …, xn with f(Xisf) = f = a + f1(x1) + f2(x2)+f3(x3)+, …, +fn(xn). Very briefly , the GAsee Wood  divides f = α + f1(xage) + b2xsex + b3xICV.To model non-linear age effects, as a prerequisite for lifespan studies, Pomponio et al.,  used plaThe main advantage of ComBat-GAM is the option to model and thereby preserve non-linear covariate effects in a flexible way during the site correction process. Pomponio et al.,  present In terms of disadvantages, the method inherits the general limitations of ComBat. Mainly, ComBat-GAM cannot preserve effects of site-confounded covariates that are not specified. Another problem might be overfitting of non-linear effects which could hamper the transfer of the model to unseen cases.CovBat  extends CovBat first calculates the ComBat-adjusted residuals, by running the default version of ComBat:eisf are assumed to have a mean of 0, however, their covariance matrices Σi may still differ across sites. In a second step, principal components analysis (PCA) is performed on those residuals eisf. PCA identifies covariance patterns in high dimensional data and compresses them by reducing the number of its dimensions. Conceptually, PCA consists of finding q orthogonal axes ϕk:After this first step, the Implementations in both R and Python are available and are clearly explained, with a call syntax highly similar to ComBat and no relevant computational burden. CovBat has been validated in one main report so far : here, tProblems with CovBat reportedly  arise int to the model, making it time variant and allowing one to specify multiple scanning time points along with constant or time-varying covariates:Longitudinal ComBat  specificIn addition to the parameter estimates for mean only mode of traditional ComBat . If. If40)].via analytical formulae   . So far,-scores) , quantil-scores) , general-scores) , 62, sup-scores) . The ver-scores) . Using tf will be dropped:Normative modeling involves estimating a z-statistic for each feature independently, which under Gaussian assumptions can be computed using the formula below, where the feature index i is the predictive mean from a (possibly nonlinear) function mapping the the noise-free prediction for each individual sample i based on the specified norm, σ2 is the noise variance and i in turn, we see that this variable is described as a combination of a 1 × p vector βj containing weights associated biological variance components and a 1 × q vector us containing weights related to site variation.Here, μi is an optional component in order to add non-linearity, for example via a Gaussian process or B-splines. Finally, this noise free prediction is linked to the noisy observation via:γ0 over both variance components: us ~ InvΓ. All free parameters, summarized in θ are estimated by performing Bayesian inference:The model in our recently specified implementation adds hierarchy by placing shared priors and hyperpriors θvia site-specific means and variances). The return of predictive mean and variance also allows to validate the predictions of NM, and thus the goodness of the subsequently calculated z-scores .Normative modeling has several advantages, the most important of them being that it does not remove any biological variance of interest that might be confounded with site variance. This characteristic makes normative modeling also applicable in a setting when biological information is not uniformly distributed across sites . Rather, NM results in direct predictions of the mean and the variance for the observed distribution, allowing to calibrate data to population centiles. This step places all data in a common reference space, whilst retaining the ability to relate these centiles back to the original scale of the data )  for for83)] To begin with, the target audience for this manuscript is researchers who plan to work with open, public or consortium data. This type of data is usually only accessible and being pooled after data collection has been concluded—and sometimes after several pre-processing steps have already been taken—meaning that the user has little or no control over those steps. Thus, we prioritized methods and strategies that can be applied after the data has already been collected and minimally pre-processed.Secondly, the origin of site effects is heterogeneous and not necessarily independent of biological variation; the extent of any interaction is not always fully known. For example, they can arise from scanner related factors, such as scanner platform and sequence details, which could potentially be alleviated using phantom scans. Yet, other factors affecting the postprocessing  equally introduce biases, and even QC procedures deserve a harmonization step. Given this complexity and the fact that some image modality specific intermediate steps are needed to transform the phantom or traveling subject information into an effective site correction, we refer the reader to another overview paper on this matter (see introduction of deep learning Section Adjustment of raw imaging data for the purpose of site harmonization by deep learning methods).For multi-site neuroimaging analyses, different methods to attenuate site effects have become available, comprising different statistical principles, degrees of use, level of validation, and readiness level. Well-founded approaches comprise the ComBat family and, more recently, normative modeling that by its flexible concept is increasingly adapted for site effect correction. Newer developments such as adjusting raw multisite MRI data through deep learning techniques show promise, but call for more accessible implementations and validation. We encourage the open sharing of site effect correction software and a detailed explanation in the methods sections of future manuscripts. We encourage the open sharing of site effect correction software and a detailed explanation in the methods sections of future manuscripts. Given that a chosen site adjustment method can significantly influence the outcome of the final analysis, future studies comparing results from multiple site correction methods could improve the replication and generalizability of large-scale, multisite neuroimaging studies.JB: conceptualization, methodology, software, writing—original draft, writing—review and editing, visualization, and project administration. PT: conceptualization, methodology, writing—original draft, writing—review and editing, and funding acquisition. CC: methodology and writing—review and editing. ML: methodology, visualization, and writing—review and editing. AC, NJ, and AM: writing—review and editing. AP: visualization. LS: conceptualization, writing—review and editing, project administration, supervision, and funding acquisition. PS: conceptualization, methodology, software, writing—original draft, writing—review and editing, visualization, project administration, and supervision. All authors contributed to the article and approved the submitted version.PT was supported by NIH grants R01AG058854, U01AG068057, RF1AG057892, R01AG060610, R01MH116147, P41EB015922, R01MH121246, R01NS107513, R01MH123163, and U.S. Alzheimer's Association grant ZEN-20-644609. He was also supported by NIA T32AG058507 and NIH grant U54EB020403 from the Big Data to Knowledge (BD2K) Program. PT also received partial research support from Biogen, Inc.  for work unrelated to the topic of this manuscript. AC was supported by the National Institute of Neurological Disorders and Stroke (grant numbers R01 NS085211 and R01 NS060910), the National Multiple Sclerosis Society (RG-1707-28586), and a seed grant from the University of Pennsylvania Center for Biomedical Image Computing and Analytics (CBICA). AM gratefully acknowledges support from the European Research Council , the Wellcome Trust under a Digital Innovator award  and the Dutch Organization for Scientific Research (016.156.415). NJ was supported by NIH grants R01AG059874 and R01MH11760. LS was supported by the National Institute of Mental Health of the National Institutes of Health (R01MH117601) and by a NHMRC Career Development Fellowship (1140764).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Millet-based dryland agriculture is the traditional mode of agricultural cultivation in northern China and has been of great significance to the emergence and development of Chinese civilization. However, although they are both millet-based agricultural production methods, with various subtypes in different regions of northern China. In the Songhua River Region in northeast China, the ecological environment and abundant natural resources led to the slow development of agriculture, and it was only after the Liaojin Dynasties that a mature farming industry was formed.We used the plant flotation instrument to flotation the soil samples unearthed in the Luotong Mountain City, a Liaojin period site in Songhua River Region, northeast China, and collected the charred plant seeds. Then observing them with the electron microscope, we identified and counted the plant seeds in this site.It was found that this region is still a millet-based crop utilization structure, and a total of 11 types of charred agricultural crop seeds were excavated from flotation at the Luotong Mountain City site. And the barnyard millet crops occupy a prominent advantage, with ubiquity of more than 91%.The ancestors of this region were still engaged in a millet-based agricultural strategy during this period, with a certain lag compared to the Central Plains’agricultural strategy where Triticeae crops had become dominant. In addition, the crop structure with the millet-based agriculture of the region is also somewhat different from that of the Central Plains. Through comparative studies of surrounding sites and reference to historical documents, it was found that this difference in crop structure is a phenomenon unique to the Songhua River Region and is related to the dietary habits of the local settled Jurchen nomads, who ate barnyard millet meal. The development of settled agriculture contributed to the emergence of civilization. During the formation of human society, many plants have been domesticated, including rice, wheat, corn, millet, barley and many other food crops. However, due to geographical and climatic constraints, the domestication of plant species was different, and the pattern and extent of agricultural development were also unlike anywhere else in the world. As a result, different centres of agricultural origin have arisen around the world, one of which is East Asia . In EastOryza sativa) were found at the Shangshan site in Zhejiang Province (Panicum miliaceum) and foxtail millet seeds  were found at the Donghulin site in Beijing  to northern China approximately 4000 years ago led to a transformation of this major crop  site  in the SRR and studied the agricultural use patterns in this region.Echinochloa utilis) as a food crop occurred in Japan, where it is known as “Japanese millet” , are more frequently seen in archaeological sites. The earliest charred wild barnyard grass seeds have been found by Chinese researchers at the Shizitan Palaeolithic site  in Shanxi  wrote during the Western Han Dynasty (206 BC-8 AD) about the planting advantages and economic value of barnyard millet as a food crop, “Barnyard millet can be grown in both flooded and dry conditions, and as long as it is planted, it will be harvested. It is also particularly luxuriant and suitable for planting in preparation for famine. The seeds of barnyard millet have grain, and when mature, they can be pounded out and cooked as barnyard meals, which is no worse than sorghum meal. It can also be used to make Baijiu ” ” .Qimin Yaoshu , that “Barnyard millets can be used to make Baijiu, and the smell is both fragrant and strong, much more than broomcorn millet wine and sorghum wine. Emperor Wudi of Wei Dynasty (155-220 AD) once ordered the chief agricultural officer to plant barnyard millets … The grains of barnyard millet could be ground into flour to feed the hungry in bad years; in good years, it could be used to feed cattle, horses, pigs and sheep” (Jia Sixie of the Northern Wei Dynasty (386-534 AD) added, in his agriculture book, d sheep” .In the Western Han Dynasty, ancient Chinese farmers already had mature knowledge of the barnyard millet and sometimes used it as human food. By the Wei Jin Dynasties (220-420 AD), the value of the barnyard millet as a brewing crop and livestock feed was more valued, and it was only consumed as human food during some famine periods. However, after the Northern Wei Dynasty, there are no more records of barnyard millets in agricultural books. It is clear that after the Northern Wei Dynasty barnyard millets gradually left the Chinese table.Northeast China, with its rich natural environment, has been of great importance and is one of the birthplaces of Chinese civilization. Since the Neolithic period, it has been an important settlement for nomadic fishing and hunting crowds and has developed a different form of civilization than that of the Central Plains. Before the establishment of the Liao Dynasty, the region was dominated by non-agricultural production methods . TherefoHowever, the Liaojin Dynasties were also a period when agriculture flourished in north-eastern China. According to historical records, large-scale population migration occurred in the northeast during the Liaojin Dynasties. During the period when the Liao Kingdom and Jin Kingdom were confronted, one after another, with the Song Kingdom, the cultural exchange between the two sides also intensified. At the same time, the Liao and Jin governments realized the importance of agriculture and moved a large number of Han Chinese and Bohai people to the SRR to strengthen the development of agriculture in this region, which brought advanced farming techniques and abundant food crops to the region, resulting in a highly developed agricultural process . In thisThe LMC site is located at the intersection of Liuhe County, Huinan County and Meihekou County in Jilin Province, between 125°17′ ~126°34′ east longitude and 41°53′~42°35′ north latitude .In addition, researchers have found that the site contains not only historical relics from the Goguryeo period, but also remains from the Liaojin Dynasties and even unearthed copper coins from the Song dynasty. It is clear that the city continued to be used for some time after the fall of Goguryeo and had close ties with the Central Plains. The discovery of this site is valuable not only for the study of Goguryeo history, but also for the transformation of the business model, ethnic integration and cultural exchange in northeast China.From 2006 to 2009, the Institute of Cultural Relics and Archaeology of Jilin Province organized relevant personnel to systematically conduct a survey and three excavations on the LMC site. During the excavation work, the unearthed artefacts included ironworks, potteries, porcelain, jade artefacts, etc. The ironworks included arrowheads, spear blades, chisels, armor pieces, pots and pans. The pottery included pots, jars, and bowls; the jade includes rings and beads; and the porcelain included bottles. It is a very typical ancient Chinese city site . The matThe archaeological excavation area was small, so the diversity of the collected flotation samples was somewhat limited. Therefore, a directional sampling method was chosen to sample a total of 13 house sites, one city gate and one drainage ditch excavated from the Liaojin Dynasties in the LMC site. Among them, 13 house sites of the Liaojin Dynasties were identified as the focus of sampling. Due to the shallow burial of the relics, which were quite close to the surface, there is a higher possibility of late human interference, and in consideration of maintaining the purity of the sample collection, no sampling was conducted on the stratum within the rectangular unit. After counting, the total number of samples collected by flotation reached 23, with a total of 337 L of soil samples and an average soil volume of 15 L per sample.The samples collected from this excavation were separated in the field using a water wave flotation device  and an 8Charred wood chips are the remnants of wood that has been burned, the main source of which is fuel, wood buildings, and wood that has been burned for other purposes. The total amount of charred wood chips obtained from flotation at the LMC site was 125.74 g through screening using a 1 mm pore size splitting sieve. The average amount of charred wood chips per sample reached 5.47 g , broomcorn millet (Panicum miliaceum), barnyard millet (Echinochloa esculenta), sorghum (Sorghum bicolor), barley (Hordeum uhulgare), oats (Avena sativa), buckwheat (Fagopyrum esculentum), soybean (Glycine max), cowpea (Vigna), bristlegrass (Setaria), grass family (Poaceae), legumes (Leguminosae), polygonum (Polygonaceae), Euphorbiaceae (Euphorbiaceae), cannabis (Cannabis Sativa), Perilla (Perilla frutescens), raspberry (Rubus), Huangbai (Phellodendron chinense), chenopodium (Chenopodium), cocklebur (Xanthium sibiricum), elderberry (Sambucus chinensis), kochia scoparia (Kochia scoparia), etc. In addition, there were a small number of unknown species of plant seeds that were not clearly characterized or had lost their key characteristic parts for identification due to more severe charring, so the species could not be accurately identified. The number of seeds of charred plants excavated is shown in A total of 6,715 charred plant seeds were found in the 23 flotation samples collected from the site of LMC site  in Heilongjiang, Bayantala site  in Inner Mongolia , LichunjMao Zhai Zi Xu (Autobiography in Maozhai) recorded his own mission to the Jin kingdom about the food situation: “From Xianzhou (咸州) to the area north of the Songhua River, people do not grow common millets, all they grow are barnyard millets, which are pounded into grain and made into barnyard millet meal. Once I saw the Jin emperor (Wan-yen A-ku-ta) and the chiefs of the Jin country eat together, and put a low table on the kang (a heatable brick bed in North China). Each person holds a bowl of barnyard millet meal, which is the so-called Jin kingdom royal banquet.” , which also mentioned the barnyard millet: “Since the change of Jingkang in the Northern Song Dynasty, all the people who were captured by the Jin Kingdom, regardless of the royals, or the officials of government, all of them were relegated to slavery and driven into servitude by the Jin Kingdom. Each person was given five Dou of barnyard millets (unhusked) in a month, and they were asked to pound the barnyard millets themselves to get one Dou and eight litres of the grains of barnyard millet as a month’s ration” (. “LiaoShi”(The History of Liao) also recorded the scene of the Liao royal sacrifice using barnyard: “One day, the shaman inserted a willow branch  in the southeast of the canopy and toasted it with wine, broomcorn millets and barnyard millets to pray for blessings”  The high latitude and cold climate of northeastern China limited the types of crops suitable for cultivation, so the barnyard millet became the primary choice of the Jurchen people because of their low requirements for the growing environment. (2) The rich plant and animal resources and relatively sparse population in the SRR made the relationship between people and land harmonious. In this environmental context, inhabitants did not need to vigorously cultivate crops and develop agriculture to obtain living resources. Therefore, the barnyard millet is already sufficient to meet the inhabitants’ food crop needs. (3) This tradition may also be related to the Goguryeo that once settled in the area. However, the relationship between the two is not yet clear due to the limitations of excavated materials.Combining the research results of the surrounding sites, we argue that eating barnyard millet meals tradition of Jurchen in the SRR is the result of a combination of the above three reasons. At the same time, this dietary tradition is strongly associated with Jurchen people, even with the expansion of the dominion of the Jurchen people, affecting areas further south . HoweverThe government of the Liao Dynasty implemented an immigration policy to promote agriculture in the region, moving agricultural people from the Central Plains and the Bohai Kingdom into the SRR. And probably under the effect of migration of the agricultural population, the dietary tradition of eating barnyard millet meals was also declined. The differences in the number of charred barnyard millet seeds excavated from different archaeological sites in the region reflect the changes on dietary traditions. And because of its remote location and the fact that it was once a city of Goguryeo, LMC site has preserved the most primitive tradition of eating barnyard millet and has unearthed the largest number of charred barnyard millet seeds.At the LMC site, barnyard millet is the most abundant and is the main source of grain; foxtail millet is the next most abundant, and broomcorn millet accounts for the smallest proportion of these three crops. However, among the three sites of Bayantala, Yongping, and Lichunjiang, foxtail millet excavation was the most abundant and dominant, and although there is also a certain amount of barnyard millet, its quantity was less than that of the LMC site.At the Bayantara site, the number of Charred foxtail millet and broomcorn millet plant seeds excavated was comparable, 213 and 205, respectively, and the number of barnyard millet was extremely small, with only 4 being observed; at the Yongping site, the number of foxtail millet was the most abundant at 565, followed by 265 for millet and only 15 for barnyard millet; at the Lichunjiang site, the number of foxtail millet excavated was absolutely dominant, 1123, followed by 57 for barnyard millet, and extremely small for broomcorn millet, with only 1 being observed. The comparative study with the surrounding sites of the same period, Bayantala site, Yongping site and Lichunjiang site, reveals that in these four sites during the Liaojin Dynasties in the SRR, millet crops were still predominant, with foxtail millet as the main crop, followed by broomcorn millet and barnyard millet.Wheat crops are in stark contrast to millet crops. Only 16 barley seeds were unearthed at the LMC site, and no wheat was observed. This situation also occurs in the surrounding sites. At the Bayantala site, only 3 grains of barley were unearthed, and no wheat was found; at the Yongping site, only 4 seeds of wheat were found; and at the Lichunjiang site, no wheat crop was found. This shows that wheat crops were not widely promoted and used in the SRR during the Liaojin Dynasties.In addition, Charred seeds of sorghum, buckwheat, oats, and soybean miscellaneous grains have been found at the LMC site. Sorghum was also found at the Lichunjiang and Yongping sites, with only individual seeds. Similarly, buckwheat was also found at the Yongping site and Bayantala site, and soybeans were only excavated at the Yongping site. Such miscellaneous grains in the SRR during the Liaojin Dynasties should also occupy a certain proportion of people’s preferred recipes.Moreover, charred cannabis seeds were excavated from the Liaojin Dynasties LMC site, Yongping site and Bayantala site in the SRR, and the number of cannabis charred seeds excavated from the Bayantala site even reaching 167. From this, it can be inferred that cannabis, an economic crop, was already commonly cultivated in the SRR during the Liaojin Dynasties.All of the above provides evidence for the speculation on crop utilization during the Liaojin Dynasties in the SRR. The region is still a millet-based agricultural utilization model, but the proportion of the three types of foxtail millet, broomcorn millet and barnyard millet, varies from site to site, probably due to regional dietary differences. Wheat was not yet commonly cultivated in the region during the Liaojin Dynasties, and only a few seeds have been unearthed sporadically, which has a certain lag compared with the Central Plains during the same period, proving that the SRR may have been a relatively closed agricultural production unit during the Liaojin Dynasties. However, the excavation of non-staple food crops such as sorghum, oat, buckwheat, barley and soybean, proved that the region had a rich variety of crops, and this diversity of crops was obviously the result of cultural exchange.This contradictory situation corresponded to the stage of agricultural development in the region at that time. The Liaojin Dynasties was a key period in the transformation of the production pattern of the SRR. Before the Liaojin Dynasties, the SRR was dominated by agricultural production methods, and during the Liaojin Dynasties, agriculture developed rapidly under the influence of the government’s policy of vigorously developing agriculture. Against this background, a relatively independent, but inclusive, pattern of crop utilization emerged and is expressed in archaeological sites, such as the LMC site, Bayantala site, Yongping site, and Lichunjiang site.Based on the study of the flotation charred crop seed remains excavated from the LMC site, and combined with the charred crop seed remains excavated from surrounding sites of the same period, the following conclusions can be drawn:The millet-based crop cultivation strategy is still the main strategy for agricultural production in the city and even in the region during the Liaojin Dynasties, supplemented by sorghum, buckwheat, oats and other miscellaneous cereal cultivation. Among the three millet crops, barnyard millet has a very important position. This strategy may be related to the superior natural environment of Northeast China and the social environment of mixed ethnic groups.During the Liaojin Dynasties, the crop structure of the entire SRR showed a certain regional independence. However, within this seemingly relatively independent area, there are some differences between sites, showing different characteristics of agricultural production. This contradiction is due to the historical background of the migration of the agricultural population during the Liaojin Dynasties.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CW, CY and LB designed the study. CY, CW, LB and XL conducted the study. CY, DL, KX and XG carried out the flotation. CY, LB and CW identified charred plant seeds. CW, CY and LB wrote the main manuscript text. CW, LB and XL prepared figures. CY and LB have contributed equally to this paper and are co-first authors of this paper. All authors contributed to the article and approved the submitted version.This work was supported by the Fundamental Research Funds for the Central Universities, “Migration, Cultural Integration and Environmental Adaptation of Ancient Populations along the Belt and Road Based on the Bioarchaeological Perspective” (No.2022CXTD17) and the Graduate Innovation Fund of Jilin University (No.2022219).We thank archaeological excavators at Institute of Cultural Relics and Archaeology of Jilin Province for their careful excavation work. We thank Qiang Hu from the Chinese Academy of Environmental Sciences for his help with the mapping. We also thank Shu Wang, Han Dong, Hongyu Zhang, Puyu Chen, Rongyan Li from the Bioarchaeological Laboratory of Jilin University for their help with this work. We are very grateful to the editor Jianping Zhang and two reviewers for reviewing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TWSG1 knockout mice showed neural, craniofacial, and mammary defects. TWSG1 also regulated erythropoiesis and thymocyte development. Furthermore, the relationship between TWSG1 and cancer has been elucidated. Allelic loss of TWSG1 was detected in colorectal cancer. TWSG1 expression was upregulated in papillary thyroid carcinoma and glioblastoma but downregulated in gastric and endometrial cancers. TWSG1 suppressed BMP7-enhanced sphere formation and migration in endometrial cancer cells, indicating its tumor-suppressive role. Further studies are required to clarify the TWSG1 function and its association with BMP signaling in cancer development. Finally, TWSG1 is abundantly expressed in human and mouse ovaries and sustains follicular growth in rodent ovaries. Thus, TWSG1 has various functions ranging from fertility to cancer. Therefore, TWSG1 signaling modulation may be beneficial in treating specific diseases such as cancer.Bone morphogenetic proteins (BMPs) play an important role in development. Twisted gastrulation BMP signaling modulator 1 (TWSG1) was initially identified as a regulator of the dorsoventral axis formation in Drosophila. The mechanism of BMP signaling modulation by TWSG1 is complex. TWSG1 inhibits BMP signaling by binding to BMP ligands including BMP4, whereas it enhances signaling by interacting with Chordin, a BMP antagonist. Therefore, TWSG1 can act as both a BMP agonist and antagonist. TWSG1 has various functions ranging from embryogenesis to cancer progression.  Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily. BMPs are secreted extracellular matrix-associated proteins that perform crucial roles in the morphogenesis of several organs, including the musculoskeletal, cardiovascular, and neural systems . In addiThe twisted gastrulation BMP signaling modulator 1 (TWSG1) gene encodes a secreted protein, which modulates BMP signaling. TWSG1 is a secretory BMP-binding glycoprotein that regulates the activity of BMP ligands in the extracellular matrix and is associated with a variety of diseases . TWSG1 iTSG homologs share 72–98% homology in amino acid sequences and retain 24 cysteine residues in Drosophila . TSG expBMP signaling is tightly regulated at multiple stages . BMP ligConsidering that BMP ligands and antagonists are distributed throughout the body, TWSG1 could have a wide range of functions. In this review, we focus on TWSG1 biology ranging from embryogenesis to cancer development .TSG, a TWSG1 homolog, regulates dorsoventral axis formation in Drosophila, fly, frog, and fish embryos . The TSGTWSG1 gene [TWSG1 mutations have rarely been detected in human HPE [TWSG1 has previously been reported to be expressed in multiple tissues during the gastrulation, neurulation, and organogenesis stages such as the neural plate, neural tube, somite, branchial arches, surface ectoderm, choroid plexus, hippocampus, and endoderm. TWSG1-mutant mice exhibit neural arch deletions in the cervical vertebrae and show a high mortality rate of up to about 50% . As prevSG1 gene . Howeveruman HPE . Among auman HPE . In addiuman HPE , suggestBecause BMP signaling is indispensable for osteogenesis , TWSG1 mTWSG1 plays an important role in T-cell differentiation and erythropoiesis. Both T and B cells dynamically express TWSG1 . TWSG1-dThe previous report showed that the BMP endothelial cell precursor-derived regulator (BMPER), one of the extracellular BMP modulators, acts proangiogenically on endothelial cells in a concentration-dependent manner. In addition, TWSG1 enhances endothelial cell ingrowth, migration, and sprouting, as confirmed by a mouse Matrigel plug and human umbilical vein endothelial cells (HUVEC) sprouting assay . AccordiTWSG1 induces the phosphorylation of SMAD1/5/8, AKT, and extracellular signal-regulated kinase (ERK) , indicathttps://www.proteinatlas.org/ accessed on 11 October 2022), TWSG mRNA levels are different in various cancers (TWSG1 have been observed in familial colorectal cancer [TWSG1 mRNA expression [According to the Human Protein Atlas ( cancers . The rel cancers . A loss l cancer . TWSG1 el cancer ,52,55,56pression , indicatpression . TherefoTWSG1 expression was initially detected in adult sheep ovaries . JuvenilTWSG1 is also indispensable for mammary gland development in mice. Regulation of BMP signaling by TWSG1 is required for normal ductal elongation, branching of the ductal tree, lumen formation, and myoepithelial compartmentalization in the postnatal mammary gland . UnderstAlthough TWSG1 plays important roles in many aspects, the mechanism of how TWSG1 is regulated remains to be elucidated. Once the regulation mechanism is understood, we could suppress the progression of cancers and specific diseases by modulating its expression. Considering its abundant expression in ovaries, TWSG1 could be a new marker of fertility. In addition, recombinant TWSG1 has an advantage over conventional BMP receptor kinase inhibitors in that it has no effect on other receptors. Therefore, recombinant TWSG1 could be used as a novel BMP inhibitor.In conclusion, TWSG1 has a variety of functions ranging from fertility to cancer in multiple organs. However, the detailed mechanisms underlying TWSG1 functions have not yet been fully elucidated. Although further studies are needed to clarify these mechanisms, the modulation of TWSG1 signaling could be promising for treating specific diseases and cancers.


Reabsorption is driven by the Na,K‐pump that is acutely stimulated by Ang II, which requires phosphorylation of serine‐938 (S938). This site is present in humans and only known to phosphorylated by PKA. Yet, activation of AT1 decreases cAMP required to activate PKA and inhibiting PKA does not block Ang II‐dependent phosphorylation of S938. We tested the hypothesis that Ang II‐dependent activation is mediated via increased phosphorylation at S938 through a PI3K/AKT‐dependent pathway. Experiments were conducted using opossum kidney cells, a proximal tubule cell line, stably co‐expressing the AT1 receptor and either the wild‐type (α‐1.wild‐type) or an alanine substituted (α‐1.S938A) form of rat kidney Na,K‐pump. A 5‐min exposure to 10 pM Ang II significantly activated Na,K‐pump activity (56%) measured as short‐circuit current across polarized α‐1.wild‐type cells. Wortmannin, at a concentration that selectively inhibits PI3K, blocked that Ang II‐dependent activation. Ang II did not stimulate Na,K‐pump activity in α‐1.S938A cells. Ang II at 10 and 100 pM increased phosphorylation at S938 in α‐1.wild‐type cells measured in whole cell lysates. The increase was inhibited by wortmannin plus H‐89, an inhibitor of PKA, not by either alone. Ang II activated AKT inhibited by wortmannin, not H‐89. These data support our hypothesis and show that Ang II‐dependent phosphorylation at S938 stimulates Na,K‐pump activity and transcellular sodium transport.Angiotensin II (Ang II)‐dependent stimulation of the AT Angiotensin II‐dependent stimulation of the rat kidney Na,K‐pump in proximal tubules is mediated at least in part via an increase in the phosphorylation of the Na,K‐pump at S938 through a PI3K/AKT(PKB)‐dependent pathway, not by PKA. S938 is a conserved site of phosphorylation on all four isoforms of the human Na,K‐pump and was thought to be exclusively phosphorylated by PKA. The Na,K‐pump is the active transport mechanism that drives sodium reabsorption in the proximal tubule, which increases blood pressure. Therefore, the identification of this signaling pathway to phosphorylate the Na,K‐pump at S938 in response to angiotensin II is potentially relevant to human physiology. The approach used was similar to that previously developed  to give a final concentration of 1, 10, or 100 pM was added to the basolateral side of the cells. An equal volume of buffer alone was added to the apical side of the cells. Five minutes later nystatin at a final concentration of ~595 USP Units/ml   was added to the apical side of the cells. The plate was put back in the microscope and the fluorescence was measured for 5 min. The time between the addition of the nystatin solution and the beginning of the live confocal recording was approximately 1 min.2, 15 mM Hepes free acid, 3.0 mM CaCl2 and 5 μM gramicidin , pH 7.4. The gramicidin forms channels in the plasma membrane equally permeable to sodium and potassium  of the rat Na‐K‐ATPase α1‐isoform was developed by Cell Signaling Technology and previously characterized  was purchased from Sigma–Aldrich. Secondary antibodies were from Jackson ImmunoResearch: donkey anti‐mouse (# 715‐035‐150) and anti‐rabbit (# 711‐035‐152) both coupled to horseradish peroxidase (HRP). H‐89 was from LC Laboratories. SDS‐PAGE reagents were from Fisher Scientific (Hanover Park); acrylamide was from Bio‐Rad Laboratories; polyvinylidene difluoride was from Millipore; KPL chemiluminescence reagents were from Insight Biotechnology; phosphatase inhibitors (microcystin and okadaic acid) were from Axxora; the molecular weight marker MagicMark was from Invitrogen. CPT‐2Me‐cAMP (CPT) was from Tocris. All other reagents were from Sigma‐Aldrich.Anti‐P‐Ser‐473 Akt (#9271) and anti‐AKT antibodies (#9272) were from Cell Signaling Technology. The phosphospecific antibody to the α2.6p ≤ 0.05. In each experiment the value of n is equal to the number of independent experiments.The results were analyzed by a one‐way ANOVA followed by Bonferroni's post‐hoc multiple comparison test using GraphPad Prism 5 software. Results were considered significant if 3The direct effect of Ang II on the Na,K‐pump in the basolateral membrane of polarized proximal tubule cells was measured with the apical membrane made highly permeable to sodium to avoid confusing a direct effect of Ang II on Na,K‐pump activity with secondary activation. The latter is caused by Ang II first stimulating NHE3 in the apical membrane, increasing the concentration of intracellular sodium, which is the rate‐limiting substrate for the Na,K‐pump. The experimental basis for our approach to measure Na,K‐pump activity is introduced in Figure We measured the concentration of intracellular sodium during the protocol used for the experiments shown in Figure Thus, the results in Figures Adding Ang II 5 min before the addition of nystatin significantly stimulated Na,K‐pump activity in α‐1.wild‐type cells Figure . The posp = 0.03) and wortmannin increased the phosphorylation of S938 compared to control  as analyzed by a paired t‐test. Likewise, Ang II increased the phosphorylation at S938 in the presence of H‐89 compared to control  and increased the phosphorylation at S938 in the presence of wortmannin compared to control , and the combination of H‐89 and wortmannin inhibited the phosphorylation of S938 observed in the presence of Ang II and H‐89 . A negative control for anti‐P‐Ser938 was presented previously , a synthetic activator of EPAC (exchange factor directly activated by cAMP), at 10 μM for 20 min also increased the phosphorylation of AKT at S473 Figure , lane 9,4The results of this study support our hypothesis that acute Ang II‐dependent activation of the rat kidney‐pump in the proximal tubule is mediated at least in part via increased phosphorylation of the Na,K‐pump at S938 via a PI3K/AKT(PKB)‐dependent pathway. Our data show that preventing phosphorylation of S938 blocks the ability of Ang II to acutely stimulate the Na,K‐pump and increase the net movement of sodium from the apical to basolateral side of differentiated cultured proximal tubules. Previous studies on the same cells not differentiated into distinct apical and basolateral membranes showed that phosphorylation of S938 was required for Ang II to rapidly (<5 min) increase the amount of Na,K‐pump in the plasma membrane and thereby stimulate Na,K‐pump activity  mediated increases in phosphorylation at S938 play a significant role in the mechanism by which Ang II acutely stimulates Na,K‐pump activity in the proximal tubule Figure . It has Based on our results we propose that AKT phosphorylates Na,K‐pumps that are in intracellular vesicles and PKA phosphorylates Na,K‐pumps that are in the plasma membrane. Using our model of reciprocal inhibition between AKT and PKA Figure , one wouWe note that the ouabain‐sensitive change in Isc in α‐1.S938 cells in the absence of Ang II Figure  is aboutAs specified above the interpretation of our phosphorylation results in terms of signaling pathways is based on the expectation that we have used wortmannin under conditions that it selectively inhibits PI3K and H‐89 under conditions that it selectively inhibits PKA. On the other hand, exposing murine cortical collecting duct cells to 100 nM wortmannin for hours reduces epithelial integrity, with some effects observed in as little as 30 min  pathway in rat proximal tubule cells  in the proximal tubule  and R01‐CA81150 (to R.R.M.)]. Confocal microscopy was performed through the Wayne State University and Karmanos Cancer Institute Microscopy, Imaging and Cytometry Resources Core, which is supported in part by National Institutes Health center grant P30 CA22453.The authors declare that they have no conflict of interest.


N-methyl-D-aspartate receptors (NMDARs) in the central and peripheral nervous systems promotes neuropathic pain. Amitriptyline (ATL) and desipramine (DES) are tricyclic antidepressants (TCAs) whose anti-NMDAR properties contribute to their analgetic effects. At therapeutic concentrations <1 µM, these medicines inhibit NMDARs by enhancing their calcium-dependent desensitization (CDD). Li+, which suppresses the sodium–calcium exchanger (NCX) and enhances NMDAR CDD, also exhibits analgesia. Here, the effects of different [Li+]s on TCA inhibition of currents through native NMDARs in rat cortical neurons recorded by the patch-clamp technique were investigated. We demonstrated that the therapeutic [Li+]s of 0.5–1 mM cause an increase in ATL and DES IC50s of ~10 folds and ~4 folds, respectively, for the Ca2+-dependent NMDAR inhibition. The Ca2+-resistant component of NMDAR inhibition by TCAs, the open-channel block, was not affected by Li+. In agreement, clomipramine providing exclusively the NMDAR open-channel block is not sensitive to Li+. This Ca2+-dependent interplay between Li+, ATL, and DES could be determined by their competition for the same molecular target. Thus, submillimolar [Li+]s may weaken ATL and DES effects during combined therapy. The data suggest that Li+, ATL, and DES can enhance NMDAR CDD through NCX inhibition. This ability implies a drug–drug or ion–drug interaction when these medicines are used together therapeutically.The facilitated activity of  N-methyl-D-aspartate receptors (NMDARs), which belong to a large family of glutamate-activated ionotropic receptors, participate in CNS functioning both in health and neurological and neurodegenerative diseases [2+ as a voltage-dependent blocker of NMDARs inhibits central sensitization and thereby decreases pain hypersensitivity [2+-independent and voltage-dependent channel block of NMDARs [2+ [2+]) lowers the IC50 for NMDAR inhibition by ATL  and, presumably, may interfere with Ca2+-dependent inhibition of NMDARs by TCAs. At high doses, lithium carbonate causes body intoxication ) may interfere with the TCA action on NMDARs, here the effects of different [Li+]s on the TCA inhibition of currents through native di- and triheteromeric NMDARs composed of GluN1, GluN2A, and GluN2B subunits [2+] the therapeutically relevant [Li+]s strongly elevate ATL and DES IC50s for NMDAR inhibition. Another TCA, clomipramine (CLO), causes inhibition of NMDAR currents, which is not affected by Li+. This property coincides with the observation that the open-channel block of NMDARs by CLO is not Ca2+-dependent  were studied. With this goal, ATL concentrations ([ATL]) from 1 to 600 µM were sequentially applied with an increment to the steady state of NMDA-activated currents in the presence of 1 or 2 mM Ca2+ in Li+- or Na+-containing bathing solutions  coincides well with the previous data [50 with the [Ca2+] elevation from 1 to 2 mM  of 1.4 ± 0.57 mM, which is similar to therapeutic [Li+]s  induced maximal effects on both DES and ATL NMDAR inhibition  EC50s of Li+ effects to increase the IC50 of ATL and DES were within 0.8–2.0 mM, which overlaps the therapeutic range of Li+ concentrations (0.5–1 mM) found in the blood plasma, and (iii) Li+ does not change the parameters of CLO effect on NMDARs, which performs exclusively the open-channel block and is Ca2+-independent in contrast to ATL and DES. Overall, this type of pharmacology is consistent with the competitive action of Li+ and ATL or DES. In general, the main observations of our investigation could be defined as (i) the Li+ and ATL or DES could be explained by the competition for the same molecular targets, which results in a considerable weakening of the ATL and DES effects on NMDARs. Because NMDAR CDD is the only Ca2+-dependent process in the kinetics of the NMDAR activation and the NMDAR inhibition by Li+ [2+], the CDD seems to represent a most likely process that could be targeted by these medicines. Therefore, any molecule involved in the CDD regulation may in principle represent a molecular target for Li+ and TCA action. As we previously demonstrated s s ,12. In o+]s above 0.5 mM cause the development of NMDAR CDD so that ATL and DES could not add anything to further induction of CDD. This explains an inability of 10 µM ATL to inhibit NMDA-activated currents in the presence of 0.5 mM Li+. In contrast, CLO does not exhibit Ca2+ dependence of NMDAR inhibition h/IC50h).To determine the blocking potency of TCAs, NMDA-elicited currents were measured in the absence and presence of different TCA concentrations. Amplitudes of currents measured in the presence of blocker (In) refers to the number of recorded cells. Data pairs were compared using an unpaired Student’s two-tailed t-test. Multiple groups were compared using one-way AVOVA and post hoc Tukey’s test. Statistical significance is reported in the figures according to the following symbols *, **, ***, and ****, which indicate p values below (<) 0.05, 0.01, 0.001, and 0.0001, respectively. In addition, p values for each comparison are indicated. Curve fitting was performed using OriginPro software (OriginLab Corp.). IC50 values obtained from individual experiments performed under the same experimental conditions were averaged to get mean ± SEM values.Data are shown as representative measurements and mean values ± standard error of the mean (SEM). (


Our case study at Ga-Mohana Hill in South Africa then offers practical approaches and methodologies that the fields of cultural heritage documentation and archaeological visualisation can employ to address their recurring issues as identified in the critical analysis. We present an online, interactive 3D digital replica of a living heritage and archaeological place that we believe responds appropriately to its political, cultural, and social context along with communicating its archaeological significance.Creating and sharing 3D digital replicas of archaeological sites online has become increasingly common. They are being integrated in excavation workflows, used to foster public engagement with the site, and provide communication and outreach of research, which now happen on digital media platforms. However, there has been little introspection by the community involved in the 3D documentation field, which has resulted in problematic practices. We critique the western paradigm of archaeological visualisation and propose recommendations for inclusive, decolonised visualisations of living heritage and archaeological places. To begin, we define in broad terms what an archaeological site is, and then we describe how these sites have been recorded and represented using the latest technology for digital re-production, namely laser scanning and photogrammetry. Following that we provide a critical analysis of current 3D visualisations of archaeological sites and develop an approach to ensure that the significance, meaning, and  Archaeological visualisation is an essential practice that facilitates the flow of archaeological information and interpretation from the academic world to the public. The power of visual over textual presentation has been widely embraced and acknowledged by archaeological visualisers  world of possibilities, providing access to the sites as never before, and representing the site to a larger audience. Since the models are created in a digital environment, there are opportunities to insert interpretative devices that cannot be created at the physical site, but that would enhance the visitors’ experience of the place. When the models are given interactive functionality, they also allow engagement with the representation, giving agency to the viewer. The models can additionally be accompanied by annotations and multimodal contextualisation, which provides insights into objects context and biographies . Some of their aims include creating records of the physical sites for future generations as a response to the threat of natural and man-made destruction, as well as creating awareness for the site and creating materials for education and tourism Rüther . UniversIn general, the public are limited to 3D models that they can access for free online of sites with the minimum technological means . As the Covid-19 pandemic closed borders and classrooms, many educators scrambled for digital models of objects and places that could be safe substitutes for in-person experiences. There are many platforms that host interactive 3D models of archaeological and heritage sites  Agency—having agency is the ability to act upon people to give and receive meaning. (2) Multi-vocality—being multi-vocal seeks to accommodate different perspectives, past and present, of a place and supports a decolonised, de-centred, and inclusive representation of a place that is relevant and appropriate today. (3) Proximity—the significance of an archaeological and heritage site is its ability to provide proximity to the people who used the place in the past and who use it in the present. Digital replicas must, therefore, seek to provide a faithful experience of proximity to the physical place and the people who use it. We aim to implement these three aspects using the objectives below.AgencyShow the place as a living entityShow the place is not static and has a biography from its distant past to the presentRe-humanise the 3D modelEnable engagement with the place via its 3D replicaMulti-vocalRespect the site and the current users of the placeRepresent the multiple perspectives of the siteBring to the centre other uses, past and present, of the site besides its scientific significanceCreate a decolonised visualisation of the placeProximity3D model of the site and artefacts must be good quality  to provoke the feeling that the virtual visitor is there at the siteFacilitate inclusive access to the siteMediate the experience to preserve the atmosphere of the place, and communicate its narrative appropriately3D model must be given authority and authenticityWe embarked on a case study approach that attempts to overcome the many challenges outlined in the critical analysis, and to show how the above aspects can be practically implemented.Ga-Mohana Hill and rock shelters in the Northern Cape province of South Africa was chosen as our case study location. We present a visualisation of Ga-Mohana Hill as an online interactive 3D model residing on Sketchfab.Ga-Mohana possesses significant archaeological value as a record of early human origins and evolution  and southeast (Ga-Mohana Hill South Rockshelter) sit at opposite sides of the hill. The region is characterised as semi-arid savannah. The hill is located on common/tribal land owned by the Baga Motlhware Traditional Council.This case study project includes four authors from three different disciplines and backgrounds the goal of which is to intentionally bring different perspectives to the visualisation. SW is a geomatician with a background in digital 3D re-production of heritage sites and objects. SM is an architect who brings philosophical insight into indigenous knowledge and representation. SM grew up in Kuruman where he is a member of the local community and has done extensive research into ritual practices in the area. JW and BS are archaeologists who lead excavations at the Ga-Mohana Hill North and South shelters.For some local residents of Kuruman and the surrounding area Ga-Mohana Hill is regarded as a spiritual and cultural place and is used for prayer and ritual Maape . Myths oDrawing creepy places: Representing liminal ritual spaces of Kuruman—South Africa’ , surrounding area of shelter (north and south), hill-top, and landscape.The most relevant archaeological artefacts for this model were identified as coming from the dark brown silty roofspall unit (DBSR) in excavation Area A of Ga-Mohana Hill North Rockshelter, which dates to 105,000 years ago  which encourages thoughts about human’s unique place in the universe, being alive and conscious to experience it, as well as to think about the different cosmologies that are represented at the site.Our initial plan was to incorporate into the model the drawings SM had done for his project “Drawing Creepy places” Maape . These dThis idea was tested, but abandoned in favour of a more subtle approach. We did not want to impose too much onto the landscape, as we were aiming to be multi-vocal in our approach, and felt that by imposing one view we were emulating the colonial project. We wanted the site to speak for itself, and we felt the drawings would interfere with people’s perception of the place as they navigated around the 3D model. We wanted to encourage people to generate their own feelings as they explored the site.We decided on the metaphor of light and darkness as the general theme of the visualisation. The colonial project sought to illuminate the dark continent of Africa, to lift the veil of its mysteries and transform its people to the so-called civilised culture of the West. In contrast to this idea, many indigenous rituals make explicit use of darkness as a means to incite a particular feeling. Fear, secrecy, and mystery are all associated with the dark. Illuminating the dark, therefore, strips the potency from these ritual places Maape .We, therefore, decided to embrace the darkness, and carefully arranged our lighting of the site to keep certain areas in shadow. We also wanted to acknowledge the role and presence of the Great Snake in the site. As SM has pointed out “Any representation of sites associated with the snake must therefore address the difference between objective and animistic space and find other ways to represent their nature as being the snake” Maape , 57. TheTo represent this notion, we again employed the metaphor of light and dark. We attached a light source to the viewer, but pointed it at right angles to the viewer's forward view. This device then creates an unpredictable result when rotating in the 3D environment. The viewer gets the feeling that they are not in direct control of what is illuminated and what is left in shadow, but also creates a means for the cave to be lit up, just maybe not when it is intended. This dynamic lighting, therefore, created agency for the shelter and represents the snake. It gives the site a feeling of being alive by casting shadows haphazardly around the shelter.Next, we developed the flow of the movement the viewer experiences while navigating through the site. The flow is created by annotations that are built into the functionality of Sketchfab. Using the annotations, the viewer can easily move between the points of interest that we created around Ga-Mohana. The points of interest are numbered to encourage a sequential movement, but this is not enforced. The viewer is given free control and agency of how to view the site. However, each point of interest is carefully curated with one aspect of the curation being to define the focus point of each point of interest, which was achieved by choosing a particular point of rotation for each annotation. The rotation point will either remain at the centre of the view, such as when we wish to focus attention on particular objects such as the archaeological artefacts, or the point of rotation is located at the viewer's location, which results in sweeping views where nothing is fixed or focused on Figure  .Figure 1Realising there is no way to avoid using text in the visualisation to provide the information we wanted to provide we created unique, rotating sign boards. This was partly to create movement that captures the viewer's eye, who will be drawn to read the text. It also serves as a device to display text, but not all at once, which would otherwise block out the view of the model. The unique designs on the rotating text objects were inspired by archaeological artefacts discovered in South Africa at Diepkloof rock shelter , where the archaeological excavations have uncovered significant finds  who inhabited the shelter in the Later Stone Age. The rock art is highlighted by offsetting a panel in the wall, which is given a higher resolution to the rest of the shelter  followed for the creation of the 3D model are almost identical  to the objective approach it is the curation of the model, and the emphasis on co-production and multi-vocality, which saw the most change and affected the 3D visualisation that is presented to the public. The difference between the two approaches is in the shift away from  a focus creating perfect copies of space, to the end result which is representing that space as meaningful place to a public audience. This focus has been lacking in the 3D documentation community, but it is vital to the preservation of cultures as archaeological and living heritage sites enter the digital realm.To establish agency in the 3D model a combination of visual and navigational devices was used with the aim of giving the model life. This was achieved by: the unpredictable (mind of its own) nature of the shadow when navigating; the presence of the Great Snake via its shadow moving across the wall which clearly announced its conceptual presence; animating the sparks of the fire, the waterfalls, engravings, and the rotating sign boards which presented the model as non-static and alive; showing the evidence of people at the place  which re-humanised the model; and finally by allowing active engagement rather than passive presentation the model is able to create unique experience for all visitors, thereby creating new meaning, which is essential for the place to maintain its significance.To achieve multi-vocality in the model, we adopted an ethical data capture policy that respects the site and the people who use it. We also aimed to represent the different significant uses of the place in a way that gave equal voice to all. This proved to be challenging in regards to the current uses as it became apparent that indigenous practices at Ga-Mohana are neglected and fragmented. From field work observations, people in the community, rather than being a homogenous group who are custodians of the site, are in fact cut-off from the site, not physically, but perhaps more problematically, culturally. Sites like these were places where moral and ethical values were imparted Maape , which aTherefore, the aim of making the 3D model, and some of the decisions made regarding its representation, are influenced by this severing of people from their culture, and lack of advocacy for protecting the site. The place, in essence, belongs to all that use it, who as mentioned are a diverse group. So, an over emphasis on one representation of the site, has the potential of doing precisely what we believe is the risk, namely the exclusion of others. As a response to this, we have tried to include all the diverse elements, particularly the symbolic elements of the site that are present there, including the shadow of the snake, as an element that is not physically present like the symbols of the churches and the healers but is represented in the myths. From observing the new ways in which the youth are claiming the site for activities that address their current economic needs, we have proceeded to draw the site into new forms of engagement, and therefore, instead of creating complete secrecy and prohibition, are aiming to use this model as a way of creating new meaning, but specifically towards establishing contemporary forms of value and preservation. SM, who is a co-author of the 3D model, is exercising his rights as a member of this community, who has spent over 10 years advocating for the protection and reestablishment of the value of the site, to re-imagine the site in a digital form, representing as far as possible, the interests of the various groups mentioned above as perhaps a custodian of the site in both its physical and digital forms.Proximity was established by creating a good quality digital re-production of the site using state-of-the-art photogrammetry capture and processing techniques with a focus to digitise the site as realistically and accurately as possible. To ensure the 3D model was inclusive and easily accessible meant we were forced to limit how much detail could be contained in the model due to file size limitations that were imposed to allow low-bandwidth and low-spec devices access. To mediate the experience the atmosphere of the site was recreated in the model using light and shadow. The website created to prepare viewers before entry into the model, and to provide contextualisation of the model, creates a sense of authority for the model, as museums do for artefacts. By providing information on how and who created the 3D model the chain of events leading back to the original is established, which along with the authority provided by the websites gives the model authenticity.With the ever-advancing digital realm enveloping our lives and cultures, we are tending to view digital media as an important and necessary component and counterpart to physical things and places. We need to begin to shift our framing, understanding, and expectations of digitised entities from trivial digital copy, to valuable digital manifestation. We have shown that 3D models can offer a digital counterpart to a physical place and even offer a far richer experience, especially if the visitor is able to experience both the physical and digital manifestations of the place. We believe our approach is a step in the right direction to preserve and manifest the original potency of significant places in digital form, while at the same time creating decolonised and de-centred representations. Finally, we have shown that 3D models can also help with the preservation of indigenous cultures that are neglected, and we hope even revitalise them and for younger generations and help people re-connect with their past.Following the current trajectory of the digital trend, future 3D digital replicas of places will exist as persistent places in the metaverse—an interconnected, embodied 3D digital virtual reality, currently being conceptualised, and touted as the new internet. Through the metaverse, we will be able to access cultural sites digitally in ever more realistic representations using immersive Mixed Reality technology . This concept is alive today but is not yet fully implemented. To prepare for this world, future work should continuously question how archaeological and heritage sites are presented and engaged with to ensure their meaning, memory, and potency are transferred to an embodied internet.


Black pepper powder (BPP) and cinnamon powder (CP) are traditionally used as food ingredients and can apply related to developing a functional product. In this study, BPP and CP were used as an ingredient in mixed-spice ice cream (MSIC). The physicochemical properties, textural properties, and sensory qualities were investigated as key points. BPP (0.51–17.49 g) and CP (8.79–51.21 g) were combined using a central composite design (CCD) with 2 centerpoints. The optimized BPP and CP for the MSIC were 15.00 g and 34.00 g, which exhibited firmness and overrun at 3210.65 ± 105.74 g.force and 61.63 ± 0.60%. The MSIC with optimized BPP and CP also provides high bioactive compounds and antioxidant activities with cinnamaldehyde and piperine as flavor characteristics. The findings indicated that BPP and CP can enhance the functional properties and provide alternative flavors in the food product, providing an innovative approach to deliver health-beneficial combinations for consumer satisfaction. Ice cream is one of the most consumed dairy products in the world. However, ice cream is commonly poor in natural compounds exhibiting polyphenol, antioxidants, and even color. An interesting field of research to investigate is the possibility to enhance and increase the nutritional values of ice cream using ingredients beneficial for health, such as herbs and spices3.Many dairy products can act as successful carriers in delivering phytochemicals and nutrients that exhibit health-beneficial effects in our system of nutrition and food5.Herbs and Spices are natural additives that contribute to creating flavor in foods. In addition to contributing to the taste of food, spices exhibit nutritional value and health-related beneficial properties. The incorporation of herbs, spices, and/or extracts to various dairy products, including ice cream, can make these products suitable carriers for nutraceuticalsPiper nigrum L.) and cinnamon (Cinnamomum zeylanicum Linn.) are well-known spices that are used for various medicinal and flavor-enhancing purposes throughout the world. Black pepper is a medicinal plant from the family Piperaceae which is currently employed in many aspects of the food and pharmaceutical industries. Cinnamon is a prominent spice used in the aroma and flavor industry that can be applied to food, fragrance, and medicinal products. They contained various bioactive compounds such as carotenoids (lutein and beta-carotene) and flavonoids , which plays a role in the antioxidant, antiallergic, and anti-inflammatory activities6. The monoterpenes including linalool, α-phellandrene, limonene, α- and β-pinene, myrcene, methylpropanal, and 3-methylbutanal are the important odorants of black pepper, while mouldy and musty off-flavor is 2,3-diethyl-5-methylpyrazine and 2-isoprophyl-3-methoxypyrazine7. While cinnamon volatile flavor was cinnamaldehyde, copaene, caryophyllene, and bornyl acetate8.Black pepper (The study on the incorporation of black pepper powder (BPP) and cinnamon powder (CP) into an ice cream product can be a novelty and innovative way to deliver health-beneficial compounds to consumers. Thus, the objectives of this study were to optimize the amount of BPP and CP for mixed spice ice cream (MSIC) and evaluate its effect on the physicochemical properties and sensory qualities.All the ice cream ingredients were purchased from Tesco Lotus, Thailand: pasteurized milk ; skim milk powder ; whipped cream ; sugar ; BPP and CP . Guar gum was obtained from Chemipan Corporation Co., Ltd., Bangkok, Thailand. All ice cream samples were prepared in the pilot plant at the Faculty of Agro-Industry, Prince of Songkla University (Hat Yai Campus), Songkhla, Thailand.3.6H2O (PubChem CID 16211236), sodium carbonate (PubChem CID 10340), and sodium nitrite (PubChem CID 233668193), purchased from Kemaus, Australia; Folin–Ciocalteau reagent and NaOH (PubChem CID 14798) procured from Loba Chemie Pvt. Ltd., India; ABTS (PubChem CID 16240279), DPPH (PubChem CID 57654141), potassium persulfate (PubChem CID 24412), and TPTZ (PubChem CID 77258) acquired from Sigma-Aldrich, Chemie GmbH, Germany. All chemicals were analytical grade.The chemicals used in this experiment were: methanol (PubChem CID 887), acquired from Avantor Performance Materials, LLC., Poland; aluminium chloride (PubChem CID 24564), FeCl10 with slight modification. The constant ingredients of MSIC consisted of pasteurized milk (62.50%), whipped cream (22.20%), sugar (8.58%), inulin (4.02%), skim milk powder (2.30%), and guar gum (0.30%). The BPP and CP were varied in the range of 3.00–15.00 g and 15.00–45.00 g using the central composite design with 2 center points  were mixed thoroughly, added into the liquid ice cream mix when the temperature reached 60 °C and stirred into a homogeneous mixture using a laboratory homogenizer  for 2 min at 3350 g. The liquid ice cream mixes were pasteurized for 30 s at 80 ± 1 °C, homogenized using a laboratory homogenizer  at 3350 g for 5 min at 60 °C and then cooled to 20 °C. To ensure the complete hydration of the ingredients, the ice cream mixes were stored for 24 h at 4 °C. The ice cream mixes were frozen for 30 min in a batch ice cream laboratory freezer . The ice cream samples were separated into 2 portions, in a 175 mL plastic container for physical properties and ice cream characteristic analysis, and 10 mL plastic cups for sensory evaluation. All samples were taken to harden at − 30 °C for 24 h and stored at − 18 °C before the analysis.After the storage at − 18 °C, the MSIC samples were immediately placed in a chamber in which they were uniformly illuminated from all directions. Color measurement was performed using a Color Quest XE Colorimeter , according to CIEL*a*b* system, using spectral reflectance for calibration, illuminant D65, and an observation angle of 108. The L* (lightness), a* (red intensity), and b* (yellow intensity) values were measured at six different points for each sample. The color was measured in triplicate on each sample. The whiteness index (WI) was calculated with the following equation:11 with modification. The analysis was performed at 25 °C using a TA.XT plus Texture Analyzer , equipped with a vertical stainless-steel sheet (30 × 1 mm). The firmness of ice cream samples (500 mL) was measured by penetration of a vertical sheet into the ice cream to a depth of 20 mm at a constant speed of 20 mm/min. For every batch, 10 different samples were analyzed at three different places in the ice cream box.The texture profile was assessed according to Kaleda et al.9. The apparent viscosity of the ice cream mix (400 mL) at 5 ± 0.5 °C after 48 h of aging was measured in triplicate with a spindle number 63 at a shear rate of 102/s. One measurement was taken per mix. The ice cream mix temperature was controlled at 5 ± 0.5 °C using a jacketed beaker connected to a refrigerated bath  circulating a 50% aqueous solution of ethylene glycol.The concentric cylinder viscometer  was used to measure the apparent viscosity12. The samples were then placed on a 1 mm stainless steel mesh at ambient temperature (20 ± 1 °C). The weight of the melted ice cream was recorded every 10 min until the ice cream was melted completely. A plot of the percentage of melted ice cream versus time was created with the slope of the linear part of the plot indicating the melting rate (g/min). Measurement was done in triplicate.The melting rate of ice cream samples was measured using 175 mL of ice cream sample stored at − 18 °C for 7 days9. The overrun measurement was carried out in triplicate and measured by the following equation:The overrun of the MSIC was measured and performed by weighing the ice cream mix in a fixed volume container (175 mL) separately13.To analyze TPC and antioxidant activities of MSIC, the ice cream sample was mixed with methanol at a ratio of 1:20, then mixed thoroughly using a magnetic stirrer for 15 min. The MSIC extract was then filtered using Whatman filter paper No. 1 and prepared into a methanolic extract (1 mg/mL). The prepared filtrate was stored in an amber bottle at 4 °C until the further determination of TPC and antioxidant activities. All analysis was calculated from the Trolox calibration curve and expressed as mg Trolox equivalent/g of sample2CO3 (20% w/v). The mixed solution was allowed to stand for a further 30 min in the dark. The absorbance of the solution was measured at 750 nm.The TPC of the MSIC was determined using the Folin–Ciocalteau method. The 100 μL of methanolic extract was mixed with 0.5 mL of Folin–Ciocalteau phenol reagent and 2 mL of NaDPPH radical scavenging activity was measured by mixing 200 μL methanolic extract with 3.8 mL of 0.5 mM DPPH methanolic solution (0.5 mM), and allowing it to stand in the dark for 30 min. The absorbance was immediately measured at 517 nm.Ferric reducing antioxidant power (FRAP) was determined using 0.25 mL of methanolic extract mixed with 0.25 mL of FRAP reagent and incubated at 37 °C for 20 min. Subsequently, the mixture was centrifuged at 3350 g for 10 min. The supernatant (1 mL) was mixed with 1 mL of distilled water and 0.2 mL of 1% ferric chloride and the absorbance was measured at 593 nm.An ABTS assay was conducted using a spectrophotometer . A working solution was obtained by mixing ABTS stock solution . The solution was kept for 12–16 h at room temperature in the dark. The MSIC extract (150 μL) was mixed with the ABTS· + solution (2.85 mL) and measured at 734 nm.15. The relative content of the identified volatile compounds was calculated based on 1 μL of hexanal solution as an external standard reference to create a calibration curve. The relative amount was calculated from the constant ratio of hexanal and effective carbon number (ECN) of the identified volatile compounds.Before analysis, the volatiles from MSIC were extracted by carboxen/polydimethylsiloxane fiber (CAR/PDMS). A 2 g of samples were extracted with ethanol in 20 mL vials. The extraction was performed at 60 °C for 60 min. The bounded volatile compounds were analyzed by gas chromatography (GC). The GC analysis was performed using 7890B and mass Spectrometry (MS) was performed by using an Agilent 5977B system . The chromatographic column was HP-5msUI  and helium carrier gas (at a flow rate of 29.0 mL/min) was used for both analyses. A 1 μL of sample headspace was injected in split mode with a split ratio at 25:1. The injection port temperature was set at 280 °C. The temperature program was started at 50 °C for 1 min and then increased to 70 °C at 5 °C/min for 2 min. After that, the temperature was increased to 100 °C at 3 °C/min for 1 min, then the temperature was increased to 130 °C at 15 °C/min 1 min. The temperature was increased further to 140 °C at 4 °C/min and maintained for 1 min, then the temperature was increased again to 280 °C at 9 °C/min for 2 min. Finally, the temperature was increased to 300 °C at 10 °C/min and maintained for 5 min16. The MSIC sample for sensory evaluation was retained under 4 ± 1 °C until the assessment was initiated. The MSIC was evaluated using a 9-point hedonic scale on the appearance, black pepper aroma, cinnamon aroma, melt in mouth, black pepper flavor, cinnamon flavor, sweetness, creaminess, oiliness, viscosity, spiciness, and overall liking.The sensory evaluations of developed ice cream (8 formulations) and final formulation were evaluated by sixty and four hundred participants according to the Declaration of Helsinki guidelines. All sensory evaluations were performed under the approval of the Office of Human Research Ethics Committee, Health Sciences, Prince of Songkla University . The consumer was recruited from students and staff from Prince of Songkla University, Hat Yai, Thailand, and written informed consent was obtained from all participants before the evaluation. All participants signed and returned the consent form to the research team, and the consent of the participants was ensured before participating in the study. The MSIC was presented in disposable closed-lid plastic cups, each coded with a three-digit number which was evaluated by each consumer in a monadic order, following a balanced designAll measurements were done in triplicate and reported as a mean ± standard deviation. The statistical analysis was conducted using SPSS 17.0  using Duncan’s multiple range test (DMRT) at the significance level < 0.05. The response surface methodology (RSM) was applied to optimized BPP and CP using a regression equation by Design Expert 13 , with the significant responses selected to generate the optimized amount of BPP and CP for MSIC.17, which are yellow, red, and brown and were responsible for the alteration of color properties in the products. The carotenoids and flavonoids of black pepper and cinnamon are lutein and beta-carotene, and catechin, myricetin, and quercetin, which showed antioxidant, antiallergic, and anti-inflammatory activities6. However, the addition of spices was not significantly affected to whiteness index (WI) of developed ice creams (p > 0.05). These findings suggest that the addition of spices into a product with an unpigmented color base can alter the color into a darker tone with tints of yellow, red, and brown. These results were in the same direction as many pieces of research that added spices and extract them into ice cream20.The incorporation of BPP and CP in the MSIC significantly affected the physical properties of the MSIC Table . The L*,21 and Zuha et al.22 on functional frozen dairy product enrichment with fruits and vegetables. The addition of particles  into frozen dairy products decreased the firmness and increased the apparent viscosity. In this research, the alteration of firmness and apparent viscosity happened because BPP and CP released volatile oil during the process into the ice cream mixture which contributed to the differentiation of the firmness and apparent viscosity19. Changes in firmness and apparent viscosity were also found when fermented pepper powder and silver ear mushroom powder were added to ice cream because the volatile compounds and solid content from additional particles or powders can influence the initial and gradual growth of ice crystals and interrupt product native thermodynamic instability23.The firmness and apparent viscosity of the MSIC were in the range of 548.53–4519.10 g. force and 442.60–1723.77 cP , cinnamaldehyde (386.94–773.05 µg/g sample), β-pinene (4.62–27.34 µg/g sample), limonene (1.98–59.2 µg/g sample), bornyl acetate (1.44–2.81 µg/g sample), cinnamyl acetate (2.11–4.33 µg/g sample), β-caryophyllene (2.31–46.69 µg/g sample), and piperine (12.69–25.39 µg/g sample). Increasing the BPP can increase β-pinene, limonene, β-caryophyllene, and piperine whereas the increasing CP can increase benzaldehyde, cinnamaldehyde, bornyl acetate, and cinnamyl acetate due to these two different groups comprising the aromatic characteristics of black pepper and cinnamon. Those results were also indicated that the incorporation of BPP and CP in different amounts into the MSIC formulation also affected the content of these compounds significantly34.The sensory rating score of MSIC revealed that BPP and CP affected color, cinnamon aroma, and pepper aroma insignificantly whereas the rest of the sensory rating score of MSIC was significantly different Table . The ins28 on the manufacture of ice cream using basil powder as a flavoring agent. Additionally, the uneven dispersion of spice powder into MSIC can also affect flavor perception. There was a wide range of sensory rating scores of cinnamons and pepper flavor from 5.0–7.2 and 4.1–6.3. These results suggest that the increase of spiced powder into MSCI can cause the sensory rating score to increase, however, an exceedingly high increase of mixed spice can decrease the liking score of the product because of an excessively strong flavor28.The significant effect on the MSIC appearance can be attributed to the addition of BPP and CP which can affect the physical properties of ice cream mixed with spices. The differences in appearance can happen because the uneven dispersion of spice powder into the ice cream matrix affects the appearance and causes it to be significant to the panels' perception, as suggested in the research of Tridevi et al.23.Other sensory responses that were affected by BPP and CP were melting in mouth, creaminess, oiliness, and viscosity, which provided rating scores in the range of 6.0–7.3, 6.0–7.3, 5.9–7.1, and 5.0–7.0, respectively. Increasing BPP and CP altogether affected all mentioned attributes above to be increased similarly to melting rate and overrun of the MSIC. The quantity of spice powder added into the ice cream mixture can increase firmness and viscosity because the interaction of spice powder and water in the mixture causes large ice crystal formation which affects the texture state and retards the melting of ice cream which indicated the correlation of physical properties and sensory quality of MSIC36. Consequently, the variation of BPP and CP mixed into MSIC affected the sweetness and spiciness significantly. The ratio of BPP and CP was vital to the cinnamon and pepper flavor in MSIC. The mixed ratio of BPP and CP at 1:1 resulted in the highest sensory rating score of cinnamon and pepper flavor. When the ratio of BPP and CP were deviating from 60 to 96% the preference score significantly decreased. This suggests that a suitable proportion of spice powder applied in a food product can help balance the flavor compounds appropriately, increasing the preference rating score of sweetness, spiciness, and overall liking in MSIC.Since BPP and CP contain compounds that give distinctive sweet and spicy flavorsThe MSIC properties were carried forward to optimize the amount of BPP and CP using RSM Figs.  and 2. T37. The R-square value of the significant physicochemical responses was in the range of 0.8403–0.9692, with the significant level being in the range of 0.0008–0.0084.The regression equation of firmness, overrun, cinnamaldehyde, and piperine from Table The regression equation of sensory responses from Table The reason for this incident is that only a small amount of spice can affect the intensity of aroma and flavor. An exceedingly high amount of BPP will affect the preference for spiciness whereas an exceedingly high amount of CP can affect the melt in mouth preference due to the presence of irritation particles in the mouth and throat.The optimum formulation for the MSIC consists of BPP and CP based on the significant response as shown in Table 39. Besides, the addition of herbs, spices, and their extract also increased taste intensity which affected the favorable of consumers toward the optimized MISC.The MSIC with optimized BPP and CP was subjected to evaluation for consumer acceptance. The preference rating score from consumer acceptance (n = 400) showed that the MSIC exhibited an acceptance rating score in the range of ‘like slightly’ to ‘like moderately’ (6.0–7.2) (Supplementary Fig. This research concludes that the application of RSM on the optimization of BPP and CP in MSIC can create appealing properties and product acceptability. The BPP and CP can also provide exceptional flavor and bioactive compounds which can be used as favorable ingredients for dairy products to obtain compatibility products with herbs and spices. The outcomes from this research can be applied to develop dairy products innovatively fortified with herbs and spices to increase potential consumption and deliver health-beneficial compounds to consumers.Supplementary Figure 1.Supplementary Table 1.