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Animal husbandryCarukia barnesi polyps were available in culture from the James Cook University Aquarium, spawned from medusa originally collected near Double Island, North Queensland, Australia (16° 43.5′ S, 145° 41.0′ E) in 2014 and 20158. Populations exponentially increase through asexual reproduction8. Detached buds and swimming polyps were collected from the main culture, and transferred into 6-well tissue culture plates in natural filtered seawater. Plates were maintained in darkness to inhibit algae growth at 27 °C in a constant temperature cabinet. Buds and swimming polyps were left to develop and attach to well bottoms, at which point they were then fed freshly hatched Artemia nauplii and water changed 2–3 times per week. Lids remained attached to tissue culture plates to negate water evaporation and maintain a stable salinity. Polyps were maintained in this way for a minimum of 4 months before experiments began, with all individuals matured with the ability to asexually reproduce further buds. To preserve water quality15 polyps were starved for two days prior to experiment start and were not fed for the duration of the trials. One day prior to the experiment start, all immature buds and polyps were removed from wells, leaving approximately 5–10 mature polyps attached to the substrate for analysis.Preparation of reagentsReagentsSix chemicals were trialed in the current study to induce metamorphosis in C. barnesi polyps. Four indole containing compounds were chosen that have previously been trialed with other cubozoan species: 5-methoxy-2-methyl-3-indoleacetic acid, 5-methoxyindole-2-carboxylic acid, 2-methylindole16 and 5-Methoxy-2-methylindole15,16. Along with the retinoic X receptor 9-cis-retinoic acid and lugols solution.Indole compound treatmentsChemical concentrations of indoles documented in the literature were used to conduct preliminary concentration tests. Fifty mM stock solutions were prepared with 100% ethanol, which was diluted with filtered seawater to the desired experimental concentrations: 50 μM16, 20 μM and 5 μM15. Due to high fatality rates at all of these concentrations when used in this study on C. barnesi, all concentrations were diluted. Fifty mM stock solutions of 5-methoxy-2-methyl-3-indoleacetic acid, 5-methoxyindole-2-carboxylic acid, 2-methylindole and 5-Methoxy-2-methylindole were prepared with 50% ethanol (50% Milli-Q® water) and stored at − 20 °C. Fifty mM stock solutions were diluted with filtered seawater to the experimental concentrations of 5 μM, 1 μM, 0.5 μM, 0.1 μM and 0.05 μM. The carrier solution of 50% ethanol (50% Milli-Q® water) was diluted to the equivalent of the experimental concentrations listed above for use as a control, and incorporated into data as concentration 0. Seventeen ml of solution was added to polyps to fill each well of a 6-well plate.Iodine treatment (lugols solution)Aqueous iodine in the form of Lugols solution (0.37% iodine and 0.74% potassium iodide (sigma product information)) was prepared with equivalent concentrations of moles iodine/iodide: 1.5 μM, 3 μM, 6 μM, 12 μM and 24 μM. Filtered seawater only was used a control for this treatment and incorporated into data as concentration 0. 17 ml of solution was added to polyps to fill each well of a 6-well plate.Retinoid treatmentTo reduce ethanol associated fatality of polyps 0.015% ethanol in Milli-Q® water was used to prepare a 1 mM stock solution of 9-cis-Retinoic acid. The 1 mM stock solution was diluted with filtered seawater to the experimental concentrations of 5 μM, 1 μM, 0.5 μM, 0.1 μM and 0.05 μM. The carrier solution of 0.015% ethanol (Milli-Q® water) was diluted to the equivalent of the experimental concentrations listed above for use as a control, and incorporated into data as concentration 0. 17 ml of solution was added to polyps to fill each well of a 6-well plate.Metamorphosis trialsPrimary trialsExperimental concentrations of reagents were added to C. barnesi polyps growing in the wells of sterile 6-well tissue culture plates. One plate was used per chemical, per concentration, in which five wells functioned as replicates containing the chemical being trialed, whilst the sixth well contained only the control medium. Five concentrations were run for each of six chemicals; 30 plates in total.The filtered seawater the polyps were growing in was exchanged for the experimental chemical on day 0, and was not changed for the duration of the trial. Lids remained attached to tissue culture plates to negate water evaporation and hence salinity changes.Polyps in each well were photographed each day through a dissection microscope over a period of 34 days. Results were then recorded from the photographs, categorised (Fig. 6) as the number of polyps which displayed:Tentacle migration: one of the key signs of metamorphosis in this species, polyp tentacles merge, migrating to form four distinct corners in a square shape8.Detached medusa: a medusa formed and detached from the polyp, recorded regardless of health.Mobile detached medusa: a healthy medusa formed and detached from the polyp, with the ability to swim.Polyp survival: this was then used to calculate the number of polyps which survived the treatment which did not metamorphose.Optimisation trialThe optimal chemical and concentration was then deduced by choosing the combination that produced the largest percentage of healthy detached medusa, in this case 5-methoxy-2-methylindole at 1 μM. A final trial was then run with this to determine if length of chemical exposure could optimize healthy medusa yield. Three replicates of a minimum of five polyps were used per treatment, in which in 1 μM of 5-methoxy-2-methylindole (in seawater) was added to polyps for 24, 48, 72, 96 and 120 h, before the solution was changed to fresh seawater. A sea water only control was also run. The total number of healthy detached medusa were recorded each day.Data analysisAll statistical analysis was conducted in IBM SPSS Statistics Ver28. Graphs were produced in Microsoft Excel 2016 and OriginPro Graphing and Analysis 2021.Primary trialsThe effect of chemical, concentration and time was analysed using a repeated measures three-way ANOVA for four sets of data gathered during the metamorphosis process: percentage of polyps to display tentacle migration, percentage of polyps to have medusa detach, percentage of polyps to have healthy swimming medusa detach, percentage survival of polyps that did not metamorphose. Percentage data was arcsine square root transformed prior to analysis. Mauchly’s Test of Sphericity indicated that the assumption of sphericity had been violated on all four sets of data and therefore, a Greenhouse–Geisser correction was used.Optimisation trialDifferences in the mean percentage of healthy medusa produced at different exposure times was analysed using ANOVA. Differences between means were elucidated using a Post hoc Tukey pairwise comparison test (Tukey HSD alpha 0.05). More

Study areaGrassland herbage samples are from Shaerqin base, institute of grassland research of CAAS (Chinese Academy of Agricultural Sciences). We obtained the permission of the institution to take HSI of the grassland sample. Our work did not cause damage to grassland. Researcher Weihong Yan of the institute provided us with relevant information about grassland. The land use type in the study area is mainly grassland, which is composed of forage species, most of which are representative species of typical grassland. We take this area as an example to conduct research on grass classification. By enriching the relevant recognition technology, it can also be used as a reference for the pastures of other grasslands. The grass species Grass1 for the experiment is shown in Table 1. The official introduction of plant materials is detailed in the flora of China15.Table 1 Samples information for Grass1 dataset.Full size tableThe field hyperspectral platformWe assemble a system for collecting HSI in the field: HyperSpec©PTU-D48E HSI instrument, high-precision scanning PTZ, tripod, data analysis software Hyperspec, etc. The light source is natural light. The imaging instrument is in line scanning mode. Table 2 shows the technical parameters.Table 2 Technical parameters of hyperspectral instrument.Full size tableData collectionIn July 2021, the data was collected during the lush grass growth period. Collect data from 11:00 a.m. to 2:00 p.m. every day. At this time, it is sunny, cloudless and the wind force does not exceed level 2. So as to ensure the consistency of the acquisition time line and avoid the influence of different degrees of light on the reflectivity as far as possible. The measuring points are arranged facing the sun and the opposite direction of the shadow. We collect data from different angles of the grassland, which is based on the growth of various types of forages, and selects relatively concentrated places within the study area. Each shot is a single category of grass. The image resolution is 1166 × 1004 pixels (Fig. 1). The imaging spectrometer is fixed with scanning head when shooting. Data acquisition and transmission are executed on Hyperspec software. Then save it as a BIL file. The ENVI5.3 software was used to extract the forage spectrum to establish the dataset Grass1. Well balanced regions with a clear image, uniform spectral distribution are selected for further segmentation. The average value of spectral reflectance of grass pixels was taken as the reflectance spectrum of a single type of grass.Figure 1True color map of grass samples.Full size imageMethodologyIn Fig. 2, we present the framework of visible-NIR hyperspectral classification of grass based on multivariate smooth mapping and extreme active learning (MSM–EAL). Specifically, we first introduce the proposed MSM algorithm for global enhanced spectral reconstruction, which utilizes smooth manifold projection technology to alleviate the problems of difficult feature selection and redundant data. Then, the EAL framework is proposed to address the matter of hyperspectral labeled samples and spectral classification. In the following, each step of this method will be presented in detail.Figure 2Proposed MSM–EAL framework for grass HSI classification.Full size imageThe proposed MSM algorithmIn the process of field HSI acquisition, on the one hand, the surface distribution of grass is uneven and the plant height is different, causing certain scattering effect and coverage spectrum change. On the other hand, HSI is easy to be disturbed by external natural factors such as light, wind and shadow, resulting in a certain degree of distortion. Multiplicative scatter correction (MSC) is a scattering correction effect, which helps to eliminate the scattering effect caused by the above reasons and enhance the spectral variability. The moving window smooth spectral matrix (Nirmaf) belongs to the smooth effect, which improve the signal-to-noise ratio of the spectrum and reduce the influence of random noise16,17. Preprocessing methods are different and related to each other. We design an enhanced preprocessing multivariate smooth (MS) method that fusing MSC and smooth Nirmaf to target grass spectral signal features. In the follow-up, a model will be established to verify the validity of MS.Most of the high-dimensional spatial data have the characteristics of being embedded in a manifold body, so the manifold learning isometric feature mapping (Isomap) based on spectral theory is adopted. Isomap preserves the global geometric features of the initial data and extracts features by reconstructing the underlying smooth manifold of HSI. It is nonlinear dimensionality reduction based on linear and multidimensional scaling transformation18. Isomap has been applied in image and HSI classification19,20, but there is no report on visible-NIR hyperspectral classification of grass.In view of the above, we proposed the multivariate smooth mapping (MSM) spectral reconstruction algorithm, which can be represented as follows:$$ MSM_{z} { } = { }frac{{left( {P_{j} – b_{j} } right)left( {2n + 1} right) + n_{j} cdot mathop sum nolimits_{j = – n}^{n} C_{j} P_{k + j} }}{{n_{j} left( {2n + 1} right)}} + V_{Z} F_{Z}^{frac{1}{2}} { } $$
(1)
where Pj, bj, and Cj represent the raw reflectance value of spectrum j, baseline shift amount, and weight factor, respectively, k and nj represent the polynomial degree and offset, respectively. MSMz is the feature cube reconstructed to Z dimension from the spectrum calculated by 2n + 1 moving window width, V eigenvector matrix and F eigenvalue matrix.In Isomap equidistant mapping, the shortest path of edge Pi Pj needs to be solved, and the representation matrix is:$$ D_{G} = [d_{G}^{2} (P_{i} ,P_{j} )]_{i,j = 1}^{n} $$
(2)
where d (Pi, Pj) is the weight of the edge Pi Pj calculated from the neighborhood graph G and its side Pi Pj.The proposed EAL frameworkLabeling hyperspectral samples is expensive in terms of time and cost, at the same time, the lower spatial resolution and more bands increase the difficulty of labeling. Active learning (AL) provides an efficient labeling strategy, which only needs to label a relatively small number of samples to learn a more accurate model21. The pool-based AL selects the most informative samples according to the query strategy for limited labeling through iteration, so as to facilitate model improvement. Commonly used query strategies are uncertainty criteria, such as least confidence22, the bayesian active learning disagreement (BALD), the entropy sampling23, etc.Due to there is still an over-fitting problem, different strategies such as hybrid prediction and regularization need to be used for non-recursive datasets24. The research25 proposed that extreme gradient boosting algorithm (XGBoost) based on gradient boosting. As a classification method, XGBoost has been successfully applied in Kaggle competition and other fields. Its most important feature for visible-NIR hyperspectral classification is that can easily and directly classify according to features, and the physical interpretation of features can help understand the electronic nature behind spectral classification. XGBoost is a machine learning algorithm based tree structure that integrates multiple weak classifiers to achieve flexible and high-precision classification. It is an upgraded version of gradient boosting decision tree. The optimization process of XGBoost entailed: (1) Expanding the objective function to the second order, and finds a new objective function for the new base model to improve the calculation accuracy. (2) L2 regularization term is added to the loss function to prevent over-fitting. (3) Using blocks storage structure realize automatic parallel computing26,27. The algorithm steps are as follows:The objective function:$$ Lleft( Phi right) = mathop sum limits_{i} lleft( {y^{i} ,widehat{{y^{i} }}} right) + mathop sum limits_{k} Omega left( {f_{k} } right) $$
(3)
In formula (3), the first and second terms are the loss function term and the regularization term, respectively. Where,$$ Omega left( {f_{k} } right) =upgamma {text{T}} + frac{1}{2}lambda left| w right|^{2} $$
(4)
γ and λ are regularization parameters which are used to adjust complexity of the tree.Next, second derivative Taylor expansion of the objective function. Where (g_{i}) and (h_{i}) are the first derivative and second derivative, respectively.$$ L^{left( t right)} = mathop sum limits_{i = 1}^{n} lleft( {y_{i} ,widehat{{y_{i}^{t – 1} }} + f_{t} left( {x_{i} } right)} right) + Omega left( {f_{t} } right) $$
(5)
$$ g_{i} = partial_{{hat{y}_{i} (t – 1)}} lleft( {y_{i} ,widehat{{y_{i}^{t – 1} }}} right) $$
(6)
$$ h_{i} = partial_{{widehat{{y_{i} }}(t – 1)}}^{2} lleft( {y_{i} ,widehat{{y_{i}^{t – 1} }}} right) $$
(7)
$$ {text{L}}^{left( t right)} approx mathop sum limits_{i = 1}^{n} left[ {lleft( {y_{i} ,widehat{{y_{i}^{t – 1} }}} right) + g_{i} f_{i} left( {x_{i} } right) + frac{1}{2}h_{i} f_{t}^{2} left( {x_{i} } right)} right] + Omega left( {f_{t} } right) $$
(8)
Final objective function:$$ {hat{text{L}}}^{ i} left( q right) = – frac{1}{2}mathop sum limits_{j = 1}^{T} frac{{(mathop sum nolimits_{{i in I_{j} }} g_{i} )^{2} }}{{mathop sum nolimits_{{i in I_{j} }} h_{i} + lambda }} + gamma T $$
(9)
Equation (9) can be used as the fraction of tree cotyledons, and the tree structure is directly proportional to the fraction. If the result after splitting is less than the maximum value of the given parameter, the cotyledon depth stops growing24,28.AL solves the problems of limited number and high cost of grass hyperspectral labeling samples. The default model of traditional AL is logistic regression, which is mostly studied on the ideal public dataset. However, the actual data has more uncertain noise, which still poses a certain challenge to AL. Consequently, we propose the extreme active learning (EAL) framework to minimize the classification cost of visible-NIR hyperspectral. The framework replaces the logistic regression model with XGBoost. Taking advantage of AL, XGBoost can improve performance with less training marker samples. By jointing of XGBoost and AL, EAL provides significantly better results than AL in field Grassl dataset recognition. Additionally, based on the characteristics of XGBoost, EAL more intuitively enhances the physical essence behind spectral classification than AL. Algorithm 1 summarizes the workflow of EAL framework.Random forest (RF) and decision tree (DT) were used to compare with EAL. RF and DT are frequently used in the field of grassland remote sensing9,29. Furthermore, RF, DT and XGBoost have the same point is that are learning algorithms based on tree structure. DT determines the direction by judging the conditions of the decision node12. RF is an integrated learning of multiple decision trees30. More

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Stability analysis of the deterministic modelSolving (left( x(t) times left( r_{p}x(t)^{(alpha )}left( 1-big (frac{x(t)}{K}big )^{beta }right) – nx(t)^{(delta )} right) right) =0), we obtain two stable and one unstable equilibrium points for the model. One stable equilibrium is trivial, i.e., (x(t)=0), another stable equilibrium point being the non-zero satisfying (left( r_{p}x(t)^{(alpha )}left( 1-big (frac{x(t)}{K}big )^{beta }right) – nx(t)^{(delta )} right) =0). Figure 1a shows three different equilibrium points of the model. In addition to the equilibrium, the model has two inflection points (Fig. 1a). At these inflection points the absolute growth rates are minimum and maximum. The density vs relative proliferation rate (RPR) profile of the model shows that the model can attain negative RPR for a positive cell density, suggesting that the model can portray the Allee phenomenon (Fig. 1b). Figure 1c,d portray the proliferation and decay phases, respectively through the model.Figure 1Growth dynamics of the proposed model: (a) Absolute proliferation rate (APR) profile considering (r_{p}=0.13), (K=1.43), (n=0.0095), (alpha =1.15), (beta =0.99) and (delta =0.2); (b) RPR profiles for different n and other same constant model parameters; (c) Cell population survive for (r_{p}=0.13), (K=1.43), (n=0.0095), (alpha =1.15), (beta =0.99) and (delta =0.2) with the initial cell density 0.1; (d) The population goes to extinction for the initial cell density 0.06 with the same constant parameters.Full size imageThe solution of the deterministic model finally provides two theorems.
Theorem 1

(x^{*}approx K -Kleft( frac{Big (beta r_{p}K^{alpha }+n delta K^{delta }Big )-sqrt{Big (beta r_{p}K^{alpha }+n delta K^{delta }Big )^{2}-2 left( 2 alpha beta r_{p}K^{alpha } +beta (beta -1)r_{p}K^{alpha }+delta (delta -1)nK^{delta } right) nK^{delta }}}{left( 2 alpha beta r_{p}K^{alpha } +beta (beta -1)r_{p}K^{alpha }+delta (delta -1)nK^{delta } right) }right)) is the conditional MSSCD for the intercellular-interaction-induced proliferative cells. The conditional threshold density for cell-proliferation upon interaction is (x^{*}=K -Kleft( frac{Big (beta r_{p}K^{alpha }+n delta K^{delta }Big )+sqrt{Big (beta r_{p}K^{alpha }+n delta K^{delta }Big )^{2}-2 left( 2 alpha beta r_{p}K^{alpha } +beta (beta -1)r_{p}K^{alpha }+delta (delta -1)nK^{delta } right) nK^{delta }}}{left( 2 alpha beta r_{p}K^{alpha } +beta (beta -1)r_{p}K^{alpha }+delta (delta -1)nK^{delta } right) }right)) (proof is in the supplementary information).
Allee and cooperation models are the only extended logistic law other than our model to provide a threshold population size for growth or proliferation. Our proposed model is superior to the Allee and cooperation model as it can detect the conditional threshold cell density for proliferation and regulate the density by its different parameters. For example, One may reduce the conditional threshold density by either regulating the interaction between growth-inhibiting molecules and cells ((delta)) or reducing the inhibiting molecule concentration (n).The conditional MSSCD from Theorem 1 is lower than the carrying capacity of the conventional logistic model due to growth-inhibiting molecules; it provides the expected cell density during culture in a given environment. Theorem 1 also states the set of parameters to control the cell proliferation and get the desired density during such cell cultures. A further question arises knowing this set of parameters: which one of the parameters in the expression is crucial in terms of application purpose? Since the (r_{p}) is the constant proliferation rate for a given cell line, controlling the conditional MSSCD is not possible through (r_{p}). We simulate the distribution of conditional MSSCD for other parametric planes to answer this question. For this, we use the parameter values obtained from the data.

Theorem 2

The RPR is maximum at the cell density (x^{*}= K-Kleft( frac{r_{p}beta K^{alpha -1}+ndelta K^{delta -1}}{2r_{p}alpha beta K^{alpha -1}+r_{p}beta (beta -1)K^{alpha -1}+ndelta (delta -1)K^{delta -1}}right)) for the concave downward profile under the condition (r_{p}alpha (alpha -1){x^{*}}{}^{(alpha -2)}-frac{r_{p}}{K^{beta }}(alpha +beta )(alpha +beta -1){x^{*}}{}^{(alpha +beta -2)}-ndelta (delta -1){x^{*}}{}^{(delta -2)}n) (see the supplementary information). The cell population sustain with any positive initial cell density x(t) and try to stabilize at (x(t)= K(1-frac{n}{r_{p}})^frac{1}{beta }). Therefore, bimodality vanishes and unimodality is observed for the case (alpha =delta) (r_{p} >n). The RPR profile will be concave downward always with the maximum RPR value is at the inflection point (x(t)= K(frac{(r_{p}-n)alpha }{r_{p}(alpha +beta )})^frac{1}{beta }). The deterministic potential function in this case is (U(x)=-Big [(r_{p}-n)frac{x^{(alpha +2)}}{(alpha +2)}-frac{r_{p}}{K^{beta }}frac{x^{(alpha +beta +2)}}{(alpha +beta +2)} Big ]). The minima of this effective potential function will be at (x(t)= K(1-frac{n}{r_{p}})^frac{1}{beta }) which is the maximum stable cell density for (r_{p} >n).
Parameter estimationThe density-RPR and time-density fitting to the scratch assay datasets show a lower RSS for our model than the logistic one for each of the three seeding conditions. The estimated parameters from the RPR fitting through the grid-search are in Table 2. Although the RSS for the RPR fitting of the seeding 2 is very low, the data itself is too scattered in both the upper and lower range for the small cell density. Therefore, there is a chance that regardless of the low RSS value, the fitting for seeding 2 may not reflect the actual estimates of the parameters with the bias in the data set (Fig. 2b). Nevertheless, the density-RPR fittings to the other two seeding density datasets do not suffer from bias (Fig. 2a,c).Table 2 Estimated model parameters from density-RPR fitting of our model.Full size table
Figure 2Our proposed model best fitted the cell density-RPR datasets for all of the seeding conditions generated through the grid-search method.Full size image
Jin et al.1 suggested that their two phase logistic model may share similarities with the Allee effect. However, they did not fit the Allee model stating seeding 2 and 3 were large enough seeding densities. We calculated the conditional threshold density, conditional MSSCD, density at the minimum and maximum RPR for the model from our estimated parameters (Table 3). The conditional threshold cell density calculated from our estimated parameters confirms that the smallest initial seeding density of the dataset was greater than the conditional threshold cell density.Table 3 Calculated cell densities from estimated parameters from our model fitting.Full size tableFigure 3 compares the portrayal of the data through our model with the fitting by Jin et al.1. The blue dashed line is the time-series fitting of the proposed model, and the red-colored line is the time-series fitting of the logistic model to the scratch assay data sets in the Fig. 3. The carrying capacity values are unexpectedly very high in the logistic fit, keeping the model near the exponential phase for the entire dataset. Thus the overall and two phase logistic fits are unrealistic compared to the highest cell density observed in the assay. Also, logistic fitting of the RPR profiles to the data after 18 h does not capture the whole scenario. The green solid and the violet dashed line represent the logistic time-density fit after and before 18 h density profiles respectively. The orange-colored lines in the Fig. 3 are the expected population density as per estimated parameters from the RPR fitting after 18 h data sets. Table 4 enlists all parameters for a comparison between logistic and our model fitting.Figure 3Time series solution of the proposed model and logistic law with comparative RSS for all three seeding conditions.Full size imageTable 4 Logistic model fitting with the Jin et al.1 estimates used in Fig. 3 with the specific colors.Full size tableTrends in cell densities under deterministic set upThe (r_{p}) is fixed for a cell line among all the determining parameters of the conditional MSSCD. n and K vary together with the culture media, flask, and environmental setup. On the other hand, the (alpha), (beta), and (delta) vary together with intercellular-interactions and cellular-interaction with growth-inhibitory molecules, which depend on the medium’s initial cell density per well and fluidity. We observe that the distribution of the conditional MSSCD depends more on the K than the n. There is a chance of overproliferation in the deterministic setup under low n but high K. The cells may die under high n. The cell density at maximum RPR also depends more on K than n (Fig. 4). So the cells should be cultured in the larger flask to achieve maximum proliferativeness.Figure 4The distribution of conditional MSSCD and cell density at maximum RPR in n-K parametric plane.Full size imageThe conditional MSSCD depends more on (beta) than (alpha) (Fig. 5a). The cells may tend to overproliferate under both high (alpha) and (beta). The conditional MSSCD does not exist for a high (delta) and low (beta) depending more on (delta) than (beta). The cells may overproliferate only under a high (beta) and low (delta) (Fig. 5b). The conditional MSSCD also depends more on (delta) than (alpha) showing mostly underproliferation of cells in the (delta ~-alpha) parametric plane. Therefore, the proliferation can be controlled via regulating the interaction between the growth-inhibitory molecules and cells followed by density-regulation through contact-inhibition and cell-cell cooperation (Fig. 5c).Figure 5The distribution of the conditional MSSCD in parametric plane of regulators in the growth law: (a) dependence of the conditional MSSCD on (alpha) and (beta) parameters; (b) dependence of the conditional MSSCD on (delta) and (beta) parameters; (c) dependence of the conditional MSSCD on (alpha) and (delta) parameters.Full size imageThe new cell fitness measure, i.e. cell density at maximum RPR depends more on the (alpha) than the (beta) (Fig. 6a). The cells achieve maximum RPR at a great cell density under the high value of these two parameters. Figure 6b,c suggest that cell density depends only a little on the (delta) under high (alpha) and (beta). Under the low value of these two regulators, a high (delta) always reduces the cell density attaining the maximum RPR, resulting a poor cell-fitness.Figure 6The distribution of cell density at maximum RPR in parametric plane of regulators in the growth law: (a) dependence on (alpha) and (beta) parameters; (b) dependence on (alpha) and (delta) parameters; (c) dependence on (delta) and (beta) parameters.Full size imageStochastic model analysisOur proposed stochastic model (3) can be compared with the general stratonovich stochastic differential equation (frac{dx}{dt}=f(x)+g_{1}(x)epsilon (t)+g_{2}(x)Gamma (t)). Comparing it with our proposed stochastic model we obtain (g_{1}(x)=-x^{delta +1}) and (g_{2}(x)=1). Using the help of47, we get noise induced drift (A(x)=r_{p}x^{alpha +1}left( 1-Big (frac{x}{K}Big )^{beta } right) -nx^{(delta +1)}+D(delta +1)x^{(2delta +1)}-lambda sqrt{DQ}(delta +1)x^{delta }) and noise induced diffusion coefficient (B(x)=Dx^{(2delta +2)}-2lambda sqrt{DQ}x^{(delta +1)}+Q). The cell density at long run can be obtained from the steady state probability density function (SSPDF). The analytical expression of the SSPDF is obtained from the Fokker-Planck equation. The Fokker-Planck equation is (frac{partial P(x, t)}{partial t} =- frac{partial big [ A(x) P(x, t)big ]}{partial x}+ frac{partial ^{2} big [B(x) P(x, t)big ]}{partial x^{2}}), where P(x,t) is the probability density function of the cell population at the time point t. Solving the Fokker-Planck equation we get the SSPDF as (P_{st} (x)= frac{N^{prime }}{B(x)} exp left( int _{x} frac{A(x^{prime })}{B(x^{prime })} dx^{prime }right)) with the normalizing constant (N^{prime }). The value of (N^{prime }) can be obtained from (int _{0}^{infty } P_{st} (x)dx=1).This SSPDF (P_{st} (x)) helps to understand the validity of the proposed stochastic model. Since the number of the data points is too low to fit the stochastic model to the data directly, validation of the stochastic model is challenging in this case. The dataset we used is a time series with 15 data points with three replicates only. An experiment must have many replicates to have a sample with a large sample size so that the SSPDF of cell densities obtained from theoretical findings can be validated with the real observation of cell densities at the steady state. Such datasets with many replicates are rare.So, we generate 2000 sample paths with the help of numerical simulation based on stochastic model 3. We use the parameter values estimated from the fittings of the deterministic model to the seeding condition 1, and we consider some particular values for the two noise intensities and correlation strength ((lambda)) to get a simulated dataset. To achieve the stationary state, we consider sufficiently large time points, and the cell densities at the final time point are used as the data set for the stationary state. We compare the frequency density of cell densities at steady-state of a simulated dataset of 2000 sample paths with the SSPDF obtained from the analytical solution. This comparison shows that the cell density distribution at the steady state matches the steady state probability density function obtained analytically (Fig. 7).In addition, we illustrated the time series generated with the help of stochastic model 3 through numerical technique (Fig. 8). We have plotted the time series data thus obtained for each of the three seeding conditions and in the same figure we also plotted the observed cell densities. The red dots (o) represent the original/experimental dataset of Jin et al.1. The blue dots ((*)) represent the simulated dataset obtained from the stochastic model. This Fig. 8 clarifies our claim that the proposed stochastic model is in good agreement with the actual observation.Figure 7The histogram shows the distribution of cell densities at steady state under additive and multiplicative noises. The blue curve is the SSPDF. The function SSPDF and the distribution of cell densities matches to each other.Full size imageFigure 8The red dots (o) in each sub-figures represent the experimental data of Jin et al.1. The blue dots ((*)) are obtained from the stochastic model (3) considering: (a) The seeding 1 estimated model parameters with (D= 0.002), (Q= 0.06) and (lambda = 0.4). (b) The seeding 2 estimated model parameters with (D= 0.01), (Q= 0.15) and (lambda = 0.6). (c) The seeding 3 estimated model parameters with (D= 0.002), (Q= 0.2) and (lambda = 0.4).Full size imageFigures 7 and 8 suggest that the stochastic model is valid. So the model can be further analyzed to meet the first objective. Differentiating (P_{st} (x)), we obtain (frac{dP_{st} (x)}{dx}=frac{N^{prime }}{[B(x)]^2} exp left( int frac{A(x)}{B(x)}dx right) left( A(x)-frac{dB(x)}{dx} right)) and (frac{d^{2}P_{st} (x)}{dx^{2}}= frac{N^{prime }}{[B(x)]^{2}}exp left( int frac{A(x)}{B(x)}dx right) left( frac{dA(x)}{dx}-frac{d^{2}B(x)}{dx^{2}} right) +frac{N^{prime }}{[B(x)]^{2}} left( A(x)-frac{dB(x)}{dx} right) exp left( int frac{A(x)}{B(x)}dx right) frac{A(x)}{B(x)}-frac{2}{[B(x)]^3}N^{prime } exp left( int frac{A(x)}{B(x)}dx right) left( A(x)-frac{dB(x)}{dx} right) frac{dB(x)}{dx}). At the extrema of the SSPDF, we must have (frac{dP_{st} (x)}{dx}=0) i.e. (left( A(x)-frac{dB(x)}{dx} right) =0).

Theorem 3

(x^{*}approx K-K left( frac{nK^{delta +1}+D(delta +1) K^{2delta +1}-lambda sqrt{DQ}(delta +1)K^{delta }}{beta r K^{alpha +1}+n(delta +1) K^{(delta +1)}+D(delta +1) (2delta +1)K^{(2delta +1)}-lambda sqrt{DQ}delta (delta +1)K^{delta }} right)) is the conditional MSSCD due to the correlated additive and multiplicative noises under the condition (r_{p}(alpha +1)x^{*}{}^{alpha }-frac{r_{p}}{K^{beta }}(alpha +beta +1)x^{*}{}^{(alpha +beta )} -n(delta +1)x^{*}{}^{delta }-D(delta +1)(2delta +1)x^{*}{}^{(2delta )}+lambda sqrt{Dalpha }delta (delta +1)x^{*}{}^{(delta -1)} < 0) (proof is in the supplementary information). Figure 9 visualizes the effect of noise strength and correlation strength on the conditional MSSCD. The conditional MSSCD increases with the additive noise strength (Q) and decreases with the multiplicative noise strength (D) when the other model parameters are fixed (Fig. 9a). There is a high chance of overproliferation for a low D and a high Q (Fig. 9a). Again, there is a high chance of extinction for the low Q and high D. The conditional MSSCD depends more on D than (lambda) (Fig. 9b), and more on (lambda) than Q (Fig. 9c). The conditional MSSCD increases with (lambda) and Q; there is a high chance of overproliferation for high (lambda) and Q. The extinction risk of cells from the culture increases with low (lambda) and Q.Figure 9The change in the conditional MSSCD value for different noise strengths and correlation strength using the parameters estimated for seeding 1: (a) the conditional MSSCD values in (D-Q) noise strength plane with highest correlation ((lambda =1)); (b) the conditional MSSCD values in (D-lambda) noise plane with (Q=0.01); (c) the conditional MSSCD values in (Q-lambda) noise plane with (D=0.01).Full size imageDue to the difficulty and complicated expression of the analytical expression of the SSPDF, we use numerical simulation to study the steady-state behavior in the long run under correlated noises. We draw a histogram of the cell densities based on 500 normal sample paths at the final time points. We use seeding 1 fitting estimates as the initial parameter values for this simulation. The cell population is stable and steady at either 0 cell density or at the conditional MSSCD. The distribution is symmetric around the conditional MSSCD for (lambda =1) (Fig. 10a). There is a loss in the symmetry for the decreasing (lambda). For (lambda =0.5), there is a mode at the zero states with another mode at conditional MSSCD (Fig. 10b). The histogram shows a bi-modality for low values of (lambda). The mode at the zero state is highest for (lambda =0) (Fig. 10c). Therefore, the extinction chance increases for zero noise correlation between the additive and the multiplicative noises.Figure 10Distribution of cell density for (r_{p}=0.13), (K=1.43), (n=0.0095), (alpha =1.15), (beta =0.99), (delta =0.2), (D=0.01), (Q=0.01), and variable correlation between additive and multiplicative noises: (a) (lambda =1), (b) (lambda =0.5) and (c) (lambda =0).Full size imageThe sustainability of the cell population depends on the strength of the two noises, like the correlation strength between them. For the zero strength multiplicative noise, the population has the mode at around the conditional MSSCD value (Fig. 11). Therefore, the population sustains in this case and tries to stabilize at the conditional MSSCD value. For (D=0.02), there is a bimodality, where the highest mode is at the zero cell density. For (D=0.05), we observe only one mode at (x=0). Therefore, with the increasing values of the multiplicative noise strengths (D), the chance of extinction increases for (lambda =0.5), (Q=0.01), and other constant model parameters for the seeding condition 1. Similar things happen for increasing Q values considering (D=0.01), (lambda =0.5), and other constant model parameters (Fig. 12).Figure 11Distribution of cell density for (r_{p}=0.13), (K=1.43), (n=0.0095), (alpha =1.15), (beta =0.99), (delta =0.2), (lambda =0.5), (Q=0.01), and variable strength of multiplicative noise: (a) (D=0.05), (b) (D=0.02) and (c) (D=0).Full size imageFigure 12Distribution of cell density for (r_{p}=0.13), (K=1.43), (n=0.0095), (alpha =1.15), (beta =0.99), (delta =0.2), (lambda =0.5), (D=0.01), and variable correlation between multiplicative noise: (a) (Q=0.05), (b) (Q=0.02) and (c) (Q=0).Full size image Remark 5 We have previously discussed the scenario for (alpha =delta) for deterministic case in Remark 4. It is important to understand the scenario under stochastic case too. For (alpha =delta) the proposed stochastic model 3 becomes (frac{dx(t)}{dt}=r_{p}x(t)^{(alpha +1)}left( 1-big (frac{x(t)}{K}big )^{beta }right) - nx(t)^{(alpha +1)}-x(t)^{(alpha +1)} epsilon (t)+ Gamma (t)). For this stochastic model (g_{1}(x)=-x^{alpha +1}) and (g_{2}(x)=1). We get, (A(x)=r_{p}x^{alpha +1}left( 1-Big (frac{x}{K}Big )^{beta } right) -nx^{(alpha +1)}+D(alpha +1)x^{(2alpha +1)}-lambda sqrt{DQ}(alpha +1)x^{alpha }) and (B(x)=Dx^{(2alpha +2)}-2lambda sqrt{DQ}x^{(alpha +1)}+Q). The extrema of the SPDF (big (x(t)=x^{*}big )) must satisfy the growth equation (r_{p}{x^{*}}^{alpha +1}-frac{r_{p}}{K^{beta }}(x^{*})^{alpha +beta +1}-n(x^{*})^{alpha +1}-D(alpha +1)(x^{*})^{2alpha +1}+lambda sqrt{D~Q}(alpha +1)(x^{*})^{alpha }=0). Therefore, for (alpha =delta) the conditional MSSCD is (x^{*}= K-Kfrac{nK^{(alpha +1)}+D(alpha +1)K^{(2alpha +1)}-lambda sqrt{DQ}(alpha +1)K^{alpha }}{beta r_{p}K^{(alpha +1)}+nK^{(alpha +1)}(alpha +1)+D(alpha +1)(2alpha +1)K^{(2alpha +1)}-alpha lambda sqrt{DQ}(alpha +1)K^{alpha }}) under the condition ((r_{p}-n)(alpha +1)(x^{*})^{alpha }-frac{r_{p}}{K^{beta }}(alpha +beta +1)(x^{*})^{(alpha + beta )}-(alpha +1)(2alpha +1)D(x^{*})^{2alpha }+lambda sqrt{DQ}(alpha +1)alpha (x^{*})^{(alpha -1)} More

SubjectsThe study comprised 34 men (19–26 years, mean 23 years; body mass index 19–31 kg/m2, mean 24 kg/m2) with no physical disabilities or mental disorders, including color blindness and claustrophobia30,31. All subjects were informed of the aims, the study procedure, and the financial compensation for participation, and were asked to follow the rules of the study. Prior to each experiment, subjects underwent short-term starvation31,54 (18–20 h; no food except pure water after lunch (12–1 pm) or dinner (6–7 pm), no later than 1 pm or 7 pm, respectively, one the day before the test), no medical treatments, and normal sleep (at least 6 h, between 10 pm the day before the test day to 8 am on the test day)31. Prior to starting each experiment, subjects were stabilized on a chair for ~ 10 min in a room next to the testing room. Based on an assessment with a pre-experiment questionnaire and the first blood glucose level, measured before starting the experiment (see “Geomagnetic orientation assay” section below), subjects who had not followed these rules were not allowed to take the test on the day and the test was postponed. The study was approved by the Institutional Review Board of Kyungpook National University and all the procedures followed the regulations for human subject research. Informed consent was obtained from all subjects.Modulation of GMFThe ambient GMF in the testing room had a total intensity 45.0 μT, inclination 53°, and declination − 7° (Daegu city, Republic of Korea); the total intensity of 50.0 μT in our previous study31 was changed due to a reconstruction of the building; 45.0 μT was maintained throughout the period of this study. To provide the subjects with various GMF-like magnetic fields (i.e., by modulating of total intensity, inclination, or direction of magnetic north), the coil system from our previous studies6,7,31 was used. It comprised three double-wrapped, orthogonal, rectangular Helmholtz coils (1.890 × 1.890 m, 1.890 × 1.800 m, and 1.980 × 1.980 m for the north–south, east–west, and vertical axes, respectively), electrically-grounded with copper mesh shielding. The subject was seated on a rotatable plastic chair with no metal components, at the center of the three-dimensional coils with his head positioned in the middle space of the vertical axis of the coils. To modulate the geomagnetic north, each pair of coils was supplied with direct current from a power supply (MK3003P; MKPOWER, Republic of Korea). The magnetic field was measured using a 3-axis magnetometer (MGM 3AXIS; ALPHALAB, USA); the field homogeneity at the position of the subject’s head was found to be 95%. The testing room was shielded by a six-sided Faraday cage comprising 10 mm thick aluminum plates, and was grounded during the entire experiment40. Background electromagnetic noise was measured inside the coils at the start and the end of each experimental day. It was attenuated by the Faraday cage more than 200-fold over the range from 500 Hz to 100 MHz as described in detail in our previous study31. The 60 Hz power frequency magnetic field was no more than 2 nT (3D NF Analyzer NFA 1000; Gigahertz Solutions, Germany). All electronic devices were placed outside the Faraday cage during the experiments, with the exception of the switch button module for GMF modulation and the antenna for generating the oscillating magnetic fields. The temperature experienced by the subjects was maintained at 25 ± 0.5 °C (Data logger 98,581; MIC Meter Industrial, Taiwan) by air circulation through the honeycomb on the ceiling of the Faraday cage31.Geomagnetic orientation assayAdopting a two-alternative forced choice (2-AFC) paradigm33,34, a geomagnetic orientation assay was conducted similar to our previous study31. Experiments were performed at 09:30–11:30 am or 1:00–5:00 pm (local time, UTC + 09:00) (each experiment: 50 min–1 h 10 min; mean ≈ 1 h, which was shorter by approximately 30 min than that in the previous study: 1 h 20 min–1 h 40 min; mean ≈ 1 h 30 min). Depending on the experiment, starved or unstarved subjects were tested individually. Prior to each experiment, the subjects were asked to remain with their heads facing the front, with eyes closed and earmuffs on during the experiment. In particular, they were asked to concentrate on sensing, if they could, the ambient geomagnetic north during the association phase, and to use the sensed information, depending on the experiment, to orient toward one of the two modulated magnetic norths (0°/180° for magnetic north–south axis or 90°/270° for magnetic east–west axis, rotated clockwise with respect to the ambient geomagnetic north) during the test phase. Subjects were instructed to avoid distracting thoughts and to think immediately “which direction is modulated magnetic north?” whenever they were distracted during the test phase, or felt they were being biased by experiences from earlier experiments. While seated on the rotatable chair, the subject’s blood glucose level was measured shortly before the first session and immediately after each session with eyes open except in the ‘dark’ experiment (Accu-Chek Guide; Roche, Germany)31. If the determined value before the first session varied by more than 15% relative to the mean (Table S2)31, the experiment was postponed and repeated at a later date (approximately 2% of experiments). The subjects were stabilized with eyes closed for 2 min before the first trial in the absence of visual, auditory, olfactory, and haptic sensory cues. For the ‘dark’ experiment (light intensity ≈ 0 lx), subjects wore home-made ‘blind’ goggles and were stabilized with eyes closed for 5 min55,56, and then asked whether they could see any light. If they could, the goggles were adjusted to prevent leakage of light, and the subject then had another 5 min of stabilization with eyes closed before starting the experiment. The subjects were illuminated with light from a filtered/non-filtered diffused light-emitting diode, depending on the experiment (Table S1). The home-made filter goggles were worn throughout the experiment, including the association and test phase, when required. The goggles contained glass filters (Tae Young Optics, Republic of Korea) to provide the eyes with particular wavelengths of light (Spectrometer USB4000-UV-VIS, Ocean Optics, USA) (Fig. S1). Each experiment consisted of 16 sequential trials for ‘no-association’ and ‘food-association’. For the food-association, a subject facing toward the ambient geomagnetic north was gently provided with a chocolate chip31 on his right palm by an experimenter, and given 30 s to eat it, while during no-association trials, food was not provided during the association phase. After a subsequent 5 s interval, the experimenter gently touched the subject’s right thenar area using a paper rod, as the cue to start the test. One of the two modulated magnetic north directions, depending on the experiment, was randomly provided 3 s before the cue for the test. Each of the modulated magnetic north directions was provided eight times for the no-association and food-association sessions. Subjects were informed of the nearly equal probability for each of the modulated magnetic north directions before each experiment. With the touch cue, subjects were asked to rotate freely toward any direction (clockwise or counterclockwise) by themselves (1–4 cycles of two-thirds rotation) and try to sense the direction of the modulated magnetic north during a 1 min period. Rotation was allowed within the rotation angle (− 30° to 210° for the magnetic north–south axis or − 120° to 120° for the east–west axis, depending on experiments, with respect to the ambient magnetic north), which was confined by the plastic stool (Fig. 1A) touching the left or right ankle of the subjects. When subjects determined the direction of the magnetic north, they stopped rotating to face toward the direction and lifted their right hand to indicate the direction to the experimenter. The direction was measured by the experimenter at 10° intervals using the scale on the walls of the Faraday cage31. A prerequisite for correct orientation was that the subject indicated the direction within the range of 30° to the both sides with respect to the magnetic cardinal directions, which was instructed to the subjects before each experiment. When the direction the subjects indicated was out of the 30° range, the trial was not included in the data and was repeated (approximately 0.63% of trials). Before the subsequent trial, the subject was gently rotated to face toward the ambient geomagnetic north and then rested for 5 s. For the ‘dark’ experiment, subjects were asked whether they could see any leaked light immediately after the last measurement of blood glucose level at the end of experiment. If the subject could see leaked light, the experiment was nullified and repeated later on (approximately 3% of experiments; 2/68). All experiments were performed in a double-blind fashion. The experimenter who conducted the orientation assay knew whether a subject was starved or not, wearing filter goggles, and food-associated or not, but did not know the random magnetic north sequences that were controlled by the personal computer (PC) system. Another experimenter responsible for analyzing the data did not know whether the subject was starved or not, the experimental conditions, including light wavelengths, or whether an oscillating magnetic field had been provided to the subjects. Thus, none of the experimenters were aware of all the subject information and data during the experiments and data analysis. The correct orientation rate was calculated by (the number of correct orientation trials/total number of trials) (raw data, Appendix S3). All the subjects participated in all the experiments performed in random order with an interval of at least 3 days between experiments. After each experiment, the subjects were asked to answer a post-experiment questionnaire about whether they closed their eyes when required during the entire period of the experiment. In cases when a subject did not maintain closed eyes, the experiment was repeated (approximately 1% of experiments). For each subject, a preliminary experiment on the “magnetic north–south axis” was conducted twice (unstarved and starved for each) with no goggles for adaption to the experimental procedure. These data were not included in the results.Experiments with oscillating magnetic fieldsExperiments with oscillating magnetic fields were performed using the standard geomagnetic orientation assay described above. To produce the oscillating magnetic fields, oscillating currents from a function generator (AFG3021; Tektronix, USA. For each magnetic field, sweep of 500 ms; interval of 1 ms. See Fig. S6A) were amplified (ENI 2100L RF power amplifier; Bell Electronics, USA) and fed into a calibrated coil antenna (30 cm diameter, 6509 loop antenna; ETS-LINDGREN, USA) mounted on a wooden frame, comprised of a single winding of coaxial cable. The oscillating magnetic fields were measured daily, before the first and after the last experiment of the day, using a spectrum analyzer (SPA-921TG; Com-Power, USA) with a calibrated loop antenna (48 cm diameter, AL-130R; Com-Power, USA) and a calibrated magnetometer (Probe HF 3061, NBM-550; Narda, Germany). Magnetic field intensities were measured on the glabella of the subjects; variations in intensity between subjects due to different seating heights were less than 10% of the average values (Table S3). The function generator and amplifier were placed outside the Faraday cage, and switched on during the dummy load control experiments with no signal from the PC system. The band widths of the monochromatic magnetic fields, i.e., 1.260 and 1.890 MHz were 0.020 and 0.019 MHz (“average”, √3 kHz), respectively, at the bottoms of the peaks. During the test phase, the maximum values of magnetic noise on the glabella of subjects including the dummy load did not exceed the following values: (1) 5 Hz–9 kHz; 2 nT/√ 2 kHz of “average” and 8 nT/√ 9 kHz of “max-hold” (0.05 nT/√ 2 kHz of “average” and 5 nT/√ 9 kHz of “max-hold” in the dummy load) (3D NF Analyzer NFA 1000; Gigahertz Solutions, Germany); (2) 9 kHz–500 kHz; 5 nT/√ 3 kHz of “average” and 8 nT/√ 3 kHz of “max-hold” (≈ 0 nT/√ 3 kHz of “average” and ≈ 1 nT/√ 3 kHz of “max-hold” in the dummy load) (the AL-130R antenna) (Fig. S6C); and (3) 500 kHz–30 MHz; 0.006 nT of 3.780 MHz harmonic in the 1.260 MHz, 0.03 nT of 5.670 MHz harmonic in the 1.890 MHz, and ≈ 0 nT in the dummy load (/√ 10 kHz of “average”) (Fig. S6B), and 0.15 nT/√ 10 kHz of “max-hold” at the same frequencies above and ≈ 0 nT in the dummy load (the AL-130R antenna).Statistical analysisTo determine the significance of orientation data from the 2-AFC paradigm, a one-sample t-test (test mean: 0.5), paired sample t-test, or two-sample t-test was performed using Origin software 11 (Origin, USA). To verify the reasonability of the t-tests, all data sets were checked using the Anderson–Darling test if the data follow a normal distribution (Appendix S4). To evaluate the difference between the means of two data sets when at least one of them did not show a normal distribution, the percentile bootstrap method57 was used (95% confidence interval, see Fig. S2, Appendices S1 and S2 for raw data). To analyze the blood glucose data, a paired sample t-test was used. Based on the results of previous study31, to describe different response groups of magnetic orientation in the 2-AFC paradigm, a principal component analysis36,37 was conducted on correct orientation rates by starved subjects, with no association/food-association under the full wavelength or  > 400 nm light conditions using SPSS 23 (IBM, USA). Following the principal component analysis calculation, the k-means clustering algorithm—one of the unsupervised learning methods—was used to objectively classify the groups58. The number of groups was two, and the distance between the center of the cluster and all points was Euclidean distance. The classification boundary was marked with the perpendicular bisector from the centers of the two groups. The first two principal components accounted for a significant portion of the total variance (73.1%; PC1 = 40.8%, PC2 = 32.3%). Statistical values are presented as mean ± SEM. More

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