A Multi-Variate framework to assess reliability and discrimination power of Bayesian estimation of Intravoxel Incoherent Motion parameters

PurposeTo propose a multivariate multi-step framework for a systematic assessment of the estimation reliability and discriminability of Intravoxel Incoherent Motion (IVIM) model parameters.MethodsMonte-Carlo simulations were generated on a range of SNRs and in different IVIM combinations considering: i) a dense discretization with 24 b-values; ii) a discretization with 9 b-values. A state-of-the-art Bayesian fitting method was adopted. The framework assessed: i) the best model between mono- and bi-exponential, through the BIC index; ii) the fitting accuracy; iii) the power in discriminating two different IVIM parameters distributions of estimated coefficients, using a multivariate test. Exemplificative oncologic cases were also presented.ResultsThe bi-exponential fitting was reliable for perfusion fraction higher than 5%, with high accuracy in D estimation, acceptable error for f, but high uncertainty in D*. The discrimination of two distributions is generally feasible if differences in D values (at least 0.3 x10^-3 mm²/s) are present; in the case of similar D values, a minimal difference of 5% in f can be discriminated just in case of balanced sample size and dense b-values discretization, whereas the impact of D* is quite negligible. These results were also supported by clinical examples.ConclusionsIVIM model is generally accurate in estimating diffusion, but uncertainties related to perfusion estimation are not negligible and compromise the discrimination power when different populations should be differentiated. The proposed framework should be adopted as interpretative guidelines to better understand when IVIM model applied on real data can provide reliable findings.     

Initial guesses generation for fluorescence intensity distribution analysis

The growing number of applications of Fluorescence Intensity Distribution Analysis (FIDA) demands for new approaches in data processing, aiming at increased speed and robustness. Iterative algorithms of parameter estimation, although proven to be universal and accurate, require some initial guesses (IG) of the unknown parameters. An essential component of any data processing technology, IG become especially important in case of FIDA, since even with apparently reasonable, and physically admissible but randomly chosen IG, the iterative procedure may converge to situations where the FIDA model cannot be evaluated correctly. In the present work we introduce an approach for IG generation in FIDA experiments based on the method of moments. IG are generated for the sample parameters: brightness, concentration, and for the parameters related to experimental set-up: background, observation volume profile. A number of analytical simplifications were introduced in order to increase the accuracy and robustness of the numerical algorithms. The performance of the developed method has been tested on number of simulations and experimental data. Iterative fitting with generated IG proved to be more robust and at least five times faster than with an arbitrarily chosen IG. Applicability of the proposed method for quick estimation of brightness and concentrations is discussed.     

Modified Gaussian estimation for correlated binary data

In this paper, we develop a Gaussian estimation (GE) procedure to estimate the parameters of a regression model for correlated (longitudinal) binary response data using a working correlation matrix. A two‐step iterative procedure is proposed for estimating the regression parameters by the GE method and the correlation parameters by the method of moments. Consistency properties of the estimators are discussed. A simulation study was conducted to compare 11 estimators of the regression parameters, namely, four versions of the GE, five versions of the generalized estimating equations (GEEs), and two versions of the weighted GEE. Simulations show that (i) the Gaussian estimates have the smallest mean square error and best coverage probability if the working correlation structure is correctly specified and (ii) when the working correlation structure is correctly specified, the GE and the GEE with exchangeable correlation structure perform best as opposed to when the correlation structure is misspecified.     

Respiratory pattern transitions in three species of Glossina (Diptera, Glossinidae)

Glossina exhibit cyclic (^CYCGE) or continuous gas exchange (^CONGE) patterns at rest. However, the factors influencing the transition from one pattern to another are not well understood for these or other insect species. This study examines which factors could aid in predicting the presence or absence of ^CYCGE in adults of three Glossina species: G. palpalis, G. brevipalpis and G. austeni. We report the results of temperature effects on VCO₂, pattern type and the proportion of a population showing ^CYCGE, and the prediction of ^CYCGE versus ^CONGE in Glossina. First, we investigated the influence of temperature on VCO₂ and found significant elevation in resting metabolic rate (RMR) with higher temperature in all three species (P < 0.001). Temperature-induced increases in VCO₂ were modulated by increased burst volume and by cycle frequency, except in G. brevipalpis which only appeared to modulate burst volume. These results are largely in keeping with VCO₂ modulation reported for other Glossina species previously. Second, elevating temperature resulted in significantly reduced numbers of individuals showing ^CYCGE (P < 0.001 for all three species) contrary to previous reports for other Glossing species. Finally, we examined a range of variables as potential predictors of presence or absence of ^CYCGE in these three species. Using an information theoretic approach (Akaike weights) to select the best explanatory combination of variables which predicts likelihood of ^CYCGE, we found that results varied among species. When species were pooled, the simplest, best-fit model (ΔAIC < 2 from the best model, 44.4% probability of being the best model) for predicting pattern type variation was RMR. Overall these results suggest that RMR is a key variable driving pattern type and that elevated temperature reduces the number of individuals showing cyclic patterns through elevation of RMR in these species. This study supports the idea that an interaction between cellular metabolic demand, morphological features of the gas exchange system (e.g. tracheal and spiracular conductances), and CO₂ buffer capacity likely determine gas exchange pattern variation over short time-scales.     

Biology of communities estimation of species richness of a community by its species structure,as illustrated by the macrophytes of Vityaz Bay in the Sea of Japan

Five methods of computing species richness in community are discussed using the example of a well-studied macrophytic taxocoen: (1) fitting an ogive for lognormal distribution; (2) model fitting of a rank curve for this distribution; (3) iterative computation of statistical moments using Hald’s method; (4) computation of these moments by explicit functions; and (5) model fitting of a “species-area” curve. According to the repeatability of results and simplicity of computing, the best method is the computation of statistical moments by explicit functions. The degree of our knowledge about a particular community could be considered as the ratio of the number of actually distinguished species to the greatest expected number.     

Kinetics of impurity charge-state distributions in tokamak plasmas

An analysis of impurity behavior in tokamak plasmas with the use of the observation results on impurity emission shows that it is necessary to distinguish between the ion dynamics (for example, ion transport) and ion kinetics, i.e., the processes related to the motion of ions on the charge states and/or excited states due to atomic processes in plasma. This paper presents a systematic analysis of the kinetics of impurity chargestate distributions and the related effects, as well as their typical scales and conditions for their observation. The quantitative analysis is performed in terms of the lowest moments of charge-state distributions such as the average charge m and dispersion D. Analytic approaches to solving charge-state kinetic equations are considered. An approach based on the symmetry properties of the kinetic matrix is proposed for the first time. The simplest types of impurity charge-state kinetics and the most important limiting cases are considered. A detailed analysis of the nonstationary behavior of the function of the moments D(m) of the charge-state distribution is presented. A quantitative analysis of the available experimental and model charge-state distributions of C, O, Ne, and Ar impurities in the JET, DIII-D, TORE SUPRA, ALCATOR-C, TEXTOR, PLT, TFR, and DAMAVAND tokamaks is performed in terms of the moments D(m). It is shown that the moments D(m)of the model charge-state distributions of the above impurities in the plasma core are essentially insensitive to the empirical diffusion coefficient. The equivalent curves D(m) obtained for the plasma periphery can be attributed to the convective fluxes of ionizing and/or recombining impurity ions.     

Model life table fitting by maximum likelihood estimation: a procedure to reconstruct paleodemographic characteristics from skeletal age distributions

A procedure is presented that uses the regression coefficients for the Coale and Demeny west model life tables to model selected demographic characteristics from skeletal age-at-death distributions. Model death distributions were constructed and compared to a given skeletal distribution, using methods of maximum likelihood estimation to determine the best fit. Two chi-square tests are employed to evaluate the degree of fit. The resulting model includes estimates of demographic characteristics including gross reproductive rate, crude birth rate and life expectancy. The procedure is applied to three archaeological skeletal samples as test cases: two from eastern North America and one from Mexico. These display a range of correspondence (between the best fitting model and the data) from good to poor. The proposed procedure is a potentially powerful tool for both reconstructing paleodemographic rates and illuminating differences between typical human patterns and those found in archaeological populations.     

Plant community diversity influences allocation to direct chemical defence in Plantago lanceolata

Background

Forecasting the consequences of accelerating rates of changes in biodiversity for ecosystem functioning requires a mechanistic understanding of the relationships between the structure of biological communities and variation in plant functional characteristics. So far, experimental data of how plant species diversity influences the investment of individual plants in direct chemical defences against herbivores and pathogens is lacking.

Methodology/Principal Findings

We used Plantago lanceolata as a model species in experimental grasslands differing in species richness and composition (Jena Experiment) to investigate foliar concentrations of the iridoid glycosides (IG), catalpol and its biosynthetic precursor aucubin. Total IG and aucubin concentrations decreased, while catalpol concentrations increased with increasing plant diversity in terms of species or functional group richness. Negative plant diversity effects on total IG and aucubin concentrations correlated with increasing specific leaf area of P. lanceolata, suggesting that greater allocation to light acquisition reduced the investment into these carbon-based defence components. In contrast, increasing leaf nitrogen concentrations best explained increasing concentrations of the biosynthetically more advanced IG, catalpol. Observed levels of leaf damage explained a significant proportion of variation in total IG and aucubin concentrations, but did not account for variance in catalpol concentrations.

Conclusions/Significance

Our results clearly show that plants growing in communities of varying species richness and composition differ in their defensive chemistry, which may modulate plant susceptibility to enemy attack and consequently their interactions with higher trophic level organisms.     

An improved method of genomic in situ hybridization (GISH) for distinguishing closely related genomes of tetraploid and hexaploid wheat species

An improved modification of genomic in situ hybridization (GISH) was proposed. It allows clear and reproducible discrimination between closely related genomes of both tetraploid and hexaploid wheat species due to preannealing of labeled DNA probes and prehybridization of chromosomal samples with blocking DNA. The method was applied to analyze intergenomic translocations 6A:6B and 1A:6B identified in the IG46147 and IG116188 samples of tetraploid wheat Triticum dicoccoides by C-banding. The structure of the rearranged chromosomes was defined for two translocation variants, and the breakpoints were identified on the chromosome arms. Possible application of the developed GISH variant to study genome reorganizations during speciation of allopolyploid plants in evolution is discussed.     

A Quantile Regression Approach to Estimating the Distribution of Anesthetic Procedure Time during Induction

Although procedure time analyses are important for operating room management, it is not easy to extract useful information from clinical procedure time data. A novel approach was proposed to analyze procedure time during anesthetic induction. A two-step regression analysis was performed to explore influential factors of anesthetic induction time (AIT). Linear regression with stepwise model selection was used to select significant correlates of AIT and then quantile regression was employed to illustrate the dynamic relationships between AIT and selected variables at distinct quantiles. A total of 1,060 patients were analyzed. The first and second-year residents (R1-R2) required longer AIT than the third and fourth-year residents and attending anesthesiologists (p = 0.006). Factors prolonging AIT included American Society of Anesthesiologist physical status ≧ III, arterial, central venous and epidural catheterization, and use of bronchoscopy. Presence of surgeon before induction would decrease AIT (p < 0.001). Types of surgery also had significant influence on AIT. Quantile regression satisfactorily estimated extra time needed to complete induction for each influential factor at distinct quantiles. Our analysis on AIT demonstrated the benefit of quantile regression analysis to provide more comprehensive view of the relationships between procedure time and related factors. This novel two-step regression approach has potential applications to procedure time analysis in operating room management.     

Phenotypic plasticity in anti-intraguild predator strategies: mite larvae adjust their behaviours according to vulnerability and predation risk

Interspecific threat-sensitivity allows prey to maximize the net benefit of antipredator strategies by adjusting the type and intensity of their response to the level of predation risk. This is well documented for classical prey-predator interactions but less so for intraguild predation (IGP). We examined threat-sensitivity in antipredator behaviour of larvae in a predatory mite guild sharing spider mites as prey. The guild consisted of the highly vulnerable intraguild (IG) prey and weak IG predator Phytoseiulus persimilis, the moderately vulnerable IG prey and moderate IG predator Neoseiulus californicus and the little vulnerable IG prey and strong IG predator Amblyseius andersoni. We videotaped the behaviour of the IG prey larvae of the three species in presence of either a low- or a high-risk IG predator female or predator absence and analysed time, distance, path shape and interaction parameters of predators and prey. The least vulnerable IG prey A. andersoni was insensitive to differing IGP risks but the moderately vulnerable IG prey N. californicus and the highly vulnerable IG prey P. persimilis responded in a threat-sensitive manner. Predator presence triggered threat-sensitive behavioural changes in one out of ten measured traits in N. californicus larvae but in four traits in P. persimilis larvae. Low-risk IG predator presence induced a typical escape response in P. persimilis larvae, whereas they reduced their activity in the high-risk IG predator presence. We argue that interspecific threat-sensitivity may promote co-existence of IG predators and IG prey and should be common in predator guilds with long co-evolutionary history.     

Effect of ginger extract ingestion on skeletal muscle glycogen contents and endurance exercise in male rats

[Purpose]Skeletal muscle glycogen is a determinant of endurance capacity for some athletes. Ginger is well known to possess nutritional effects, such as anti-diabetic effects. We hypothesized that ginger extract (GE) ingestion increases skeletal muscle glycogen by enhancing fat oxidation. Thus, we investigated the effect of GE ingestion on exercise capacity, skeletal muscle glycogen, and certain blood metabolites in exercised rats. [Methods]First, we evaluated the influence of GE ingestion on body weight and elevation of exercise performance in rats fed with different volumes of GE. Next, we measured the skeletal muscle glycogen content and free fatty acid (FFA) levels in GE-fed rats. Finally, we demonstrated that GE ingestion contributes to endurance capacity during intermittent exercise to exhaustion. [Results]We confirmed that GE ingestion increased exercise performance (p<0.05) and elevated the skeletal muscle glycogen content compared to the non-GE-fed (CE, control exercise) group before exercise (Soleus: p<0.01, Plantaris: p<0.01, Gastrocnemius: p<0.05). Blood FFA levels in the GE group were significantly higher than those in the CE group after exercise (p<0.05). Moreover, we demonstrated that exercise capacity was maintained in the CE group during intermittent exercise (p<0.05). [Conclusion]These findings indicate that GE ingestion increases skeletal muscle glycogen content and exercise performance through the upregulation of fat oxidation.     

Analysis of phosphorescence in heterogeneous systems using distributions of quencher concentration.

A continuous distribution approach, instead of the traditional mono- and multiexponential analysis, for determining quencher concentration in a heterogeneous system has been developed. A mathematical model of phosphorescence decay inside a volume with homogeneous concentration of phosphor and heterogeneous concentration of quencher was formulated to obtain pulse-response fitting functions for four different distributions of quencher concentration: rectangular, normal (Gaussian), gamma, and multimodal. The analysis was applied to parameter estimates of a heterogeneous distribution of oxygen tension (PO2) within a volume. Simulated phosphorescence decay data were randomly generated for different distributions and heterogeneity of PO2 inside the excitation/emission volume, consisting of 200 domains, and then fit with equations developed for the four models. Analysis using a monoexponential fit yielded a systematic error (underestimate) in mean PO2 that increased with the degree of heterogeneity. The fitting procedures based on the continuous distribution approach returned more accurate values for parameters of the generated PO2 distribution than did the monoexponential fit. The parameters of the fit (M = mean; sigma = standard deviation) were investigated as a function of signal-to-noise ratio (SNR = maximum signal amplitude/peak-to-peak noise). The best-fit parameter values were stable when SNR > or = 20. All four fitting models returned accurate values of M and sigma for different PO2 distributions. The ability of our procedures to resolve two different heterogeneous compartments was also demonstrated using a bimodal fitting model. An approximate scheme was formulated to allow calculation of the first moments of a spatial distribution of quencher without specifying the distribution. In addition, a procedure for the recovery of a histogram, representing the quencher concentration distribution, was developed and successfully tested.     

Gastric distension attenuates the hypotensive effect of intraduodenal glucose in healthy older subjects

Postprandial hypotension occurs frequently, and current management is suboptimal. Recent studies suggest that the magnitude of the fall in postprandial blood pressure (BP) may be attenuated by gastric distension. The aim of this study was to determine the effect of gastric distension on the hypotensive response to intraduodenal (ID) glucose. Eight healthy subjects (5 males, 3 females, aged 65-76 years) received an ID infusion of either 1) 50 g glucose in 300 ml saline (ID glucose) over 60 min (t=0-60 min), 2) 50 g glucose in 300 ml saline over 60 min and intragastric (4) infusion of 500 ml water between t=7-10 min (IG water and ID glucose), or 3) ID saline (0.9%) infusion over 60 min and IG infusion of 500 ml water (IG water and ID saline) all followed by ID saline infusion for another 60 min (t=60-120 min) on three separate days. BP and heart rate (HR) were measured. Gastric emptying (GE) of the IG water was quantified by two-dimensional ultrasonography. Between t=0-60 min, systolic and diastolic BP was greater (P<0.05 for both) with IG water and ID saline compared with IG water and ID glucose, and less (P<0.05 for both) with ID glucose compared with IG water and ID glucose. These effects were evident at relatively low IG volumes (approximately 300 ml). GE was faster with IG water and ID saline when compared with IG water and ID glucose. We conclude that, in healthy older subjects, IG administration of water markedly attenuates the hypotensive response to ID glucose, presumably as a result of gastric distension.     

A simple and sensitive colorimetric method for the determination of long-chain free fatty acids in subcellular organelles

A colorimetric assay for the determination of long-chain free fatty acids (FFA) is described. The FFA were extracted from subcellular organelles with chloroform:heptane:methanol. The copper soaps of FFA were determined colorimetrically with diphenylcarbazide. There are three advantages to employing the present modified procedure. (a) The sensitivity has been increased approximately twofold over that of the previous procedure of K. Falholt, B. Lund, and W. Falholt (1973, Clin. Chim. Acta46, 105–111); (b) it takes less time to complete the assay compared to the tedious procedures currently available; and (c) the presence of bovine serum albumin, a known FAA-binding protein, does not interfere with the assay procedure. The assay shows a linear response over the range of 10 to 130 nmol of FFA. The recovery of free fatty acids from mitochondria is 99%.     

基于分位数回归的长白落叶松人工林最大密度线

基于东北地区378块固定样地和415块临时样地的调查数据和Reineke方程,利用线性分位数回归技术建立了不同分位点(τ=0.90、0.95、0.99)下的长白落叶松人工林最大林分密度与林木平均胸径的关系模型,选出拟合长白落叶松人工林最大密度线的最优模型. 利用人为选取最大的拟合数据,采用最小二乘(OLS)和最大似然(ML)回归同时建立最大密度线模型. 采用极值统计理论的广义Pareto模型推算现实林分特定径阶的极限最大株数,进一步建立极限密度线模型. 将线性分位数回归模型与其他方法进行对比.结果表明: 在全部径阶范围内选取5个最大数据点拟合的方法能够得到现实林分的最大密度线,选取的样点过多会使模拟结果偏离最大密度线,且ML法要优于OLS法. 分位点为0.99的线性分位数回归模型能够取得与ML接近的拟合结果,但分位数回归模型参数的估计结果更稳定. 人为选取拟合数据具有一定的人为性,最终选取分位点为0.99的分位数回归模型为拟合最大密度线的最优模型,参数估计结果为k=11.790、β=-1.586,极限密度线模型的参数估计结果为k=11.820、β=-1.594. 所确定的极限密度线位置略高于最大密度线,但二者差异不明显. 由固定样地数据的验证结果可知,所建立的最大林分密度线及极限密度线能够对现实林分的最大密度及极限密度进行预测,为长白落叶松人工林的合理经营提供依据.     

Analysis of the probability distribution of small random samples by nonlinear fitting of integrated probabilities

Small random samples of biochemical and biological data are often representative of complex distribution functions and are difficult to analyze in detail by conventional means. The common approaches reduce the data to a few representative parameters (such as their moments) or combine the data into a histogram plot. Both approaches reduce the information content of the data. By fitting the empirical cumulative distribution function itself with models of integrated probability distributions, the information content of the raw data can be fully utilized. This approach, distribution analysis by nonlinear fitting of integrated probabilities, allows analysis of normally distributed samples, truncated data sets, and multimodal distributions with a single, powerful data processing procedure.     

Sediment Delivery Ratio of Single Flood Events and the Influencing Factors in a Headwater Basin of the Chinese Loess Plateau

Little is known about the sediment delivery of single flood events although it has been well known that the sediment delivery ratio at the inter-annual time scale is close to 1 in the Chinese Loess Plateau. This study examined the sediment delivery of single flood events and the influencing factors in a headwater basin of the Loess Plateau, where hyperconcentrated flows are dominant. Data observed from plot to subwatershed over the period from 1959 to 1969 were presented. Sediment delivery ratio of a single event (SDR _e) was calculated as the ratio of sediment output from the subwatershed to sediment input into the channel. It was found that SDR _e varies greatly for small events (runoff depth <5 mm or rainfall depth <30 mm) and remains fairly constant (approximately between 1.1 and 1.3) for large events (runoff depth >5 mm or rainfall depth >30 mm). We examined 11 factors of rainfall (rainfall amount, rainfall intensity, rainfall kinetic energy, rainfall erosivity and rainfall duration), flood (area-specific sediment yield, runoff depth, peak flow discharge, peak sediment concentration and flood duration) and antecedent land surface (antecedent precipitation) in relation to SDR _e. Only the peak sediment concentration significantly correlates with SDR _e. Contrary to popular belief, channel scour tends to occur in cases of higher peak sediment concentrations. Because small events also have chances to attain a high sediment concentration, many small events (rainfall depth <20 mm) are characterized by channel scour with an SDR _e larger than 1. Such observations can be related to hyperconcentrated flows, which behave quite differently from normal stream flows. Our finding that large events have a nearly constant SDR _e is useful for sediment yield predictions in the Loess Plateau and other regions where hyperconcentrated flows are well developed.     

Upper limb joint kinetics during manual wheelchair propulsion in patients with different levels of spinal cord injury

The purpose of this study was to compare the forces and moments of the whole upper limb, analyzing forces and moments at the shoulder, elbow and wrist joints simultaneously during manual wheelchair propulsion of persons with different levels of spinal cord injury (SCI) on a treadmill. Fifty-one people participated in this study and were grouped by their level of SCI: C6 tetraplegia (G1), C7 tetraplegia (G2), high paraplegia (G3), and low paraplegia (G4). An inverse dynamic model was defined to compute net joint forces and moments from segment kinematics, the forces acting on the pushrim, and subject anthropometrics. Right side, upper limb kinematic data were collected with four camcorders (Kinescan–IBV). Kinetic data were recorded by replacing the wheels with SmartWheels (Three Rivers Holdings, LLC). All participants propelled the wheelchair at 3 km/h for 1 min. The most noteworthy findings in both our tetraplegic groups in relation to paraplegic groups were increased superior joint forces in the shoulder (G1 and G2 vs G3 p<0.001; G1 and G2 vs G4 p<0.01), elbow (G1 vs G3 p<0.001; G1 vs G4 p<0.05) and wrist (G1 vs G4 p<0.001), an increased adduction moment in the shoulder (G1 vs G3 p<0.001; G1 vs G4 p<0.01; G2 vs G3 and G4 p<0.05) and the constancy of the moments of force of the wrist the fact that they reached their lowest values in the tetraplegic groups. This pattern may increase the risk of developing upper limb overuse injuries in tetraplegic subjects.     

Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis: IDENTIFICATION AND CHARACTERIZATION OF THE GE81112 BIOSYNTHETIC GENE CLUSTER*

The GE81112 tetrapeptides (1–3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.