Supervised classification combined with genetic algorithm variable selection for a fast identification of polymeric microdebris using infrared reflectance.

Pollution caused by plastics and, in particular, microplastics has become a source of environmental concern for Society. Their ubiquity, with millions of tons of plastic debris spilled in both land and sea, requires efficient technological improvements in the ways residues are collected, handled, characterized and recycled. For reliable decision-making, dependable chemical information is essential to assess both the nature of the plastics found in the environment and their fate. In this work an efficient method to identify the polymeric composition of microplastic fragments is proposed. It combines infrared reflectance spectra and chemometric methods. A breakthrough result is that the models include polymers weathered under both dry (shoreline) and submerged (in sea water) conditions and, hence, they are very promising as a starting point for eventual practical applications. In addition, no spectral processing is required after the initial measurement. SYNOPSIS: This approach to identify microplastics in aquatic environments combines infrared measurements and multivariate data analysis to fight against (micro)plastic pollution.

PLX4032 resistance of patient-derived melanoma cells: crucial role of oxidative metabolism.

Background: Malignant melanoma is the most lethal form of skin cancer which shows BRAF mutation in 50% of patients. In this context, the identification of BRAFV600E mutation led to the development of specific inhibitors like PLX4032. Nevertheless, although its initial success, its clinical efficacy is reduced after six-months of therapy leading to cancer relapse due to the onset of drug resistance. Therefore, investigating the mechanisms underlying PLX4032 resistance is fundamental to improve therapy efficacy. In this context, several models of PLX4032 resistance have been developed, but the discrepancy between in vitro and in vivo results often limits their clinical translation. Methods: The herein reported model has been realized by treating with PLX4032, for six months, patient-derived BRAF-mutated melanoma cells in order to obtain a reliable model of acquired PLX4032 resistance that could be predictive of patient's treatment responses. Metabolic analyses were performed by evaluating glucose consumption, ATP synthesis, oxygen consumption rate, P/O ratio, ATP/AMP ratio, lactate release, lactate dehydrogenase activity, NAD+/NADH ratio and pyruvate dehydrogenase activity in parental and drug resistant melanoma cells. The intracellular oxidative state was analyzed in terms of reactive oxygen species production, glutathione levels and NADPH/NADP+ ratio. In addition, a principal component analysis was conducted in order to identify the variables responsible for the acquisition of targeted therapy resistance. Results: Collectively, our results demonstrate, for the first time in patient-derived melanoma cells, that the rewiring of oxidative phosphorylation and the maintenance of pyruvate dehydrogenase activity and of high glutathione levels contribute to trigger the onset of PLX4032 resistance. Conclusion: Therefore, it is possible to hypothesize that inhibitors of glutathione biosynthesis and/or pyruvate dehydrogenase activity could be used in combination with PLX4032 to overcome drug resistance of BRAF-mutated melanoma patients. However, the identification of new adjuvant targets related to drug-induced metabolic reprogramming could be crucial to counteract the failure of targeted therapy in metastatic melanoma.

Intra-source provenance study on Monte Arci (Sardinia) obsidian by pXRF: Role of the data acquisition and analysis tools.

In this work, a detailed study of Monte Arci obsidian sub-sources using the increasingly accessible technique of pXRF is presented based upon a large dataset of 68 geological samples, for the development of X-ray fluorescence-based analytical standardless procedure. In addition, a non-conventional (for obsidian provenance study) direct application of multivariate analysis on XRF spectra (continuous variables), rather than absolute concentrations or intensity ratios (discrete variables) is proposed. Results from different softwares and data analysis approaches (bi-/trivariate versus multivariate) were compared. In a blind test, the bi-/trivariate approach led to the correct assignment for the main SA, SB, and SC sub-sources, taking into account averaged values of intensity ratios with their standard deviation obtained from three independent measurements. A high intra-source variability for the SB subgroups was detected (almost 13% of error in the assignment, 9 samples out of 68). A non-conventional application of multivariate analysis was carried out directly on the XRF spectra and correct assignments were obtained for SA, SB1, SC groups, while 71% of the SB2 samples were correctly identified. The non-destructive analysis on 14 archaeological samples from Su Carroppu (Carbonia, southwestern Sardinia) rockshelter and from the Middle Neolithic (MN) 422 structure of the open-air dwelling site at Cuccuru is Arrius (Cabras, central-western Sardinia) permitted to test the method and hypothesise their provenance. The comparison with visual characterization or previous analyses by Particle Induced X-Ray Emission (PIXE) permitted to verify the correct provenance assignment of all artifacts for the bi-/trivariate method, and for 12/14 samples in the case of the multivariate one. The standardless analytical approach proposed in this work can represent a more general method exploitable for other obsidian sources, other glassy materials, besides other materials of archaeological interest.

A Preliminary Color Study of Different Basil-Based Semi-Finished Products during Their Storage.

Basil-based semi-finished products, which are mainly used as an intermediate to produce the typical pesto sauce, are prepared and exported all over the world. Color is a fundamental organoleptic requirement for the acceptability of these semi-finished products by the manufacturers of the pesto sauce. Some alternative formulations, which adjust the typical industrial recipe by both changing the preservative agent (ascorbic acid, citric acid, or a mixture of both) and introducing a preliminary thermic treatment (blast chilling), were evaluated. In this work, a fast and non-destructive spectrophotometric analysis, to monitor the color variations in these food products during their shelf-life, was proposed. The raw diffuse reflectance spectra (380-900 nm) obtained by a UV-visible spectrophotometer, endowed with an integrating sphere, together with the CIELab parameters (L*, a*, b*) automatically obtained from these, were considered, and elaborated using multivariate statistical analysis (principal component analysis). From this preliminary study, blast chilling, together with the use of ascorbic acid, proved to be the best solution to better preserve the color of these products during their shelf-life.

Multi-Elemental Analysis as a Tool to Ascertain the Safety and the Origin of Beehive Products: Development, Validation, and Application of an ICP-MS Method on Four Unifloral Honeys Produced in Sardinia, Italy.

Despite unifloral honeys from Sardinia, Italy, being appreciated worldwide for their peculiar organoleptic features, their elemental signature has only partly been investigated. Hence, the principal aim of this study was to measure the concentration of trace and toxic elements (i.e., Ag, As, Ba, Be, Bi, Cd, Co, Cr, Cu, Fe, Hg, Li, Mn, Mo, Ni, Pb, Sb, Sn, Sr, Te, Tl, V, and Zn) in four unifloral honeys produced in Sardinia. For this purpose, an original ICP-MS method was developed, fully validated, and applied on unifloral honeys from asphodel, eucalyptus, strawberry tree, and thistle. Particular attention was paid to the method's development: factorial design was applied for the optimization of the acid microwave digestion, whereas the instrumental parameters were tuned to minimize the polyatomic interferences. Most of the analytes' concentration ranged between the relevant LoDs and few mg kg-1, while toxic elements were present in negligible amounts. The elemental signatures of asphodel and thistle honeys were measured for the first time, whereas those of eucalyptus and strawberry tree honeys suggested a geographical differentiation if compared with the literature. Chemometric analysis allowed for the botanical discrimination of honeys through their elemental signature, whereas linear discriminant analysis provided an accuracy level of 87.1%.

Effects of visual training on motor performance in young tennis players using FitLight trainer.

BACKGROUND: Tennis is an open-skill sport in which the athletes have a short period of time to elaborate all the information coming from the surrounding environment and produce a motor answer based on them. The aim of this study was divided in two hypotheses: 1) to assess if belonging to a certain category, athlete, or non-athlete, older or younger, can affect the development of reaction time on children; and 2) if a protocol based on visual training (VT) of 6 weeks could improve the motor performance on the field in young tennis players using FitLight Trainer (Medical Graphics, Milan, Italy). METHODS: In this evidence a group of young children (N.=40) have been tested on light board through reaction test then some young tennis players (N.=15, age: 7-12 years old) were taken as reference for the second hypothesis. They were divided in two groups: 7 of them were in the group Under-10 (U10) while 8 in a second group (U12). They performed a VT protocol once a week for at least 40 minutes for 6 weeks. They were tested at baseline (T0) and follow-up (T6) to evaluate the reaction time, time in specific lateral shift and precision about forehand and backhand. RESULTS: The development of reaction time of the athletes is principally caused by their growth (P<0.05). Principal components analysis (PCA) showed significant improvements in the Under-10 category in all the tests while in the Under-12 category not every individual showed a significant result in terms of performance. CONCLUSIONS: The developing of reaction time and coordination eye-hand is mainly due to the growth of young athletes. Also, performing a 6-week VT using FitLight Trainer is possible improve the reaction time and the motor performance on the field especially in young tennis players under 10 years old.

Background, Applications and Issues of the Experimental Designs for Mixture in the Food Sector.

BACKGROUND: Mixtures play a key role in Food Science and Technology. For studying them, rational approaches should be used. In detail, the experimental designs for mixtures are useful tools for studying the effects of ingredients/components in formulations. RESULTS: Food Science and Technology is the fourth category among the total records considered in this review. The applications span from food formulation to the composition of modified atmosphere, shelf-life improvement and bioactives extraction. However, the majority of the studies regards few products and ingredients. Simplex-lattice and simplex-centroid designs are the most common used, although some optimal designs, such as the D-optimal, have also interesting applications. Finally, some issues are highlighted, which basically regard the interpretation of the models coefficients and the lack of model validation. CONCLUSION: In the last decade, mixture designs have been fairly used in the field of Food Science and Technology. Modeling the response(s) allows researchers to achieve a global knowledge of the system under study within the defined experimental domain. However, the majority of application has regarded limited classes of products, and thus an increase in the spectrum of applications is desired.

PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis.

Cancer stem cells (CSCs) are a limited cell population inside a tumor bulk characterized by high levels of glutathione (GSH), the most important antioxidant thiol of which cysteine is the limiting amino acid for GSH biosynthesis. In fact, CSCs over-express xCT, a cystine transporter stabilized on cell membrane through interaction with CD44, a stemness marker whose expression is modulated by protein kinase Cα (PKCα). Since many chemotherapeutic drugs, such as Etoposide, exert their cytotoxic action by increasing reactive oxygen species (ROS) production, the presence of high antioxidant defenses confers to CSCs a crucial role in chemoresistance. In this study, Etoposide-sensitive and -resistant neuroblastoma CSCs were chronically treated with Etoposide, given alone or in combination with Sulfasalazine (SSZ) or with an inhibitor of PKCα (C2-4), which target xCT directly or indirectly, respectively. Both combined approaches are able to sensitize CSCs to Etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, down-regulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis. Our results suggest, for the first time, that PKCα inhibition inducing ferroptosis might be a useful strategy with which to fight CSC chemoresistance.

Potential Role of miRNAs in the Acquisition of Chemoresistance in Neuroblastoma.

Neuroblastoma (NB) accounts for about 8-10% of pediatric cancers, and the main causes of death are the presence of metastases and the acquisition of chemoresistance. Metastatic NB is characterized by MYCN amplification that correlates with changes in the expression of miRNAs, which are small non-coding RNA sequences, playing a crucial role in NB development and chemoresistance. In the present study, miRNA expression was analyzed in two human MYCN-amplified NB cell lines, one sensitive (HTLA-230) and one resistant to Etoposide (ER-HTLA), by microarray and RT-qPCR techniques. These analyses showed that miRNA-15a, -16-1, -19b, -218, and -338 were down-regulated in ER-HTLA cells. In order to validate the presence of this down-regulation in vivo, the expression of these miRNAs was analyzed in primary tumors, metastases, and bone marrow of therapy responder and non-responder pediatric patients. Principal component analysis data showed that the expression of miRNA-19b, -218, and -338 influenced metastases, and that the expression levels of all miRNAs analyzed were higher in therapy responders in respect to non-responders. Collectively, these findings suggest that these miRNAs might be involved in the regulation of the drug response, and could be employed for therapeutic purposes.

Effect of the irrigation method and genotype on the bioaccumulation of toxic and trace elements in rice.

The total concentration of three toxic elements (As, Cd and Pb) and five oligoelements (Cu, Mn, Mo, Ni and Se) has been determined using an original and completely validated ICP-MS method. This was applied to rice grains from 26 different genotypes cultivated in the same soil and irrigated with the same water in three different ways: by the traditional continuous flooding (CF) and by two intermittent methods, the sprinkler irrigation (SP) and the periodical saturation of the soil (SA). The adoption of SP hugely minimizes the average amounts of almost all elements in kernels (-98% for As, -90% for Se and Mn, -60% for Mo, -50% for Cd and Pb), with the only exception of Ni, whose concentration increases the average amount found in the CF rice by 7.5 times. Also SA irrigation is able to reduce the amounts of As, Mo and Pb in kernels but it significantly increases the amounts of Mn, Ni and - mainly - Cd. Also the nature of the genotype determined a wide variability of data within each irrigation method. Genotypes belonging to Indica subspecies are the best bioaccumulators of elements in both CF and SP methods and, never, the worst bioaccumulators for any element/irrigation method combination. In the principal component analysis, PC1 can differentiate samples irrigated by SP by those irrigated by CF and SA, whereas PC2 provides differentiation of CF samples by SA samples. When looking at the loading plot Ni is negatively correlated to the majority of the other elements, except Cu and Cd having negative loadings on PC2. These results allow to envisage that a proper combination of the irrigation method and the nature of rice genotype might be a very valuable tool in order to successfully achieve specific objectives of food safety or the attainment of functional properties.

Chemometric challenges in development of paper-based analytical devices: Optimization and image processing.

Although microfluidic paper-based analytical devices (µPADs) get a lot of attention in the scientific literature, they rarely reach the level of commercialization. One possible reason for this is a lack of application of machine learning techniques supporting the design, optimization and fabrication of such devices. This work demonstrates the potential of two chemometric techniques including design of experiments (DoE) and digital image processing to support the production of µPADs. On the example of a simple colorimetric assay for isoniazid relying on the protonation equilibrium of methyl orange, the experimental conditions were optimized using a D-optimal design (DO) and the impact of multiple factors on the µPAD response was investigated. In addition, this work demonstrates the impact of automatic image processing on accelerating color value analysis and on minimizing errors caused by manual detection area selection. The employed algorithm is based on morphological recognition and allows the analysis of RGB (red, green, and blue) values in a repeatable way. In our belief, DoE and digital image processing methodologies are keys to overcome some of the remaining weaknesses in µPAD development to facilitate their future market entry.

Electrochemical preconcentration coupled with spectroscopic techniques for trace lead analysis in olive oils.

In this paper we present a novel combined electrochemical-spectroscopic approach suitable to monitor trace levels of heavy metals directly in edible oils. The method is based on the electrochemical preconcentration/extraction of the analyte from the tested real matrix by cathodic deposition onto a Pt working electrode, then transfer and anodic re-oxidation of the metallic deposit to a "clean" aqueous solution, suitable for the subsequent spectroscopic analysis. The procedure has been here focused to the determination of lead in extra virgin olive oil (EVOO), performed by applying ICP-QMS or GFAAS techniques. To this aim, the EVOO samples were mixed with proper amounts of the room temperature ionic liquid (RTIL) [P14,6,6,6]+[NTf2]-, in order to obtain a non-aqueous supporting electrolyte suitable for the electrodeposition process. The feasibility and performance of the analytical strategy were at first tested in standard solutions of Pb(II) in RTIL, produced by anodic dissolution of lead in the RTIL, as well as in olive oil samples mixed with 0.5 M RTIL and spiked with known amounts of Pb(II). The optimisation of the electrochemical parameters was achieved by applying a D-Optimal Design, properly set up to optimise the efficiency of the deposition and re-oxidation steps, quantitative recovery and measurement time. Finally, the analytical procedure was applied to the determination of Pb content in some Italian EVOOs, without any need of performing mineralization pretreatments. Data obtained with the proposed procedure satisfactorily agree with those achieved by ICP-QMS analysis after microwave digestion, being differences between the two approaches within 10%, with the advantage of reducing to half the pretreatment time, operating at room temperature and avoiding the use of aggressive solvents.

Assessing the influence of pH and cationic strength on i-motif DNA structure.

The i-motif is a biologically relevant non-canonical DNA structure formed by cytosine-rich sequences. Despite the importance of the factors affecting the formation/stability of such a structure, like pH, cation type and concentration, no systematic study that simultaneously analysed their effect on the i-motif in vitro has been carried out so far. Therefore, here we report a systematic study that aims to evaluate the effect of these factors, and their possible interaction, on the formation of an i-motif structure. Our results confirm that pH plays the main role in i-motif formation. However, we demonstrate that the effect of the cation concentration on the i-motif is strictly dependent on the pH, while no significant differences are observed among the investigated cation types. Graphical abstract.

An innovative green extraction and re-use strategy to valorize food supplement by-products: Castanea sativa bud preparations as case study.

This research takes place in the context of an Alcotra Italy-France trans-frontier project called FINNOVER, which includes among its objectives the "green" innovation of agro-industrial chains. Bud-derivatives are a category of natural products produced macerating meristematic tissues of trees and plants. They are quite expensive compared to other botanicals, since the collection period of their raw materials is extremely limited over the time. Pulsed Ultrasound-Assisted Extraction has been employed to extract further valuable material from the buds by-products remaining after the production of Castanea sativa Glyceric Macerates. UV-Visible spectra coupled with chemometrics were employed, as untargeted phytochemical fingerprints, to quickly screen the best experimental conditions of extraction: a duty cycle of 80%, an extraction time of 15 min and a solvent/ratio of 1/10. Targeted phytochemical fingerprints by HPLC have been used to identify and quantify the main bioactive compounds of the most promising marcs extract comparing it with the corresponding commercial Castanea sativa Glyceric Macerate. An innovative extraction and re-use strategy to obtain value-added products from botanicals by-products was developed in alternative to incineration or composting. It was applied to Castanea sativa buds production as case study, but it could be analogously applied for other herbal preparations.

Fabrication of paper-based analytical devices optimized by central composite design.

In this work, an application of a design of experiments approach for the optimization of an isoniazid assay on a single-area inkjet-printed paper-based analytical device (PAD) is described. For this purpose, a central composite design was used for evaluation of the effect of device geometry and amount of assay reagents on the efficiency of the proposed device. The factors of interest were printed length, width, and sampling volume as factors related to device geometry, and amounts of the assay reagents polyvinyl alcohol (PVA), NH4OH, and AgNO3. Deposition of the assay reagents was performed by a thermal inkjet printer. The colorimetric assay mechanism of this device is based on the chemical interaction of isoniazid, ammonium hydroxide, and PVA with silver ions to induce the formation of yellow silver nanoparticles (AgNPs). The in situ-formed AgNPs can be easily detected by the naked eye or with a simple flat-bed scanner. Under optimal conditions, the calibration curve was linear in the isoniazid concentration range 0.03-10 mmol L-1 with a relative standard deviation of 3.4% (n = 5 for determination of 1.0 mmol L-1). Finally, the application of the proposed device for isoniazid determination in pharmaceutical preparations produced satisfactory results.

Optimization of the Ultrasonic-Assisted Extraction of Phenolic Compounds from Oryza Sativa L. 'Violet Nori' and Determination of the Antioxidant Properties of its Caryopses and Leaves.

'Violet Nori' is a new spontaneous growing violet rice variety showing a peculiar violet color in its fresh leaves as well. In this paper, the antioxidant properties and the content of total phenols, radical scavengers, and anthocyanins in 'Violet Nori' caryopses, flour, and leaves are explored and compared. Ultrasonic-Assisted Extraction (UAE) is employed for the extraction of phenolic compounds, improving the extraction conditions by Design of Experiments (DoE). The obtained results show that the Radical Scavenging Activities (RSAs), the Total Phenolic Contents (TPCs), and the anthocyanin amounts (1000-1500 µg/g, expressed as cyanidin-3-glucoside) of 'Violet Nori' caryopses are higher than those in the other analyzed colored rice samples (300-900 µg/g as cyanidin-3-glucoside), with the exception of the cultivars 'Artemide' and 'Nerone', which show comparable values of RSAs and TPCs. The study of 'Violet Nori' leaves at different plant maturation stages shows that their anthocyanin content is 2-3 times higher than in the caryopses and in the flour, reaching the highest levels at about 60 days from seeding. Thus, the estimated extraction yield of 4 kg anthocyanins/t makes fresh leaves very interesting for the extraction of anthocyanins on an industrial scale, whereas violet caryopses are a very interesting dietetic source of valuable anthocyanins and other antioxidant compounds.

Olive fruits and vacuum impregnation, an interesting combination for dietetic iron enrichment.

In this study vacuum impregnation (VI) was employed for the iron enrichment of olive fruits, which are very interesting as food vehicle for VI mineral supplementation for the porosity of their pulp. NaFeEDTA was chosen for olives fortification since it prevents iron from binding with compounds that could hinder it from being efficiently absorbed and since it causes few organoleptic problems. In order to improve the efficiency of the VI process, several parameters of the whole process were studied by design of experiment techniques. First of all D-optimal design was employed for a preliminary screening of the most significant process variables and showed that the concentration of VI solution was by far the most significant process variable, though its time in contact with olives was also significant. A factorial design was then applied to the remaining variables and it showed that the speed of the addition of VI solution was also significant. Finally, the application of a face centered composite design to the three selected variables allowed to detect processing conditions leading to final iron contents of 1.5-3 mg/g, corresponding to an introduction of 10-15 mg Fe with four or five fortified olive fruits. No effect on olive taste was observed at these concentrations. The results showed that olive fruits were the most interesting vehicles for the supplementation of both iron and other minerals.

Tools based on multivariate statistical analysis for classification of soil and groundwater in Apulian agricultural sites.

In this paper, the results obtained from multivariate statistical techniques such as PCA (Principal component analysis) and LDA (Linear discriminant analysis) applied to a wide soil data set are presented. The results have been compared with those obtained on a groundwater data set, whose samples were collected together with soil ones, within the project "Improvement of the Regional Agro-meteorological Monitoring Network (2004-2007)". LDA, applied to soil data, has allowed to distinguish the geographical origin of the sample from either one of the two macroaeras: Bari and Foggia provinces vs Brindisi, Lecce e Taranto provinces, with a percentage of correct prediction in cross validation of 87%. In the case of the groundwater data set, the best classification was obtained when the samples were grouped into three macroareas: Foggia province, Bari province and Brindisi, Lecce and Taranto provinces, by reaching a percentage of correct predictions in cross validation of 84%. The obtained information can be very useful in supporting soil and water resource management, such as the reduction of water consumption and the reduction of energy and chemical (nutrients and pesticides) inputs in agriculture.

Oxysterol mixture and, in particular, 27-hydroxycholesterol drive M2 polarization of human macrophages.

Macrophages play a crucial role in atherosclerosis progression. Classically activated M1 macrophages have been found in rupture-prone atherosclerotic plaques whereas alternatively activated macrophages, M2, localize in stable plaque. Macrophage accumulation of cholesterol and of its oxidized derivatives (oxysterols) leads to the formation of foam cells, a hallmark of atherosclerotic lesions. In this study, the effects of oxysterols in determining the functional polarization of human macrophages were investigated. Monocytes, purified from peripheral blood mononuclear cells of healthy donors, were differentiated into macrophages (M0) and treated with an oxysterol mixture, cholesterol, or ethanol, every 4 H for a total of 4, 8, and 12 H. The administration of the compounds was repeated in order to maintain the levels of oxysterols constant throughout the treatment. Compared with ethanol treatment, the oxysterol mixture decreased the surface expression of CD36 and CD204 scavenger receptors and reduced the amount of reactive oxygen species whereas it did not affect either cell viability or matrix metalloprotease-9 activity. Moreover, the oxysterol mixture increased the expression of both liver X receptor α and ATP-binding cassette transporter 1. An enhanced secretion of the immunoregulatory cytokine IL-10 accompanied these events. The results supported the hypothesis that the constant levels of oxysterols and, in particular, of 27-hydroxycholesterol stimulate macrophage polarization toward the M2 immunomodulatory functional phenotype, contributing to the stabilization of atherosclerotic plaques.

Fingerprint detection and process prediction by multivariate analysis of fed-batch monoclonal antibody cell culture data.

This work presents a sequential data analysis path, which was successfully applied to identify important patterns (fingerprints) in mammalian cell culture process data regarding process variables, time evolution and process response. The data set incorporates 116 fed-batch cultivation experiments for the production of a Fc-Fusion protein. Having precharacterized the evolutions of the investigated variables and manipulated parameters with univariate analysis, principal component analysis (PCA) and partial least squares regression (PLSR) are used for further investigation. The first major objective is to capture and understand the interaction structure and dynamic behavior of the process variables and the titer (process response) using different models. The second major objective is to evaluate those models regarding their capability to characterize and predict the titer production. Moreover, the effects of data unfolding, imputation of missing data, phase separation, and variable transformation on the performance of the models are evaluated.