**4. Conclusions**

*Z*-Isomerization of carotenoids can cause changes in the bioavailability, antioxidant activity, and other functionalities (such as anticancer and antiatherogenic activities), and it may result in "positive" or "negative" effects, which vary according to the type of carotenoid. Although more than 1100 carotenoids are found in nature, only the eight carotenoids discussed above have been investigated in terms of these effects. Thus, further progress in this research area is expected. Furthermore, most investigations have focused on the effects of *Z*-isomerization of carotenoids on the bioavailability and antioxidant activity, but the *Z*-isomerization has been shown to enhance the anticancer and antiatherogenic activities of β-carotene and fucoxanthin. Since these data provide important evidence for the roles of carotenoid *Z*-isomerization in human health, examination of other carotenoids is expected in the future.


*5.3. Further Research*

The Simulator mode allows direct user input and feedback during the simulation through a simple graphical interface. Although the current demonstrator is limited, all operation chart values can easily be added and manipulated during the simulation and additional settings such as compositions of charged materials for each basket could be added. All intermediate results of the model, such as temperatures, heat flows, and masses, are available during the simulation.

A target value is selected (for example 100 MW of electrical power) for both the automatic control and the simulator modes, and that exact value was used during the simulation, whereas in reality many operation chart values, especially the electrical power, current, and voltage, will fluctuate due to random influences such as the behavior of the electric arcs. In further work, a stochastic element could be added to introduce such fluctuations during simulations and produce more realistic behavior. Furthermore, it may be necessary to adjust the density of the scrap for each basket separately to better reproduce the behavior seen in real-life operation of the furnace.

In addition, further improvement of the underlying process model itself is of course possible. For example, a better representation of the melting behavior of scrap and adjustments for the use of other input materials, such as direct reduced iron, hot briquetted iron, or hot metal; improved

description of the foaming of the slag; as well as the automatic adjustment of model parameters for different furnaces are possible areas of further research.

#### **6. Conclusions**

An automatic control and a simulator were developed on the basis of a comprehensive dynamic EAF process model that has previously been validated exhaustively using data from industrial furnaces. The automatic control is capable of reproducing real-live operation charts and adjusting them for different operating conditions. It can be used to evaluate various scenarios such as new control strategies, different materials for injection and charging, or the installation of new equipment. The rules currently used are partially based on parameters that cannot be measured or observed directly in the real-live process, and adjusted rules that are based more closely on the actual parameters used in process control such as total electric energy input and off-gas composition may be more useful for future work. The rules and parameters can be adjusted easily to allow different furnaces and operating strategies to be replicated and evaluated.

The simulator was shown to be stable and fast enough to run simulations based on user input in real-time as well as with higher speeds selected by the user with no noticeable delay apart from a few seconds during initialization after charging. Additional inputs and outputs can be added to make the simulator more versatile. For use in teaching and training, a more refined user interface and documentation would also be useful.

Due to the modular structure of the EAF process model simulations based on measured data, automatic control and simulator input can be run using the same basic model and structure with changes only necessary to the input module and the evaluation and output of the results, with the flexible solver algorithm allowing for stable and fast simulations for all cases. The model can therefore be used to evaluate the current state of the process and identify potential for improvement, to create and assess different scenarios and operating strategies, and for training and teaching using the simulator.

**Author Contributions:** Conceptualization, methodology, software, and writing—original draft preparation by T.H.; supervision, project administration, and writing—review and editing by T.E.; writing—review and editing by V.-V.V.

**Funding:** This research received no external funding

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**3. Conventional and RR genetically modified soybeans: Some results in Brazil**

#### **3.1 Physiological quality and lignin content in the seed coats submitted to different harvest times**

The viability period of the soybean seed is extremely variable, depending both on genetic characteristics and environmental effects during the phases of development, harvest, processing and storage. Once unfavorable conditions occur in some of these phases, physiological damages may result in losses to seed quality, with the intensity of these damages varying with the genetic factors intrinsic to each cultivar. Various researchers have emphasized the possibility of use of the seed with seed coat with a certain degree of impermeability to water as an alternative for avoiding loss of quality in the field (Gilioli & França Neto, 1982; Peske & Pereira, 1983; Hartwig & Potts, 1987), with delay in harvest and determination of the lignin content in the seed coat being methodologies suggested for genetic breeding programs for evaluation of the quality of soybean seeds (França Neto & Krzyzanowski, 2003).

Within this context, the work presented below (Gris et al., 2010) was conducted with the purpose of evaluating the physiological quality and lignin content in the seed coat of the conventional and RR soybean seeds collected at three different times in Lavras (MG), Brazil. Thus, the seeds of ten cultivars collected at stages R7, R8 and 20 days of harvest delay (R8+20) were submitted to tests for evaluation of physiological quality and lignin content. Harvest stages were determined according to Fehr & Caviness (1977).

We observed differences in the physiological quality of seeds among the different harvest times for the cultivars BRS 134, BRS 247 RR, Conquista, Jataí and Silvânia RR, with reduction in viability with harvest delay (R8 + 20). In a similar way, when submitted to accelerated aging, the seeds of the cultivars BRS 245 RR, BRS 134, BRS Jataí and Silvânia RR also underwent a reduction in vigor with harvest delay (Table 1). Braccini et al. (2003), studying the response of 15 genotypes of soybeans to harvest delay, also observed a significant reduction in germination percentage and vigor of seeds when they were submitted to harvest 30 days after the R8 stage of development.

Means followed by the same letter in the line for each determination do not differ among themselves by the Scott-Knott test at the 5% significance level.

Table 1. Means of the germination and accelerated aging test (% of normal seedlings) from seeds of soybean cultivars and their respective RR genetically modified forms, 2007/08 harvest. UFLA, Lavras, MG, Brazil.

We observed that the greatest decreases in seed vigor by the accelerated aging test (Table 1), when the harvest delay and the mean of stages R7 and R8 are contrasted, occurred for the

impermeability to water as an alternative for avoiding loss of quality in the field (Gilioli & França Neto, 1982; Peske & Pereira, 1983; Hartwig & Potts, 1987), with delay in harvest and determination of the lignin content in the seed coat being methodologies suggested for genetic breeding programs for evaluation of the quality of soybean seeds (França Neto &

Within this context, the work presented below (Gris et al., 2010) was conducted with the purpose of evaluating the physiological quality and lignin content in the seed coat of the conventional and RR soybean seeds collected at three different times in Lavras (MG), Brazil. Thus, the seeds of ten cultivars collected at stages R7, R8 and 20 days of harvest delay (R8+20) were submitted to tests for evaluation of physiological quality and lignin content.

We observed differences in the physiological quality of seeds among the different harvest times for the cultivars BRS 134, BRS 247 RR, Conquista, Jataí and Silvânia RR, with reduction in viability with harvest delay (R8 + 20). In a similar way, when submitted to accelerated aging, the seeds of the cultivars BRS 245 RR, BRS 134, BRS Jataí and Silvânia RR also underwent a reduction in vigor with harvest delay (Table 1). Braccini et al. (2003), studying the response of 15 genotypes of soybeans to harvest delay, also observed a significant reduction in germination percentage and vigor of seeds when they were

Celeste 94.75a 96.50a 95.50a 94.75a 97.50a 91.50a 77.01a 82.42a 94.76a Baliza RR 94.25a 93.00a 91.00a 91.50a 88.50a 84.00a 83.47b 90.86b 118.01a BRS 133 91.25a 93.00a 88.00a 91.25a 96.50a 87.50a 93.66b 82.61b 107.15a

RR 91.75a 96.50a 90.50a 97.75a 99.50a 87.50b 94.79a 99.11a 97.37a BRS 134 91.75a 90.50a 79.00b 94.00a 95.00a 75.50b 86.81a 87.02a 97.65a

RR 96.50a 98.50a 87.50b 94.00a 96.00a 87.00a 76.38a 85.18b 102.44b Conquista 85.75a 90.00a 78.00b 89.75a 88.50a 84.00a 93.87b 85.23b 118.25a Valiosa RR 89.75a 83.00a 84.50a 87.50a 92.00a 87.50a 98.15b 90.01b 112.56a Jataí 91.50a 89.00a 76.50b 93.25a 87.00a 64.00b 83.42b 88.43b 152.70a

RR 93.00a 91.50a 82.00b 92.50a 92.00a 71.00b 92.92b 89.61b 143.74a Means followed by the same letter in the line for each determination do not differ among themselves by

Table 1. Means of the germination and accelerated aging test (% of normal seedlings) from seeds of soybean cultivars and their respective RR genetically modified forms, 2007/08

We observed that the greatest decreases in seed vigor by the accelerated aging test (Table 1), when the harvest delay and the mean of stages R7 and R8 are contrasted, occurred for the

Germination Accelerated Aging Electrical Conductivity R7 R8 R8 + 20 R7 R8 R8 + 20 R7 R8 R8 + 20

Harvest stages were determined according to Fehr & Caviness (1977).

submitted to harvest 30 days after the R8 stage of development.

Krzyzanowski, 2003).

Cultivars

BRS 245

BRS 247

Silvânia

the Scott-Knott test at the 5% significance level.

harvest. UFLA, Lavras, MG, Brazil.

cultivars Jataí and Silvânia RR, which presented, on average, losses in vigor of 40.82% and 29.93% respectively, indicating that not always cultivars that have high seed quality when collected near physiological maturity have greater tolerance to deterioration with delay of harvest. And, moreover, the greatest values of electrical conductivity were observed for the majority of seeds of the cultivars collected 20 days after the R8 stage, with exception of the cultivar BRS 247 RR, in which reduction in seed vigor was observed as of the R8 stage, and of the cultivars Celeste, BRS 245 RR and BRS 134 that did not undergo any alterations with the time of harvest.

As degradation of the cellular membranes is constituted hypothetically in the first event of the deterioration process (Delouche & Baskin, 1973), tests that evaluate membrane integrity, such as the electrical conductivity test, would theoretically be the most sensitive for estimating seed vigor, which is in agreement with the results obtained in this study, in which said test stood out in detecting differences of viability between the harvest times in seven of the ten cultivars evaluated. We emphasize that the electrical conductivity values observed in this study were situated from 77.01 μS cm-1 g-1 to 98.15 μS cm-1 g-1 for the R7 harvest time, 82.42 μS.cm-1.g-1 to 99.11 μS.cm-1.g-1 for the R8 harvest time and 94.76 μS.cm-1.g-1 and 152.70 μS.cm-1.g-1 for the 20 days after R8, values which demonstrate the growing trend of leachates released by the seeds with delay in harvest.

When we analyze the percentage of mechanical damage in seeds (Table 2), we observe the greatest values with delay of harvest for the cultivars Conquista (12.5%), Jataí (16.0%) and Silvânia RR (15.0%), which was not observed for the other cultivars studied. In addition, we also observed that by the germination test of seeds submitted to the water immersion test, three of the ten cultivars evaluated were differentiated in regard to the percentage of normal seedlings, however, with distinct responses. The lowest germination values when collected in R8 were observed in seeds of the cultivar BRS 245 RR; in those of the cultivar BRS 247 RR there was a reduction in germination when collected in R8 and R8 + 20; and finally in those of the cultivar Silvânia RR the lowest germinative power was verified when collected in R7 and R8. Various authors emphasize that soybean cultivars and lines behave differently in regard to degree of tolerance to delay of harvest (Lin & Severo, 1982; Rocha, 1982; Boldt, 1984), indicating that this trait may influence maintenance of the physiological quality of the seeds.

For the lignin content in the soybean seed coat, we can observe greater lignin content in the seed coat of seeds collected in the R7 and R8 + 20 stages, as well as for the cultivar Silvânia RR, when contrasted with its conventional version Jataí (Table 3).

When we observe the data of percentage of deformed abnormal seedlings, characterized by root curling, typical of damage by rapid imbibition, we observe a smaller number of abnormal seedlings due to the greater number of dead seeds with harvest delay. Giurizatto et al. (2003) affirm that the deteriorated seeds imbibe more rapidly and are therefore more prone to greater damage through imbibition, which is in agreement with the results obtained in this study.

According to Alpert & Oliver (2002) the cellular membranes have two main states, one more fluid or "crystalline liquid" and another less fluid or "gel", remaining, when organized, in the crystalline phase. In a dry seed, the membranes are found in the gel phase and therefore do not constitute an efficient barrier to contain the release of solutes. When the seeds are exposed to rapid imbibitions, the water penetrates before the membrane can be reverted to the crystalline liquid phase, with damage occurring to the cells; thus, the transition between these two phases in the configuration of the membrane constitutes the fundamental cause of possible injuries during imbibition of seeds, which makes the study of the role of lignin in the seed coat even more important.

Means followed by the same letter in the line for each determination do not differ among themselves by the Scott-Knott test at the 5% significance level.

Table 2. Means obtained for mechanical damage (%) and germination after water immersion (% of normal seedlings and abnormal curled) of soybean cultivar seeds and their genetically modified RR forms, 2007/08 harvest. UFLA, Lavras, MG, Brazil.

These differences observed for the lignin content among the harvest times are not biologically explainable, having possibly been detected due to the low coefficient of variation (CV) obtained for this variable. When we analyze the sole significant contrast, for its part, the genetically modified cultivar Silvânia RR presented greater lignin content in the seed coat than its respective conventional cultivar Jataí. Nevertheless, as an isolated fact, among the five RR combinations versus the conventional versions tested, in our view it does not justify a greater inference regarding pleiotropy of the RR transgene.

Means followed by the same letter in the column do not differ among themselves by the Scott-Knott test at the 5% significance level.

Table 3. Means with a significant difference obtained for lignin content in the soybean seed coat (%), 2007/08 harvest, UFLA, Lavras, MG, Brazil.

In general, we can conclude that in spite of there being behavioral differences in regard to tolerance to harvest delay among the different cultivars evaluated, we did not observe consistent results in regard to a comparison of the RR versus conventional cultivar, not indicating, for the conditions of this test, any sign of pleiotropy.

Celeste 3.50a 2.50a 3.00a 62.50a 70.50a 62.00a 20.00a 14.50a 11.00a Baliza RR 3.00a 3.00a 6.00a 50.00a 46.50a 44.50a 17.50a 24.00a 12.00a BRS 133 3.00a 1.00a 2.00a 55.00a 49.50a 43.50a 14.50b 26.00a 17.00b BRS 245 RR 2.50a 2.50a 5.00a 46.00a 22.50b 43.50a 19.00a 17.00b 32.00b BRS 134 1.50a 1.50a 1.00a 51.00a 47.50a 36.00a 26.00a 26.50a 23.00a BRS 247 RR 1.50a 1.00a 3.50a 63.00a 50.50b 41.00b 15.50b 32.00a 23.00b Conquista 6.00b 4.50b 12.50a 38.00a 33.00a 35.00a 10.50a 6.00a 1.00a Valiosa RR 5.50a 4.50a 5.50a 35.50a 25.50a 36.00a 9.00a 4.50a 3.50a Jataí 2.50b 3.50b 16.00a 20.50a 29.50a 26.00a 32.00a 41.50a 2.50b Silvânia RR 4.50b 5.00b 15.00a 21.50b 28.50b 40.50a 30.00a 26.00a 1.50b Means followed by the same letter in the line for each determination do not differ among themselves by

Table 2. Means obtained for mechanical damage (%) and germination after water immersion (% of normal seedlings and abnormal curled) of soybean cultivar seeds and their genetically

These differences observed for the lignin content among the harvest times are not biologically explainable, having possibly been detected due to the low coefficient of variation (CV) obtained for this variable. When we analyze the sole significant contrast, for its part, the genetically modified cultivar Silvânia RR presented greater lignin content in the seed coat than its respective conventional cultivar Jataí. Nevertheless, as an isolated fact, among the five RR combinations versus the conventional versions tested, in our view it does

Lignin Content Harvest Stages Cultivars R7 R8 R8 + 20 Jataí Silvânia RR 0.2685a 0.2385b 0.2615a 0.3008b 0.4167a Means followed by the same letter in the column do not differ among themselves by the Scott-Knott test

Table 3. Means with a significant difference obtained for lignin content in the soybean seed

In general, we can conclude that in spite of there being behavioral differences in regard to tolerance to harvest delay among the different cultivars evaluated, we did not observe consistent results in regard to a comparison of the RR versus conventional cultivar, not

Germination after Immersion Normal seedlings

R7 R8 R8 + 20 R7 R8 R8 + 20 R7 R8 R8 + 20

Germination after Immersion Abnormal curled seedlings

Cultivars

Mechanical Damage

the Scott-Knott test at the 5% significance level.

at the 5% significance level.

modified RR forms, 2007/08 harvest. UFLA, Lavras, MG, Brazil.

not justify a greater inference regarding pleiotropy of the RR transgene.

coat (%), 2007/08 harvest, UFLA, Lavras, MG, Brazil.

indicating, for the conditions of this test, any sign of pleiotropy.

#### **3.2 Physiological quality and lignin content in the plants submitted to spraying with glyphosate**

Glyphosate (N-phosphonomethyl glycine) is one of the most used herbicides in weed control throughout the world, making up nearly 12% of global herbicide sales and presenting more than 150 commercial brands (Kruse et al., 2000). The emergence of RR genetically modified soybeans increased the use of this molecule in soybeans crops in a considerable way and, along with this, also the environmental concern due to exclusive and indiscriminate use of this herbicide.

According to Sanino et al. (1999), although pesticides (especially glyphosate) may have a beneficial effect on agricultural productivity, the potential risk of these chemical compounds in the environment must be considered, which makes greater studies regarding the behavior of glyphosate under tropical conditions relevant. Within this context we aimed to evaluate the physiological quality of genetically modified RR soybean seeds and the lignin contents of plants submitted to spraying with glyphosate herbicide (Gris, 2009).

In Tables 4 and 5 we present the mean results for the variables analyzed when the soybean plants were submitted to spraying with glyphosate herbicide and water (greenhouse test) and spraying with glyphosate herbicide or manual weeding (field test) respectively.

Means followed by the same letter in the line for each determination do not differ among themselves by the Scott-Knott test at the 5% significance level.

Table 4. Means of germination and Accelerated aging (% of normal seedlings) and lignin content in the seed coat (%) of genetically modified RR soybean seeds submitted to spraying with water and glyphosate herbicide, 2007/08 harvest, Lavras, MG, Brazil, greenhouse test.

We observe that application of the glyphosate herbicide did not alter the physiological quality of the soybean seeds nor the lignin contents in the seed coat and in the plant for the two tests evaluated. These results are not in agreement with those obtained by Sanino et al. (1999), who studying the effect of application of glyphosate herbicide in soybeans observed, in a general way, reduction in the physiological quality of the RR seeds, as well as considerable reduction in activity of the enzyme α-amylase in terms of time. It is worth emphasizing that such a study was carried out comparing only 2 soybean cultivars, one conventional and one genetically modified RR variety, and that the two did not represent the same genotype, since they originated from different parentages.

In this study (Gris, 2009) we obtained a significant response only for the interaction cultivar versus treatments, when the values of electrical conductivity of the seeds produced in the field test were evaluated (Table 5), in which we observed that seeds of the cultivars Baliza RR and BRS 247 RR had their values reduced and increased respectively when the same spraying was performed. Such a differential response may possibly be explained by the different capacity of the genes inserted in the RR cultivars in expressing tolerance to the glyphosate herbicide, which according to Lacerda & Matallo (2008) may or may not occur in a homogeneous manner among cultivars and even within the same cultivar, as well as other factors inherent to the genetics of each cultivar.

Means followed by the same letter in the line for each determination do not differ among themselves by the Scott-Knott test at the 5% significance level.

Table 5. Means of germination and Accelerated aging (% of normal seedlings), Mechanical damage (%), Emergence speed index – ESI (days), Electrical conductivity (µS.cm-1.g-1), Lignin content in the seed coat, pod and stem (%) of genetically modified RR soybean cultivars submitted to manual weeding and spraying with glyphosate herbicide, 2007/08 harvest, Lavras, MG, Brazil, field test.

It is worth emphasizing that since degradation of the cellular membranes is constituted hypothetically in the first event of the deterioration process (Delouche & Baskin, 1973), tests such as electrical conductivity that evaluate membrane integrity are theoretically most sensitive for estimating seed vigor, which possibly, allied with the affirmations of Lacerda & Matallo (2008), would explain the alterations only in the conductivity values.

The absence of a significant response for treatments with weeding and spraying with the glyphosate herbicide indicate that in a general way they did not influence the physiological quality of the seeds, nor the lignin content in the soybean plants. According to Cole & Cerdeira (1982) the blocking of the shikimate pathway due to the action of the glyphosate leads to the accumulation of shikimic acid with many physiological and ecological implications, which, according to Duke & Hoagland (1985) and Becerril et al. (1989), may result in synthesis of indol acetic acid of other plant hormones, chlorophyll synthesis, phytoalexin and lignin synthesis and protein synthesis, and affect photosynthesis, respiration, transpiration, permeability of membranes and other factors.

In addition, other studies have shown that applications of glyphosate in crops interfere in nutrient absorption, increase pests and diseases, reducing crop vigor and yield (Antoniou et al., 2010). According to compilation of data made by these authors, glyphosate reduces nutrient absorption by plants, immobilizing trace elements such as iron and manganese in the soil, as well as avoiding their transport from the roots to the above ground part

Baliza RR 93.50a 96.50a 88.00a 90.50a 0.75a 0.75a 7.07a 7.14a BRS 245 RR 89.00a 93.50a 97.00a 95.50a 1.00a 0.00a 7.24a 7.12a BRS 247 RR 96.50a 97.75a 94.50a 91.50a 0.75a 1.75a 7.19a 7.07a Silvânia RR 93.00a 93.00a 87.00a 88.00a 3.00a 2.00a 7.44a 7.00a Valiosa RR 86.00a 89.00a 84.50a 83.50a 2.00a 2.25a 7.42a 7.39a

Baliza RR 61.0a 48.0b 0.24a 0.23a 8.66a 8.09a 12.71a 13.61a BRS 245 RR 69.0a 70.0a 0.19a 0.19a 8.41a 7.81a 12.20a 13.07a BRS 247 RR 52.0b 70.0a 0.20a 0.20a 9.26a 8.57a 13.43a 12.91a Silvânia RR 69.0a 69.0a 0.29a 0.30a 9.61a 9.03a 20.23a 18.50a Valiosa RR 46.0a 40.0a 0.27a 0.30a 7.87a 7.70a 14.62a 13.35a Means followed by the same letter in the line for each determination do not differ among themselves by

Table 5. Means of germination and Accelerated aging (% of normal seedlings), Mechanical damage (%), Emergence speed index – ESI (days), Electrical conductivity (µS.cm-1.g-1), Lignin content in the seed coat, pod and stem (%) of genetically modified RR soybean cultivars submitted to manual weeding and spraying with glyphosate herbicide, 2007/08

It is worth emphasizing that since degradation of the cellular membranes is constituted hypothetically in the first event of the deterioration process (Delouche & Baskin, 1973), tests such as electrical conductivity that evaluate membrane integrity are theoretically most sensitive for estimating seed vigor, which possibly, allied with the affirmations of Lacerda &

The absence of a significant response for treatments with weeding and spraying with the glyphosate herbicide indicate that in a general way they did not influence the physiological quality of the seeds, nor the lignin content in the soybean plants. According to Cole & Cerdeira (1982) the blocking of the shikimate pathway due to the action of the glyphosate leads to the accumulation of shikimic acid with many physiological and ecological implications, which, according to Duke & Hoagland (1985) and Becerril et al. (1989), may result in synthesis of indol acetic acid of other plant hormones, chlorophyll synthesis, phytoalexin and lignin synthesis and protein synthesis, and affect photosynthesis,

In addition, other studies have shown that applications of glyphosate in crops interfere in nutrient absorption, increase pests and diseases, reducing crop vigor and yield (Antoniou et al., 2010). According to compilation of data made by these authors, glyphosate reduces nutrient absorption by plants, immobilizing trace elements such as iron and manganese in the soil, as well as avoiding their transport from the roots to the above ground part

Matallo (2008), would explain the alterations only in the conductivity values.

respiration, transpiration, permeability of membranes and other factors.

Germination Accelerated Aging Mechanical Damage ESI

Electrical Conduct. Seed Coat Lignin Pod Lignin Stem Lignin

Herbicide Weeding Herbicide Weedin

Herbicide Weeding Herbicide Weedin Herbicide

g

Herbicide

Cultivars

Cultivars

Weeding Herbicide Weedin

Weeding Herbicide Weedin

the Scott-Knott test at the 5% significance level.

harvest, Lavras, MG, Brazil, field test.

g

g

(Strautman, 2007). As a result, RR soybean plants treated with glyphosate have lower levels of manganese and other nutrients and reduction in growth of budding and roots (Zobiole et al., 2010). It is worth emphasizing that the seeds produced in the two tests described in this secondary heading are being tested in regard to variation in chemical composition, data which should soon be published.

Both in the field test and in the greenhouse test, it was not possible to relate physiological quality of the seeds and lignin content in their seed coat. We observed significant differences only among the cultivars evaluated, which presented different responses when submitted to the different vigor tests, as well as lignin content, which was already expected, in terms of the great genetic variability among them.

We conclude from these tests that there is a differential response for the electrical conductivity values of the seeds when the plants of different soybean cultivars are submitted to spraying with the glyphosate herbicide; nevertheless, we did not observe a difference in the lignin contents in the stem, in the pod and in the seed coat of the soybean seeds in the cultivars evaluated when submitted to spraying with the glyphosate herbicide.

#### **3.3 Agronomic characteristics and quality of soybean seeds produced at different times**

It is known that different planting times, influenced by different environmental conditions, may be determining factors for the development of seed deterioration tolerance mechanisms and therefore for the quality of soybean seeds. Considered as a seed deterioration tolerance mechanism, the impermeability of the seed coat, characterized principally by seeds with greater lignin content, hinders water penetration in the seed coat. In a similar way to alterations in the germination process and in manifestation of vigor, in terms of the climate in the seed production phase, environmental conditions may also in some way affect the metabolism and chemical constitution of the seeds.

As we have already seen in this chapter, according to some authors, overproduction of lignin in RR soybean plants may be associated with the presence of water deficit and high temperatures during cropping, indicating that the environmental conditions found in the field during crop development may affect lignin production in the plant in an expressive way.

With this objective, we compared agronomic traits of the plant, physiological quality and seed health and lignin content in the seed coat of RR and conventional seeds produced in different time periods, summer and winter (Gris, 2009), with the determinations: plant height, height of insertion of the first pod and number of pods per plant, weight of 1000 seeds (Brasil, 1992), lignin content in the seed coat (Capeleti et al., 2005), incidence of mechanical damage (Marcos Filho et al., 1987), germination and dry matter of normal seedling from germination (Brasil, 1992), emergence speed index and germination speed index (Edmond & Drapala, 1958), final stand in the seed bed (counting at 24 days after seeding), accelerated aging at 42oC for 72h (Marcos Filho, 1999), electrical conductivity (Vieira, 1994), water immersion test of seeds and seed health, evaluating the infestation percentage (Machado, 2000) and intensity of the inoculums. The data of inoculum density were weighted by the McKinney formula (1923):

$$\Pi(\%) = \frac{\sum \left( \mathbf{F} \times \mathbf{n} \right) \times 100}{\left( N \times M \right)}$$

In which: II = inoculum intensity, F = number of seeds with a determined score, n = score observed, N = total number of seeds evaluated and M = maximum score of the scale.

In Table 6, we present a summary of the mean results for the variables in which the contrasts (RR cultivar versus conventional cultivar) presented a significant difference, for both harvests, in which among all the characteristics evaluated, significant results for the contrasts evaluated were few.

For the electrical conductivity test, we observed a greater value for the conventional cultivar Jataí (76.54 µS.cm-1.g-1) when compared to the cultivar Silvânia RR (100.25 µS.cm-1.g-1). According to Vieira & Krzyzanowski (1999) for lots of high vigor soybean seeds, the standard conductivity values should be situated at most up to 70-80 µS.cm-1.g-1, however with a strong trend to present medium vigor. Nevertheless, in spite of the high value of electrical conductivity observed in seeds of the cultivar Silvânia RR, we did not observe differences between the two cultivars in the germination and vigor tests, which, according to José et al. (2004), may indicate that there are cultivars with greater efficiency in membrane reorganization, not resulting in damages, strictly speaking.

Capital letters followed by the same letter in the line do not differ among themselves by the Scheffe Test, at the 5% significance level.

Table 6. Mean values for some variables in which the contrasts between the conventional soybean cultivar and its genetically modified RR version presented significance, summer and winter harvest, Lavras, MG, Brazil.

Panobianco (1997) upon reporting variation in electrical conductivity of soybean seeds and the lignin content in their seed coat affirms that the genotype may alter the electrical conductivity for seeds with the same standard of physiological quality. Nevertheless, we did not observe significant differences between the cultivars Jataí and Silvânia RR in regard to lignin content in the seed coat, indicating that, in this case, it may not have been responsible for the variation in electrical conductivity observed. In the same way, it was not possible to relate the difference in the lignin contents in the seed coat, observed between the cultivars Celeste (0.20%) and Baliza RR (0.26%), and the results of physiological quality of the two, produced in the summer harvest, since they differed only for this characteristic. It is worth highlighting that in spite of the differences found for these two cultivars, it was not possible through the incidence of mechanical damage to detect any differences between the cultivars studied.

Upon observing the contrasts established between the RR and conventional cultivars, we can infer that the cultivars Jataí and Silvânia RR presented the greatest number of significant differences among the variable studied (Table 6), not only in relation to the physiological quality of the seeds, but also in regard to agronomic traits, such as plant height and number of pods per plant.

In Table 6, we present a summary of the mean results for the variables in which the contrasts (RR cultivar versus conventional cultivar) presented a significant difference, for both harvests, in which among all the characteristics evaluated, significant results for the

For the electrical conductivity test, we observed a greater value for the conventional cultivar Jataí (76.54 µS.cm-1.g-1) when compared to the cultivar Silvânia RR (100.25 µS.cm-1.g-1). According to Vieira & Krzyzanowski (1999) for lots of high vigor soybean seeds, the standard conductivity values should be situated at most up to 70-80 µS.cm-1.g-1, however with a strong trend to present medium vigor. Nevertheless, in spite of the high value of electrical conductivity observed in seeds of the cultivar Silvânia RR, we did not observe differences between the two cultivars in the germination and vigor tests, which, according to José et al. (2004), may indicate that there are cultivars with greater efficiency in membrane

Variables Means – Summer 2006/07 harvest

Variables Means – Winter 2007 harvest

Capital letters followed by the same letter in the line do not differ among themselves by the Scheffe

Table 6. Mean values for some variables in which the contrasts between the conventional soybean cultivar and its genetically modified RR version presented significance, summer

Panobianco (1997) upon reporting variation in electrical conductivity of soybean seeds and the lignin content in their seed coat affirms that the genotype may alter the electrical conductivity for seeds with the same standard of physiological quality. Nevertheless, we did not observe significant differences between the cultivars Jataí and Silvânia RR in regard to lignin content in the seed coat, indicating that, in this case, it may not have been responsible for the variation in electrical conductivity observed. In the same way, it was not possible to relate the difference in the lignin contents in the seed coat, observed between the cultivars Celeste (0.20%) and Baliza RR (0.26%), and the results of physiological quality of the two, produced in the summer harvest, since they differed only for this characteristic. It is worth highlighting that in spite of the differences found for these two cultivars, it was not possible through the incidence of

Upon observing the contrasts established between the RR and conventional cultivars, we can infer that the cultivars Jataí and Silvânia RR presented the greatest number of significant differences among the variable studied (Table 6), not only in relation to the physiological quality of the seeds, but also in regard to agronomic traits, such as plant height and number

mechanical damage to detect any differences between the cultivars studied.

Plant height (m) Jataí 1.56 a vs Silvânia RR 1.41 b Number Pods/plant Jataí 110.00 a vs Silvânia RR 57.50 b Germination (%) BRS 133 95.50 a vs BRS 245 RR 87.25 b Weight of 1000 seeds (g) BRS 134 155.50 a vs BRS 247 RR 142.70 b Emergence Speed Index BRS 134 7.16 b vs BRS 247 RR 7.55 a Lignin Seed Coat (%) Celeste 0.20 b vs Baliza RR 0.26 a

(µS.cm-1.g-1) Jataí 76.54 b vs Silvânia RR 100.25 a

contrasts evaluated were few.

Electrical conductivity

Test, at the 5% significance level.

of pods per plant.

and winter harvest, Lavras, MG, Brazil.

reorganization, not resulting in damages, strictly speaking.

When we analyze the mean values of plant height and number of pods per plant, we verify once more that the conventional cultivar Jataí showed superiority to the cultivar Silvânia RR, such that for number of pods/plant, these values were up to 91.3% greater. Nevertheless, it is worth emphasizing that for these two cultivars in field conditions, we observed the greatest variations in regard to the phenological cycle, with greater uniformity in maturation and a shorter cycle, around 10 days, of the conventional cultivar Jataí in relation to the genetically modified RR cultivar. It is fitting to highlight that in spite of the RR cultivars tested in this study being essentially derivatives of the respective conventional cultivars, by means of backcrossings, the genotype of the recurrent genitor is not always recovered, due to number fewer recurrence cycles which may consequently result in variations between both materials. Nevertheless, for these cultivars, there is no information on the number of backcrossing cycles used.

When we evaluate the physiological quality of the seeds by means of the germination test in the summer harvest and of the germination speed index (IGV) in the winter harvest, we do not observe a relationship between the significant results for these variables, with the contrasts BRS 133 versus BRS 245 RR and Conquista versus Valiosa RR being differentiated respectively. For both results, the conventional cultivars showed superiority to the genetically modified RR cultivars, with the conventional cultivar BRS 133, with 95% of normal seedlings, overcoming the cultivar BRS 245 RR, with 87%, by approximately 9.5%, when they were produced in the summer harvest. Nevertheless, by the results in reference to the Emergence Speed Index, we observe a lower value for the genetically modified cultivar BRS 247 RR (7.55 days) in comparison with the conventional cultivar BRS 134 (7.16), which once more shows the inconsistency of data that justify a pleiotropic effect of the RR gene on lignin production.

It is worth emphasizing that in spite of the results found in this study, with exception of the variables Emergence Speed Index and lignin in the seed coat, the RR cultivars stood out in relation to the conventional cultivars; most of the significant contrasts, were seen to be isolated, in only one of the harvests or one of the tests in the midst of various comparisons among physiological quality of the seeds, therefore not indicating substantial differences of quality between the RR and conventional materials.

According to Menezes (2008) the physiological quality of soybean seeds is influenced by the maternal or extra-chromosome effect, just as is the cytoplasmatic inheritance, with the physical characteristics of the seed coat, of maternal origin, not being sole determinants of the physiological quality of the seeds. According to this author, the study of genetic control for seed quality indicates the effect of the general and specific combination capacity, which suggests the presence of additive and non-additive gene effects for physiological quality of soybean seeds. Therefore, the quality of seeds may not be attributed only to their seed coat and consequently to their lignin contents, but also to genes present in the nucleus.

When we analyze the results obtained in the seed health test (Figure 6), we observe that the cultivars BRS 133, BRS 245 RR, BRS 134 and BRS 247 RR presented the lowest percentages of infection and infection indexes (severity), when produced in the summer, indicating that the environmental conditions during the seed maturation period were responsible for seed health quality. In these cultivars a shorter phenological cycle and semi-early maturity was observed, which provided for the maturation period outside of the rainy period.

According to Delouche (1975), the alternating of dry and wet days during the maturation phase until harvest, which occurs with greater facility in the summer, can increase the incidence of diseases in a differentiated way at the end of the cycle of the seeds produced. Within this context, the seed becomes not only an easy target for the action of microorganisms, which considerably reduce its viability, but they also come to be efficient vehicles for dissemination of pathogens (Machado, 2000). This situation may be visualized principally for the cultivars Jataí and Silvânia RR, which remained for a greater period in the field, and presented the greatest percentages of infection, 39% and 38% (Figure 6A), and also the greatest indexes of infection by the pathogen Phomopsis, 35% and 26% (Figure 6B), respectively. It is worth emphasizing that when produced in winter conditions, under a controlled irrigation system, without rains in the seed maturation period, the presence of pathogens was not observed for any seeds.

Fig. 6. Average values for infection percentage (A) and infection indexes - severity (B) in the seed health test of conventional soybeans and the genetically modified RR versions, summer harvest.

In relation to the RR versus conventional contrasts, we observe that in spite of the cultivars tested in this study having their origin in the same genotype by successive backcrossings, when observed in the field, we verified that some presented perceptible cycle variations, maintaining the cultivars Conquista and Celeste for more days in the field in relation to the cultivars Valiosa RR and Baliza RR, respectively; enough so that the first, subjected to rains and high temperatures, presented slightly greater values in the seed health and severity test. In this case, we cannot attribute the differences of RR versus conventional contrast, observed in Figure 6A and 6B, to the effect of the RR transgene, but rather to environmental conditions associated with difference of cycle.

In view of the above, in spite of some authors suggesting the pleiotropic effect of the transgene CP4 EPSPS on lignin overproduction in the plant, it was not possible for us to identify the pleiotropic effect in the cultivars studied in this and in the other studies described here, which indicates that the alterations of lignin content in the plant, observed by those authors under normal climatic conditions, are not due to the fact of the lignin molecule precursors being formed in the shikimic acid pathway. Thus, the sequence CP4 EPSPS, introduced in the genome of commercial soybean cultivars, responsible for the production of the protein CP4 enolpyruvylshikimate-3-phosphate-synthase (EPSPS), an enzyme that participates in the biosynthesis of aromatic amino acids in plants and microorganisms, seems not to be associated with lignin contents in the plant and in the soybean seed coat, and it seems that there are no substantial differences in regard to the agronomic traits and physiological quality of seeds between conventional and genetically modified RR cultivars.


**5. Effects of digestate on crop yield**

On the bases of the plant reaction on the digestate treatment, plants could be classify into the sensitive (alfalfa, sunflower, soybean) and the non-sensitive (winter wheat, triticale,

Among the different organic wastes like compost, biogas residue, sewage sludge and different manures with and without mineral N, the biogas residue was more efficient for promoting the soil microbiological activity. The high amount of easy-degradable carbon increased the substrate induced respiration (SIR), which was enhanced by the higher carbon content resulted from the higher litter and root exudates of higher plant growth. In accordance with these results, the largest proportion of active microorganisms was found in the digestate treated samples (Odlare et al., 2008; Kirchmann, 1991). Similarly, the activity of invertase was significantly higher in the digestate treated samples than that in control ones

Besides the macro- and micronutrient content of digestate which are important not for the crops but for soil microorganisms too, it contains growth promoters and hormones, also. Therefore it could be used for stubble remains to facilitate their decomposing. Makádi et al. (2007) compared the effect of digestate and Phylazonit MC bacterial manure on the growth of silage maize (*Zea mays* L. 'Coralba') as a second crop after winter wheat and on the enzyme activities of soil. Digestate was used at the rate of 50% of the total N demand of silage maize while the Phylazonit MC was used at 5 L ha-1 dose. Their results of the changes

16/08/2006. 27/09/2006

Treatments Enzyme activity (mean±S.D.)

a) Control 5,6181,392a 3,7672,030b b) Phylazonit MC 7,4371,945a 4,0950,901b c) Phylazonit MC+digestate 6,6132,230a 1,5840,748a d) Digestate 6,0241,486a 6,2060,997c

a) Control 1,4680,118b 1,7970,289b b) Phylazonit MC 1,1600,144ab 1,4100,050a c) Phylazonit MC+digestate 0,9830,275a 1,2050,117a d) Digestate 1,9610,395c 1,2880,063a

Table 6. Invertase and catalase activities of soil on the 3rd and 9th week after digestate and Phylazonit MC treatment (*Data from Makádi et al., 2007*). a, b, c indexes mean the different

The maximum of the degradation of disaccharides, indicated by the invertase activity, was found in the 3rd week after Phylazonit MC treatment, while it was found only after the 9th week in the digestate treated soil samples. The Phylazonit MC contains only bacteria and promoting agents of bacterial activity for degrading the soil OM. Contrarily, in the digestate treated samples the degradation of disaccharides takes place at similar rate through 9 weeks because of the OM content of digestate used. Changes in catalase activity indicate the effect

On the bases of the plant reaction on the digestate treatment, plants could be classify into the sensitive (alfalfa, sunflower, soybean) and the non-sensitive (winter wheat, triticale,

(Makádi et al., 2006).

in enzyme activities are summarized in Table 6.

*Invertase activity (mg glucose 1 g-1soil 4 h-1)*

*Catalase activity (mg O2 1 g-1 dry soil 1 h-1)*

statistical groups according to Tukey's test (p<0.05).

**5. Effects of digestate on crop yield**

of nutrient content of digestate to the increasing microbial metabolism.

sweet corn, silage maize) groups. The sensitive plants can be treated by digestate only in their certain life stages, for example, young alfalfa is very sensitive after sowing while old alfalfa is very sensitive before cutting. In the case of sensitive plants the burning effect of digestate can be observed but it follows a strong and quick recovering process. For the nonsensitive plants the digestate can be used in any developmental stage. It is favourable, because for example, in rainy period the digestate technically could not be applied (Makádi et al., 2008).

The right application rate of liquid or solid digestate depends on the plant nitrogen demand. It should be applied when plant N demand arises. This time for non-legume scpecies is the late winter and spring (Stinner et al., 2008). Similarly, Wulf et al. (2006) used 70% of the digestate in spring and 30% in autumn, while Makádi et al. (2008) and Nyord et al. (2008) split into two and three the applied rate through the vegetation period.

Because of its high available nutrient content, digestate application resulted in significantly higher aboveground biomass yields in the case of winter wheat and spring wheat than the farmyard manure and undigested slurry treatment. The effectiveness of a digestate depends on the composition of co-digestied material, the treated plant species and the treatment methodology. Co-digestion of different organic materials results in more effective digestate. (Möller et al., 2008; Stinner et al., 2008).

After the burning effect of digestate the soybean plants recovered and grew more, but lower sprouts. These sprouts were very productive, the number of pods was also higher in the treated samples, therefore the yield and thousand seed weight were also higher (Table 7, Makádi et al., 2006)

Table 7. Yield parameters of soybean after digestate treatment (*Data from Makádi et al., 2008).* a, b, c indexes mean the different statistical groups according to Tukey's test (p<0.05).

These yield parameters are close correlations with some soil parameters changing after digestate amendment. Increasing in important nutrient contents contribute to the better development of plants (Makádi et al., 2008b, Table 8).

Comparing the effect of liquid digestate and the equal quantity of water to the yield of sweet corn and silage maize, significantly higher yields were found in the digestate treatment. In this case the applied digestate on the bases of plants N demand was split into two parts (Makádi et al., 2006). That means that the favourable effects of digestate are caused by its soluble macro- and micronutrient content.

Digestate: A New Nutrient Source – Review 305

respectively. Changes in amino acid composition of test plants were also very favourable, because almost every essential and non-essential amino acid quantity was increased significantly after digestate treatment. In line with these results the oil content of the treated

> Carbohydrate (%)

Straw (100%) 21.56 28.81 10.43 0

biomanure 29.81 20.21 13.73 38.3

biomanure 33.57 21.45 7.96 55.7

biomanure 33.84 21.79 13.93 57.0

contents of mushroom (*Pleurotus sajor caju*). (*Data from Banik and Nandi, 2004*)

Table 10. Effect of supplementation of rice straw with solid digestate on major nutrient

Qi et al (2005) examined the effect of fermented waste as organic manure in cucumber and tomato production in North China. Before the vegetables transplantation, the diluted fermented residual dreg was applied 20-30 cm below the soil surface at a rate of 37,500 kg ha-1, while liquid digestate was sprinkled to the soil surface in three vegetables growing stages and on the vegetable leaves once time. They found increasing yield (18.4% and 17.8%) and vitamin C content (16.6% and 21.5%) of treated cucumber and tomato, respectively.

As the results show, the digestate application in solid or liquid form could result significant improvement in the quality of foods without damaging the environment, which is very

Sustainable recycling of organic wastes demands clear regulations of recycled wastes, the used recycling methods and the controlling of products. These regulation processes for the digestate are different in certain countries, respected the elaboration and the used limits.

In Hungary, the digestate is regarded as other non-hazardous waste if the ingestate does not contain sewage or sewage sludge, while in the presence of these materials the conditions of

In Scotland the BSI PAS110:2010 digestate quality assurance scheme is applied. If a digestate complies with the standards for the quality, the usage criteria and the certification system stated in the worked scheme, the Scottish Environment Protection Agency (SEPA) does not

In Swiss the digestate which suits the limits, can be used as soil conditioner and fertilizer in

In Germany the origin of the input materials determines the quality label of digestate product by biowaste and renewable energy crops. Digestates have to fulfil the minimum quality criteria for liquid and solid types which determine the minimum of nutrients and the

Lipid (%)

Increase of protein over control (%)

plants decreased significantly.

Straw + cowdung

Straw + poultry litter

Straw + jute caddis

Treatments Protein

(%)

important for the sustainable environment and healthy life.

**7. Legislation of digestate utilization in agriculture**

the digestate utilisation depend on the quality of the given material.

apply the waste regulatory control for it.

"bio"-agriculture.

\* Correlation is significant at the 0.05 level; \*\* Correlation is significant at the 0.01 level.

Table 8. Correlations between soil and plant parameters in digestate treatment experiment. (*Data from Makádi et al., 2008b)*

Comparing the effect of digestate and a bacterial manure (Phylazonit MC, the experimental conditions can be found in the section 4.5). The Phylazonit MC treatment increased the green weight of silage maize by 47.18% while the digestate by 142.34%, comparing to the control. The results obtained can be seen in Table 9 (Makádi et al., 2007).

Table 9. Green weight of silage maize as a second crop after digestate and Phylazonit MC treatment of stubble. (*Data from Makádi et al., 2007*). a, b, c indexes mean the different statistical groups according to Tukey's test (p<0.05).

The positive effect of Phylazonit MC treatment was the result of its microbes, plant growth promoters and microelement content, while the favourable effect of digestate treatment was caused by its macro- and microelement and high water content and the increase of soluble macroelement content of soil because of the increased microbial activity.


**Author details**

Qiang Hao, Tingting Liu and Heping Zeng\*

\*Address all correspondence to<EMAIL_ADDRESS>
Shanghai Key Laboratory of Modern Optical System, Engineering Research Center of Optical Instrument and System (Ministry of Education), School of Optical-Electrical and Computer Engineering, University of Shanghai for Science and Technology, Shanghai, China


*3.4. Deactivation Model*

In general, the five intrinsic mechanisms of catalyst decay are poisoning, fouling, thermal degradation, chemical degradation, and mechanical failure [27]. Poisoning and thermal degradation are generally slow and irreversible, while fouling by coke and carbon is rapid and reversible. Generally, one of the main challenges in the acetylene hydrogenation process is catalyst deactivation, by coking and increasing acetylene concentration in the product stream. Typically butene and butadiene, as side products in acetylene hydrogenation, has led to oligomer and green oil formation on the catalyst surface [28]. The adsorbed acetylene and produced 1,3-butadiene react on the catalyst surface and green oil is produced. The deposited oligomers and green oil on the catalyst gradually reduce the catalyst activation during the process run-time [29]. The proposed correlations in the literature, that predict catalyst activity lack accuracy, so applying these activity equations in the model results in a notable error between simulation results and plant data. In this research, a power low decay model, modified by feed concentration, to account for coke formation, is proposed to calculate the catalyst activity. The considered deactivation model is as follows:

$$\frac{da}{dt} = k\_d e^{-(\frac{E\_d}{RT})} \times a^n \times \mathbb{C}^m \tag{22}$$

The proposed decay model is applied in the dynamic model and the available plant data are used to calculate the activity parameters, considering the absolute difference between plant data and simulation results as the objective function.


*Review* **Contemporary Biomarkers in Pulmonary Embolism Diagnosis: Moving beyond D-Dimers**

**Androniki Gkana 1,†, Androniki Papadopoulou 1,†, Maria Mermiri 2,\* , Eleftherios Beltsios 3 , Dimitrios Chatzis 4 , Foteini Malli 5 , Antonis Adamou 1 , Konstantinos Gourgoulianis 5 , Georgios Mavrovounis 1 and Ioannis Pantazopoulos 1**

**Abstract:** Pulmonary embolism (PE) is a rather common cardiovascular disorder constituting one of the major manifestations of venous thromboembolism (VTE). It is associated with high mortality and substantial recurrence rates, and its diagnosis may be challenging, especially in patients with respiratory comorbidities. Therefore, providing a prompt and accurate diagnosis for PE through developing highly sensitive and specific diagnostic algorithms would be of paramount importance. There is sound evidence supporting the use of biomarkers to enhance the diagnosis and predict the recurrence risk in patients with PE. Therefore, several novel biomarkers, such as factor VIII, Ischemia Modified Albumin, and fibrinogen, as well as several MicroRNAs and microparticles, have been investigated for the diagnosis of this clinical entity. The present review targets to comprehensively present the literature regarding the novel diagnostic biomarkers for PE, as well as to discuss the evidence for their use in daily routine.

**Keywords:** pulmonary embolism; diagnosis; D-dimers; biomarkers; ischemia modified albumin; microparticles; microRNAs; Factor VIII

#### **1. Introduction**

Pulmonary embolism (PE) is a relatively common cardiovascular disorder that constitutes one of the two major manifestations of venous thromboembolism (VTE). PE may be associated with high mortality, especially in untreated cases; undiagnosed PE carries a 30% mortality rate, which falls to 8% when diagnosed [1]. PE diagnosis may be challenging, especially in patients with respiratory comorbidities [1]. Therefore, it is crucial to provide a prompt and accurate diagnosis for PE by developing highly sensitive and specific diagnostic algorithms.

The diagnosis of the disease is based on the individual patient's clinical probability for PE combined with laboratory and non-invasive imaging methods, mainly Computed Tomography Pulmonary Angiography (CTPA), which serves as the gold standard [2]. Since PE pathophysiology is rather complex, incorporating both thrombotic and inflammatory components, a plethora of "novel" biomarkers for PE diagnosis is currently under investigation with the ultimate goal to increase diagnostic accuracy, leading to the effective and appropriate management of this potentially lethal clinical entity.

The purpose of the present narrative review is to discuss some of the novel diagnostic biomarkers for PE and illustrate the evidence for their use in everyday clinical practice.

**Citation:** Gkana, A.; Papadopoulou, A.; Mermiri, M.; Beltsios, E.; Chatzis, D.; Malli, F.; Adamou, A.; Gourgoulianis, K.; Mavrovounis, G.; Pantazopoulos, I. Contemporary Biomarkers in Pulmonary Embolism Diagnosis: Moving beyond D-Dimers. *J. Pers. Med.* **2022**, *12*, 1604. https:// doi.org/10.3390/jpm12101604

Academic Editor: Eumorfia Kondili

Received: 13 August 2022 Accepted: 26 September 2022 Published: 29 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Furthermore, we provide an updated review of the contemporary approaches concerning D-dimer testing for the diagnosis of PE.

#### **2. Methods**

To identify relevant articles for the present literature review, we performed an electronic search of the PubMed (MEDLINE), Google Scholar, and Scopus databases. We used the following keywords combined with the Boolean operators "AND" and "OR", as appropriate: "Pulmonary Embolism", "Diagnosis", "D-dimers", "Biomarkers", "Ischemia Modified Albumin", "MicroRNAs", "Fibrinogen", "Factor VIII", and "Microparticles". The results were limited to those written in English. The last literature search was performed on 1 March 2022. Three independent investigators (A.G., A.P., M.M) screened the titles and abstracts to select potentially relevant articles for inclusion. The full text of the selected articles was thoroughly examined, and the studies presenting original data on PE diagnosis and different biomarkers were finally included.

#### **3. Fibrinogen**

Fibrinogen is a large, complex, fibrous glycoprotein, which is converted into fibrin during the coagulation cascade, yielding the fibrin clot for hemostasis [3,4]. Moreover, fibrinogen is produced as an acute phase reactant by the liver in response to inflammation or ischemia. The cleaving of fibrin by plasmin results in the production of D-dimers, which represent the expression of fibrin degradation occurring during the fibrinolytic activity of clot breakdown [5]. Due to its nature as an acute phase reactant, as well as a significant part of the coagulation cascade, the measurement of fibrinogen levels combined with D-dimer levels has been proposed as a valuable diagnostic tool for the diagnosis of PE.

A prospective study assessed the D-dimer and fibrinogen levels in 191 outpatients with suspected PE and observed that patients suffering from PE had a lower fibrinogen and higher D-dimer/fibrinogen (D/F) ratio versus those without PE [6]. The inverse relation of D-dimer and fibrinogen is indicative of activated coagulation leading to fibrinogen consumption and the simultaneous activation of endogenous fibrinolysis, resulting in D-dimer elevation. Moreover, low fibrinogen levels can be explained by the impaired fibrinogen synthesis due to liver congestion caused by right ventricular failure in patients with PE. At the cut-off point of 100% specificity, the true PPV of D/F ratio > 1.04 × 103 was approximately twice as high when compared with D-dimer > 7000 mg/L (57.6% vs. 29.4%). As such, the authors supported that a D/F ratio > 1000 is highly specific for acute PE and might be used as a "rule in" test. [6]. In the same context, Kara et al. [4] demonstrated that patients with PE had q significantly increased D/F ratio compared to controls. However, this change was attributed to increased D-dimer levels, since fibrinogen did not differ between groups. Importantly, the D/F ratio displayed greater specificity than D-dimer levels alone for PE diagnosis (37% vs. 27%, respectively) [4].

Interestingly, in a large Danish study of 77,608 individuals, high fibrinogen levels were observed in patients with PE in combination with DVT; fibrinogen levels ≥ 4.6 g/L were associated with a multivariate-adjusted odds ratio of 2.1 [7]. On the other hand, to make matters more complicated, in a prospective study of 40 PE patients, one-third of the patients had a fibrinogen level out of the normal range and the study did not reveal lower fibrinogen levels in patients with a positive D-dimer test [8].

Another argument that has been raised is whether the D/F ratio may be utilized for the diagnosis of PE in specific clinical settings, such as Intensive Care Unit (ICU) patients. Critically ill patients may have elevated D-dimer and low fibrinogen levels due to several factors, such as infections, malignancies, or severe cardiac or respiratory diseases, rendering them unreliable for the diagnosis of PE [9,10]. In these instances, the D/F ratio may be preferable instead. Indeed, Hajsadeghi et al. [10] suggested that the D/F ratio in ICU patients was significantly higher when PE was present, having almost the same AUC with D-dimer for diagnosing patients with PE (0.710 vs. 0.714 for D/F ratio and D-dimers, respectively), while in contrast, the fibrinogen levels did not differ significantly

(536.73 ± 186.32 vs. 586.33 ± 211.06, *p* = 0.298). More specifically, a D/F cut-off ratio of 0.233 × 10−3 had the highest accuracy in the diagnosis of PE in an ICU setting (sensitivity 70%, specificity 67.1%) [10].

In conclusion, the utility of the D-dimer/fibrinogen ratio as a diagnostic tool for the diagnosis of PE, although promising, remains controversial. The data found in the literature are not conclusive and are derived from small cohorts. One has to take into account that the aforementioned data lack external validation and cannot be safely applied in clinical practice before further larger randomized clinical trials confirm their findings [4,6,10]. Table 1 summarized the available data on the use of fibrinogen in the diagnosis of PE.

**Table 1.** Summary of the available studies on the use of fibrinogen in the diagnosis of PE [4,6,8,10].

#### **4. Ischemia-Modified Albumin (IMA)**

Ischemia-Modified Albumin (IMA) is a molecule formed by the modification of albumin by reactive oxygen radicals [11]. Human serum albumins consist of 585 amino acids and the first 3 amino acids in the N-terminus, Asp-Ala-His, constitute a specific binding site for transition metals, which is susceptible to degradation. IMA is formed through the modification of this protein region due to the effect of reactive oxygen radicals produced by ischemia [12]. Serum IMA levels may increase in several acute conditions, namely, acute coronary syndrome, cardiac arrest, stroke, and mesenteric ischemia [11,13,14].

Published data from small cohort studies suggest that IMA levels are higher in PE patients vs. controls, and thus IMA has been proposed as a potential diagnostic biomarker for PE [15]. Turedi et al. [16] studied 30 PE patients and 30 healthy volunteers and demonstrated a statistically significant increase in serum IMA levels above 0.540 Absorbance Units in 97.7% of PE patients. In line with the aforementioned findings, a serum IMA above 0.4 had a sensitivity of 53.8% and specificity of 89.6% for PE, and the IMA levels were positively correlated with shock index and heart rate but failed to predict RV dysfunction in an experimental animal study [11]. A consequent study suggested that the IMA levels, in combination with clinical probability scores, had similar negative predictive value (NPV) and sensitivity to D-dimer testing [15]. However, IMA had a greater positive predictive value (PPV) compared to D-dimer (79.4% vs. 69.4%) but was not high enough to confirm the diagnosis of PE without additional investigation [15].

Despite that the aforementioned data favor a role of IMA in PE diagnosis, they should be interpreted with caution since they derive from small retrospective cohorts (i.e., patients were not followed-up for subsequent development of PE). The fact that there is no evidence about IMA's role in PE in the last 10 years highlights the need for more studies with greater sample sizes in order to conclude more reliable results. Furthermore, IMA may be elevated in many other conditions (e.g., exercise, congestive heart failure), thus performing multivariate analysis for possible confounders is crucial. Table 2 summarizes the available on the use of IMA in the diagnosis of PE.

**Table 2.** Summary of the available studies on the use of IMA in the diagnosis of PE [12,16].

#### **5. Factor VIII**

Factor VIII (FVIII) is a glycoprotein produced in liver sinusoidal cells and endothelial cells which is essential in the coagulation cascade [17]. Activated Factor VIII (FVIIIa) is derived by limited proteolysis, catalyzed by thrombin or activated factor X. FVIIIa increases the catalytic efficiency of activated factor IX in the activation of factor X. FVIII accelerates, through Xa, the conversion of prothrombin to thrombin, which converts fibrinogen to fibrin. Additionally, high FVIII levels may increase the thrombotic risk by decreasing responsiveness to activated protein C (APC) [18]. Consequently, FVIII plays an important role in the amplification of the clotting cascade at sites of vascular injury [19]. Studies indicate a direct relationship between high plasma levels of FVIII and arterial or venous thrombosis and PE [20–30].

Several studies reported that elevated plasma FVIII levels may represent a significant, independent risk factor for PE in a quantitative dependent pattern [20,21,23,24,26–31]. In 1995, Koster et al. reported the independent quantitative response association between FVIII levels and DVT (*p* < 0.001). In patients with FVIII levels above 1500 IU/L, there was a dose–response relation of FVIII levels with the risk of thrombosis (OR = 4.8) [20]. Other investigators also confirmed this independent and quantitative relationship. Rietveld et al. in 2019 [31], performed a large case–control study that assessed the levels of coagulation factors in relation to the risk for VTE. Their results showed that FVIII levels, as well as Von Willebrand Factor levels, had the strongest association of all coagulation factors with VTE, and this association was found to be independent of BMI, major illness, or CRP levels. The relative risks were similar for patients with provoked and unprovoked (idiopathic) VTE. The relative risks were found to be 15.0 (95% CI 8.6–26.1) for PE, 27.8 (95% CI 16.9–45.8) for DVT, and 43.2 (95% CI 16.6–122.5) for PE with DVT, suggesting that FVIII levels play different roles in the DVT and PE etiology [31]. Payne et al. also supported the independent relationship between FVIII levels and VTE [30]. In addition, it has been shown that Von Willebrand Factor levels correlate with FVIII levels, and this combination elevates, even more, the risk for VTE [24,30,31]. Interestingly, O'Donnell J et al. reported that elevated FVIII:C levels following VTE are persistent and independent of the acute phase reaction [23]. This result was further supported by Sane et al. who measured FVIII levels during PE diagnosis in 63 patients and after a 7-month follow-up period. The levels of FVIII were higher during PE diagnosis compared with the follow-up levels (167.2% vs. 155.1%) but the difference was not statistically significant (*p* = 0.07) [32]. Moreover, Oger et al. demonstrated a quantitative relationship between FVIII levels and VTE, not only in young adults but elderly patients (>70 years old) as well [26].

Regarding PE, Erkekol et al. supported the existence of a quantitative correlation between factor VIII levels and thrombosis, since high plasma levels of FVIII (>168 U/dL) were found in 53.3% (OR 11.04; 95% CI 3.65–33.35) of isolated PE patients and 55.0% (OR 11.81; CI 3.49–39.92) of patients with a combined form of PE and DVT compared with

9.4% in control patients. The risk was not affected after adjustment for other possible risk factors [27]. Heerink et al. found that the levels of FVIII were higher in patients with PE when compared with healthy controls, but the population of PE patients in this study was small (*n* = 11) [33]. In contrast to the previous results pointing to the specificity of FVIII levels, Kamphuisen et al. found high FVIII levels (≥150 IU/dL) at the acute phase in both PE patients and subjects with various etiologic substrates (pneumonia, heart failure, or malignancy) [25].

While several studies have investigated the possible use of FVIII levels as a biomarker for the diagnosis of PE, their importance remains unclear. The aforementioned observations need further confirmation by studies focusing on the potential influence of comorbidities (such as cancer, heart disease, or lung disease) and the acute phase reactions at the FVIII plasma level.

#### **6. MicroRNAs**

MicroRNAs (miRNAs) are non-coding RNAs with a length of approximately 22 nucleotides that are involved in important cellular pathways such as development, proliferation, and apoptosis [34]. They are present in various body fluids, being remarkably stable due to carrier-protein binding [35] and consequently, in recent years, they have been studied as non-invasive biomarkers for various disorders such as cancer, cardiovascular, and cerebrovascular diseases [36,37]. Various miRNAs have been reported to regulate several hemostatic factors (fibrinogen, factor XI, etc.), modulate platelet activation and aggregation, and have been found to be dysregulated in venous thrombosis [38,39].

In 2011, Xiao et al. were the first group that investigated the possibility of identifying some miRNAs as potential biomarkers for the diagnosis of acute PE. Specifically, they reported that miRNA-134 was significantly elevated in patients with acute PE when compared with healthy individuals, reporting a sensitivity of 68.8% and specificity of 68.2% [40]. In 2016, Deng et al. [41] published a systematic review and meta-analysis based on three studies [40,42,43], concluding that, although more research is needed to validate their role as diagnostic biomarkers for acute PE, miRNAs seem to represent reliable novel biomarker candidates (pooled sensitivity: 83%, pooled specificity: 85%). Since then, more original studies have been published [44–46], reporting that various miRNAs are upregulated during acute PE episodes and can be an additional tool available to the physician. An interesting study reported that miRNA-1233 and miRNA-134 can be potentially used to identify patients with acute exacerbation of chronic obstructive pulmonary disease complicated by acute PE [46].

It is important to note that some groups have investigated the possibility of combining various miRNAs with each other and with other established biomarkers such as D-dimers to increase their diagnostic efficacy [45]. Combining miRNA-27a/b with Ddimers significantly increases the capacity for diagnosing acute PE [45]. In more detail, combining miRNA-27a or miRNA-27b with D-dimers resulted in a significant increase in the area under the receiver operating characteristic (ROC) curve, reaching 0.909 and 0.867, respectively.

Collectively, these findings support the hypothesis that miRNAs may serve as a novel biomarker for the diagnosis of acute PE; however, further research in this field has to be performed, as studies up to this date have limited statistical power and reproducibility. Table 3 summarizes the available data on the use of miRNAs in the diagnosis of PE.

**Table 3.** Summary of the available studies on the use of miRNAs in the diagnosis of PE [40,42–46].

#### **7. Microparticles**

Microparticles (MPs) are small vesicles of <1 micron in size, derived from various cell types, including platelets, monocytes, endothelial, and cancer cells [5,47]. MPs are formed from membrane vesicles released from the cell surface by the proteolytic cleavage of the cytoskeleton. MPs provide procoagulant molecules, especially anionic phospholipids (particularly phosphatidylserine) and tissue factor (TF) protein, for the assembly of components of the coagulation cascade [47]. As a result, many studies aimed to investigate the role of MPs in inflammation, cancer, and other diseases [48]. Due to their involvement in the formation of thrombi, MPs have been proposed as potential biomarkers for the diagnosis of VTE and acute PE [48,49].

In a study by Rezania S et al. plasma MP levels were evaluated in PE patients and healthy volunteers using standard fluorescent polystyrene beads. A relative abundance of plasma MPs was noted in the PE patients [50]. Furthermore, there was a correlation between the PE and Platelet-Derived MPs (PDMPs) plasma levels; the PE patients exhibited higher levels of PDMPs as compared to their healthy counterparts [51]. The association between acute PE and procoagulant MPs levels was also highlighted by Bal et al. [52]. Specifically, circulating MPs and platelet MPs were significantly elevated in PE patients compared to healthy controls, but this correlation was not statistically significant when PE

patients were compared with controls with cardiovascular risk factors such as hypertension and diabetes [52].

The correlation between MP levels and cardiovascular diseases, such as hypertension and coronary artery disease, has been previously reported [52,53]. These results highlight the importance of adjusting the measurement of MPs in patients with cardiac comorbidities when PE is suspected.

In conclusion, these studies provide evidence in favor of the clinical use of MPs for diagnosis, but potentially after adjustment for other comorbidities (cancer, cardiovascular diseases) which may also increase the levels of MPs in patients' serum.

#### **8. Other Biomarkers and PE**

There are several more novel biomarkers that have been proposed as potential biomarkers for the diagnosis of PE. Limited data on those biomarkers are available; thus, their use in clinical practice is still considered questionable.

Endothelial cell-specific molecule 1, or endocan, a soluble dermatan sulfate proteoglycan [54], has been associated with PE, predicting the severity of pulmonary artery occlusion [55,56]. Güzel et al. measured serum endocan levels in 46 PE patients and reported a significant difference in the serum endocan levels between PE patients and healthy controls 321.93 vs. 192.77 mg/L (*p* < 0.03) [55]. On the contrary, Mosevoll et al. dispute the diagnostic value of endocan in PE, showcasing that no difference in endocan levels between patients with PE and healthy controls was noted in their study [57]. Thus, the use of endocan as a PE biomarker has to be examined further before definite conclusions can be drawn.

The soluble urokinase-type plasminogen activator receptor (suPAR) is the soluble form of the urokinase-type plasminogen activator receptor, which is a glycosyl-phosphatidylinositol (GPI)-linked membrane protein [58] and an integral part of the fibrinolytic system. SuPAR has also been related to VTE. The incidence of VTE in 5203 subjects was higher in patients with high suPAR levels independently of several potential confounding factors (age, sex, BMI, smoking, systolic blood pressure, cholesterol, HDL, leukocyte count, diabetes, history of atrial fibrillation, history of cardiovascular disease) during a 15.7-year follow-up [58]. However, suPARs' role in PE is still unclear and needs further examination before definite conclusions can be drawn.

C-reactive protein (CRP) is a biomarker of systemic inflammation which has also been evaluated as a diagnostic marker in PE. CRP levels had a sensitivity of 95.7% [95% confidence interval (CI): 90–100] and an NPV of 98.4% (96–100). CRP < 5 mg/L with a clinical probability score indicating 'PE unlikely', had a sensitivity of 96.7% (90–100), specificity of 43.0% (37–49), and NPV 99.1% (97–100) [59]. Moreover, lower CRP levels have been shown to relate to unprovoked (idiopathic) PE [60], and changes in serum high-sensitivity CRP (Hs-CRP) levels could help in the severity categorization of PE (i.e., massive or minor PE) patients, as well as outcome prediction [61]. Thus, it has been proposed that CRP levels could be used to safely exclude PE, either alone or combined with clinical probability assessment [59,61].

A contemporary working hypothesis suggests that proteomic analysis could be used to identify potential biomarkers for PE. According to Granholm F et al. [62], the use of proteomic analysis showed that Complement component 9, Complement factor H, and Leucine-rich α-2-glycoprotein were increased in patients with PE, whereas Carboxylic ester hydrolase, Antithrombin-III, Procollagen C-endopeptidase enhancer, Serpin peptidase inhibitor, clade A, member 4, Afamin, and, *N*-acetylmuramoyl-L-alanine amidase, among others, were decreased. These findings consider proteins of general inflammation, atherosclerosis, and hemostasis as possible biomarkers in the diagnosis of PE while further research is needed to confirm it [62]. In the same context, data acquisition mass spectrometry and antibody microarray studies revealed that serum amyloid A1, calprotectin, and tenascin-C have promising value in PE diagnosis with acceptable sensitivity and specificity [63]. Few data support the use of haptoglobin (a hemoglobin-binding protein that

serves as an acute-phase protein) as a biomarker of PE since its levels are increased in PE patients; the cut-off of 256.74 mg/L had a sensitivity of 62% and specificity of 83% for PE diagnosis [64].

#### **9. Cost-Effectiveness Perspectives**

Another significant parameter regarding the biomarkers in PE diagnosis is the evaluation of cost-effectiveness in health care systems worldwide. In fact, the use of an ageadjusted D-dimer cutoff of <500 ng/mL up to 50 years, then <age × 10 ng/mL, increases the cost savings by more than USD 80 million per year for the United States health care system [65]. Moreover, the D-dimer test and lower-limb compression ultrasonography are not only cost-effective in the diagnosis of PE but also easily available, thus allowing centers devoid of CTPAs to screen patients with suspected PE and avoid costly referrals [66]. On the other hand, novel biomarkers are usually expensive, and in an increasingly cost-conscious health care environment, regulatory approval will not be a guarantee of clinical adoption. Researchers must prove that a specific biomarker can change clinical practice and reduce costs by eliminating time-consuming, expensive, and sometimes ineffective diagnostic tests.

Table 4 summarizes the available data on the use of novel biomarkers in the diagnosis of PE, according to our literature search.

**Table 4.** Summary of the use of novel biomarkers in the diagnosis of PE.

#### **10. Conclusions**

There is evidence supporting the use of biomarkers to enhance the diagnosis and predict the recurrence risk in patients with PE. There are several molecules related to both thrombotic and inflammatory PE-associated processes under investigation. So far, only D-dimer levels have been widely employed for the diagnosis of PE in daily clinical practice and have been implemented in international guidelines for PE diagnosis. This review was conducted in order to shed more light on non-D-dimer biomarkers, such as fibrinogen, IMA, factor VIII, microRNAs, and microparticles, which are present in the current bibliography, even though there are not yet randomized control trials to confirm and advance the existing evidence. Major caveats that should be addressed before the incorporation of biomarkers in clinical decision algorithms include the estimation of optimal cut-off values, the need for adjustment for cofounders (i.e., renal function), and the absence of studies that perform external validation. Further studies addressing the role of the aforementioned candidate biomarkers in PE should be designed to elucidate their importance in the diagnostic algorithm of the disease.

**Author Contributions:** Conceptualization, A.G., A.P., M.M., G.M. and I.P.; methodology E.B., A.A., A.G., A.P., M.M., G.M. and I.P.; writing—original draft preparation, A.G., A.P., M.M., E.B. and G.M.; writing—review and editing, E.B., A.A., D.C., F.M., K.G. and I.P.; supervision, K.G. and I.P.; project administration, I.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** No institutional Review acceptance was needed since this study is a narrative review.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**1. Introduction**

Frailty and Mild Cognitive Impairment are common and inevitable conditions in the elderly citizen population defined as premature or accelerated physical and mental declines. These conditions are often an early indicator of more severe states, such as Alzheimer's disease. Control (delaying or decelerating) of the onset and progression of MCI/frailty is becoming one of the major tasks of global efforts in maintaining the functional independence and quality of life of the globally growing elderly population. In 2016, for the first time in history, estimated majority of the world population can expect to live into their sixties and beyond. Beside the global initiatives, strategies and action plans on healthy ageing conducted by the relevant key organizations such as World Health Organization (WHO), or United Nations (UN), the growing market trend for the so-called "silver economy" sector is booming. The increase of population aged 65 and over is projected to reach 28.1% of the whole population in the EU by 2050, they have a spending power higher than the generation segment aged 18 to 39, and they account for approx. 60% of total expenditures in the US and 50% in the UK, generating demand for new services and products, ranging from personalized care to age-friendly technologies and other solutions that enable the maintenance and prolongation of healthy, independent lives. Technologies and systems supporting innovative ways of influencing people's behavior and lifestyles at all ages also present a significant economic and business opportunity.

Montpellier and Singapore. **The main goal** of this research and City4Age project is to develop a framework for predictive and preventive risk control of Frailty and MCI, as one of the core system infrastructure assets of age-friendly cities. **Specific goals** are development of methods based on smart cities IoT data for early risk detection and enrichment of traditional geriatric instruments. In order to achieve these goals, we are faced with several **challenges**: (1) identification and characterization of temporal behavioral patterns from sensor data, (2) Identification of behavior changes (transitions) and (3) Anomalous behavior and anomalous data detection. Given that IoT data are collected from smart devices in form of unlabeled data streams, for initial behavior variation detection models, unsupervised machine learning techniques have to be employed. From data analytics point of view, behavior can be defined as alternating pattern of sequences of activities. Based on this definition, clustering is identified as natural technique for behavioral pattern recognition, change and anomaly detection. Clustering techniques that allow grouping objects into homogenous groups where objects in the same group are similar (intra-cluster distance is low) and objects between groups are dissimilar (intercluster distance is high). For building cluster models, we employed Hidden Markov models (HMMs), since they allow direct modeling of time series, provide framework for anomaly detection and have high degree of interpretability. Interpretability is very important property for incorporation of data driven models in healthcare practice and integration with domain

Temporal Clustering for Behavior Variation and Anomaly Detection from Data Acquired…

http://dx.doi.org/10.5772/intechopen.75203

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Contributions of this chapter are twofold: (1) we propose a framework for behavior characterization, change and anomaly detection in IoT data in smart cities environment and (2) we provide first experimental evidence of usefulness of data driven modeling of behavioral data

World Health Organization (WHO) had recognized the importance as well as human and economic impact of age-friendly environments and launched the age-friendly Cities and Communities Programme that introduced the terms in 2006/2007, as the foundation initiative aimed for local and metropolitan governance and development levels. The European Commission (EC) supports the pursues of goals and objectives of age-friendly environments and sustainable development by numerous different instruments, primarily through R&D funding programs such as Horizon 2020 or specialized Active and Assistive Living (AAL) Programme. Important higher-level EC initiatives that foster innovation and concentrate

• European Innovation Partnership on Smart Cities and Communities (EIP on SCC), involving almost 400 committed cities and other partners, with a marketplace of specialized ini-

• European Innovation Partnership on active and healthy ageing (EIP on AHA), first established EIP, in 2011, with specialized dedicated groups A3 for Functional decline & frailty,

knowledge of geriatricians.

on collected City4Age IoT data.

stakeholder efforts are the recently established:

and D4 for age-friendly environments, among others.

tiatives, solutions and tools.

**2. State-of-the-art**

Geriatric practice has in this aim utilized different standardized instruments based on traditional data collection methods (administration of questionnaires, meter-based measurement or direct observation in controlled conditions) which are in most cases intrusive and demand citizens' presence in geriatric centers and a lot of time for data collection. More importantly, these methods do not enable real time monitoring of behavioral changes (e.g., data from questionnaires are collected on semi-annual or annual intervals) and thus prevent predictive and preventive interventions. Finally, data collected from such method is often subjective or incomplete.

With the goal to overcome the stated drawbacks**, many technological instruments and methods emerged**, aiming to automate as much as possible the detection and mitigation of behavior deteriorations and anomalies. Particularly, the recent development and expansion of wearable technologies/devices and Internet of Things (IoT) has enabled the build-up of infrastructures of smart devices that collect vast and heterogeneous volumes of various sensory data in smart cities. These social and technological infrastructural advances potentiate the public health and prevention aspects of smart cities, transforming the urban public health from a reactive to a predictive system. In the specific area of support for (active and healthy) ageing, the transformation and progress direction of particular interest is the expansion of concept of **ambient-assisted environments**, from currently predominant implementations in residential and social indoor spaces (homes, elderly care/community centers) to outdoor and public environments. However, there is still a large gap between potential and actual IoT data exploitation because of many challenges that have to be overcome before putting data driven predictive and preventive models in geriatric or healthcare practice [1, 2].

Research presented in this paper is mainly the part of **City4Age project** (www.city4ageproject.eu) that develops age-friendly **Cities and Environments in** deployments in six different prominent pilot smart cities in EU and beyond—Athens, Birmingham, Lecce, Madrid, Montpellier and Singapore. **The main goal** of this research and City4Age project is to develop a framework for predictive and preventive risk control of Frailty and MCI, as one of the core system infrastructure assets of age-friendly cities. **Specific goals** are development of methods based on smart cities IoT data for early risk detection and enrichment of traditional geriatric instruments. In order to achieve these goals, we are faced with several **challenges**: (1) identification and characterization of temporal behavioral patterns from sensor data, (2) Identification of behavior changes (transitions) and (3) Anomalous behavior and anomalous data detection.

Given that IoT data are collected from smart devices in form of unlabeled data streams, for initial behavior variation detection models, unsupervised machine learning techniques have to be employed. From data analytics point of view, behavior can be defined as alternating pattern of sequences of activities. Based on this definition, clustering is identified as natural technique for behavioral pattern recognition, change and anomaly detection. Clustering techniques that allow grouping objects into homogenous groups where objects in the same group are similar (intra-cluster distance is low) and objects between groups are dissimilar (intercluster distance is high). For building cluster models, we employed Hidden Markov models (HMMs), since they allow direct modeling of time series, provide framework for anomaly detection and have high degree of interpretability. Interpretability is very important property for incorporation of data driven models in healthcare practice and integration with domain knowledge of geriatricians.

Contributions of this chapter are twofold: (1) we propose a framework for behavior characterization, change and anomaly detection in IoT data in smart cities environment and (2) we provide first experimental evidence of usefulness of data driven modeling of behavioral data on collected City4Age IoT data.