**6. Genetic diversity and evolution of Fusarium wilt of bananas**

*Fusarium oxysporum* Species Complex (FOSC) are widely distributed and it is mostly non-pathogenic and it is commonly found in roots and soil associated fungus in asymptomatic crop plants. It has been found to be associated with plants as endophyte, saprophyte or just latent in agro-ecosystems [30]. Both, studies on FOSC isolated from non-cultivated species and form cultivated crops have reported a considerable variability based on the morphology of the asexual reproductive structures [31] and latter at the DNA sequence [32, 33]. Understanding its genetic variability is relevant to implement an earlier detection system and implement a proper disease surveillance program.

Recent studies on molecular genetics of *Fusarium* from cultivated plants have shown a high diversity and this variation relays on environmental conditions and are classified in groups and vegetative compatible groups (VCG) as described latter in this chapter. *Fusarium* has evolved heavily depending on its interaction with plant genotypes, such is the case for both 'tropical' and 'subtropical' race 4, which attacks different cultivars, depending of the geographical region [34] as well as for those FOSC from non-cultivated species [30].

Knowledge of the genetic diversity of populations of phytopathogenic fungi and their mode of reproduction are important for the application of management strategies, this with the aim of reducing the impact of the disease [35]. In the case of Foc, this pathogen shows a relatively diverse population genetic structure for a fungus apparently of asexual reproduction and is composed of different evolutionary lineages [33], which has 24 groups of vegetative compatibility (VCGs, VCG0120 to VCG0126 and VCG0128 to VCG01224) distributed worldwide [34, 36–40].

However, in recent samplings in Latin America it was possible to identify 20 new VCGs (new VCG 1 to new VCG 20), these were distributed over the three main clades (clade 1, clade 2 and clade 3), these results show that the majority of the new VCG are grouped in clade 3 and these originate from Latin America [41], this supports the hypothesis on the evolution of Foc, in which it is mentioned that the local populations of *F. oxysporum* evolved and they became pathogenic in the introduced bananas [36, 42–44].

Studying VCGs has been a useful means of subdividing Foc into genetically isolated groups, but it does not, however, measure the genetic relationship between

**135**

*Genetic Diversity of Fusarium Wilt Disease of Banana DOI: http://dx.doi.org/10.5772/intechopen.94158*

the pathogen can potentially reproduce by sexual means.

other molecular tools.

the appearance of Foc-TR4.

considered to be part of Foc [5].

genome B.

the isolates. Furthermore, VCG are phenotypic markers that can undergo a selection process. Direct identification of VCG is a relatively objective, but time-consuming test, and the results indicate genetic similarity rather than genetic difference [31]. Therefore, VCGs represent good phenotypic traits for assessing diversity within populations, but the genetic relationships between VCGs must be assessed using

Fourie and collaborators [39], classified Foc into two clades, clade 1 and clade 2, these based mainly on their evolutionary origins. In the case of clade A, the Foc groups that co-evolved with bananas of genome A belong, while those that belong to clade B evolved with their hosts having genome B or both genome A and

The teleomorph for Foc has never been reported and the pathogen is likely to manifest mutations or parasexualism, as the main basis for its genetic diversity. Although PCR analysis has shown the presence of both MAT idiomorphs, therefore

The race concept has been widely used in the *F. oxysporum* classification system

Three races of Foc are known; but nevertheless, the term race is used in a less formal way in relation to this pathosystem (*Musa*-Foc), since the genetic bases of susceptibility and resistance have not yet been characterized. The Foc races currently described refer to strains of the pathogen, which have been found to be pathogenic to specific cultivars in the field [9, 38]. For example, race (R1) is pathogenic to cultivars of 'Silk', 'Manzano' (AAB) and 'Gros Michel' (AAA). While race 2 (R2) is pathogenic to cooking bananas such as 'Bluggoe' and 'Pear' (ABB) and race 4 (R4) affects all cultivars of the Cavendish subgroup (AAA) and those susceptible to R1 and R2 [4, 5, 45]. Previously, a population of *Fusarium oxysporum* in Central America was considered as race 3 causing wilt in *Heliconia* spp., but is no longer

A Foc race 4 variant was reported in Taiwan affecting Cavendish cultivars in the tropics in 1967 [4, 38]. Therefore, it was necessary to separate the populations that only affected Cavendish cultivars in the subtropics from those populations that affected in the tropics, so two divisions of Foc R4 were generated: race 4 sutropical (STR4) and race 4 tropical (TR4) [38], however, TR4 was pathogenic under tropical and subtropical conditions affecting Cavendish cultivars [25, 39]. In the case of VCGs, they have been associated with STR4 (0120, 01201, 01202, 01209, 01210, 01211, 01215, 0120/15; 0129/11), while only one VCG to TR4 (01213/16) [25, 40]. Visser and collaborators [46], carried out a study on the characterization of tropical Foc race 4 populations affecting 'Cavendish' plantations in South Africa. Only VCG 0120 and idiomorph MAT-2 could be identified, while phylogenetic analysis of the TEF sequence revealed that the isolates from South Africa were pooled with other isolates belonging to VCG 0120 from Australia and Asia. Suggesting, the

introduction and dispersal mainly by infected material within the country.

In Latin America and the Caribbean, the composition of the populations has been limitedly studied. For example, the Cuban populations belong to VCG 01210 (mostly race 1), 0124, 0124/0125 and 0128 (mostly race 2); the isolates did not produce lacinias in K2 medium and the production of volatiles was independent of the race, while in Venezuela VCG 01215 and race 1 are reported. A study using AFLP markers grouped VCG 01210 into a subgroup and showed the presence of common

by plant pathologists. Based on the published data, it can be inferred that the Foc-TR4 isolates recently evolved from the predecessors in Foc-R1. Foc-R1 showed greater phylogenetic diversity than Foc-TR4. Once established, both races apparently co-evolved in the same region, which means that possible horizontal gene transfer could be involved in the high level of diversity seen in Foc-R1, as well as in *Genetic Variation*

Foc has the ability to spread in the soil, which indirectly contaminates in and around plantations, but unfortunately it is also used in nurseries for the propagation of seedlings used for field establishment [25]. Surface waters are easily polluted and use for irrigation of polluted river or pond water is highly risky. In addition, Foc is spread by contaminated tools (shovels, machetes, hoes, etc.), agricultural machinery, clothing and footwear [9, 28]. Any or all of these ways can facilitate the spread of Foc in and around a plantation, and may be possible through other means [6, 28]. Studies carried out in Australia detected TR4 spores in the exoskeletons of the banana weevil (*Cosmopolites sordidus*) and suggested that the insect could be a

The recent transcontinental disseminations of TR4, suggest that something other than vegetative material (suckers) was responsible for these long-distance disseminations. Although these outbreaks may have been the result of something as simple as workers' boots impregnated with soil contaminated by Foc spores from plantations in Southeast Asia, or some other means could be responsible such as the entry of machinery from affected areas. Better knowledge is needed to understand

**6. Genetic diversity and evolution of Fusarium wilt of bananas**

*Fusarium oxysporum* Species Complex (FOSC) are widely distributed and it is mostly non-pathogenic and it is commonly found in roots and soil associated fungus in asymptomatic crop plants. It has been found to be associated with plants as endophyte, saprophyte or just latent in agro-ecosystems [30]. Both, studies on FOSC isolated from non-cultivated species and form cultivated crops have reported a considerable variability based on the morphology of the asexual reproductive structures [31] and latter at the DNA sequence [32, 33]. Understanding its genetic variability is relevant to implement an earlier detection system and implement a

Recent studies on molecular genetics of *Fusarium* from cultivated plants have shown a high diversity and this variation relays on environmental conditions and are classified in groups and vegetative compatible groups (VCG) as described latter in this chapter. *Fusarium* has evolved heavily depending on its interaction with plant genotypes, such is the case for both 'tropical' and 'subtropical' race 4, which attacks different cultivars, depending of the geographical region [34] as well as for those

Knowledge of the genetic diversity of populations of phytopathogenic fungi and their mode of reproduction are important for the application of management strategies, this with the aim of reducing the impact of the disease [35]. In the case of Foc, this pathogen shows a relatively diverse population genetic structure for a fungus apparently of asexual reproduction and is composed of different evolutionary lineages [33], which has 24 groups of vegetative compatibility (VCGs, VCG0120 to

However, in recent samplings in Latin America it was possible to identify 20 new VCGs (new VCG 1 to new VCG 20), these were distributed over the three main clades (clade 1, clade 2 and clade 3), these results show that the majority of the new VCG are grouped in clade 3 and these originate from Latin America [41], this supports the hypothesis on the evolution of Foc, in which it is mentioned that the local populations of *F. oxysporum* evolved and they became pathogenic in the introduced

Studying VCGs has been a useful means of subdividing Foc into genetically isolated groups, but it does not, however, measure the genetic relationship between

VCG0126 and VCG0128 to VCG01224) distributed worldwide [34, 36–40].

predisposing agent as a vector of the disease [29].

the long-distance spread of this pathogen [6].

proper disease surveillance program.

FOSC from non-cultivated species [30].

**134**

bananas [36, 42–44].

the isolates. Furthermore, VCG are phenotypic markers that can undergo a selection process. Direct identification of VCG is a relatively objective, but time-consuming test, and the results indicate genetic similarity rather than genetic difference [31]. Therefore, VCGs represent good phenotypic traits for assessing diversity within populations, but the genetic relationships between VCGs must be assessed using other molecular tools.

Fourie and collaborators [39], classified Foc into two clades, clade 1 and clade 2, these based mainly on their evolutionary origins. In the case of clade A, the Foc groups that co-evolved with bananas of genome A belong, while those that belong to clade B evolved with their hosts having genome B or both genome A and genome B.

The teleomorph for Foc has never been reported and the pathogen is likely to manifest mutations or parasexualism, as the main basis for its genetic diversity. Although PCR analysis has shown the presence of both MAT idiomorphs, therefore the pathogen can potentially reproduce by sexual means.

The race concept has been widely used in the *F. oxysporum* classification system by plant pathologists. Based on the published data, it can be inferred that the Foc-TR4 isolates recently evolved from the predecessors in Foc-R1. Foc-R1 showed greater phylogenetic diversity than Foc-TR4. Once established, both races apparently co-evolved in the same region, which means that possible horizontal gene transfer could be involved in the high level of diversity seen in Foc-R1, as well as in the appearance of Foc-TR4.

Three races of Foc are known; but nevertheless, the term race is used in a less formal way in relation to this pathosystem (*Musa*-Foc), since the genetic bases of susceptibility and resistance have not yet been characterized. The Foc races currently described refer to strains of the pathogen, which have been found to be pathogenic to specific cultivars in the field [9, 38]. For example, race (R1) is pathogenic to cultivars of 'Silk', 'Manzano' (AAB) and 'Gros Michel' (AAA). While race 2 (R2) is pathogenic to cooking bananas such as 'Bluggoe' and 'Pear' (ABB) and race 4 (R4) affects all cultivars of the Cavendish subgroup (AAA) and those susceptible to R1 and R2 [4, 5, 45]. Previously, a population of *Fusarium oxysporum* in Central America was considered as race 3 causing wilt in *Heliconia* spp., but is no longer considered to be part of Foc [5].

A Foc race 4 variant was reported in Taiwan affecting Cavendish cultivars in the tropics in 1967 [4, 38]. Therefore, it was necessary to separate the populations that only affected Cavendish cultivars in the subtropics from those populations that affected in the tropics, so two divisions of Foc R4 were generated: race 4 sutropical (STR4) and race 4 tropical (TR4) [38], however, TR4 was pathogenic under tropical and subtropical conditions affecting Cavendish cultivars [25, 39]. In the case of VCGs, they have been associated with STR4 (0120, 01201, 01202, 01209, 01210, 01211, 01215, 0120/15; 0129/11), while only one VCG to TR4 (01213/16) [25, 40].

Visser and collaborators [46], carried out a study on the characterization of tropical Foc race 4 populations affecting 'Cavendish' plantations in South Africa. Only VCG 0120 and idiomorph MAT-2 could be identified, while phylogenetic analysis of the TEF sequence revealed that the isolates from South Africa were pooled with other isolates belonging to VCG 0120 from Australia and Asia. Suggesting, the introduction and dispersal mainly by infected material within the country.

In Latin America and the Caribbean, the composition of the populations has been limitedly studied. For example, the Cuban populations belong to VCG 01210 (mostly race 1), 0124, 0124/0125 and 0128 (mostly race 2); the isolates did not produce lacinias in K2 medium and the production of volatiles was independent of the race, while in Venezuela VCG 01215 and race 1 are reported. A study using AFLP markers grouped VCG 01210 into a subgroup and showed the presence of common

alleles with VCG 0124 [47]. On the other hand, the pathogenicity studies with representative isolates of each VCG in Cuba, showed a differentiated aggressiveness on different clones between VCG 0124 and 0128, belonging to race 2, indicating lack of genetic sense in the racial classification. It is required to determine in Latin America and the Caribbean the VCG present in the different countries and the pathogenic relationships between them.

In order to better understand how races 1 and 4 are related, genome and transcriptome analysis of *F. oxysporum* f. sp. *cubense* has shown common sequences of single-copy genes from Race 1 and Race 4, showing that there is a close relationship and suggesting that they share a common ancestor. Furthermore, a comparative genomics study among *F. oxysporum* f. sp. *licoperci*, *F. graminearum* and *F. verticillioides* showed that there is transfer of lineage-specific (LS) genomic regions that have pathogenicity related genes with distinct evolutionary profiles, indicative of horizontal acquisition and suggesting that there is transfer of LS chromosomes between genetically isolated *Fusarium* species. This is of high relevance and of particular concern for agricultural systems, because non-pathogenic *F. oxysporum* strains that are already endophytic to crop plants could suddenly become pathogenic [48] and give origin to new pathogenic lineages of *F. oxysporum*. It is clear that in the last decade a large amount of DNA sequence information has been published on *F. oxysporum*, but there is a lack of consistency in the data and a larger study needs to be conducted in which DNA sequences of isolates from non-cultivated species is included and even from *Fusarium* species that are thought not to be related.

Foc genetic diversity studies were initiated using various molecular methods, including random amplified polymorphic DNA markers (RAPDs) [49]; Restriction Fragment Length Polymorphisms (RFLP) [43]; Amplified fragment length polymorphism (AFLP) [50]; DNA sequence analysis [32, 44]; microsatellites or simple repetitive sequences [51]; simple repetitive inter sequence (ISSR) [52]. These studies showed that the population of this fungus in Southeast Asia shows a high degree of variation, suggesting that the Foc lineages evolved together with their hosts in Southeast Asia.

Alternatively, Foc has been suggested to have multiple independent evolutionary origins, both within and outside the *Musa* genetic center [36]. Using the phylogenetic genealogical approach, [32] identified five Foc-independent genetic lineages in a global population. Using a similar approach and additional data, [44] found three additional lineages. However, none of these studies included Indonesian populations, and therefore there is only limited information available on Foc diversity at the center of origin of bananas.

*F. oxysporum* f.sp. *cubense* probably coevolved with its host species within its center of origin [32, 36, 44]. For example, various studies that have used deoxyribonucleic acid (DNA) markers have revealed the polyphyletic origin of Foc and the separation of two main clades and eight to ten lineages, as some VCGs are taxonomically closer to other special forms of *F. oxysporum* than some Foc VCGs [32, 36, 42, 44, 50, 53].

Furthermore, strains belonging to various VCGs infect particular banana cultivars and, therefore, were grouped in the same race, suggesting that the pathogenicity towards a specific cultivar evolved in a convergent way [32, 38, 44] or as a result of horizontal gene transfer between members of the *F. oxysporum* complex [48, 54].

High resolution genotyping sequencing analyzes using (DArTseq) validated and expanded these findings [55]. According to the DArTseq markers of 24 Foc strains (representing all the known VCG so far) they were divided into two groups. These results strongly corroborate the clades mentioned in previous studies, except VCG0123, VCG01210, VCG01212 and VCG01214, which were occasionally grouped into opposing clades, VCG 01221 and 01224, which were never classified before but now clearly belong to clade 2 [55].

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*Genetic Diversity of Fusarium Wilt Disease of Banana DOI: http://dx.doi.org/10.5772/intechopen.94158*

lack of chromosome stability [57].

*F. odoratissimum* II-5 comprises TR4 [58].

two large clades and nine clonal lineages.

**7. Conclusions**

In the advent of high throughput DNA sequencing technology [56] has allowed scientist to better understand the molecular weaponry used by this pathogen. The pathogen molecular tools include genes involved in root attachment, cell degradation, detoxification of toxins produced by the plant's defense mechanism and signal transduction, among others [16]. In Ref. [57], the authors have reported a predicted genome size for several *F. oxysporum* f. sp. *cubense* with a size of 48.56 Mb for Foc Race 1 and 48.81 for Foc Race 4, comprising and estimated of 15,865 and 14,506 genes, respectively. This genome information was compared and aligned to 11 of the 15 chromosomes contained in *F. oxysporum* f. sp. *licopersici*, including those regions reach in transposable elements; which might explain its high genetic variability and

Recently, in a study samples of musaceae with wilt symptoms were collected in the regions of Indonesia, Java, Sumatra, Kalimantan, Sulawesi, Papua and Nusa Tenggara, this demonstrated by phylogenetic analysis that the Foc lineages were genetically different, and it was achieved to identify 11 new species of *Fusarium* affecting musaceae, these were: *Fusarium cugenangense*, *F. duoseptatum*, *F. grosmichelii*, *F. hexaseptatum*, *F. kalimantanense, F. odoratissimum, F. phialophorum, F. purpurascens, F. sangayamense, F. tardichlamydosporum,* and *F. tardicrescens*, placing them in the Banana Fusarium Complex (FOBC), as well as showing that

Fusarium wilt disease of banana caused by soil-born pathogen *Fusarium oxysporum* f.sp. *cubense* (Foc) is considered of the most destructive diseases of bananas and plantains worldwide. Foc produces three types of asexual spores, these are macroconidia, microconidia and chlamydospores, which function as mechanisms of dispersal, reproduction and survival. Foc is a genetically diverse pathogen, although the available evidence so far indicates that it does not use the mechanisms of sexual reproduction, such as recombination, to increase its genetic diversity. Furthermore, the population of this fungus in Southeast Asia shows a high degree of variation, suggesting that Foc lineages evolved together with their hosts in Southeast Asia. Alternatively, it has been suggested that Foc has multiple independent evolutionary origins, both within and outside of the Musaceae origin center. Actually, more than 24 vegetative compatibility groups and three races have been reported. This genetic diversity is accommodated in

*Genetic Diversity of Fusarium Wilt Disease of Banana DOI: http://dx.doi.org/10.5772/intechopen.94158*

In the advent of high throughput DNA sequencing technology [56] has allowed scientist to better understand the molecular weaponry used by this pathogen. The pathogen molecular tools include genes involved in root attachment, cell degradation, detoxification of toxins produced by the plant's defense mechanism and signal transduction, among others [16]. In Ref. [57], the authors have reported a predicted genome size for several *F. oxysporum* f. sp. *cubense* with a size of 48.56 Mb for Foc Race 1 and 48.81 for Foc Race 4, comprising and estimated of 15,865 and 14,506 genes, respectively. This genome information was compared and aligned to 11 of the 15 chromosomes contained in *F. oxysporum* f. sp. *licopersici*, including those regions reach in transposable elements; which might explain its high genetic variability and lack of chromosome stability [57].

Recently, in a study samples of musaceae with wilt symptoms were collected in the regions of Indonesia, Java, Sumatra, Kalimantan, Sulawesi, Papua and Nusa Tenggara, this demonstrated by phylogenetic analysis that the Foc lineages were genetically different, and it was achieved to identify 11 new species of *Fusarium* affecting musaceae, these were: *Fusarium cugenangense*, *F. duoseptatum*, *F. grosmichelii*, *F. hexaseptatum*, *F. kalimantanense, F. odoratissimum, F. phialophorum, F. purpurascens, F. sangayamense, F. tardichlamydosporum,* and *F. tardicrescens*, placing them in the Banana Fusarium Complex (FOBC), as well as showing that *F. odoratissimum* II-5 comprises TR4 [58].


**Promoting Research and Landscape Experience in the Management of the Archaeological Networks. A Project-Valuation Experiment in Italy**

**Salvatore Giu**ff**rida 1,\*, Filippo Gagliano 2, Enrico Giannitrapani 3, Carmelo Marisca 4, Grazia Napoli 5 and Maria Rosa Trovato 1**

Received: 15 April 2020; Accepted: 10 May 2020; Published: 14 May 2020

**Abstract:** Archaeological sites are part of the history and identity of a community playing a strategic role on the different scales of the cultural and economic common life. Whereas on the one end the most famous archaeological sites attract huge flows of tourists and investment, on the other hand, many minor archaeological sites remain almost ignored and neglected. This study proposes a project-evaluation approach devoted to the "minor" archaeological site development, outlining a territorial, socio-economic, and landscape communication pattern aimed at creating an archaeological network integrating other cultural and natural resources. As such, these networks get able to match the demand of customers who shy away from iper-consumerist tourism and want to deepen their knowledge of a place. The proposed approach integrates knowledge, evaluation, and design in a multiscale pattern whose scope is to foster and extend the archaeological research program, involving public and private stake/stockholders to widen the cultural-contemplative experience and promote further educational events concerning the themes of the local identity. With reference to the archaeological basin of Tornambè, Italy, a Web-GIS knowledge system has been drawn to provide the territorial information requested by the economic-evaluation multiscale pattern implemented to verify the cost-effectiveness of the project. The expected negative results of the economic valuation supported the allocation pattern of the considerable investment costs, as well as the hypothetic scenarios about the evolution of the cultural-contemplative experience due to the extension of the archaeological estate. Some disciplinary remarks propose a heterodox approach for a further interpretation of the economic results and financial indexes, by introducing the monetary dimension of such a social capital asset.

**Keywords:** archaeological basins; Web-GIS and Geodatabases; territorial marketing; cultural economics; land economy; tourism experience management; cultural estate; landscape heritage

### **1. Introduction**

The management of the cultural real estate heritage is de facto a central issue of territorial policies as this heritage plays an important role in creating the community identity, supporting local economic development [1], driving sustainable innovation of local businesses, and redefining the very concept of social well-being. The preservation and enhancement of cultural heritage, as a 'common good passed from previous generations as a legacy for those to come' [2,3], is promoted on global, European,

and national levels through specific programs, laws, and actions. The European Framework for Action on Cultural Heritage promotes an integrated and participatory approach to cultural heritage and—supported by the European Parliament—contributes to the mainstreaming of cultural heritage across European Union policies [4]. In Italy, where a great cultural heritage and an extraordinary archaeological heritage is concentrated, the high number of exceptional sites has contributed to spreading a general interest for art and culture, creating the basis for the concept of cultural basins [5] in which minor archaeological sites, that otherwise would remain marginal, form networks together with other resources.

The tourist sites of global or national importance attract huge financial resources and millions of tourists from all over the world every year, but they often generate territorial polarization that accentuates an unfair wealth distribution and affects peripheral areas. These sites face the impacts of overtourism [6–9], such as pollution and congestion, so much some limits on tourists access and stay need to be imposed for the preservation of the site itself, as in the case of the archaeological site of Machu Picchu in Peru, or in high naturalistic interest areas such as the Galapagos Islands in Ecuador. Conversely, the minor archaeological sites risk being abandoned due to the small tourist flows and the consequent public fund reduction.

The stratification and combination of multiple values and functions [10–12] in a territorial network connecting archaeological as well as ethno-anthropological and architectural features is coherent with the World Tourism Organization suggestions [13] on an alternative to predatory mass tourism, responding to the demand for slow tourism valuing the profound knowledge of a place, such as local traditions and products. Accordingly, alternative touristic itineraries should be identified as a success factor for reducing the effects of competition form the art cities and the most important tourist sites.

Among alternative models of tourism, such as Sustainable tourism—that can assure long term benefits to the local community and environment, as well as the tourists and tourist operators—, or Ecotourism—focused on travelling to wild and fragile areas and has the ethic goal of educating the traveller to the respect toward different cultures—, Slow tourism allows the traveller to get possession of time again, to be in tune with whatever surrounds him, interact to with local people, history and tradition [14,15]. Adopting a model of slow tourism allows putting into network different type of resources connected to a common theme and/or to a same territorial system in order to promote local resources and products which, otherwise, would not be economically feasible, as well as minor touristic sites which would not be able to attract flows of tourists. The implementation of this model requires appropriate marketing strategies, to build the supply and put it in the national and international tourism market in order to meet the demand. The marketing strategy should apply a systematic analysis of the slow tourist profile's and the most favourite activities, and verify strengths and weaknesses of the proposed territorial network in order to select the best actions to improve the supply. An application of this approach was proposed for the valorization of slow tourist itineraries between Italy and Slovenia in a project financed by the European Union and Slovenia within a cross-border cooperation program [16]. Moreover, marketing strategy could include the building of a brand to allow immediate identification of the 'territorial product', as proposed for the archaeological sites of the Pompeian pole [17,18].

So that demand for territorial networks evaluation and management tools [19,20] from the public institutions has grown, as well as, in response, the commitment from the science of appraisal and valuation in supporting political-administrative decision-making processes on the planning, programming and designing scale [21–28].

The strong connection of knowledge, valuation and project allows planners and decision-makers to generate multiple strategies aimed at promoting public consensus thus stimulating the participation of local communities [29], and at the same time to verify the consistency between outputs and outcomes, in terms of economic results [30].

The administration of the archaeological territory—concerning research (surface research, findings analysis and excavation campaigns programming) administration (the archaeological constraint subjection of the areas to be investigated) technical management (asset worksite and remains restoration) protection/promotion (on/off-site musealization of the remains)—requires specific professional and managerial know-how, and in Italy it is the exclusive responsibility of Superintendence of Cultural Heritage, applying generalized top-down decision-making processes. Some studies, however, promote the participation of tourists supporting a demand-oriented heritage management process complementary to the traditional supply-oriented one [31–33]. Such an approach focuses on the heterogeneous tourists' expectations outlining customer-oriented ways of fruition aimed at enhancing the customer service supply [34,35]. Other researches remark the need to include in management process other stakeholder groups and especially local communities [36] mostly concerned about the integration of the cultural heritage issue in the local socio-economic development [37,38]. Such an extension of the consensus deals with the natural conflict of values, expectations and scopes supported by different types of stakeholders (internal and external, private and public, economic and cultural, professional and common, etc.) to be involved [39] in the social communication process (public hearings, focus groups, citizen review panels, surveys, etc.) for new proposals ideas and perspectives [40,41].

In general, the economic profile of the archaeological assets is characterized by high investment costs and low and deferred streams of benefits [42] increasing over time. With the exception of some important archaeological parks, the negative results of their economic management is covered by public expenditure, whose amount can be considered the monetary measurement of the value a local community attributes [43–45] to the memory of its "ancient greatness", as well as a cultural-historic identity indicator.

The archaeological assets are quite different from primary goods, as so far as:

In the ground of the cultural heritage economics and in the prospect of a "circular economy" aimed at the progressive dematerialization of production and consume and mainly intended to create a culture of sustainability, valuation is committed to verifying how, in which extent, by and for whom economic development of land cultural assets characterized by mainly unspoiled potential and contextual territorial value, can be feasible.

In such prospect, this study proposes an integrated approach to knowledge, evaluation and design on two scales: on the territorial scale, a Web-GIS [46–49] Geodatabase-based knowledge approach allowed us to outline the landscape profile of the sites over the Province of Enna. On the local scale, it provided the spatial data describing the main "Value bearers" [50], involved in the project of research extension powering the cultural-contemplative experience supporting the promotion of the local economic activities and players.

Accordingly, the economic-financial analysis [51–54] aimed at outlining the efficiency profile of the development project proposed, although performed from the private player's perspective, tries to stress the favourable conditions of a coordinated land-economy-oriented policy integrating the visions and the missions of multiple, mostly public, players providing the socio-economic and political-decision context favourable to develop the archaeological basins culture and economy.

An archaeological basin is a territorial-landscape unit having a specific identity whose anthropology (including the economic framework) can be still related to the fundamental historic permanency as integrated into the multiple landscape dimensions [55]. So that the different basins constituting the archaeological asset of territory, should be defined as externally (from the landscape point of view) and internally (from an economic point of view) coherent territorial entities.

In such a prospect—that we assume as the political, institutional and economic condition for the sustainable development of low-value density wide territorial areas—the entities responsible for the protection and valorisation of the cultural asset, also according to the Italian Code of Cultural Heritage and Landscape currently in charge, demand behavioural patterns integrating multiple scales, subjects, scopes, and skills toward the common overall end of identifying in the permanence of the main outcrops of the ancient local civilization, the programmatic references of the socio-economic development of the settled communities.

Accordingly, the proposed approach outlines a project for the enhancement and musealization of a wide Sicilian inland archaeological basin (whose original core is the identified in the site of Tornambé in the Province of Enna, Italy). The general objective is the definition of the different dimensions of the project, involving the wide, intermediate and detail scales, public and private corporate players, and different kinds of actions, targeted to archaeological research development, to the opportunities of cultural and contemplative experiences, and to the promotion of entrepreneurial initiatives aimed at better finalizing the aforesaid activities. The specific object is the critical application of the cost-effectiveness analysis tools taking into account multiple scenarios validated by the prospect of connecting the multiple purposes of the subject involved, starting from the accounting of the investment and operating costs [56], revenues, and proposing the comparison of two different and somehow complementary interpretations of the specific findings. In fact, the application of the Discount Cash Flow Analysis confirmed that, despite the synergistic participation of public and private capitals and of various stakeholders, the economic feasibility remains a critical issue and the contribution of public expenditure is necessary. Rather, the socio-territorial and landscape context, powered by the public [57,58] intentionality and illuminated by the prevailing of the collective intelligence characterizing the local community identity, are assumed as the decisional long-run prospect supporting a more challenging entrepreneurial action in spite of the modest economic short-run results.

This share of public expenditure, which is a non-productive investment according to the market law [59], can be defined as a sort of equalization monetary transfer from major archaeological sites, that have active balance sheets, to minor archaeological heritage, and would implicitly constitute the minimum social value [60] given to historical and identity heritage of local communities.

The paper presents, in Section 2, the basic descriptive aspects of the basin of Tornambè. In Section 3, the methodological approach, articulated according to three dimensions of communication—territorial, socio-economic and landscape—and focusing on the Discounted Cash Flow Analysis (DCFA) carried out according to the arrangement of public/private commitments. In Section 4, the description of the results in the above-mentioned triple communicative pattern. In Section 5, a critical and proactive discussion of the results of the valuation output based on the comparison between two different (orthodox and heterodox) approaches. In Section 6, the conclusions that frame the different points of view and highlight in the valuation the prospects outlined based on the related results, concerning the commitment of public-private players in the creation of the identity of this landscape unit.


**2. Structure of vitamin K**

Both vitamin K forms have 2‐methyl‐1,4‐naphthoquinone, also called menadione or vitamin K3, as a common ring structure. However, they differ from each other in the length and degree of saturation of the polyisoprenoid side chain attached to the 3‐position.

Phylloquinone (vitamin K1) possesses a phytyl side chain, which consists of four isoprene units, and one of them is unsaturated. Phylloquinone is found primarily in plants in association with chlorophyll, whereas menaquinone (vitamin K2) is principally synthesized by bacteria. Menaquinone contains side chains of varying length, for most the part of a polymer of repeating unsaturated 5‐carbon prenyl units. Depending on the microorganism by which the chain is synthesized, the chain length generally ranges from 4 to 13 prenyl units. Menaquinones are classified according to the number of prenyl units. The number of units is given in a suffix (‐*n*), that is, menaquinone‐*n* and often abbreviated as MK‐*n* [2, 6, 7]. Some bacteria produce isoprenologues in which one or more of the prenyl units are saturated. The additional hydrogen atoms are indicated with the prefix dihydro‐, tetrahydro‐, and so on and are abbreviated MK‐*n*(H2), MK‐*n*(H4), etc. [8].

 The melting points of menaquinones vary from 35°C to 62°C depend‐ ing on the length of the multiprenyl side chain. Menaquinones are stable to heat and air but are very sensitive to alkali and ultraviolet (UV) irradiation [9].


**7. Conclusions**

The results of this study provide empirical evidence of the positive effects of social support on the emotional well-being of young immigrants and refugees. Specifically, the potential of mentoring programmes to cushion the stressful events to which they are subjected is made evident. The social support perceived during the Nightingale project contributed to improve some aspects related to the psychological well-being of the mentees, who saw their levels of personal satisfaction increase in a short period of time.

The data gleaned from this study suggest that mentoring is associated with more positive indices of personal well-being, although it can produce different effects in women and men, as a review of the relevant literature has shown [63,70]. While scientific research has increasingly focused on observing the effects of mentoring on social inclusion, empirical work addressing the influence of sex on the process and impact of such a relationship is scarce and mostly comes from studies on school-based mentoring programmes [66,71,72]. Therefore, interdisciplinary work with a gender perspective is needed that can address the gaps that remain in the evaluation of youth mentoring and its effects differentiated by sex.

The results reveal the importance of school counsellors, psychologists and social workers providing immigrant minors with programmes that encourage the building of social networks and the promotion of social support. We believe that prioritising policies and services that ensure a socially supportive environment in the reception of young people of foreign origin may help reduce the stressors associated with the migration process, which place minors in a vulnerable situation. The post-mentoring outcomes support the scientific consensus in the field of youth mentoring regarding the key role that relationships with non-parental adults play in supporting the social inclusion and subjective well-being of young immigrants.

With the aim of promoting the implementation of social mentoring programmes in schools, the findings of this study will be presented to primary and secondary school teachers. Educational institutions must show concern about the reality of students of foreign origin, at risk of exclusion, so as to help them overcome the adversities that arise from the adaptation process, avoiding leaving scars that affect emotional stability and lead to poor academic performance. From what is stated in this study, it can be seen that there is a need to promote the implementation of mentoring programmes as a reception plan that fosters the development of supportive relationships between adults and minors that serve the latter as a resource to try to adapt to the new environment, learn the language, build local networks and plan for the future.

A. and A.B.-E.; resources and data curation, Ò.P.-F.; writing—original draft preparation, A.S.-A.; writing—review and editing, A.S.-A., A.B.-E. and Ò.P.-F.; funding acquisition, A.B.-E. and Ò.P.-F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Secretariat of Universities and Research of the Government of Catalonia's Ministry of Business and Knowledge, the European Union and European Social Fund. (FSE) (2020 FI-B1 00109).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of the University of Girona under the code: CEBRU0001-2018 (6th of April 2018).

**Informed Consent Statement:** Informed consent was obtained from the parents of all subjects involved in the study as well as their assent.

**Data Availability Statement:** The data presented in this study are openly available in Harvard Dataserve repository at https://doi.org/10.7910/DVN/ZXC8RC.

**Acknowledgments:** This study is part of the Project RECERCAIXA2017UdG, Applying Mentoring: Social and technological innovations for the social inclusion of immigrants and refugees, funded by the RecerCaixa programme, a collaboration of "La Caixa" Welfare Projects and the Catalan Association of Public Universities. We would also like to express our gratitude to the Chair for Social Inclusion of Rovira i Virgili University for its assistance.

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


**2. ICPS non-medical software (ORI FIT-MET™)**

#### **2.1 Description of the process models**

Here we introduce ICPS ORI FIT-MET™ for the purpose to achieve fitness and prevent prediabetes, DM2 and complications such as CVD and AD. Uniquely, ICPS can construct trajectories of SV's (metrics) quasi real time in three domains of health: 1. Cardiometabolic Functioning and Disease (CMD), 2. Cardiovascular Functioning and Disease (CVD), and 3. Cardio-vegetative Functioning and Stress (CVS) with major implications to morbidity/mortality risks. Each of these domains have their mathematical process models to estimate the SV's (metrics). ICPS uses the predictive Kalman filter to predict future changes based on serially measured input data and using the respective predictive model calculation.

Ad 1. For CMD we use our Cardiometabolic Function Model (CMFM) which utilizes our Self-Adaptive Model of the Human Energy Metabolism (SAM-HEM) [1–4]; the Weight, Fat weight, Energy Balance model calculation (WFE); and the de novo lipogenesis, adaptive thermogenesis, and 24 hr. respiratory quotient model calculation WFE-DNL-AT [6]. The metric for insulin resistance in terms of R- or Rw-ratio carries the power of allowing to estimate the fat vs. carbohydrate burning and it is reflective of overall oxidative stress and inflammation. The CMFM modeling can calculate and predict the following physiological SV's: weight, fat mass, lean mass, ECW, ICW, R-ratio, Rw-ratio, Fat vs. Carbohydrate Oxidation, and 24 h non-protein respiratory quotient. With precise calorie counting the estimations of utilized macronutrient energy intake, de novo lipogenesis DNL and adaptive thermogenesis AT is possible.

Ad 2. For CVD process modeling ODI uses a cardiovascular fitness model (CVFM) in which the maximum oxygen uptake capacity (VO2max) is estimated

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

ment and recognize inadvertent side effects.

*and cardiovascular disease progression***.**

the daily macronutrient calorie intake.

to pharmacological interventions could be gauged, helping to track results of treat-

To our knowledge there is no noninvasive tool or monitoring device available to measure increased oxidative stress, inflammation, or insulin resistance in the user's natural environment. However, these pathological processes are strongly interlinked, leading to among others DM2, CMD, MS, CVD, ASCD, AD, Chronic Anemia of CKD, and HF. Importantly, ICPS is built on the holistic modeling approach of considering the entire human energy metabolism and insulin resistance. The latter can be viewed also as a surrogate marker for whole body oxidative stress and inflammation [15]. The bio-physical principle behind the proposed conceptual framework of ICPS and for process models is the recognition that the changes of the body composition (lean mass and fat mass) and the energy flow in and out of the body are governed by the fat vs. carbohydrate burning ratio and are strongly linked to insulin resistance [16, 17]. The significance of this is that an impaired mitochondrial lipid oxidation is a major anomaly in the chain of metabolic events leading to obesity and increase of insulin resistance [18]. High insulin resistance is associated with high respiratory quotient (RQ ) reflecting lower fat burning than normal [19]. We have no non-invasive measuring technique for Oxidative Stress. However, there is a strong connection between Oxidative Stress and Insulin Resistance [20]. Similarly, there are strong connections between inflammation and insulin resistance [21] but there is no non-invasive tool available currently to monitor whole body inflammation. Therefore, we intend to use the R- and Rw ratio to give at least a qualitative signal tool if the trends of changes in the metabolism are in the right or wrong direction in terms oxidative stress and inflammation. *Our central hypothesis is that by improving insulin resistance with the use of ICPS, we can ameliorate the condition of oxidative stress, overall inflammation, fat vs. carbohydrate oxidation,*

To our knowledge ODI is the first in using the principle of "least action/ stationary action" as a principle for finding key physiological parameters of the energy metabolism [5, 6]. This is instrumental to estimate noninvasively the HOMA- IR linked marker of insulin resistance R- or Rw-ratio which are defined as R = ΔL/ΔF and Rw = ΔW/ΔF where ΔL, ΔW and ΔF are lean mass, weight and fat mass change over 24 hrs. For monitoring of insulin resistance, we were able to prove the feasibility of this concept [5–6]. Further, we have shown that our Weight, Fat weight, Energy Balance (WFE) model can estimate changes of Rw without mandatory calorie counting by serially measuring weight, fat weight, and energy balance [6]. Our extended model of WFE calculation is called WFE-DNL-AT [6] and allows also for estimating for the first time noninvasively in the user's natural environment the otherwise difficult or impossible to measure changes of state variables (SV's) of the metabolism such as 24 h nonprotein respiratory quotient (24hRQ ), utilized macronutrient energy intake, fat vs. carbohydrate oxidation rate (Fox/Cox), de novo lipogenesis (DNL), and adaptive thermogenesis (AT). However, WFE-DNL-AT calculations require knowledge of

For measuring daily changes of fat mass F, lean body mass L, the measurement of intracellular water mass (ICW) as well as extracellular water mass (ECW) are also needed. Unfortunately, bioimpedance measurement technologies are not suitable for clinical use in current form due to significant interindividual variations mainly due to lack of reliable bio-electrical modeling of electrical properties of a body segment. On the other hand, bioimpedance measurements are quite well suited for individualized measurements or serial measurement as the intraindividual variation is small. The electrical modeling issue can be improved with using the principle of "maximum information entropy" [9, 10]. Therefore, ODI developed

**410**

from heart rate and measuring maximal activity energy expenditure (aEEmax) during graded exercise. The VO2max calculation model uses multiple linear regression with data on age, sex, height, percent body fat, aEEmax, and the slope between HR and physical activity as in [29]. CVFM is self-adapting (self-learning) from the daily incoming data and assesses changes of VO2max, exercise capacity, and heart rate reserve. We adopted the Critical Power model from [30] which is defined as the maximal sustainable aerobic power not causing "fatigue" to measure exercise capacity.

Ad 3. For CVS modeling ODI uses its Cardio-vegetative Stress Model (CVSM) which calculates the state variables (SV's) measuring functioning of the autonomous nervous system and estimating imbalance between sympathetic vs. parasympathetic activity. The time domain measure is the standard deviation of R-R intervals (SDNN) and the frequency domain power spectrum indicators are the low frequency spectral power of HRV (LFr), the high frequency spectral power of HRV (HFr), and their ratio LFr/HFr [12–13, 28].

#### **2.2 Data flow**

The usage of ICPS ORI FIT-MET™ is centered around data flowing in and out of the system. ICPS works with a wearable Sensor System (SS) to provide input data for the process models to arrive at metrics regarding CMD, CVD, CVS. The heart rate and physical activity energy expenditure related input data come from a wearable wristwatch-type fitness tracker like Garmin's smart watch. The body composition and hydration status related input data come from Garmin's Index scale. Alternatively, ODI developed its own fitness tracker, the sensor belt (SB) [7], and the BC-HS-A stand up scale [8, 9]. During regular use, ICPS updates every day the SV's and creates metrics allowing for trend prediction. The input and result data can be displayed on MHM or MST.

#### **2.3 Analysis and interpretation**

ODI's proposition is that MC modeling can provide special insight into physiological/ pathophysiological processes. MC modeling gives the expected direction of change of a variable in the future i.e. by connecting the data points and drawing a trajectory of the predicted changes. The benefit is that instead of comparing the user's data against a group average, the individualized modeling and data trajectory creation allows for self-comparison to historical data, capturing individual characteristics and facilitating individualized interventions. The MC models are generating metrics and trajectories allowing for tracking progress and facilitating dynamic behavioral changes. The undeniable advantage of modern portable electronics is that they can provide the resources and powerful data for self-healing in a non-judgmental way. The self-explaining context of SV's have the potential to raise self-awareness and draw attention to risk reduction and individual responsibility in the fight against modifiable noncommunicative disease processes. The derived metrics provided by ICPS have the potential to give the opportunity for education and learning about risks for health, development of new skills to fight risks, building motivation, as well as measuring self-efficacy in the fight against modifiable risks. The same ICPS metrics can be used by a personal trainer/primary provider for teaching and guiding needed changes of lifestyle or behavior. Importantly, it must be emphasized that the most important tool in our armamentarium to enhance insulin sensitivity and along with-it fat burning is endurance training [31] and it works even if no weight loss is achieved.

**413**

*Integrated Cyber-Physical System to Support Early Diagnosis and Prevention of Prediabetes…*

Inspired by the call for person-centered, outcome-driven treatment as a new paradigm for treatment of type 2 diabetes in primary care [14] we present here our vision of how MC type approaches could significantly help goals set forth by the academic authors in [14]. Target points for outcome in [14] are ASCD, CKD, and HF. For a practicing primary physician, it is desirable to offer non-invasive monitoring for patients in their natural environment not just for early detection of deterioration but also to improve patients' handling of rising issues with

Here we offer a preview about ICPS as a Medical Software and show how we can construct trajectories of SV's quasi real time in five domains of disease processes: 1. CMD and Metabolic Syndrome (MS), 2. Atherosclerotic Cardiovascular Disease (ASCD), 3. CVS and Autonomic Dysfunction (AD), 4. Chronic Anemia due to CKD, and 5. Heart failure (HF). It appears natural to extend the use of ICPS non-Medical Software with the areas of Chronic Anemia due to CKD and Heart failure.

Ad1. The MC model for CMD and MS remains the same Cardiometabolic Function Model (CMFM) as in ICPS ORI FIT-MET™. Response to the therapies of metabolic syndrome could be tracked and compared with baseline for de novo lipogenesis DNL, Fat vs. Carbohydrate Oxidation, and 24 h non-protein respiratory quotient. These metrics can supply valuable feedback in terms of ongoing diet and

Ad 2. For ASCD we want to extend CVFM. In the modeling of the maximum oxygen uptake capacity (VO2max) we also want to consider modeling oxygen delivery which depends on hemoglobin concentration (Hb), total hemoglobin mass due to chronic anemia of CKD and cardiac output. For modeling of oxygen delivery and oxygen consumption we use the model equations in [32]. For process modeling of hemoglobin concentration, total hemoglobin mass, and cardiac output see also

Ad 3. For CVS modeling ODI uses CVSM. For quantifying AD, the rationale is that there are strong associations between central adiposity (which is a marker of insulin resistance) and autonomic dysfunction [33] and there is an increased sympathetic system activity in metabolic syndrome [34]. We plan on using promising markers beyond SDNN, LFr, and HFr to recognize AD such as heart rate recovery time [33]. For the prediction of sudden cardiac death, we want to also use

Ad 4. We want to build a modeling platform for Chronic Anemia due to CKD. The main rationale is that anemia is a recognized risk factor for cardiovascular disease [36]. This is potentially important because iron deficiency anemia, if corrected, may in fact improve endothelial function and potentially improve morbidity and mortality [36]. Not surprisingly, anemia and insulin resistance and type 2 diabetes are interlinked [37] through various inflammatory processes which play crucial roles in the development of insulin resistance. There is also an inverse correlation between iron levels and HbA1c [38]. The reasons for this include kidney complications, neuropathy, and malabsorption occurring in the setting of advanced DM2. The elevated blood sugar will, over time, damage small blood vessels in the kidneys leading also to CKD. The erythropoietin production by the kidney goes down and along with it the production of red blood cells by bone marrow. Several studies show that diabetics with reduced renal function are more likely to end up with iron deficiency anemia than those without reduced

*DOI: http://dx.doi.org/10.5772/intechopen.94232*

**3.1 Description of the process models**

The respective process models are the following:

exercise habits with implications to spur needed change.

the correlation dimension of R-R intervals D2 [35].

**3. ICPS medical software**

appropriate behaviors.

Ad 4. and 5.

*Integrated Cyber-Physical System to Support Early Diagnosis and Prevention of Prediabetes… DOI: http://dx.doi.org/10.5772/intechopen.94232*


**1. Introduction**

In the developing world, energy crisis is the main reason for less progress and development. Renewable and sustainable energy may be of bright future for scientific lagging and low-income countries; further, sustainability through smart materials got a huge potential; so, hereby keeping in view the energy crisis which developing world is facing for many decades, there exists a possibility for obtaining energy through cheap, sustainable, and smart carbon materials. The nanostructures which are made up of sp2-hybridized carbon such as fullerene, carbon nanotubes, and graphene remain pivot point of advanced carbon functional material research [1]. As we look into the reversible electrochemical strength keeping in view of their great electric conductivity, chemical and mechanical durability, and the custom fitted structures that can be developed from advanced carbon functional materials, carbon materials have been the most significant anode material for the Li-ion batteries [2]. Advanced carbon functional materials may include graphene, carbon nanotubes, and fullerenes, respectively. Moreover, CNTs and graphene with high explicit surface territories can store higher charge capacity by the adsorption/desorption of particles at the anode/electrolyte interface. The higher charge capacity is hence incredible subject to the qualities of the carbon material, for example, its pore structure, doping, and defects. In this way, the advancement of electrochemical capacity in carbon needs a judiciously planned structure. One issue identified with the utilization of CNTs and graphene for electrochemical storage is the agglomeration of the nanostructures that prompts diminished efficiency [3, 4]. There has been progressive demand for the electrochemical energy storage with high energy density and remarkable rate performance. Electrical double layer capacitors (EDLCs), additionally known as supercapacitors (SCs), have attracted a worldwide attention because of their long cycle life and high power density but comparatively low energy density of commercially available carbon-based SCs. Graphene has attracted an in-depth attention in energy storage applications relating to its distinctive options of high surface space, flexibility, chemical stability, and remarkable electrical and thermal conduction [5]. Energy storage capacity defines advanced energy technologies. Further, superior energy storage may be obtained through various routes like using pyrrolic (N5) and pyridinic (N6) doping in carbon materials, or superior energy by KOH activation in carbon materials, or through carbonization in organic matter, respectively. Further, energy storage using pyrrolic (N5) and pyridinic (N6) doping, or KOH activation, or through carbonization in organic matter will be discussed one by one.


Advances in Green Electronics Technologies in 2023

*Edited by Albert Sabban*

Published in London, United Kingdom

Advances in Green Electronics Technologies in 2023 http://dx.doi.org/10.5772/intechopen.100759 Edited by Albert Sabban

#### Contributors

Fernando Vélez Varela, Carlos Daniel Valencia Rincón, Daniel Revelo Alvarado, Hammadi el Farissi, Prasanna Venkatesh Ramesh, Prajnya Ray, Aji Kunnath Devadas, Akshay Surendranp, Tensingh Joshua, Shruthy Vaishali Ramesh, Meena Kumari Ramesh, Ramesh Rajasekaran, Rajashree Lodh, Subhashis Chowdhury, Souvik Chakraborty, Shireen Ibrahim Mohammed, Giovanni Campeol, Lorella Biasio, Silvia Foffano, Davide Scarpa, Giulio Copparoni, Daniel Llarens, Laura Souilla, Santiago A. Masiriz, Gastón R. Lestard, Albert Sabban

#### © The Editor(s) and the Author(s) 2023

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#### Notice

Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book.

First published in London, United Kingdom, 2023 by IntechOpen IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: 11086078, 5 Princes Gate Court, London, SW7 2QJ, United Kingdom

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3.1.21. (3*S*,4*S*)-7-(aminomethyl)-4-methoxy-5,8-bis(methoxymethoxy)-3-pentylisochroman-1-one (**14a**)

To a solution of diMOM azide derivative **13a** (3.3 mg, 7.8 μmol) in MeOH (0.78 mL) was added Et3N (0.10 mL, 7.35 mmol) and Pd/C (1.6 mg, 1.5 μmol) at room temperature. After stirring for 1 h at the same temperature, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified with PTLC (MeOH:CH2Cl2 = 1:9) to give diMOM amine derivative **14a** (2.0 mg, 65%) as brown oil. 1H-NMR (400 MHz, CDCl3) δ 7.49 (1H, s), 5.26 (2H, s), 5.16 (1H, d, *J* = 7.2 Hz), 5.07 (1H, d, *J* = 6.8 Hz), 4.61 (1H, d, *J* = 1.2 Hz), 4.27 (1H, ddd, *J* = 1.2, 6.0, 7.6 Hz), 4.00 (2H, s), 3.61 (3H, s), 3.50 (3H, s), 3.32 (3H, s), 2.59 (1H, br-s), 2.03 (1H, m), 1.82 (1H, m), 1.57 (1H, m), 1.43 (1H, m), 1.40-1.25 (1H, overlapped), 0.91 (3H, t, *J* = 6.8 Hz); 13C-NMR (125 MHz, CDCl3) δ 162.6, 152.6, 150.5, 128.1, 120.0, 119.0, 102.4, 95.4, 81.2, 68.5, 57.9, 57.2, 56.8, 42.5, 32.0, 30.9, 30.0, 25.2, 22.9, 14.4.; IR (KBr) 2957, 2925, 2857, 2827, 1726, 1470, 1153, 1005 cm−1; HRMS (ESI) *m*/*z* (M + H)+ calculated for (C20H32NO7) + 398.2179, found 398.2178.

#### 3.1.22. (3*S*,4*S*)-7-(aminomethyl)-5,8-dihydroxy-4-methoxy-3-pentylisochroman-1-one (**14**)

To a solution of diMOM amine derivative **14a** (4.4 mg, 11.1 μmol) in MeOH (0.83 mL) was added 6 M aqueous HCl (0.28 mL) at 0 ◦C. After stirring for 5 h at room temperature, the reaction was quenched by adding saturated aqueous NaHCO3 at 0 ◦C. The mixture was extracted with the mixture of MeOH and CH2Cl2 (MeOH:CH2Cl2 = 1:4) (×4) and the combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified with PTLC (MeOH:CHCl3 saturated with NH3 = 1:9) to give amiomethyl derivative **14** (1.1 mg, 32%) as brown solid. m.p. 78–80 ◦C; 1H-NMR (400 MHz, CDCl3) δ 6.98 (1H, s), 4.59 (1H, d, *J* = 1.8 Hz), 4.35 (1H, ddd, *J* = 1.8, 6.0, 8.0 Hz), 3.97 (1H, d, *J* = 13.3 Hz), 3.88 (1H, d, *J* = 13.3 Hz), 3.19 (3H, s), 1.98 (1H, m), 1.83 (1H, m), 1.56 (1H, m), 1.43 (1H, m), 1.40–1.25 (4H, overlapped), 0.90 (3H, t, *J* = 7.0 Hz) ; 13C-NMR (125 MHz, CDCl3) δ 169.9, 154.2, 146.2, 130.0, 125.8, 122.9, 108.1, 82.8, 68.5, 56.9, 42.3, 31.9, 30.6, 25.1, 22.8, 14,3.; IR (KBr) 2956, 2921, 2857, 1676, 1441, 1171 cm−1; HRMS (ESI) *m*/*z* (M + Na)+ calculated for (C16H23NO5Na)+ 332.1474, found 332.1474.


**1. Introduction**

The major role of skin is the protection of our body from external stimuli. Skin also plays an important role in maintaining homeostasis, including protection against loss of moisture and adjustment of body temperature [1]. Furthermore, aging-associated changes of skin conditions, such as the increased wrinkles and decreased skin elasticity, are a major concern for maintaining quality of life. Skin is composed of epidermis, dermis, and subcutaneous tissue. Collagen and elastin in the dermis maintain the structure of skin and create its elasticity [2,3]. Notably, the fibrous protein collagen, which plays a major role in maintaining the mechanical strength of skin, constitutes the majority of the dermis. The collagen molecule is formed by the three polypeptides, named alpha chains. The alpha chains are composed of high levels of glycine, hydroxyproline, proline and alanine [4]. Thus, collagen has unique amino acid composition. These collagen molecules assemble to form collagen fibrils by cross linking. Then, collagen fibrils assemble to form large collagen fiber. There are several types of collagen, and type I and type III collagen are the most abundant collagen in the skin [5]. Because collagen synthesis decreases with aging, decreased collagen content is one of the major causes of aging-associated changes of skin conditions [6,7]. As skin aging and nutrition states are linked [8], the improvement of skin conditions by dietary supplementation is a topic of increasing public interest [9].

The effects of growth hormone, a peptide hormone secreted from the pituitary gland (especially during the first few hours of sleep), cover a broad range of biological phenomena including cell growth, proliferation, regeneration, and metabolism [10–12]. For skin, severe growth hormone deficiency results in early aging, such as wrinkling and dryness [13]. In addition to the direct effects of growth hormone to several tissues, it also elicits indirect effects mediated by insulin-like growth factor-1 (IGF-1) [12]. Plasma IGF-1 levels correlate with plasma growth hormone levels, as production of IGF-1 by the liver is stimulated by growth hormone [14,15]. Similar to growth hormone, IGF-1 activates cell growth in several tissues including bone [16], muscle [17], and skin [18,19], whereby it contributes to both epidermal or dermal skin development and maintenance. Secretion of growth hormone is decreased with aging, suggesting that aging-associated changes of skin conditions are mediated, at least in part, by decreased levels of growth hormone, or its associated decrease of IGF-1 [20]. Thus, these previous studies suggest that aging-associated changes of skin conditions might be improved by increasing growth hormone and/or IGF-1 levels.

In recent years, many products containing collagen or a denatured form of collagen have been used for a wide variety of purposes, including cosmetics and food. With regard to food or supplements, the effects of collagen have been controversial, as orally ingested native collagen or its partially hydrolyzed form, gelatin, are not efficiently absorbed [21]. However, several lines of evidence revealed the beneficial role of collagen-derived small peptides, which exhibit high absorbability compared with native collagen [21,22], for a wide variety of tissues including bone [23], joint [24], muscle [25], tendon [26], and skin [27–29] in humans. Collagen has been isolated from many marine, brackish water and freshwater sources such as fishes [30–32] and mollusks [33–35]. Compared with collagen peptide derived from land animals, collagen peptide derived from these aquatic sources has unique molecular and biological properties for amino acid composition, antioxidant activity, neuroprotective activity and anti-skin aging activity, because of low temperature and/or high salt condition in the surrounding environment [36–39]. Furthermore, a previous study showed that collagen derived from sea- and freshwater-rainbow trout had quite similar amino acid composition and molecular weight properties [40]. Collagen derived from two marine demosponges, *Axinella cannabina* and *Suberites carnosus*, collected from the Aegean and the Ionian Seas, respectively, had low imino acid content, and showed lower or similar denaturation temperatures compared with collagen derived from other marine organisms such as tropical fish [41], suggesting the universal properties of collagen derived from aquatic sources. In addition to collagen peptide, ornithine is a non-essential, non-protein amino acid contained in various foods such as freshwater clams. Recent studies have highlighted ornithine as a functional food for improving sleep quality [42] and recovery from fatigue [43,44] in human.

The beneficial effects of collagen peptide for skin conditions have been analyzed by several groups both in rodents [39] and humans [27–29,45]. However, to our knowledge, the effects of collagen peptide, ornithine or the combined effects of collagen peptide and ornithine (CPO) on the increase of growth hormone and/or IGF-1 levels, and the subsequent improvements of skin conditions have not been investigated. A previous study showed the increase of plasma growth hormone levels after ingestion of ornithine [46], though its relationship to the improvements of skin conditions have not been investigated. In this study, we anticipated the combinational effects of fish-derived collagen peptide and ornithine on skin conditions, and plasma growth hormone and/or IGF-1 levels in healthy Japanese people. We hypothesized that orally administered CPO induced an increase of plasma growth hormone and/or IGF-1 levels, which exerted subsequent improvements of skin conditions including elasticity, moisture and transepidermal water loss (TEWL).

#### **2. Results**

#### *2.1. Participants*

Forty participants were recruited from the Osaka area, of whom 22 participants (aged from 31 to 48 years, 18 females and 4 males) exhibiting low skin moisture and elasticity were enrolled. Participants were recruited from September to October 2017. Included participants were assigned to either the CPO group (*n* = 11) or placebo group (*n* = 11). All participants completed the study. One participant in the CPO group was excluded from analysis because of aberrant blood ureic acid levels both before and after supplementation (Figure 1).

**Figure 1.** Flow diagram of participants.

Finally, 21 participants (aged from 31 to 48 years, 17 females and 4 males) were analyzed. Thus, per protocol set analysis was performed. Statistically significant differences between originally included participants and finally analyzed participants were not observed for baseline scores including age, skin elasticity, and moisture. This study consisted of an 8-week administration period from October to December 2017. CPO and placebo groups were matched according to age, gender, body mass index (BMI), skin elasticity, and moisture at baseline (Tables 1 and 2).

**Table 1.** Baseline characteristics of participants who completed 8-week test.

Average ingestion rate was 100 ± 0% and 99.8 ± 0.5% in CPO and placebo groups, respectively. All subjects had a more than 98% ingestion rate. No statistically significant differences were observed for ingestion rate between CPO group and placebo groups.


**1. Introduction**

The application of light-emitting diodes (LED) in horticultural lighting systems provides new possibilities for light intensity and light spectrum fine regulation along with a significant reduction in energy consumption [1–3]. A breathtaking possibility to modulate the LED lighting spectrum can also help in promoting the accumulation of important plant metabolites, which are often associated with nutraceutical properties, as has been shown in various crops, including lettuce [4]. The set-up of plant-specific light protocols for their cultivation is a critical phase in improving the sustainability of indoor growing systems [2].

Besides photosynthesis, plants are capable of perceiving and processing information with light signals from their biotic and abiotic surroundings for optimal growth and development [5]. Reviews of studies on light quality effects on plant growth and development can be found elsewhere [6–8]. Red and blue are generally recognized as the most important light regions necessary for plant development and growth [3]. However, other wavelengths

**Citation:** Tarakanov, I.G.; Tovstyko, D.A.; Lomakin, M.P.; Shmakov, A.S.; Sleptsov, N.N.; Shmarev, A.N.; Litvinskiy, V.A.; Ivlev, A.A. Effects of Light Spectral Quality on Photosynthetic Activity, Biomass Production, and Carbon Isotope Fractionation in Lettuce, *Lactuca sativa* L., Plants. *Plants* **2022**, *11*, 441. https://doi.org/10.3390/plants11030441

Academic Editors: Valeria Cavallaro and Rosario Muleo

Received: 23 December 2021 Accepted: 3 February 2022 Published: 5 February 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/).

(such as those corresponding to yellow or green colors) could also have a role in affecting the quality of crops [6]. Blue light is involved in a wide range of plant processes such as phototropism, photomorphogenesis, stomatal opening, and leaf photosynthetic functioning [9]. Most studies assessing the effects of blue light (blue LEDs) on the leaf or whole plants have either compared their response to a broadband light source with the response to blue-deficient light [10] or plants grown under red light alone [11,12]. On the other hand, red LEDs emit a narrow spectrum of light (660 nm) that is close to the maximum absorbance for both chlorophyll and phytochromes. The absorption of blue and red light (LEDs) by plants has been measured as 90% [13], which indicates that plant development and physiology is strongly influenced by blue and red light [6]. The effects of green light tend to reverse the processes established by red and/or blue light. In this way, green light may be functioning in a manner similar to far-red light, informing the plant of photosynthetically unfavorable conditions and triggering adaptative responses [14]. Many studies have been reported on several crops grown under deficiency/efficiency or using a combination of red and blue light at different wavelengths [15,16] to investigate their effects on plant growth and development. While red light promotes biomass accumulation, growth, and photosynthesis in lettuce, blue LED light is effective in stimulating photomorphogenesis and adaptive phenomena such as the stomata-opening/closing-regulation mechanism, as well as biomass accumulation and chlorophyll and anthocyanin biosynthesis [3,17]. A positive growth response to the combination of blue and red light was confirmed in Batavia lettuce plants [18]. Green LED light regulates leaf expansion, stem stretching, and stomatal conductance. Moreover, it has been shown that green LED light addition leads to greater dry mass accumulation and growth stimulation [19].

The plant perceives light environment signals by means of photosynthetic apparatus (PSA) and specific photoreceptors sensitive to different light spectral regions. Blue and red light are not equal in their effects on photoreceptors: red light is perceived in addition to PSA by phytochromes only, and blue light is absorbed by both phytochromes and blue-light receptors (cryptochromes, phototropins) [20]. Blue light influences a greater number of photoreceptors and is functionally more versatile. It is most effective in stimulating the transcription of photosynthesis-related genes (via cryptochromes and phytochromes) [21]. Interestingly, barley plants grown with monochromatic red light demonstrated specific organization of chloroplast membranes (shaggy-formed grana) and light-harvesting complexes (increased energy transfer to PSI, possibly due to spillover promoted by this particular granum structure) [20]. These specific responses can be related to contradictory information from the photoreceptors; the signals from the phytochromes and photosynthetic apparatus indicate the incidence of light, while the lack of a signal from the blue-light receptors can be misinterpreted as darkness [20]. Most of the negative monochromatic red-light effects can be avoided by the addition of blue light [22–24]. Furthermore, a combination of red and blue light in certain cases can result in synergetic effects in biomass accumulation [25,26] Plant photosynthesis and growth, directly or indirectly, can also be mediated by the photoreceptor response. Additionally, chloroplasts play an important role in photoreceptor-mediated control of photomorphogenic responses [27]. The main obstacle in the transition to LED lighting in crop production is that it involves a complex system change beyond lighting (e.g., plant light recipes, which are species- and often cultivardependent), resulting in serious associated costs [28]. Lighting systems using specific wavelengths are capable of target compound biosynthesis fortification; however, special attention has to be paid to the stress the artificial light may cause in the photosynthesis and biomass accumulation [29]. To explore the action mode of different light spectrum regions, various experimental approaches are used. Thus, in the studies on the blue-light effects, plant responses to a broadband light source with a response to blue-region-deficient light were compared [10] with plants grown under red light alone [11]. So, the experimental set up can include studies on the effects of monochromatic irradiation. Additionally, plant responses to photosynthetically active radiation (PAR) missing distinct spectrum regions can be investigated [30].

In our studies with lettuce plants, we have used both screens mentioned above, emphasizing research on light spectral quality effects on the carbon isotope composition of plant biomass (Section 2.3). It is known that plant cells are able to fractionate carbon isotopes in the light and in the dark [31,32]. The carbon isotope composition of plant leaf biomass is mainly related to the light processes, CO2 assimilation, and photorespiration [32,33]. The 12C enrichment of plant biomass during CO2 assimilation occurs at Calvin cycle entry during RuBP carboxylation. During photorespiration, carbon isotope fractionation occurs with the opposite sign, thus reducing the effect of CO2 assimilation and enriching biomass with 13C. The isotope effect of photosynthetic assimilation and photorespiration are coupled by a key photosynthetic enzyme, Rubisco, that oscillates from CO2 assimilation to photorespiration and back [34,35]. The effects of monochromatic light and other unique artificial light treatments on the carbon isotope fractioning have not been investigated until now. In our studies with lettuce plants, we have used both screens mentioned above, emphasizing research on light spectral quality effects on the carbon isotope composition of plant biomass (Section 2.3). It is known that plant cells are able to fractionate carbon isotopes in the light and in the dark [31,32]. The carbon isotope composition of plant leaf biomass is mainly related to the light processes, CO2 assimilation, and photorespiration [32,33]. The 12C enrichment of plant biomass during CO2 assimilation occurs at Calvin cycle entry during RuBP carboxylation. During photorespiration, carbon isotope fractionation occurs with the opposite sign, thus reducing the effect of CO2 assimilation and enriching biomass with 13C. The isotope effect of photosynthetic assimilation and photorespiration are coupled by a key photosynthetic enzyme, Rubisco, that oscillates from CO2 assimilation to photorespiration and back [34,35]. The effects of monochromatic light and other unique artificial light treatments on the carbon isotope fractioning have not been investigated until now.

blue-light effects, plant responses to a broadband light source with a response to blueregion-deficient light were compared [10] with plants grown under red light alone [11]. So, the experimental set up can include studies on the effects of monochromatic irradiation. Additionally, plant responses to photosynthetically active radiation (PAR) missing

*Plants* **2022**, *11*, x FOR PEER REVIEW 3 of 17

distinct spectrum regions can be investigated [30].

#### **2. Materials and Methods 2. Materials and Methods**

#### *2.1. Plant Material 2.1. Plant Material*

Lettuce, *Lactuca sativa* L., plants of the Aficion RZ cultivar were used in our studies. This is Batavia-type lettuce, leaves with strongly wavy edge, light green. Batavia lettuce is highly appreciated in the market due to the variability in shape, color, texture, and taste. As for the nutritional value, it is a source of vitamin A, niacin, riboflavin, thiamine, Ca, Fe, K, Mn, Se, and β-carotene [36]. Aficion cultivar is widely grown in greenhouses and vertical farms with artificial lighting. Lettuce, *Lactuca sativa* L., plants of the Aficion RZ cultivar were used in our studies. This is Batavia-type lettuce, leaves with strongly wavy edge, light green. Batavia lettuce is highly appreciated in the market due to the variability in shape, color, texture, and taste. As for the nutritional value, it is a source of vitamin A, niacin, riboflavin, thiamine, Ca, Fe, K, Mn, Se, and β-carotene [36]. Aficion cultivar is widely grown in greenhouses and vertical farms with artificial lighting.

#### *2.2. Cultivation Conditions 2.2. Cultivation Conditions*

Plants were grown in growth chambers (Urbangrower 150, China; Figure 1) with various light treatments according to experimental layout described in Section 2.3. Each chamber had dimensions of 1.50 × 0.90 × 2.00, 2.7 m3 , with gloss white walls. Chambers were supplied with fans; day/night temperature was 20/18 ◦C, with less than 1 ◦C variation over time and 1 ◦C variation among chambers. Plants were grown in growth chambers (Urbangrower 150, China; Figure 1) with various light treatments according to experimental layout described in Section 2.3. Each chamber had dimensions of 1.50 × 0.90 × 2.00, 2.7 m3, with gloss white walls. Chambers were supplied with fans; day/night temperature was 20/18 °C, with less than 1 °C variation over time and 1 °C variation among chambers.

**Figure 1. Figure 1.**  Plant-growing chambers with various light environments. Plant-growing chambers with various light environments.

Plants were grown in 2 L vegetational vessels (3 plants in each container). Seeds were sown directly into the commercial neutralized peat-based substrate "Agrobalt-C" (Pindstrup, Pskov region, Russia) with pH 6.0–6.5 and complete macro- and micronutrient supply including 150 mg L−1 [NH4 + andNO3 −], 270 mg L−1 P2O5, and 300 mg L−1

K2O. Substrate humidity was maintained at 70% of full water capacity, watering up to calculated weight. PPFD at ±5%. To provide uniform PPFD, plant pots were moved and rotated within the marked uniform light platform every second day.

Plants were grown in 2 L vegetational vessels (3 plants in each container). Seeds were sown directly into the commercial neutralized peat-based substrate "Agrobalt-C" (Pindstrup, Pskov region, Russia) with pH 6.0–6.5 and complete macro- and micronutrient supply including 150 mg L−1 [NH4+ andNO3−], 270 mg L−1 P2O5, and 300 mg L−1 K2O. Substrate humidity was maintained at 70% of full water capacity, watering up to calculated

Plant chambers were illuminated with lamps consisting of various light-emitting diode (LED) bars specifically designed to provide a custom spectrum in each chamber. Fixtures consisted of light modules with tunable light-emitting diodes varying in wavelength

Four types of high-performance narrow-band 3-Watt LEDs (Estar Technology, Changchun, China) were used: short-wave red (∆λ0.5 = 623 ÷ 641 nm, λmax = 632 nm), longwave red (∆λ0.5 = 646 ÷ 674 nm, λmax = 660 nm), far-red (∆λ0.5 = 727 ÷ 751 nm, λmax = 739 nm), and blue (∆λ0.5 = 452 ÷ 477 nm, λmax = 465 nm). The control light treatment included all 4 types of LEDs, and in each of the other regimes one of them was excluded (except shortwave red) in order to elucidate the wavelength that affected distinct crop physiological processes. Short-wave red was used as an additional background spectral region to provide chlorophyll *a* excitation in the absence of long-wave red light. The same daily light integral (DLI) of 9.72 mol m−2 d−1 was maintained in all the treatments with photosynthetic photon flux density (PPFD) 150 µmol m−2 s−1 , photoperiod 18 h. Spectra of the resulting lamp systems were measured with a spectrometer UPRtek PG100N (Taiwan). To measure the PPFD in the PAR region, an LI-191R quantum sensor with an LI-250A data logger (LI-COR Biosciences, NE, USA) was used. It was measured at the top of the plant canopy (the distance from the light source was ≥50 cm), and each chamber was adjusted to maintain

and spectral composition of the emitted light over wide ranges (Figure 2).

#### *2.3. Light Treatments* In the experiment on the red and blue monochromatic light effects, two types of tunable LEDs (Cree, USA) were used: red (∆λ0.5 = 647 ÷ 671 nm, λmax = 659 nm) and blue (∆λ0.5

weight.

*2.3. Light Treatments*

Plant chambers were illuminated with lamps consisting of various light-emitting diode (LED) bars specifically designed to provide a custom spectrum in each chamber. Fixtures consisted of light modules with tunable light-emitting diodes varying in wavelength and spectral composition of the emitted light over wide ranges (Figure 2). = 438 ÷ 462 nm, λmax = 450 nm). To provide easy reading of the figure legends, wavelengths representing figures for combined-spectra regions (blue, short-wave red, long-wave red, and far-red) are "rounded" to 460, 640, 660, and 730, respectively.

*Plants* **2022**, *11*, x FOR PEER REVIEW 4 of 17

**Figure 2.** Light treatments: (**1**) "460 + 640 + 660 + 730"—4-peak reference treatment; (**2**) "460 + 640 + 730"—3-peak treatment missing red-light R660 region; (**3**) "460 + 640 + 660"—3-peak treatment missing far-red-light FR730 region; (**4**) "640 + 660 + 730"—3-peak treatment missing blue-light B460 region; (**5**) "450"—monochromatic blue-light B450 region; (**6**) "659" monochromatic red-light R659 region. **Figure 2.** Light treatments: (**1**) "460 + 640 + 660 + 730"—4-peak reference treatment; (**2**) "460 + 640 + 730" —3-peak treatment missing red-light R660 region; (**3**) "460 + 640 + 660"—3-peak treatment missing far-red-light FR730 region; (**4**) "640 + 660 + 730"—3-peak treatment missing blue-light B460 region; (**5**) "450"—monochromatic blue-light B450 region; (**6**) "659" monochromatic red-light R659 region.

*2.4. Plant Growth Parameters Analyses*  Four plants per each treatment were destructively harvested 30 days after emergence. The number of leaves (>1 cm) per plant was counted, and total leaf area was measured using a leaf area meter LI-3000A (LI-COR Biosciences, NE, USA). Shoot fresh weight was measured using an electronic balance. Subsequently, shoots were oven-dried to a constant weight at 70 °C for dry weight determination. Specific leaf weight (SLW) was calculated by dividing leaf weight by leaf area (dry weight per unit leaf area). Four types of high-performance narrow-band 3-Watt LEDs (Estar Technology, Changchun, China) were used: short-wave red (∆λ0.5 = 623 ÷ 641 nm, λmax = 632 nm), long-wave red (∆λ0.5 = 646 ÷ 674 nm, λmax = 660 nm), far-red (∆λ0.5 = 727 ÷ 751 nm, λmax = 739 nm), and blue (∆λ0.5 = 452 ÷ 477 nm, λmax = 465 nm). The control light treatment included all 4 types of LEDs, and in each of the other regimes one of them was excluded (except shortwave red) in order to elucidate the wavelength that affected distinct crop physiological processes. Short-wave red was used as an additional background spectral region to provide chlorophyll *a* excitation in the absence of long-wave red light. The same daily light integral (DLI) of 9.72 mol m−2 d −1 was maintained in all the treatments with photosynthetic photon flux density (PPFD) 150 µmol m−2 s −1 , photoperiod 18 h. Spectra of the resulting lamp systems were measured with a spectrometer UPRtek PG100N (Taiwan). To measure the PPFD in the PAR region, an LI-191R quantum sensor with an LI-250A data logger (LI-COR Biosciences, NE, USA) was used. It was measured at the top of the plant canopy (the distance from the light source was ≥50 cm), and each chamber was adjusted to maintain PPFD at ±5%. To provide uniform PPFD, plant pots were moved and rotated within the marked uniform light platform every second day.

In the experiment on the red and blue monochromatic light effects, two types of tunable LEDs (Cree, USA) were used: red (∆λ0.5 = 647 ÷ 671 nm, λmax = 659 nm) and blue (∆λ0.5 = 438 ÷ 462 nm, λmax = 450 nm).

To provide easy reading of the figure legends, wavelengths representing figures for combined-spectra regions (blue, short-wave red, long-wave red, and far-red) are "rounded" to 460, 640, 660, and 730, respectively.


*4.7. Statistical Analysis*

Values are shown as the mean ± SEM. Significant differences between the mean values of the two groups were evaluated by Student *t*-test with IBM SPSS Statistics 23. Statistical significance was indicated by a *p* value < 0.05.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/md19050278/s1, Table S1: Effect of fish oil (FO) supplementation on gene expression in liver.

**Author Contributions:** T.Y. designed the research; R.I., D.L. and T.Y. conducted the research, and T.Y. and R.I. analyzed the data. T.Y. and R.I. wrote the manuscript with contributions from D.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by a JSPS Grant-in-Aid for Scientific Research (C) Grant Number 16K01855.

**Institutional Review Board Statement:** Care of the mice followed guidelines of the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. The National Institutes of Biomedical Innovation, Health and Nutrition, Japan, reviewed and approved all animal procedures (Approval no. DS27-52R3).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are included in the corresponding sections throughout the manuscript.

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


**6. Current evidence and status of ECMO simulation**

In a recent survey from the United States, lectures (99%), water drills (99%) and bedside training (99%) remain the chief training modalities for new ECMO specialists. Forty-six per cent of ECMO centres had an ECMO simulation programme. ECMO centres with access to a simulation centre, those with higher case numbers and pediatric cardiothoracic ICU are more likely to have an ECMO simulation programme [11].

Simulation-based ECMO education is a growing research area with increasing evidence to support its effectiveness. Earlier publications were mainly descriptive and focused on the setup and content of simulation courses and the evaluation of the learner [12, 13]. Some studies reported an improvement in confidence and performance after ECMO simulation training. Su et al. reported a faster deployment time to ECPR initiation after simulation training [14], and Allan et al. reported a decrease in cannulation time for ECPR in pediatric cardiac surgery trainees [15].

a standardized curriculum. Only through the implementation of a uniform ECMO simulation programme can multi-centred studies be carried out to provide a better understanding of the

Education Curriculum on Extracorporeal Membrane Oxygenation: The Evolving Role of Simulation Training

ECMO is a low-volume but highly complex technology. The management of ECMO patients often requires an integration of cognitive, psychomotor and behavioural skills that can be addressed by simulation training. As a result, simulation is increasingly acknowledged as one of the essential learning tools in ECMO education. Further researches in simulation technology, medical science pedagogy and clinical trials are warranted to delineate its impact on

The authors would like to acknowledge all the members of the ECMO team, Dr. Chan Wai Ming, the Chief of Service of the Department of Adult Intensive Care, Queen Mary Hospital, Dr. Mark Ogino, Director, Critical Care Services Nemours/Alfred I duPont Hospital for Children and Mr. Kenneth KY Yu, Department Operation Manager of the Department of Adult Intensive Care, Queen Mary Hospital, for their zealous support of the ECMO simula-

, Wallace Ngai Chun Wai1

, Peter Lai Chi Keung1

http://dx.doi.org/10.5772/intechopen.76656

145

,

best approach in ECMO education.

**8. Conclusion**

patient outcomes.

tion programme.

**Author details**

Ricky Chan Wai Kit1

**References**

17, 2018]

Simon Sin Wai Ching1,2\*, Pauline Yeung Ng1

\*Address all correspondence to<EMAIL_ADDRESS>
ment. Englewood Cliffs, NJ: Prentice Hall; 1984

and Andy Mok Yuen Tin1

1 Department of Adult Intensive Care, Queen Mary Hospital, Hong Kong, China

2 Li Ka Shing Department of Medicine, The University of Hong Kong, Hong Kong, China

[1] Kolb DA. Experiential Learning: Experience as the Source of Learning and Develop-

[2] Fleming ND. The ADRK modalities. Available from: vark-learn.com [Accessed: March

[3] Edgar S. Kurt Lewin's change theory in the field and in the classroom: Notes toward a model of managed learning. Systemic Practice and Action Research: 1996;**9**(1):27-47

**Acknowledgements**

Recently, Zakhary et al. published the first randomized controlled trial comparing the performance after conventional water-drill training and simulation training. In this study, the simulation training group had a higher scenario score and a shorter time to critical action in certain scenarios. More importantly, this superior performance was sustained over 1 year [16]. However, it must be noted that despite the ongoing research and implementation of simulation training, there is yet evidence of association with better patient outcomes.