**6. Conclusions and research developments**

In conclusion, the critical and diachronic reading of the CSR approaches faced by the scientific community since the 1950s increasingly highlights the need to read the results of the capital investment not only in terms of financing costs, but also through the relationship risk/liability not only of the investor but of all the interest groups involved, of a private, public and public-private nature.

In this sense, "hybrid" assessment models can certainly offer innovative contributions, where, with respect to future emergencies (environmental, social, health), the following activities/phases become fundamental: 1) the identification, in forecast terms, of the changes generated by interventions, also in light of the framework of responsibility; 2) continuous monitoring of the cycle of the investment project; 3) the signing of a "liability pact" between the interested parties from the initial stages of the processes.

In all the models shown in §5.1 and 5.2 four the founding principles, which reinforce the classic CSR approach and on which to direct future research developments: 1) the adoption of a social impact oriented approach, which explicitly includes the achievement of social objectives, 2) the investigation of new and more

#### *Corporate Social Responsibility*

extensive value chains, in order to better understand the rainfall generated by the assumption of social responsibility and the prevention of any areas of conflict; 3) the environmental management is the strategic action for the top management and entrepreneurs, that in corporate decisions evaluate green solutions among the factors upstream of the company's competitiveness, its degree of innovation and research and its credibility among stakeholders; 4) the green processes and product innovation as the lever of proactive measures.


**2. Model and Formalism**

We consider a two-dimensional (2D) non-interacting pseudospin-1 Dirac–Weyl system modulated by two time-dependent electric potential barriers illustrated in Figure 1. The pseudospin-1 Dirac–Weyl fermions are charged quasiparticles originating from free electrons moving in the three-band structure consisting of gapless tip-to-tip two cones intersected by a flat band, which is shown in Figure 1c. Their dynamics are governed by the dot product of the spin-1 operator and the momentum. Matrices of the spin-1 operator *S*ˆ = *S*ˆ *x*, *S*ˆ *y*, *S*ˆ *z* in the *S*ˆ *z*-representation (the representation that *S*ˆ *z* is diagonalized) can be deduced from spin-lifting/lowering operators *S*ˆ ± = *S*ˆ *x* ± *S*ˆ *y* by *S*ˆ ± |*S*, *Sz* = (*S* ∓ *Sz*) (*S* ± *Sz* + 1)|*S*, *Sz* ± 1 [53]. Simple algebra leads to the results that

$$
\hat{S}\_x = \frac{1}{\sqrt{2}} \begin{pmatrix} 0 & 1 & 0 \\ 1 & 0 & 1 \\ 0 & 1 & 0 \end{pmatrix}, \quad \hat{S}\_y = \frac{1}{\sqrt{2}} \begin{pmatrix} 0 & -i & 0 \\ i & 0 & -i \\ 0 & i & 0 \end{pmatrix}, \quad \hat{S}\_z = \begin{pmatrix} 1 & 0 & 0 \\ 0 & 0 & 0 \\ 0 & 0 & -1 \end{pmatrix}. \tag{1}
$$

By applying AC gate voltages, Hamiltonian of the pseudospin-1 Dirac–Weyl fermions has the form

$$
\hat{H} = -i\hbar v\_{\hat{\mathcal{S}}} \hat{\mathbf{S}} \cdot \nabla + V \left( \mathbf{x}, t \right), \tag{2}
$$

where **Sˆ** is the spin-1 operator defined in Equation (1), *vg* ≈ 106 m/s is the group velocity associated with the slope of the Dirac cone. As shown in Figure 1a, the potential function has the form

$$V(\mathbf{x},t) = \begin{cases} V\_0 + V\_1(t), & 0 < \mathbf{x} < L\_{1\prime} \\ V\_0 + V\_2(t), & L\_2 < \mathbf{x} < L\_{3\prime} \\ 0, & \text{others} \end{cases} \tag{3}$$

with *V*1(*t*) = *V*1*ω*cos(*ωt* + *ϕ*) and *V*2(*t*) = *V*2*ω*cos(*ωt*). The Fermi energy of the two reservoirs to the two sides of the double-barrier structure are equalized to eliminate the external bias and secure energy-conserved tunneling. While the frequency of the potential modulation *ω* is small compared to the carrier interaction time (Wigner delay time) with the conductor, the quantum pump can be considered "adiabatic" [1,18,23]. In this case, one can employ an instant scattering matrix approach, which depends only parametrically on the time *t*. The Wigner–Smith delay time can be evaluated by *τ* = Tr( − *ih*¯ **s**† *∂***s** *∂EF* ), with **s** the scattering matrix defined in Equation (5). Calculations below show *τ* ≈ 10−14 s for all the parameter values. Thus, the adiabatic condition can be well justified when *ω* is in the order of MHz [3].

**Figure 1.** (**a**) schematics of the adiabatic quantum pump. Two time-dependent gate voltages with identical width *d* and equilibrium strength *V*0 are applied to the conductor. Time variation of the two potentials *V*1 and and *V*2 is shown in panel (**b**). *V*1 and *V*2 have a phase difference giving rise to a looped trajectory after one driving period; (**c**) two-dimensional band structure of the pseudospin-1 Dirac–Weyl fermions with a flat band intersected two Dirac cones at the apexes; (**d**) conductivity of the pseudospin-1 Dirac–Weyl fermions measured by [54] *σ* = *e*2*kF d πh π*/2 −*π*/2 |*t*(*EF*, *θ*)|2 cos *θdθ* in single-barrier tunneling junction as a function of the Fermi energy for three different values of barrier height *V*0. *kF* = *EF*/¯*hvg* is the Fermi wavevector and *t* is the transmission amplitude defined in Equation (5). It can be seen that higher barrier allowing larger conductivity occurs at the Dirac point *EF* = *V*0 and around *EF* = *V*0/2 (see the text).

For studying the transport properties, the flux normalized scattering modes in different regions can be expressed in terms of the eigenspinors as

$$\Psi = \begin{pmatrix} \Psi\_1 \\ \Psi\_2 \\ \Psi\_3 \end{pmatrix} = \begin{cases} a\_l \Psi\_{\to} + b\_l \Psi\_{\leftarrow \leftarrow} & \mathbf{x} < \mathbf{0}, \\\ a\_1 \Psi\_{1 \to} + b\_1 \Psi\_{1 \leftarrow \leftarrow} & \mathbf{0} < \mathbf{x} < L\_1, \\\ a\_2 \Psi\_{\to} + b\_2 \Psi\_{\leftarrow \leftarrow} & L\_1 < \mathbf{x} < L\_2, \\\ a\_3 \Psi\_{2 \to} + b\_3 \Psi\_{2 \leftarrow \leftarrow} & L\_2 < \mathbf{x} < L\_3, \\\ a\_r \Psi\_{\leftarrow} + b\_r \Psi\_{\rightarrow} & \mathbf{x} > L\_3. \end{cases} \tag{4}$$

where *kx* = /*E*2*F*/(*hv*¯ *g*)2 − *k*2*y* with *EF* the quasiparticle energy at the Fermi level of the reservoirs. Ψ→ = 1 2√cos *θ e*−*iθ*, √2*s*,*eiθ*T*eikx x* for *EF* = 0 (quasiparticles on the two cone bands) and √ 1 2 −*e*−*iθ*, 0,*eiθ*T (we also identify it as Ψ0 for discussions in the next section) for *EF* = 0 (quasiparticles on the flat band). *θ* = arctan(*ky*/*kx*), and *s* = sgn(*EF*). Ψ← can be obtained by replacing *kx* with −*kx* in Ψ→; Ψ*i*→/Ψ*i*← (*i* = 1, 2) can be obtained by replacing *kx* with *qxi* = /(*EF* − *V*0 − *Vi*)2/(*hv*¯ *g*)2 − *k*2*y* and *s* with *si* = sgn(*EF* − *V*0 − *Vi*) in Ψ→/Ψ←. The flux normalization factor 2√cos *θ* is obtained [55] by letting Ψ†(*∂H*ˆ /*∂kx*)Ψ = 1. *ψi* (*i* = 1, 2, 3) picks up the *i*-th row of the spinor wave function in all the five regions. Note that quasiparticles on the flat band contribute no flux in the *x*-direction. However, it must be taken into account in the pumping mechanisms while the Fermi energy lies close to the Dirac point. We will go to this point again in the next section.

The boundary conditions are that *ψ*1 + *ψ*3 and *ψ*2 are continuous at the interfaces, respectively [48]. The derivation of the boundary condition is provided in Appendix A. After some algebra, the instant scattering matrix connecting the incident and outgoing modes can be expressed as

$$
\begin{pmatrix} b\_l \\ b\_r \end{pmatrix} = \begin{pmatrix} r & t' \\ t & r' \end{pmatrix} \begin{pmatrix} a\_l \\ a\_r \end{pmatrix} = \mathbf{s}(V\_1, V\_2) \begin{pmatrix} a\_l \\ a\_r \end{pmatrix} \tag{5}
$$

where **s** is parameter-dependent. Detailed derivation of the scattering matrix is provided in Appendix B.

The DC pumped current flowing from the *α* reservoir at zero temperature could be expressed in terms of the Berry phase of the scattering matrix formed within the looped trajectory of the two varying parameters as [2,18,23]

$$I\_{\rm p\alpha} = \frac{\omega e}{2\pi} \int\_{A} \Omega\left(\alpha\right) dV\_{1} dV\_{2\prime} \tag{6}$$

where

$$\Omega\left(\alpha\right) = \sum\_{\beta} \text{Im} \frac{\partial \mathbf{s}\_{a\beta}^{\*}}{\partial V\_{1}} \frac{\partial \mathbf{s}\_{a\beta}}{\partial V\_{2}}.\tag{7}$$

*A* is the enclosed area in the *V*1-*V*2 parameter space. While the driving amplitude is small ( *Viω V*0), the Berry curvature can be considered uniform within *A* and we have

$$I\_{\mu\nu} = \frac{\omega e \sin \varphi V\_{1\omega} V\_{2\omega}}{2\pi} \Omega\left(\alpha\right). \tag{8}$$

Conservation of current flux secures that the pumped currents flowing from the left and right reservoirs are equal: *Ipl* = *Ipr*. The angle-averaged pumped current can be obtained as

$$I\_{\rm paT} = \int\_{-\pi/2}^{\pi/2} I\_{\rm pa} \cos \theta d\theta. \tag{9}$$


**3. Results**

#### *3.1. Inhibition of GA against SARS-CoV-2 Infection against S Protein-Pseudotyped Virus*

We used an S protein-pseudotyped lentivirus to determine whether GA had an antiviral effect against SARS-CoV-2. The lentivirus system is easy to construct and has been widely used for virus binding and infection assay. To this end, cells were infected with varying amounts of Lenti-S in the absence or presence of varying amounts of GA (Figure 1A). We tested GA at concentrations of 0.5–5 mM since previous studies showed that GA was previously reported active against a wide range of enveloped viruses at concentrations of 1–8 mM [10,12]. Vero E6 cells in 96-well plates were untreated or treated with GA at 0.5, 1, 2.5, and 5 mM 30 min prior to Lenti-S infection, and we found that GA treatment resulted in a reduction in luciferase activity (Figure 1B). At 2.5 and 5 mM, GA treatment reduced Lenti-S-mediated luciferase gene delivery by approximately 77% and 92%, respectively. The effect of GA on Lenti-S infection was specific since treatment of 293T cells transfected with pLenti-CMV-luc did not affect luciferase expression (Figure 1C).

**Figure 1.** *Cont*.

**Figure 1.** GA effect on Lenti-S infection. (**A**) Molecular structure of glycyrrhizic acid. (**B**) Effect of GA on Lenti-S infection. Vero E6 cells were infected with Lenti-S pseudovirus in the absence or presence of GA at indicated concentrations for 24 h. Luciferase activity was determined. Data are expressed as a percentage of untreated controls (Lenti-S). The experiment was performed twice, and data are mean ± SD of triplicate wells. Independent two-sample comparisons between non-GA treated sample and sample treated with GA at different concentrations, respectively, were determined by Student's t test. ns: no significance; \*, *p* < 0.05; \*\*, *p* < 0.01. (**C**) Effect of GA on luciferase expression in pCMV-luc-transfected cells. Monolayers of 293T cells were transfected with pCMV-luc for 16 h. The cells were then treated with GA at indicated concentrations. Luciferase activity was determined after 24 h incubation. GA treatment did not affect luciferase expression delivered by an encoding plasmid. The experiment was performed twice. The readings from untreated samples were used as a control for the calculation of relative luciferase activity. Data are mean ± SD of duplicate wells from 2 independent experiments. (**D**) Time of GA addition on Lenti-S-mediated luciferase gene delivery. Vero E6 cells were untreated or treated with 3 mM GA at 2 h prior to (−2 h), during (0), or at 2 and 4 h post Lenti-S infection. Luciferase activity was determined 24 h PI. The experiment was performed 2 times. Data from the untreated controls were used for the calculation of relative luciferase activity. \*\*, *p* < 0.01.

The time effect of GA addition was tested by treating Vero E6 cells with 3 mM GA at 2 h prior to (−2 h), during (0 h), or at 2 h and 4 h post Lenti-S infection. Luciferase expression was determined at 24 h PI. As shown in Figure 1D, GA addition prior to or during Lenti-S inoculation significantly blocked Lenti-S infection. For comparison, addition of GA at 2 and 4 h post Lenti-S inoculation showed diminished effect against Lenti-S infection. This result suggests that GA likely targeted the early stages of Lenti-S infection.


**Figure 6.**

*xCARE adapted to PAPRIKA.*

13 https://www.garmin.com/en-US/

14 http://lifevit.es/

The pilot of PAPRIKA is currently running in the 4 sites (Spain, Germany, France, and Poland). The case management system for professionals is available in each hospital thanks to its integration with the EHRs, while the self-management system is an app available in both Apple App Store and Google Play markets15. **Figure 7** shows some screenshots of the case management system for the professionals: (a) the list of prescriptions of a patient; and of the self-management system for the patients (b) the main screen with the list of tasks to be performed, (c) the performed physical activity, and (d) the communications with the team of professionals in charge.


[6] Selleslagh J, Echard A, Pécheyran C, Baudrimont M, Lobry J, Daverat F. Can analysis of *Platichthys flesus* otoliths provide relevant data on historical metal pollution in estuaries? Experimental and in situ approaches. Science of the Total Environment. 2016;**557**:20-30

[7] Cappello T, Mauceri A, Corsaro C, Maisano M, Parrino V, Paro GL, et al. Impact of environmental pollution on caged mussels *Mytilus galloprovincialis* using NMR-based metabolomics. Marine Pollution Bulletin. 2013;**77**(1-2):132-139

[8] de Souza Machado AA, Spencer K, Kloas W, Toffolon M, Zarfl C. Metal fate and effects in estuaries: A review and conceptual model for better understanding of toxicity. Science of the Total Environment. 2016;**541**:268-281

[9] Prabhakaran P, Ashraf MA, Aqma WS. Microbial stress response to heavy metals in the environment. RSC Advances. 2016;**6**(111):109862-109877

[10] Fernandez-Luqueno F, Lopez-Valdez F, Gamero-Melo P, Luna-Suarez S, Aguilera-Gonzalez EN, Martínez AI, et al. Heavy metal pollution in drinking water-a global risk for human health: A review. African Journal of Environmental Science and Technology. 2013;**7**(7):567-584

[11] Engel DW, Fowler BA. Factors influencing cadmium accumulation and its toxicity to marine organisms. Environmental Health Perspectives. 1979;**28**:81-88

[12] Nemr AE, Khaled A, Sikaily AE. Distribution and statistical analysis of leachable and total heavy metals in the sediments of the Suez Gulf. Environmental Monitoring and Assessment. 2006;**118**(1):89-112

[13] Murray CN, Murray L.

Adsorption-desorption equilibria of some radionuclides in sediment-freshwater and sediment-seawater systems. Monaco: International Atomic Energy Agency; 1973. pp. 105-124

[14] Bryan GW. The occurrence and seasonal variation of trace metals in the scallops *Pecten maximus* (L.) and *Chlamys opercularis* (L.). Journal of the Marine Biological Association of the United Kingdom. 1973;**53**(1):145-166

*Fish Otolith Microchemistry as a Biomarker of Metal Pollution in the Estuarine Ecosystem DOI: http://dx.doi.org/10.5772/intechopen.114005*

[15] Bryan GW, Hummerstone LG. Indicators of heavy-metal contamination in the Looe estuary (Cornwall) with particular regard to silver and lead. Journal of the Marine Biological Association of the United Kingdom. 1977;**57**(1):75-92

[16] Tomlinson DL, Wilson JG, Harris CR, Jeffrey DW. Problems in the assessment of heavy-metal levels in estuaries and the formation of a pollution index. Helgoländer meeresuntersuchungen. 1980;**33**(1):566-575. DOI: https://doi. org/10.1007/BF02414780

[17] Sunda WG, Engel DW, Thuotte RM. Effect of chemical speciation on toxicity of cadmium to grass shrimp, *Palaemonetes pugio*: Importance of free cadmium ion. Environmental Science & Technology. 1978;**12**(4):409-413

[18] Dauvin JC. Effects of heavy metal contamination on the macrobenthic fauna in estuaries: The case of the Seine estuary. Marine Pollution Bulletin. 2008;**57**(1-5):160-169

[19] Pan K, Wang WX. Trace metal contamination in estuarine and coastal environments in China. Science of the Total Environment. 2012;**421**:3-16

[20] Leonhard I, Agiadi K. Addressing challenges in marine conservation with fish otoliths and their death assemblages. Geological Society, London, Special Publications. 2023;**529**(1):243-262

[21] de Almeida PRC, da Costa MR, de Oliveira RSC, Almeida A, Azevedo R, Monteiro-Neto C, et al. The use of the shape and chemistry of fish otoliths as a subpopulational discrimination tool for *Eugerres brasilianus* in lagoon systems in the Southwest Atlantic Ocean. Fisheries Research. 2023;**267**:106795

[22] Jenkins JA, Musgrove M, White SJO. Outlining potential biomarkers of exposure and effect to critical minerals: Nutritionally essential trace elements and the rare earth elements. Toxics. 2023;**11**(2):188

[23] Arai T, Ohji M, Hirata T. Trace metal deposition in teleost fish otolith as an environmental indicator. Water, Air, and Soil Pollution. 2007;**179**(1):255-263

[24] Milton DA, Chenery SR. Sources and uptake of trace metals in otoliths of juvenile barramundi (*Lates calcarifer*). Journal of Experimental Marine Biology and Ecology. 2001;**264**(1):47-65

[25] Dove SG, Gillanders BM, Kingsford MJ. An investigation of chronological differences in the deposition of trace metals in the otoliths of two temperate reef fishes. Journal of Experimental Marine Biology and Ecology. 1996;**205**(1-2):15-33

[26] Coote GE, Gauldie RW, West IF. Application of a nuclear microprobe to the study of fish otoliths and scales. Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms. 1991;**54**(1-3):144-150

[27] Rana A, Attri SV, Singh S, Kaur R. Otolith microchemistry of freshwater indigenous minor carp (Bangana dero) as a biomonitoring tool to trace heavy metals in River Sutlej, Ropar Wetland (Ramsar site), Punjab, India. Environmental Monitoring and Assessment. 2023;**195**(11):1286

[28] Vrdoljak D, Matić-Skoko S, Peharda M, Uvanović H, Markulin K, Mertz-Kraus R. Otolith fingerprints reveals potential pollution exposure of newly settled juvenile Sparus aurata. Marine Pollution Bulletin. 2020;**160**:111695

[29] Pracheil BM, George R, Chakoumakos BC. Significance of otolith calcium carbonate crystal structure diversity to microchemistry studies. Reviews in Fish Biology and Fisheries. 2019;**29**:569-588

[30] Andronis C, Evans NJ, McDonald BJ, Nice HE, Gagnon MM. Otolith microchemistry: Insights into bioavailable pollutants in a man-made, urban inlet. Marine Pollution Bulletin. 2017;**118**(1-2):382-387

[31] Simpson SD, Radford AN, Nedelec SL, Ferrari MC, Chivers DP, McCormick MI, et al. Anthropogenic noise increases fish mortality by predation. Nature Communications. 2016;**7**(1):1-7

### *Edited by Ana Marta Gonçalves*

Marine ecosystems are very productive and include the open ocean, the deep-sea ocean, and coastal marine ecosystems, each of which has different physical and biological characteristics. The biodiversity of some of these ecosystems is very rich and abundant, offering unique opportunities for high-yield production of proteinaceous material, which is a source of high-quality foods. Biodiversity is fundamental to sustaining marine ecosystem services, such as food security, maintenance of water quality, and recovery from perturbations, which are currently being threatened worldwide. The main threats to marine biodiversity are habitat loss, eutrophication, overexploitation, pollution by hazardous substances, introduction of non-native species, and other human activities. Efforts to reduce these pressures are essential for coastal water quality, recovery of ecosystem services, global food security, and ecosystem stability. Bioindicators are important tools to be used as early warning signals to detect the presence of natural and chemical stressors when monitoring and managing ecosystems and thus promoting ecosystem health. The protection of biodiversity is a major target of the European Union Marine Strategy Framework Directive, requiring an assessment of the status of biodiversity on the level of species, habitats, and ecosystems, including genetic diversity and the role of biodiversity in food web structure and functioning. The restoration of marine ecosystems can support the productivity and reliability of goods and services that the ocean provides to humankind to maintain ecosystem integrity and stability. Some of the goods produced by the marine ecosystem services are fish harvests, wild plant and animal resources, water, and services that provide recreation, tourism, breeding and nursery habitats, water transport, carbon sequestration, erosion control, and habitat provision. This book comprises six chapters that discuss the ecological, economic, and social roles of a variety of organisms, from primary producers to consumers, relating these roles with water quality and conservation of estuarine and marine ecosystems and thus the contribution to human health.

### *J. Kevin Summers, Environmental Sciences Series Editor*

Published in London, UK © 2024 IntechOpen © Jian Fan / iStock

Marine Ecosystems - Biodiversity, Ecosystem Services and Human Impacts

IntechOpen Series

Environmental Sciences, Volume 15

Marine Ecosystems

Biodiversity, Ecosystem Services

and Human Impacts

*Edited by Ana Marta Gonçalves*


**3. Defense Responses of GLS to Bacterial Diseases**

The bacteria that infect cruciferous plants are all rod-shaped bacteria, which can invade the host through stomata, hydathodes, and wounds, and then be retransmitted by running water, rain, insects, etc. Bacterial diseases in cruciferous have widespread occurrence, are highly destructive, and are challenging to control. Meanwhile, because the pathogens are different from the fungal diseases, the corresponding disease resistance mechanism of host and the GLS involved in resistance may be different. Several studies have demonstrated that GLS are involved in plant defense against a variety of bacterial diseases. Similar to the case in fungal disease infection, infection by *Burkholderia cepacia*, *Pseudomonas syringae*, and *Xanthomonas campestris* pv. *campestris* (*Xcc*) led to the upregulation of the GLS biosynthesis [99]. In addition, the introduction of CYP79 influenced *Arabidopsis* disease resistance by increasing the GLS synthesis, and overexpressing the CYP79D2 from cassava increased the accumulation of the aliphatic isopropyl and methylpropyl GLS, which also enhanced resistance against the soft-rot pathogen, *Erwinia carotovora*; however, overexpressing the sorghum CYP79A1 or CYP79A2 increased the accumulation of p-hydroxybenzyl and benzyl GLS, respectively [100].

Mishina et al. (2007) observed that the knockout of PAL1 increased leaf survival after *P. syringae* infection in an analysis conducted on *Arabidopsis* mutants and wild type plants, while Truman et al. (2007) and Aires et al. (2011) observed that indole GLS biosynthesis decreased after *P. syringae* infection [101–103]. Following the transcriptional and metabolic profiling of *A. thaliana* mutants, Clay et al. (2009) reported that the PEN2 and PEN3 genes are necessary for resistance to PtoDC3000 pathogens [104]. Furthermore, Geng et al. (2012) demonstrated that coronatine, a toxin produced by *P. syringae*, suppresses the salicylic acid (SA)-independent pathway, facilitating callose deposition by reducing the accumulation of an indole GLS upstream of the PEN2 myrosinase activity [105]. In addition, a positive correlation has been reported between total GLS content and *Xcc* disease severity, and *Xcc* infection enhanced GLS biosynthesis during the early infection period [106,107]. *Pectobacterium carotovorum* ssp. *carotovorum* infection in *B. rapa* can trigger the upregulation of the JA and ethylene (ET) biosynthesis genes in sr gene mutants and increase resistance capacity via GLS accumulation [108].


**Author details**

Shanuka Senarath Department of Economics, University of Colombo, Colombo, Sri Lanka

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


*3.2. Thermochemical Investigations*

Figure 2 shows differential scanning calorimetry (DSC) thermograms of enantiopure (curve 1) and racemic (curves 2-4) samples of diol **1**. The thermogram of an enantiopure sample (*R*)-**1** is represented by a single narrow peak, which indicates its phase uniformity. The thermochemical parameters for this sample are presented in Table 3. The same parameters for the sample (*R*)-**1** obtained by crystallization from the melt practically do not differ from those for the sample crystallized from solution.

On the contrary, a thermogram of a freshly-prepared chemically-pure racemate obtained by crystallization from a hexane/ethyl acetate mixture demonstrates the complex contour of the melting process curve 2. The main peak, located in the temperature range of ~ 75 ◦C, has a leading edge with a pronounced kink, which is preceded by a minor endothermic peak, observed at a significantly lower temperature (~ 68 ◦C). This behavior is characteristic of phase-inhomogeneous systems represented by a mixture of several crystalline modifications. Intensive mixing of a suspension of such a sample in hexane for several hours at room temperature results in a thermodynamically equilibrium phase-homogeneous racemic sample, the thermogram of which is a narrow peak with a regular shape (curve 3). The demonstration of spontaneous resolution of *rac*-**1** (Section 3.1) suggests that the main crystalline form of the racemate is a conglomerate, which we denote by the symbol (*R*+*S*). The melting point of the brought to equilibrium *rac*-**1** sample, calculated as the intersection of the free energy curves of the crystalline enantiopure phase and melted (*R*+*S*)-**1** (Figure 4) was 76.2 ◦C, which practically coincides with the actually observed for conglomerate (76.3 ◦C) (Table 3).

**Figure 2.** Differential scanning calorimetry (DSC)traces of an enantiopure sample (*R*)-**1** (blue curve 1), a freshly recrystallized racemic sample *rac*-**1** (wine curve 2), the same sample after prolonged stirring of a suspension in hexane (red curve 3), and after crystallization of the racemate from the melt (olive curve 4). Sample weight ~ 1 mg.

**Table 3.** Thermochemical characteristics of the identified crystalline forms of 3-(3,4-dimethylphenoxy) propane-1,2-diol **1**.

1 In parentheses are the values calculated on the basis of the DSC data for the dependence of the thermodynamic potentials of the phases on temperature (Section 3.3, Figure 3).

If a racemic sample of diol **1** of any prehistory is melted and quickly cooled to room temperature, then the supercooled melt in the range of ~10 min undergoes spontaneous crystallization. The melting of the phase thus obtained (β-*rac*-**1**) is described by a peak at ~ 66 ◦C (curve 4), which on the temperature scale is practically in the same region as the minor endothermic peak in curve 2, Figure 2. This suggests that the β-*rac*-**1** phase is present in the samples, which initially precipitate from solutions, and the minor endothermic peak arises due to this.

The general appearance of melting curve 2 indicates that the phase behavior of the primary *rac*-**1** crystals is even more complex and, in addition to the identified (*R*+*S*)-**1** and β-*rac*-**1**, other crystalline modifications can be present in the system. Indeed, after the β-*rac*-**1** phase is heated to 72 ◦C and held at this temperature, it melts almost completely, but then crystallizes again. The melting parameters of the resulting α-*rac*-**1** phase (Figure 3, violet) are close, but not identical to those for the equilibrium racemic sample (Figure 3, red; Table 3).

Taken together, the thermochemical data show that for the enantiopure forms of diol **1** the only one crystalline phase is realized, and the racemate can be represented by a thermodynamically equilibrium conglomerate (*R*+*S*)-**1**, two polymorphs α-*rac*-**1** and β-*rac*-**1**, as well as a mixture of these modifications. Table 3 shows the thermochemical parameters of the corresponding systems.

**Figure 3.** DSC traces of an equilibrium racemic sample identified as a conglomerate (red curve), a racemic sample obtained by crystallization from melt at 72 ◦C (violet curve), and crystallization from melt upon rapid cooling to 20 ◦C (olive curve). Sample weight ~ 8 mg.


*2.5. Processing and Analysis of 1H NMR Data*

All NMR spectra were phased, manually baseline corrected and exponentially linebroadened with a 1Hz window function using TOPSPIN 3.6.1 (Bruker) software. The spectra were then imported into Chenomx 8.4. A shim correction of 1.2 Hz was applied and the region from 4.56 ppm to 4.97 ppm was deleted to eliminate water resonance peaks. Peak assignment was performed using the Chenomx profiler tool fitting the spectral line to the proposed compounds in the standard Chenomx library. The efficacies of the extraction solvents, lysis methods and incubation temperatures were then compared using molecule concentration ratios and number of metabolites ratios between the two samples (Figure S3) (e.g.: IE/M = I ethanol/I methanol).

The median, standard deviation (StDev) and coefficient of variance (CV) were all calculated to determine the reproducibility of the results. Any outliers were detected and removed from the analysis.

#### **3. Results**

In order to determine the optimal protocol to extract metabolites from *Blastocystis* ST7 for NMR analysis, a series of extraction solvents, lysis techniques and incubation temperatures were examined. The efficacy of each protocol was assessed using proton NMR and the peak intensity was compared using TOPSPIN 3.6.1 to determine which method extracted the highest concentrations of metabolites. We then developed an efficient, reproducible protocol to perform metabolomics studies on *Blastocystis* species and found that the extraction solvent and lysis method were the most important factors for metabolite extraction. The efficacy was optimised in four sets of experiments, which firstly compared solvents (MeOH versus EtOH), then compared methods (sonication vs. bead bashing) and finally the temperature regime used (−20 ◦C versus RT,) and (60 ◦C versus RT).

#### *3.1. Comparison of Steps*

Two analysis methods were used during the comparisons of pairs of processing steps to rank efficacy. These were molar concentration ratios C μMA/B for processes A and B, as measured using the standard Chenomx metabolite library against the internal DSS standard. Secondly, the ratios of the raw number of detectable metabolites extracted NA/B using the two processes (A and B), again using the Chenomx metabolite library. All analyses were made in pairs of samples in triplicates, and samples in the triplicate were denoted by Arabic numerals 1–3 and condition pairs by A and B. Therefore, for a comparison of methanol and ethanol, 1A–3A were ethanol samples and 1B–3B were methanol samples.


*Edited by Priti Kumar Roy*

This book is projected as a preliminary manuscript in Infectious Disease. It is undertaken to cover the foremost basic features of the articles. Infectious Disease and analogous phenomenon have been one of the main imperative postwar accomplishments in the world. The book expects to provide its reader, who does not make believe to be a proficient mathematician, an extensive preamble to the field of infectious disease. It may immeasurably assist the Scientists and Research Scholars for continuing their investigate workings on this discipline. Numerous productive and precise illustrated descriptions with a number of analyses have been included. The book offers a smooth and continuing evolution from the principally disease oriented lessons to a logical advance, providing the researchers with a compact groundwork for upcoming studies in this subject.

ISBN 978-953-51-0319-6

Insight and Control of Infectious Disease in Global Scenario

Photo by Rost-9D / iStock

## Insight and Control of Infectious Disease in Global Scenario

*Edited by Priti Kumar Roy*

**INSIGHT AND CONTROL**

**OF INFECTIOUS DISEASE**

**IN GLOBAL SCENARIO**

Edited by **Priti Kumar Roy**


Therefore, ascending the ordination platform in the midst of the *bhiks.un. ¯ı*s is a 'preparatory' step, but a necessary one. Only after ascending the ordination platform in the midst of the *bhiks.u*s is it decided that you have eventually become a *bhiks.un. ¯ı*. However, the master of the discipline will also stress that, as you have been ordained there, after ordination you will have to continue studying the precepts with that *upadhy ¯ ayin ¯ ¯ı*. So, this is how this system was established. It is called Dual Ordination.

所以,比丘尼當中登壇算是一個預備,但是是必要的預備。而比丘當中登壇才算 是最後,決定你最後成為比丘尼了。但是戒和尚還是說,你在這裡受了戒,以後 還是要去跟著你那個比丘尼的戒和尚學習,所以這個制度是這樣建立起來的,稱 為二部僧戒。

#### **Notes**

seeing a newly ordained *bhiks.u*, and she must pay obeisance; (2) A *bhiks.un. ¯ı* may not revile a *bhiks.u* saying that he has done something wrong; (3) A *bhiks.un. ¯ı* may not admonish a *bhiks.u*, whereas a *bhiks.u* may admonish a *bhiks.un. ¯ı*; (4) After a woman has been trained as a *´siks. ama¯n. a¯* for two years, the ordination ceremony must be carried out in both orders; (5) When a *bhiks.un. ¯ı* has committed a *sam. ghava´se ¯ s. a* offense (an offense that leads to a temporary exclusion), she has to undergo the penance in both orders; (6) Every fortnight, *bhiks.un. ¯ı*s have to ask *bhiks.u*s for instruction; (7) *Bhiks.un. ¯ı*s cannot spend the summer retreat (rainy season) in a place where there are no *bhiks.u*s; (8) At the end of the summer retreat, *bhiks.un. ¯ı*s also have to carry out the *pravara ¯ n. a¯* ceremony in the *bhiks.u* order. On the history of the beginning of the *bhiks.un. ¯ı* order, see Analayo ¯ (2016, 2019) and, for a different view, von Hinüber (2008); on the narrative of the foundation according to the *Vinaya* of the Dharmaguptaka and a comparison with the other available *Vinaya*s, see Heirman (2001, pp. 278–84).

period was also created to check the possibility of pregnancy in female candidates (Huimin 2007, p. 16; Heirman 2008, p. 108). On *´siks. aman¯ . a¯* ordination, see also Heng-Ching (2000, pp. 510–13), and Heirman (1997, pp. 36, n. 14, 45–47).

#### **References**

Aidaotang 愛道堂, ed. 2002. *Dangdai di yi biqiuni* 當代第一比丘尼. (DVD). Chengdu: Aidaotang.

Aidaotang 愛道堂, ed. 2009. *Dangdai di yi biqiuni* 當代第一比丘尼. (DVD). Chengdu: Aidaotang.

Analayo, Bhikkhu. 2013a. The Legality of Bhikkhun ¯ ¯ı Ordination. *Journal of Buddhist Ethics* 20: 310–33.

Analayo, Bhikkhu. 2013b. The Revival of the Bhikkhun ¯ ¯ı Order and the Decline of the Sasana. ¯ *Journal of Buddhist Ethics* 20: 110–93.

Analayo, Bhikkhu. 2014. On the Bhikkhun ¯ ¯ı Ordination Controversy. *Sri Lanka International Journal of Buddhist Studies* 3: 1–20.

Analayo, Bhikkhu. 2016. ¯ *The Foundation History of the Nuns' Order*. Bochum: Projektverlag, Hamburg Buddhist Studies 6.

Nenghai 能海. 1995. *Wenshu wuzi genben zhenyan niansongfa* 文殊五字根本真言念誦法. Chengdu: Zhaojuesi. First published 1936.