**Abstract**

Bone is the second most transplanted organ, just after blood. It provides structural support, protection for organs and soft tissues. It holds some critical biological processes such as the bone marrow blood forming system. It is responsible for storing and supplying minerals such calcium and phosphate. Bone is a connective tissue formed by two predominant phases: an inorganic phase containing mainly apatitic calcium and phosphate and an organic phase made of fibrous type I collagen. This natural biocomposite has many biological features such osteoconductivity, osteoinductivity, osteogenicity and is subject to a continuous remodeling process through osteoclastic and osteoblastic activities. In biomedical engineering, the restoration of damaged hard tissue with autologous bone is not always possible or even the best option. The development of some safe and low-cost alternatives such as biocomposites that mimic organic and calcified bone materials have shown very good results and offer an alternative to autologous bone implants. However, the mechanical properties of biocomposites still present a big challenge as a hard tissue substitute. This chapter reviews the properties of bone substitute materials chitosan and calcium phosphates, discusses strategies used in the treatment of calcified hard tissues as well as new approaches developed in this field.

**Keywords:** Bone, Chitosan, Calcium phosphate, Bioceramics, Biocomposites


**1. Introduction**

Many real-world systems exhibit some level or form of randomness. This is reflected in their mathematical models, which are often probabilistic. There are two basic strategies to interpret the randomness captured by probabilistic models. The frequency of occurrence of a random phenomenon is a measurable quantity that can be used to predict how likely the phenomenon can be observed in future. The other approach quantifies the uncertainty about the occurrence of random phenomenon more subjectively, that is, as a degree of belief or expectation of observing the phenomenon, given domain knowledge and the past experiences. This latter approach led to a broad area of general Bayesian methods [1], Bayesian data analyses [2], Bayesian signal processing [3], Bayesian regression [4], Bayesian machine learning [5], and

Bayesian optimization [6]. In the tasks of statistical inference, the assumption of knowing (or not) a prior distribution crucially affects the feasibility as well as the structure of estimators [7]. There is also an intimate connection between Bayesian probabilistic models and making causal inferences [8], as will be discussed in Section 3.

Bayesian methods found widespread applications in many probabilistic modeling frameworks. These methods are all rooted in the surprisingly simple Bayes's theorem, that is,

$$p(\theta|\mathbf{x}) = \frac{p(\mathbf{x}|\theta)p(\theta)}{p(\mathbf{x})} \tag{1}$$

where *p*ð Þ� denotes the (for continuous random variables) or the probability (for discrete random variables). Eq. (1) quantifies how our belief about the parameter *θ* changes from its prior, *p*ð Þ*θ* , to posterior, *p*ð Þ *θ*j*x* , after observing data *x*. The conditional term, *p x*ð Þ j*θ* , represents the likelihood of the parameter *θ*, given observations, *x*. The scaling term, *p x*ð Þ, in Eq. (1) is usually referred to as evidence. It should be noted that both the parameter and the data can be assumed in an arbitrary number of dimensions.

The majority of tasks in Bayesian inference involves explicitly or implicitly solving one of the following integrals (or sums, for discrete random variables), that is,

$$\text{marginalization:}\ p(\mathbf{x}) = \int\ p(\mathbf{x},\theta)d\theta\tag{2}$$

$$\text{sammarization: } E[f(\mathbf{x})|\theta] = \begin{cases} f(\mathbf{x}) \ p(\mathbf{x}|\theta) d\mathbf{x} \\ \end{cases} \tag{3}$$

$$\text{prediction:}\ p(\mathbf{x}\_{t+1}) = \begin{cases} p(\mathbf{x}\_{t+1}|\mathbf{x}\_t) & p(\mathbf{x}\_t)d\mathbf{x}\_t \\ \end{cases} \tag{4}$$

$$\text{do} \qquad \text{do} \qquad \begin{cases} p(\mathbf{x}\_{t+1}|\mathbf{x}\_t) & p(\mathbf{x}\_t)d\mathbf{x}\_t \end{cases} \tag{4}$$

$$p(\mathbf{x}\_{t+1}|\mathbf{x}\_t) = \int p(\mathbf{x}\_{t+1}|\theta) \ p(\theta|\mathbf{x}\_t) d\theta.$$

Unfortunately, in most but a few real-world scenarios, the expressions (2)–(4) are not mathematically tractable. In particular, the distributions often involve multiple sums and/or integrals, and their closed-form expressions cannot be obtained. The distributions are sometimes only known up to a scaling constant. Then, even numerically computing Eq. (1) can be rather challenging, since many complex distributions are multi-modal with a large number of local minima and maxima. Moreover, in online data processing, the distributions must be updated continuously as soon as the new data arrives.

The rest of this chapter is organized as follows. The strategies for performing Bayesian inferences with intractable distributions are outlined in Section 2 including sampling, filtering, approximation, and likelihood-free methods. The applications of Bayesian inferences are discussed in Section 3 including Bayesian experiment design, Bayesian hypothesis testing, Bayesian machine learning, and Bayesian optimization. Bayesian Monte Carlo simulations are reviewed in Section 4. Although the chapter mostly reviews known concepts and frameworks in Bayesian analysis of probabilistic models, Section 4 contributes a description of augmented Monte Carlo simulations. These simulations aim at providing explainability and improving information gain.

The references cited at the end of this chapter are by no means comprehensive; rather, they are suggestions of initial readings where to find further information about the topics discussed in this chapter.


New Scientific Approaches to Hypnosis

*Hypnotherapy and Hypnosis*

men: Issues for social work practice. In: Longres J, editor. Men of Color. New York: Harrington Park Press; 1996

[66] Daniel A. Hypnosis in human sexuality problems. American Journal of Clinical Hypnosis. 2005;**47**(4):229-242

[67] Unestahl LE. Alert, eyes-open sport hypnosis. American Journal of Clinical Hypnosis. 2018;**61**:159-172. DOI: 10.1080/00029157.2018.1491387

**38**

**Chapter 3**

*Rashit Tukaev*

**Abstract**

Hypnotherapy

the mindfulness effect of UH.

**1. Introduction**

**41**

of the research and practice.

efficiency of psychotherapy, mindfulness effect

The Integrative Theory of

Hypnosis in the Light of Clinical

The chapter describes the author's integrative theory of hypnosis and hypnotherapy (ITHH) and the universal hypnotherapy (UH) method. The ITHH contains neurophysiological, biological, and communicative components. (1) Hypnosis is triggered by symbolical hypnogenic situations of inability of decision-making and/or its behavioral realization. Hypnosis development results in qualitative reorganization of the brain activation system functioning from distribution to generation of activity. Hypnosis deepening is based on the increase of brain activation. Hypnosis development in right-handers is associated with a regressive reorganization of the left hemisphere to the right hemisphere functioning mode, with whole brain functioning on right hemispheric principle. (2) Hypnotization generates hypnogenic stress. Hypnotherapy activates a readaptation process, including neurohormonal, neurotransmitter secretions; activation of the immunological and biochemical responses; and spontaneous change of pain sensation. (3) Hypnotic communication styles (directive, non-directive) are (i) changing due to historical evolution of social communication styles and (ii) indirectly using the representations about hypnosis. The UH utilizes the ITHH, being close to the positive and mindfulness psychotherapeutic approaches. The complex of UH and psycho-education formed positive-dialogue psychotherapy (PDP) for the treatment of anxiety disorders. The randomized clinical trial of PDP efficiency in the therapy of panic and generalized anxiety disorders confirmed high clinical efficiency and

**Keywords:** hypnosis, theory, secondary-phenomenological approach, neurophysiology, hypnogenic situation, brain activation system, biology, communication, universal hypnotherapy, positive-dialogue psychotherapy, panic disorder, generalized anxiety disorder, randomized clinical trial,

The author's long-term work in the fields of hypnology and hypnotherapy revealed restrictions associated with the lacks of consistency and interdisciplinarity


**1. Introduction**

The collection, transportation, and disposal of solid waste (SW) produced by houses is a process known as municipal solid waste management. Waste collection and transportation are accomplished using a fleet of trucks. This conventional waste collection is based on speculation as to whether the filling levels of waste bins could vary from overflowing, partial filling, to completely emptying, which would result in unnecessary resource consumption. Improvements will reduce municipal spending as the cost of waste collection/transportation accounts for 60–80% of overall waste management system (WMS) expenses.

VRP research was conducted in 1959, and its goal was to model the distribution process and map out the most efficient pathways to deliver products to consumers [1]. In addition to the VRP's constraints, the CVRP adds the constraint of the vehicle's capacity.

A significant amount of garbage is produced due to the development in product diversity and consumption, endangering the ecosystem [2]. E-waste seriously harms the ecosystem when combine with the air and soil [3]. Burning/burying plastic garbage could endanger the land and air due to the hazardous chemical gases released [4]. Some 4 to 12 million metric tons of plastic trash are dumped into the oceans annually, leading to water contamination [5]. When medical waste is disposed of, it might cause major environmental issues [6].

It is estimated that the global daily production of medical waste due to the pandemic was over 1.5 million tons [7]. It is now necessary to collect this waste in a systematic

**Citation:** Rashid, T.; Ullah, S.; Habib, U.; Rehman, N.; Qureshi, U.R.; Jafar, F. Optimizing Waste Collection and Transportation in Islamabad: Efficient Vehicle Routing for Sustainable Waste Management. *Eng. Proc.* **2023**, *45*, 41. https://doi.org/10.3390/ engproc2023045041

Academic Editors: Mohammad Javed Hyder, Muhammad Mahabat Khan, Muhammad Irfan and Manzar Masud

Published: 14 September 2023

manner [8], in order to lessen its harmful impact [9], along with other forms of e-trash [10]. One Indian city's annual e-waste contained valuable materials worth \$65, 000 [11], showing the economic advantages of employing demolition waste, which is between 50 and 70% of the world's total SW [12].

In this study the method of optimizing routes is used not the waste. The remainder of this article is outlined as follows: Section 2 includes the acquisition of a dataset and an overview of the research methodology; Section 3 contains the model design; the simulation results are presented in Section 4; finally, the article is concluded in Section 5.

The MATLAB 2017B algorithm CVRP is used for simulation.

#### **2. Data Acquisition and Research Methodology**

In this section a brief overview of steps to acquire data and the employed research method are provided. The following steps were taken to acquire the waste bin and vehicle data from the concerned authorities: 1. visit MCI; 2. enlist their challenges and collect data; 3. locate the bins on Google Maps according to the acquired dataset; 4. import data to the web plot digitizer, draw axes, locate bins, and find coordinates; 5. create CSV and TXT files so the data becomes ready for the mathematical computing and analysis tools.

The dataset is ready, the only problem left was to employ the research method for obtaining the optimized route. Amongst the available resources, CVRP is widely used as discussed in Section 1. The following steps were taken to obtain an optimized route: 1. import geographic data into MATLAB 2017B; 2. transform data, i.e., the number of vehicles, capacities of vehicles, distances between the bins, expected expenditure, etc. into the MATLAB 2017B format; 3. locate bins and input the amount of waste in each bin into MATLAB 2017B; 4. run the CVRP simulation in MATLAB 2017B; 5. find optimal results; 6. based on these results, we plan to submit the proposal to MCI.

#### **3. Model Design**

#### *3.1. Description of the Problem*

The aim of this research is to minimize the overall distance traveled and the total comprehensive cost, including vehicle costs. The vehicles are located at a depot, and its routes begin there. When a waste truck is full, or the assigned task is completed, it returns to the depot.

Assumptions: In addition to the above, we assume the following: 1. one vehicle collects each bin once; 2. there is a single depot; 3. the vehicles leave the depot and return when the job is completed; 4. the vehicles are of various capacities; 5. the location of the depot and each bin is known.

#### *3.2. Model Construction*

In this subsection, the complete mathematical model for the optimized waste collection problem is provided. The main objectives are to minimize the vehicle distance as well as the cost of waste collection.  Constraint Equation (3) ensures that each waste bin is collected by a single vehicle. All routes will respect the maximum capacity by Equation (4) to Equation (6). Equation (7), defines the variable types, where *B* represents bins, *D* is depot, *V* is a set of vehicles, *Pfixed* is the expenses attached to each vehicle, *dmn* is the distance between bins '*m*' and '*n*', *rmn* is fuel consumption rate per unit kilometer, *Pf uel* is fuel consumption cost, *xk mn* checks whether a vehicle '*k*' moves from waste bin '*m*' to waste bin '*n*'. *qn* shows Waste collected at waste bin '*n*'. *Cp* is a vehicle's maximum carrying capacity.

#### **4. Results and Discussion**

The major results of this research are presented in this section. The map of the waste collection points and depot at G-6/1 is presented in Figure 1a. Seventy bins are located throughout the territory. The red truck represents the MCI and the blue dots indicate the waste bins.

**Figure 1.** (**a**) Locating the collection points on Google Maps; (**b**) importing to WebPlotDigitizer 4.1, drawing axes, locating collection points, and finding coordinates.

The CVRP algorithm is used for analysis to determine the exact method of solving. In Figure 1b, the Google Maps points were transferred into the WebPlotDigitizer 4.1 to transform the location of bins into a Cartesian plane dataset. The depot was defined at (0, 0), the *x* − *axis* parallel to the Kashmir Highway and the *y* − *axis* perpendicular to it. The geographic coordinates were retrieved and saved in both CSV and TXT file formats.

In Figure 2a, bins are displayed graphically within MATLAB 2017B, after the geographical coordinates were imported. The red circles are the location of the bins and the depot at (0, 0).

In Figure 2b, the yellow square shows the depot and rest of the colors show the optimized routes. Figure 2c shows a monotonic reduction, illustrating a steady decline in cost.

**Figure 2.** (**a**) Islamabad waste collection points in MATLAB 2017B; (**b**) Islamabad waste collection routes ( 70 × 8); (**c**) Islamabad waste collection optimized cost ( 70 × 8).

#### **5. Conclusions**

A case study in which the environmental problem of SWM was optimized by employing CVRP. The case involved SWM in the Islamabad Capital Territory (ICT) administered by MCI supervised by Capital Development Authority (CDA). The algorithm implemented for optimizing the routes shows a consistent and gradual decrease in cost with each iteration. It is crucial to emphasize that the algorithm ensures no further cost reduction is possible. After 1200 iterations, the algorithm identified the optimum paths, and no additional route optimization was achievable. This finding highlights the algorithm's effectiveness in enhancing SWM efficiency.

**Author Contributions:** Conceptualization, T.R., S.U. and U.R.Q.; methodology, T.R. and U.H.; software, T.R. and F.J.; validation, U.H. and N.R.; formal analysis, T.R. and U.H.; investigation, S.U.; resources, T.R. and U.R.Q.; writing—original draft preparation, T.R., N.R. and F.J.; writing—review and editing, U.H. and U.R.Q.; visualization, F.J.; supervision, S.U.; All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Dataset is available upon request.

**Acknowledgments:** The authors are thankful to the Sanitation Directorate (MCI), CDA, Islamabad for their continuous support during the course of this research.

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

#### **References**


**Acknowledgements**

This work is supported by the US Department of Energy, Office of Nuclear Energy, under DOE Idaho Operations Office Contract DE-AC07-05ID14517. Accordingly, the US Government retains a non-exclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for US Government purposes. This information was prepared as an account of work sponsored by an agency of the US Government. Neither the US Government nor any agency thereof, nor any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, com‐ pleteness, or usefulness of any information, apparatus product, or process disclosed, or represents that its use would not infringe privately owned rights. References herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise do not necessarily constitute or imply its endorsement, recommendation, or favoring by the US Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the US Government or any agency thereof.


**5. Radiation resistance of commercial GFSA components**

An external γ ray source was used to test commercially available GFSA components to analyze the effect of γ radiation. The examination was carried out on the following commercial components (1) SIEMENS (type A) gas surge arresters (nominal voltage 230 V), (2) CITEL BB (type B) bipolar ceramic gas surge arresters (DC spark overvoltage 230 V). Тhе outer dimensions and shape of all components of the same type were the same. The effects of γ radiation on following GFSA characteristics were examined:

The scheme of the test cycle for investigating the radioactive resistance of GFSA by a DC voltage is depicted in **Figure 5**.

Examination of GFSA radioactive resistance was carried out in a gamma radiation field of 60Co at the Institute of Nuclear Sciences "Vinča." The average energy of the applied gamma quantum was 1.25 MeV. The dose rate in air was 87.5, 875, and 1750 cGy/h, respectively. The distance between the radioactive source and the examined overvoltage components was 272, 86, and 60 cm, respectively. All tests were performed at room temperature, 20°C.

Test specimens, consisting of 50 commercial components of a single manufacturer, having identical characteristics, have been used in the experiment. During the formation of experimental groups consisting of 50 components each, the nominal characteristics of the tested components have been measured. When the measured values for a particular component exhibited significant discrepancy with respect to the declared values, they were excluded from further testing in accordance to the Sovene's criterion [2, 8].

The GFSA prebreakdown current as a function of applied voltage without radiation and with γ radiation is shown in **Figure 6A** and **B**, respectively. The diagrams demonstrate:

Single electrons in atomic orbitals have low effective photoionization cross sections due to the small wavelength of high energy γ photons that cause ionization through the Compton effect [21].

The GFSA resistance versus applied voltage is shown in **Figure 7A** and **B**, without radiation and in Co γ field, respectively. From the volt-ampere curve, the volt-ohm characteristic can be easily determined. Formula-defining relationship between resistance and voltage is obtained by linear regression, using least-square minimal error method. The following conclusions are made:

The main conclusions of the experiment are:

**223**

**Figure 6.**

**Figure 5.**

*Influence of Gamma Radiation on Gas-Filled Surge Arresters*

decrease of the resistance was observed.

3.In the prebreakdown regime, GFSA resistance increased linearly with the applied voltage. When the voltage reached breakdown level, an abrupt

4. γ radiation shows significant influence on GFSA performance. In prebreakdown regime, the current had one order of larger magnitude values than

5.In a γ radiation field, resistance also showed a linear increase with the applied voltage, but one order of magnitude was lower than values without radiation.

6.All observed effects of γ radiation on GFSA commercial components had reversible character. Shortly after exposure (in a matter of hours), GFSA

characteristics were the same as before irradiation.

*Scheme of the test cycle for investigating the radioactive resistance of GFSA by a DC voltage.*

*GFSA prebreakdown current without radiation (A) and under γ radiation (B) [9].*

*DOI: http://dx.doi.org/10.5772/intechopen.83371*

without radiation.

#### **Figure 5.**

*Use of Gamma Radiation Techniques in Peaceful Applications*

electrodes in the observed time frame.

irradiation.

through the Compton effect [21].

reaches breakdown level.

1.Before the breakdown in the absence of radiation, the current conducted by GFSA is constant and in the order of 0.1 nA. Upon the breakdown, the current rises very sharply (breakdown voltage being 212 V for type A components and 223 V for type B components). When voltage reaches the breakdown level, an abrupt increase of the current takes place, and current values reach μA level. Radiation causes increased numbers of electron-ion pairs in the area between electrodes, leading to the increase of prebreakdown current (consisting of the free electrons and ions reaching electrodes per unit of time). In the moment of breakdown (when one of the free electrons generated in this way becomes initial), an avalanche process generates a breakdown current, the magnitude of which is independent of the prebreakdown current. Ohm's law cannot be applied in this region, given that the observed two-electrode system saturated: all electron-ion pairs generated reach the

2.The influence of γ radiation on GFSA performance is significant. The prebreakdown current is 10 times larger than the current without radiation. The increase of the radiation dose rate causes an increase in the prebreakdown current. Breakdown occurred at lower voltages (205 V) in a γ radiation field. During the transition between nonconducting and conducting regime, the current increase is not as sharp as compared to the breakdown without

Single electrons in atomic orbitals have low effective photoionization cross sections due to the small wavelength of high energy γ photons that cause ionization

The GFSA resistance versus applied voltage is shown in **Figure 7A** and **B**, without radiation and in Co γ field, respectively. From the volt-ampere curve, the volt-ohm characteristic can be easily determined. Formula-defining relationship between resistance and voltage is obtained by linear regression, using least-square

1.GFSA resistance shows linear increase with the applied voltage in the prebreakdown regime. Increase is more prominent for type A commercial components than type B. Abrupt decrease of the resistance is observed as voltage

2.Resistance also exhibits linear increase with the applied voltage in a γ radiation field but has one order of magnitude lower values than in operation without radiation. A slight decrease of the resistance is observed at voltage values near

breakdown voltage; at breakdown voltage the decrease is pronounced.

1.In a γ radiation field, breakdown occurred at lower voltages (205 V). The values of breakdown voltage for two types of commercial GFSA have been

0.1 nA. When voltage reached breakdown level, an abrupt increase of current

determined in the presence of γ radiation and without radiation.

2.Prior to breakdown, the current had constant values, of the order of

minimal error method. The following conclusions are made:

The main conclusions of the experiment are:

was observed, current values reaching μA level.

**222**

*Scheme of the test cycle for investigating the radioactive resistance of GFSA by a DC voltage.*

**Figure 6.** *GFSA prebreakdown current without radiation (A) and under γ radiation (B) [9].*

**Figure 7.** *GFSA resistance versus applied voltage without radiation (A) and under γ radiation (B) [9].*


**1. Introduction**

To better understand the basis of the activity of any molecule with biological activity, it is important to know how this molecule interacts with its site of action, more specifically its conformational properties in solution and orientation for the interaction. Molecular recognition in biological systems relies on specific attractive and/or repulsive interactions between two partner molecules. This study seeks to identify such interactions between ligands and their host molecules, typically proteins, given their three-dimensional (3D) structures. Therefore, it is important to know about interaction geometries and approximate affinity contributions of attractive interactions. At the same time, it is necessary to be aware of the fact that molecular interactions behave in a highly non-additive fashion. The same interaction may account for different amounts of free energy in different contexts and any change in molecular structure might have multiple effects, so it is only reliable to compare similar structures. In fact, the multiple interactions present in a single two-molecule complex are a compromise between attractive and repulsive interactions. On the other hand, a molecular complex is not characterised by a single structure, as can be seen in crystal structures, but by an ensemble of structures. Furthermore, changes in the degree of freedom of both partners during an interaction have a large impact on binding free energy [Bissantz et al., 2010].

The availability of high-quality molecular graphics tools in the public domain is changing the way macromolecular structure is perceived by researchers, while computer modelling has emerged as a powerful tool for experimental and theoretical investigations. Visualisation of experimental data in a 3D, atomic-scale model can not only help to explain unexpected results but often raises new questions, thereby affecting future research. Models of sufficient quality can be set in motion in molecular dynamic (MD) simulations to move beyond a static picture and provide insight into the dynamics of important biological processes.

© 2012 Yunta, licensee InTech. This is an open access chapter 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. © 2012 Yunta, licensee InTech. This is a paper 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.

Computational methods have become increasingly important in a number of areas such as comparative or homology modelling, functional site location, characterisation of ligandbinding sites in proteins, docking of small molecules into protein binding sites, proteinprotein docking, and molecular dynamic simulations [see for example Choe & Chang, 2002]. Current results yield information that is sometimes beyond experimental possibilities and can be used to guide and improve a vast array of experiments.

Using Molecular Modelling to Study Interactions Between Molecules with Biological Activity 187

of substituents on a single bond may vary with time. Therefore, any compound containing one or several single bonds exists at every moment in many different conformers, but generally only low energy conformers are found to a large extent [Kund, 1997, as cited in

The biological activity of a drug molecule is supposed to depend on one single unique conformation amongst all the low energy conformations, the search for this so-called bioactive conformation for compound sets being one of the major tasks in Medicinal Chemistry. Searching for all low energy conformations is possible with molecular modelling studies, since molecular modelling is concerned with the description of the atomic and molecular interactions that govern microscopic and macroscopic behaviours of physical systems. These molecular interactions are classified as: (a) bonded (stretching, bending and torsion), (b) non-bonded (electrostatic (including interactions with metals), van der Waals and π-stacking), and (c) derived, as they result from the previous ones (hydrogen bonds and

Protein-ligand or, in general, molecule-molecule binding free energy differences can *a priori* be computed from first principles using free energy perturbation techniques and a full atomic detailed model with explicit solvent molecules using molecular dynamics simulations. However, these are computationally demanding. More affordable approaches use end-point molecular dynamic simulations and compute free energies accounting for solvent effects with continuum methods, such as MM-PBSA (molecular mechanics Poisson-Boltzman surface area) or MM-GBSA (generalized Born surface area) [Kollman et al., 2000; Wang et al., 2005]. One of the first approaches was comparative molecular field analysis (CoMFA) [Cramer et al., 1988], which enabled interpretation and understanding of enzyme active sites when the crystal structure was absent. However, this type of analysis was not possible until *in vitro* drug-drug interaction studies were widely used (through the 1990s).

Molecular mechanics (MM) is often the only feasible means with which to model very large and non-symmetrical chemical systems such as proteins and polymers. Molecular mechanics is a purely empirical method that neglects explicit treatment of electrons, relying instead on the laws of classical physics to predict the chemical properties of molecules . As a result, MM calculations cannot deal with problems such as bond breakage or formation, where electronic or quantum effects dominate. Furthermore, MM models are wholly system-dependent. MM energy predictions tend to be meaningless as absolute quantities, as the zero or reference value depends on the number and types of atoms and their connectivity, and so they are generally useful only for comparative studies. A force field is an empirical approximation for expressing structure-energy relationships in molecules and

Molecular mechanics have been shown to produce more realistic geometry values for the majority of organic molecules, owing to the fact that they are highly parameterised. Parameterisation of structures should be performed with care and non-"standard"

**2.1. Molecular mechanics, molecular dynamics and docking**

is usually a compromise between speed and accuracy.

Tóth et al., 2005].

hydrophobic effect).

To apply computational methods in drug design, it is always necessary to remember that to be effective, a designed drug must discriminate successfully between the macromolecular target and alternative structures present in the organism. The last few years have witnessed the emergence of different computational tools aimed at understanding and modelling this process at the molecular level. Although still rudimentary, these methods are shaping a coherent approach to help in the design of molecules with high affinity and specificity, both in lead discovery and in lead optimisation. Moreover, current information on the 3D structure of proteins and their functions provide a possibility to understand the relevant molecular interactions between a ligand and a target macromolecule. As a consequence, a comprehensive study of drug structure–activity relationships can help identify a 3D pharmacophore model as an aid for rational drug design, as a pharmacophore model can be defined as 'an ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular interactions with a specific biological target and to trigger (or block) its biological response', and a pharmacophore model can be established either in a ligandbased manner, by superposing a set of active molecules and extracting common chemical features that are essential for their bioactivity, or in a structure-based manner, by probing possible interaction points between the macromolecular target and ligands.

Molecular recognition (MR) is a general term designating non-covalent interactions between two or more compounds belonging to host-guest, enzyme-inhibitor and/or drug-receptor complexes. A rigorous approach to an MR study should involve the adoption of a computational method independent from the chemical intuition of the researcher. Drug design purposes prompt another challenging feature of such an ideal computational method, the ability to make sufficiently accurate thermodynamic predictions about the recognition process.


**Author details**

Zouhair O. Amarin\* and Omar Farouq Al tal Jordan University of Science and Technology, Irbid, Jordan

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 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.


**5. Behavior of flavonoid in soil salinity**

In the kingdom of plantae, flavonoids are the secondary metabolites that are present in the widest variety of plant species. These chemicals have a wide variety of physiological and molecular functions in plants, including acting as signaling molecules, contributing to plant defense, influencing the transport of auxin, exhibiting antioxidant activity, and scavenging free radicals [64]. According to Sirin and Aslam [65], among the nonenzymatic antioxidants, phenols and flavonoids make a substantial contribution to removing free radicals in plants, which allows plants to tolerate salt stress by storing the antioxidants in a variety of tissues. Flavonoids are a group of compounds that are found in many plants. These compounds have been studied for their

#### *Plant Adaptation to Salinity Stress: Significance of Major Metabolites DOI: http://dx.doi.org/10.5772/intechopen.111600*

potential health benefits, and many are now known to be beneficial for human health. Flavonoids are a type of polyphenol. They play essential roles in soil salinity regulation [30, 41, 66]. They can reduce the number of ions, such as sodium and chloride, present in the soil solution, which can help maintain soil health and fertility [66]. They have also been shown to promote beneficial microbial activity in the soil, including improving the growth and health of beneficial bacteria and fungi. They also have antioxidant properties, which can help protect plants from environmental stressors such as high levels of salinity. They can also act as signaling molecules, helping plants regulate their response to salt stress. In addition, flavonoids can help promote beneficial microbial activity in the soil, which can improve the soil's fertility and reduce its salt content. This can help boost the soil's fertility, improve water retention and nutrient cycling, and reduce the risk of disease-causing pathogens. They can also help protect plants from environmental stressors such as drought and extreme temperatures [67, 68].

In plant materials, polyphenols can be found in both their free and bound forms. Phenolic acids make up the bulk of the polyphenols that are found in grains and baked goods derived from cereal, and approximately 75% of these are accessible in bound form. Plants' development, reproduction, and eventual grain yield are almost entirely dependent on the leaf proteins [68]. It is not surprising that salt stress causes a decrease in protein content in plant leaves, given that proteins are known to be one of the first targets of reactive oxygen species (ROS) in living organisms. One of the principal sites of ROS damage is the chloroplast, where it leads to the degradation and inactivation of Rubisco, as well as many other changes in the thylakoid and stromal proteins [69].

Because of this, leaf protein concentration is an important indicator of salt stress [70]. One of the most debated topics in the literature is the extent to which genetic diversity affects the protein content of plant leaves when subjected to salt stress. Few studies have looked into how genetics interact with salt stress to affect protein levels. In addition, there is a lot of debate in the scientific literature concerning the way salinity has an effect. For instance, Birhanie et al. [71] found that salt stress similarly reduced total protein concentration in the shoots of two cultivars of wheat (tolerant and sensitive). In contrast, it was discovered that during salt stress, leaf protein concentration varied by genotype and increased [72, 73].

Many studies have been proposed that increasing the accumulation of suitable solutes, such as proline, can improve salt tolerance. Proline, such as other osmolytes, can eventually regulate redox potential through its effects on osmotic adjustment, membrane protection, and enzyme stability in the face of abiotic and biotic stresses [74]. Arabbeigi et al. [75] reported greater proline biosynthesis gene (P5CS) expression in *Ae. cylindrica* may be linked with salt tolerance. It is a bacterial species that is commonly found in salt marshes and coastal habitats. The P5CS gene is known to play a role in proline biosynthesis, which is a process that helps cells to build proteins. The researchers found that *Ae. cylindrica* cells that expressed high levels of the P5CS gene were more resistant to salt stress. Additionally, the study found that deleting the P5CS gene had no impact on the salt tolerance of *Ae. cylindrica* cells. The authors of the study say that the findings suggest that the P5CS gene may play a role in salt tolerance in *Ae. cylindrica*. They say that the findings could help to identify new strategies for preserving salt marsh habitats. In wheat, this finding agrees with that of Kumar et al. [76]. This is congruent to a certain extent with the findings of Ebrahim et al. [77] in barley, who also found that salt stress led to a huge buildup of proline but also found that salt-sensitive genotypes accumulated more proline in their leaves than salt-tolerant ones. Since the osmo-adaptive response includes proline buildup, whether or not it plays a special function in the resistance to abiotic and biotic stresses (such as salinity

and drought) is debatable. Several explanations come to mind for this discrepancy. Variations in genotype or species, stress level or duration, and physiological maturity or development are particularly important to note when comparing research.


# *Boiled Milk Anne Brontë's Final Journey*

michael stewart

# *Boiled Milk*

*Anne Brontë's Final Journey*

michael stewart

Published by University of Huddersfield Press

The University of Huddersfield Queensgate Huddersfield HD1 3DH

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Text © 2020

This work is licensed under a Creative Commons Attribution 4.0 International License

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The publisher is not responsible for the continued existence and accuracy of websites referenced in the text.

A CIP catalogue record for this book is available from the British Library.

ISBN: 978-1-86218-180-9

Designed by Dawn Cockcroft

This book is a special collectors' edition of a forthcoming chapter from *Walking the Invisible: The Brontës' Lives and Landscapes, Then and Now*, which will be published by HarperCollins in hardback in July 2021. A limited number of 200 copies have been published, signed and numbered, to celebrate 200 years since the birth of Anne Brontë.

# about walking the invisible

Following in the footsteps of the Brontës across meadow and moor, through village and town, award-winning writer Michael Stewart, author of *Ill Will: The Untold Story of Heathcliff*, takes a series of inspirational walks through the lives and landscapes of the Brontë family, looking at the geographical and social features that shaped their work, and comparing the north of England then with how it is now. This is a poetic study of the social and natural history that has inspired writers and walkers for hundreds of years. Firmly in the tradition of nature writing, this book also encompasses the history of the north and the changing lives of those that have lived there.

Michael Stewart is the creator of The Brontë Stones project which features the work of some of the most prominent writers today, including former poet laureate Carol Ann Duffy and singer/songwriter Kate Bush. The book includes his reflections and anecdotes.

*'The idea of being authors was as natural to us as walking.'*

Charlotte Brontë

# introduction

# *Brontë Fever*

I wasn't born with a Brontë obsession. As far as I know, it is not a congenital condition. But these past few years I've been struck with Brontë Fever. I'm not the only one. Over the course of my fanaticism, I've met others. They appear quite normal, some of them. There are no flags or bells. They walk amongst us.

I was born and brought up in Salford, a city within a city, and went to a failing comprehensive that also 'educated' most of *The Happy Mondays*. It was a school built on a marsh and made of plasterboard. There were head and boot shaped holes in the walls, where the pupils had found an outlet for their pent-up aggression. It was sinking. Actually sinking. The science labs were on the same level as the all-weather pitch. I was in the bottom class for English and was not allowed to study the 'classics'. Instead, we were given books that were written in a simple style, avoiding big words and grammatical complexity. They often had a glossary in the back pages. So I never encountered the work of the Brontës. We were told we weren't bright enough.

Shortly after I left school the building was demolished and the site flattened. At sixteen I started work in a factory in an area of Manchester called Newton Heath. Thousands of people worked there, mostly men. Though few retired. I used to visit my local library every Saturday morning and take out three books: two fiction and one non-fiction. During the bus journey to and from work, I'd read them. One of these books was *Wuthering Heights*. I already knew some of the story. I'd watched the 1939 MGM adaptation with my mother when I was a kid. But the book was very different and at first I wasn't even sure if I liked it. I'd come across the characters of Cathy and Heathcliff even earlier, in Kate Bush's 1978 debut single. I found the book a bit of a slog to begin with, but I persisted. Slowly the story and characters drew me in. Somehow they took hold of me and wouldn't let go.

Gradually, over the years, my obsession has grown. I moved to Thornton, the birthplace of the Brontës, and my interest in their literary work began to extend into their lives. I wanted to discover them for myself. I started to hunt them down. Beginning in Thornton, I imagined Patrick Brontë walking the same streets as me. He would have walked right past my door every day, and I wondered what life was like for him and his family. I read the stories he wrote during his time in Thornton. I read all of their work: the novels, the poems, and everything else. I read what others had written about them. I joined the Brontë Society and started to pore over their journal of essays. But the one book I returned to again and again was *Wuthering Heights*. I became fixated on the two gaping holes in the narrative: where had Heathcliff come from and where did he go during the missing years? And I started to write a novel that would fill in the gaps. That novel became *Ill Will*, and during my research I recreated the walk that Mr Earnshaw takes on foot, from Yorkshire to Liverpool. I also spent many hours walking the moors around Haworth, writing the book as I tromped across the landscape, talking into a Dictaphone that I always carried in my pocket.

My quest to find the landscapes that inspired the Brontës had begun. I went to Broughton-in-Furness, where it was claimed Branwell Brontë sired an illegitimate child, and from there to Thorpe Green, where he was dismissed for having an affair with his employer's wife. I went to Dentdale and Law Hill, in search of the origin myths behind Emily Brontë's only published novel. I visited North Lees Hall in Hathersage, to discover the inspiration for Charlotte Brontë's *Jane Eyre*. I retraced Anne Brontë's last days in Scarborough, and became captivated by the story of the Luddites in Charlotte's *Shirley* and how this contrasted with how they were in real life. In short, I travelled all over the north of England in search of their lives and landscapes. In doing so, I realised how important it was to get people out there too. I wanted people to engage, not just with their

## introduction 13

lives and literary works, but the places that had inspired them. Up on the moors, I had a profound understanding of the texts. I started to connect with their writings in a visceral way. It was like I had discovered another flavour, another layer opened up and I sunk further into the texts. The words and the moors were one.

This lead to my Brontë Stones project. I became aware of how their birthplace is overlooked and wanted to connect it to Haworth where they spent their formative years. I came up with the idea of a literary trail, with stones along the way, to mark the bicentenaries of the siblings, through a desire to encourage more people to experience the landscape that inspired all three sisters. The landscape that surrounded them and offered them a place of solace, but also at times must have felt like divine punishment. When the winds were wuthering and rain ripped through the sky like lead shot, with only shawls and hobnail boots to protect them from the relentless elements.

I thought about the journey they took in 1820 from Thornton where they were born, over to Haworth. They were a happy family, but very shortly after their move to Haworth tragedy struck. First the death of Maria, the mother, then the two oldest siblings.

But the Brontë Stones project wasn't conceived as a heritage enterprise. The project is about celebrating female writers now. Alongside the Bradford Literature Festival, I commissioned the then poet laureate Carol Ann Duffy, singer/songwriter Kate Bush, the Scottish Makar Jackie Kay, and award-winning novelist Jeanette Winterson. These four writers have pushed forward contemporary literature in the same ways the Brontë Sisters opened up the possibilities of the Victorian novel and Victorian poetry. Bold, experimental, playful and dark, the four poems are a recognition of what is alive about our language today.

Pip Hall, the fine letter carver I worked with on the project, emphasised the power of the poems by taking her cue stylistically from the plentiful surrounding inscriptions – in local streets and churchyards – dating from the Brontës' era. 'In drawing the lettering and devising layout, I wanted the poems to look as if they belonged in their settings. The letter shapes at that time were typically broad, bold and seriffed, with a distinct contrast between the vertical and horizontal strokes.' At other times Pip responded to the encompassing landscape, drawing on curves to follow the gentle contours of meadow and moor.

Alongside this project, I devised four walks, one from Thornton to Haworth, and three circular walks for each of the sisters. Maps of these walks are available and included in this publication. They are drawn and designed by the cartographer Chris Goddard who makes beautiful bespoke drawings, very much in the Wainwright tradition.

The Brontë Stones Walk is a characterful nine-mile route over the hills from Thornton to Haworth that takes in all four of the Brontë Stones. It is a linear trek over the moors that also includes Ogden Kirk, Denholme Beck, Nan Scar and Oxenhope, following the Brontë Way in places, but elsewhere offering interesting alternatives.

The Charlotte Brontë Walk is a simple four-miler around Thornton, the birthplace of the Brontë children and home to the Charlotte Stone. The walk takes a short loop across the hills around Thornton, starting at St James' Church, opposite the Old Bell Chapel where Patrick Brontë worked. It also takes in Thornton Hall, Hanging Fall, Thornton Viaduct and the Brontë Birthplace, and has some great views over the valley.

The Anne Brontë Walk is a varied seven-mile ramble around the lush valleys north of Haworth, taking in Newsholme Dean, the Worth Valley and Holden Park. It follows the Railway Children Walk to begin with, before climbing through Oakworth and Holden Park to charming Newsholme hamlet and Pickles Hill, then dropping down to follow the river Worth back towards Haworth and Parson's Field where the Anne Stone is placed.

The Emily Brontë Walk is, as you would expect, a strenuous and remote fifteen-mile yomp across the moors high above Oxenhope and Haworth, traversing the landscape that inspired *Wuthering Heights*. This is a hearty hike across the wild moorland Emily loved to roam. The route takes in Top Withins, Alcomden Stones and Ponden Hall, as well as various other beautiful sites.

I don't believe that anyone can really connect, can really understand, the Brontë literary oeuvre without experiencing this uniquely bleak countryside, without feeling the force of the hair-ruffling winds, the earthy smell of the peat bogs, the haunting call of curlew in the summer, and the warning rattle of red grouse all year round. And the aim of the book is to link landscape with literature, by emphasising the relationship of wandering the moors with writing fiction, as both allow the mind a creative freedom. As Charlotte herself said, 'The idea of being authors was as natural to us as walking.'

*Walking the Invisible* immerses the reader in the lives and landscapes of the family. It is a walking book in the tradition of nature writing, but it is also a social and literary history of the North. I want you to walk with me but see through their eyes, as I compare the times they lived in with the times we live in now. What follows is a sample chapter from towards the end of the book, which investigates Anne Brontë's last three days, and traces her final journey to St Mary's Church where she is buried. She is the only Brontë not buried beneath St Michael and All Angels' Church next to the parsonage in Haworth.

*Map drawn and designed by Chris Goddard*

# boiled milk

# *Anne's Final Journey*

In Scarborough on Friday 25th May 1849, sometime in the afternoon, a train pulled up at the station. It had come from York. The doors opened and Anne Brontë alighted. Beside her were her eldest and only surviving sister, Charlotte, and their friend Ellen Nussey. In three days' time Anne would be dead and Charlotte would be the only remaining Brontë sibling. Charlotte had fought hard to stop Anne's plan of returning to Scarborough, a holiday resort Anne had visited many times with the Robinsons, but in the end she had lost the fight. It was their father, Patrick, who intervened and insisted that she go. Charlotte had only recently seen, first her brother Branwell, then her sister Emily, taken to their graves.

They made their way to Wood's Lodgings at No.2, The Cliff, where Anne had stayed with Lydia and the rest of the Robinson family. The view overlooked the sea. The lodgings are no longer here – replaced shortly after by The Grand Hotel. The Grand Hotel was first devised in the early 1860s and in 1863 the building began. It opened in 1867, reportedly the 'largest and most handsomest hotel in Europe'. Anne's ghost is said to haunt its corridors.

The Scarborough they would have seen as they walked from the station to their lodgings, would have been much smaller than the town that exists today. Anne would have seen the Town Hall in St. Nicholas Street, and the Borough Gaol at the top of St. Thomas Street, which was opposite the Seaman's Hospital. There was also the theatre close by, 'the only place of amusement in town,' according to a general directory published at the time. The Customs House was situated between the piers. Scarborough exported corn, butter, hams, bacon and salt fish. It imported coal from Newcastle and Sunderland; timber, deal, hemp and flax, from the Baltic; and groceries from London. There were fifteen beer houses, one billiard room and sixty-six public houses. Twenty dressmakers, seven hatters, five stay and corset makers and one gun maker. Three boat builders, two fossil dealers and one bone crusher.

*View of Scarborough, 19th century drawing*

I'm trying to picture all this as I wait for poet and playwright Wendy Pratt, who was born and bred in Scarborough but now lives in Filey, down the road. She has written five books of poetry. She has also just completed a play about Anne's last days. She wants to show me around Anne's Scarborough and take me to the key places. We meet in Greensmith & Thackwray, which is now a coffee shop, but was once a 'colonial outfitters', as the writing above the shop still attests.

Wendy introduces herself. She is wearing a bright yellow cardigan and carries a capacious satchel. We sit and chat before agreeing on our route. The forecast of light cloud and gentle breeze has so far held up, but as we walk down St. Nicholas Street, to The Grand Hotel, the air turns icy. We're passing my van, so I nip in and get a warmer coat.

'Will you be warm enough?' I ask her.

'Yeah,' she says. 'I'm a proper Yorkshire lass.'

I wonder if Anne and Charlotte felt the same way about the breeze. We dodge kittiwake shit, which falls from the window sills above us. Kittiwakes are protected seabirds, and they have made a welcome home in the nooks and crannies of Scarborough's buildings. We stand underneath the blue plaques on the wall of the hotel, the highest of which is to mark Anne Brontë's stay here, and her death. Juliet Barker talks about how, on the Saturday, Anne went to the Bath house nearby but collapsed at the garden gate on the way back. I want to know where this was.

 *From Theakston's Guide to Scarborough. This establishment was situated at the entrance to the Cliff, and was originally opened in 1798.*

'I think the bath house must have been there.' Wendy points to the buildings opposite the Grand Hotel, which are now hotels and town houses, with a Masonic Lodge in the middle. She shows me a sketch of Travis' Baths from Theakston's 1841 *Guide to Scarborough*. The baths were made of wood and marble and were adapted for either 'plunging, sitting, or the recumbent position'. Every tide, the baths were supplied with pure sea water. There were four other bath houses in addition to Travis'.

'The garden gate must have been here.'

She points to the communal garden that still stands between these buildings and the hotel. It is still gated. It's possibly the same gate, and I imagine Anne clinging on to it, using it to pull herself upright again with arms 'no thicker than a little child's', as Charlotte described them in a letter to Ellen, when she wrote to warn her of Anne's decline. Anne was carried indoors by the hired servant, Jane Jefferson.

'Didn't she take a donkey cart ride in the afternoon?'

'Yes, she took the reins herself. She didn't like the cruel way the boy was treating the animal.'

*View of the beach, 19th century drawing*

Anne always stood up against the abuse of animals. It's in her poems and her novels. Barker recounts how Ellen Nussey came to meet Anne at the point that she was giving the boy a lecture. Although the conversation was not recorded, so we can only guess in what way she scolded the boy. There's a drawing of these cart rides in the archives of Scarborough Library, but most of the carts are being pulled by horses, not donkeys.

As we talk, we are passed by people dressed as cartoon characters: a Hulk, a Thor and a Black Widow. There has been a comic convention and it must have just finished. To the left is Spa Bridge that Anne walked with Charlotte and Ellen, which would then have been a toll bridge. A day ticket was one shilling. Below is the sandy beach where Anne strolled. The reason Anne gave for wanting to visit Scarborough again was the hope that the sea air would revive her. It was thought to be a cure for TB. She wrote to Ellen, 'the doctor says that a change of air or removal to a better climate would hardly ever fail of success in consumptive cases.' But Wendy thinks that the real reason for her visit was very different.

'She knew she was dying. She was thinking about her father, and what he'd been through.' Wendy says.

'Yeah, you think about the close succession of those deaths and what he must have gone through.'

Although, Ann Dinsdale, and other Brontë experts I have spoken to, feel strongly that Anne didn't know and was holding out for a miracle cure. Perhaps she was hedging her bets. She also wrote later, in the same letter to Ellen, 'I have no horror of death: if I thought it inevitable I think I could quietly resign myself to the prospect.' Scarborough perhaps then, held both possibilities for Anne: potential remedy and final resting place. The three women had stayed in York on the way to Scarborough, at the George Hotel in Coney Street, the site of which is now a Waterstones bookshop. We know from Ellen's detailed record of the journey that they bought bonnets and ribbons there so that they could look their best in Scarborough. For me this is evidence that can be used to bolster either case. Would she have wasted money on expensive garments if she knew she was dying? Or if she knew she was dying, why would she care about money?

We can see St. Mary's Church from where we are standing. This was Anne's final destination, and the way we are heading. In Anne's day, Scarborough was an up-and-coming spa town of great prestige. Like Bath, people came here to rest and restore their health. These days Scarborough is down on its luck. Scarborough has one of the highest mortality rates from opioid overdoses in the country. On the seafront, between the candied confectionaries, rainbow slush, and pink candyfloss, are shops selling Day-Glo bongs and skunkweed grinding machines. Poppers that are now disguised as room odorisers. It is no longer the holiday destination of the middle classes. Right now, it is April and outside holiday season. There's an edge to the town. There isn't much work for locals out of season, unless you work at McCain's up the road, or the local hospital. The bars are already starting to fill with hardened drinkers.

*The Grand Hotel, as viewed from the sea.*

Wendy explains the reason for this, 'We just don't get the sort of money coming into the town that we did, so it contributes to the poverty in a way. People who are lost and broken, people who are living in poverty often come to live in Scarborough because of nostalgic memories from the good times they've had on holidays here, which means we tend to get a higher number of impoverished people coming to the area and no jobs to support them if they are able to work. The town then revolves around squeezing money out of these impoverished people by offering many opportunities to gamble. So many betting shops.'

'And pubs selling cheap alcohol?'

'Yeah. We are basically offering them the dream that will never come true, and then anaesthetising them when they realise their life is just endless trauma. There's a reason the town is the way it is.'

We stand outside the Grand Hotel, reflecting on this contrast. I stayed here a few years ago, when I was invited to run a workshop for the National Student Drama Festival. It was more like an old people's home than a holiday resort. I came back late one night and I rang for room service, but the only thing they could offer me was a cream cheese sandwich on milk loaf bread. Going down for breakfast the next day, it was clear that the menu had been devised to be denture friendly. Porridge and mashed fruit for breakfast, soup and stew for lunch, and a dinner of shepherd's pie. You could eat anything you wanted as long as it was pre-digested and didn't require chewing. I took the lift to the top floor which wasn't supposed to be open. There was a sign saying that it was out-of-bounds. There was mould growing over the flock wallpaper, and various receptacles were laid out on the mildewed carpet, to capture fallen drops of rainwater as it leaked through the roof. There was even a plant growing out of a crack in one of the walls. The management, unable to afford the upkeep, and not needing the extra rooms, had abandoned it to the elements.

Barker writes that in the evening Anne sat and watched 'a particularly splendid sunset', but Wendy points out that this wouldn't have been possible as the view was over the east, over the sea, not the west. It's a minor quibble in what is an otherwise assiduously researched tome.

*Christ Church, now Wackers*

We walk up Vernon Road to where Christ Church used to stand. The church where Anne's funeral took place, attended by only Charlotte, Ellen and Charlotte's friend, Margaret Wooler, who had come to offer her support. She had taught Anne at Roe Head School. Where the church used to be there is now a fish restaurant called Wackers.

'Everyone worked at Wackers when I was growing up. You got a Saturday job.' Wendy says.

'It doesn't sound very appetising. When was the church knocked down?'

'I think in 1979. My mum remembers it.'We look at old photos of the church. It was a grand building with a four cornered tower and gothic windows. During Anne's time here, the church would have stood apart from any other buildings, surrounded by trees.

'Why did they knock it down?'

'To build Wackers.'

We walk up Westborough towards the train station. It's a small terminus with five platforms and three rail tracks.

'At one point we had a train to Whitby, but they took it away.'

'Why did they do that?'

'We are historically unlistened to in the town, over everything, the residents are always put second to the need to keep tourist numbers up and to save money for tourism. Apparently the line was very steep and became very slippery in the frequent sea frets leading to trains stalling a lot, which caused massive backlogs and delays so they closed it, despite Scarborough residents petitioning against the closure.'

The skeleton of the station is mostly unchanged. Its ribs are wrought iron, painted austere black.

'They say you are thirty-seven miles from civilisation if you live in Scarborough.' Wendy says.

'And that's the distance to York?'

'It's on a limb.'

We look at the map in front of us, of Scarborough and the surrounding coastland. Scarborough sticks out like an epidermal cyst or a swollen knuckle. We walk through the station. It's a generic space with a Pumpkin café, a chain that has specialised in train stations and hospitals. We pass a young mother with her son. She drinks from a foamy paper cup, as he forks huge pieces of chocolate cake into his mouth. I don't think dandelion coffee will be on the menu. A beverage that the three women purchased shortly after leaving the train. I wonder why dandelion coffee, a coffee substitute made from roasting the root, and not real coffee? Perhaps they were attracted to the novelty or its comparative cheapness?

Outside we are confronted by the Stephen Joseph Theatre, the first theatre-in-the-round in Britain, and home to Alan Ayckbourn. Its red and white curved corner and jutting brick sections, are still an impressive example of Art Deco design. It wasn't built as a theatre as Wendy explains,

'The actual first theatre in the round wasn't at the SJT as it is now, the original was in the basement of the library on Vernon Road, next to where Christ Church was. This was originally an Odeon cinema.'

The sign above the door tells us that the latest show is called *Martha, Josie and the Chinese Elvis*. I once drove the actor Richard Wilson here, famous for portraying Victor Meldrew, the grumpy pensioner and his catchphrase, 'I don't believe it!' We were late and I'd forgotten to tell him that the seatbelt was faulty. As I pulled up he was exasperated to learn that he was belted to the seat and couldn't get out. I had to pull the belt loose so he could slip underneath it. Only he got caught up in the loose webbing. As he pulled and yanked the black strap, becoming more entangled, he went red in the face. He was very close to using his catchphrase.

'It would be great if my play was produced here.' Wendy says.

'Yeah. That would be cool. Aren't they doing a scene study of it soon?' 'I'm really excited.'

'You should be. It's a really good play.'

It tells the story of Anne's last days and is structured through three interwoven monologues: Anne's, Charlotte's and Ellen's. It's funny in places, with Ellen providing most of the comedy, but ultimately it is a heartrending tale.

We walk down Westborough and turn up St. Thomas Street towards Castle Road. The terraced houses that line Castle Road are early Victorian. Anne's coffin would have been carried along here on a cart.

'Whenever I walk up this part of town, I feel like I'm following Anne's wake. Especially since working on the play, where I was immersing myself completely in this part of her life and imagining it over and over. It's like I can map out, in my head, what was and wasn't there, what the view was like, what the sounds must have been like, what the smells must have been like, to the point that this part of the town is overlaid with Anne's death.'

'There's a different feel to the town here, isn't there?'

'It's almost immediately quieter, the hustle and bustle of modern Scarborough drops away. The sounds are muffled, it feels different, it's reflective.'

We walk past Wilson's Mariners' Home, a charity that once provided almshouses for 'poor aged persons of good character being ship owners, shipmates', and other nautical folk. Built in 1836, it would have been a comparatively new building. It's an impressive design. The window surrounds are elaborately carved. The gulls screech above us. The route is well signposted.

'It's a big draw for tourism.' Wendy says.

As we approach St. Mary's we see a group of Japanese tourists coming down the hill. I assume they have been to see Anne's grave. It's hard to believe they have come all this way just to sample the £2 a pint lager bars. The church stands above the town and overlooks South Bay. The remains of the earlier chapel are still within its grounds, giving the place an eerie feel.

'Do you think Scarborough fully capitalises on the Anne Brontë link?'

'We don't embrace this part of our story here and it's always something of a surprise when you see the constant stream of tourists, who may well have deliberately come to Scarborough for the specific purpose of visiting Anne's grave. We could have a link to the parsonage. We could easily have a small museum to show the Scarborough of Anne's time here, on the East coast.'

*St Mary's Church, 19th century drawing*

We reach the grave. There isn't much left of the original headstone. The salt in the sea air eats into the soft sandstone, and many of the monuments here are pockmarked or whittled away by the weather. In 2011 the Brontë Society placed a monument at its foot, recording the original 1849 inscription, which apparently contained five errors. It was refaced three years later when Charlotte returned and discovered the errors. And yet, the most egregious error, that the inscription gave her age as twenty-eight, when in fact she was twenty-nine, was not corrected. There is no record anywhere to provide details of the other errors.

*Anne Brontë's grave*

'You've written poems about both Sylvia Plath's grave and Anne Brontë's. Sylvia Plath's grave is full of pens and poems. Little notes and letters that obsessive fans have left. But you don't get that here. Why is that? They were both poets and novelists. They both died tragically young.'

'It's partly because of the location, you have to deliberately set out to come to Scarborough, it's not on the road or a short diversion away from anywhere else. But I also think that there is a bit of a cult around Sylvia's death, suicide and mental health and the relationship problems. These are all things that people can relate to. They feel a deep sense of connection with Sylvia and want to give these little votive offerings to her to say thank you. Also, she died within living memory for a lot of people, she's still fresh, we can see pictures of her, we know her life in miniscule detail. Anne died a long time ago, and to be fair she is the lesser known Brontë, unfairly seen as quite pious and prim. Her books are not tearing themselves inside out with passion. She is not romanticised in the same way that Charlotte and Emily have been. Anne seems boring, difficult to connect to, so perhaps the votives – that connection between reader, pilgrim, writer – aren't quite as strong here. What you do get is people quietly tending her grave. There are people in Scarborough who visit every day, who make sure she is remembered as a part of our town story, our heritage.'

I notice that the daffodils at the foot of the grave are blooming. The bench we are sitting on is dedicated to a lost loved one.

'In memory of, and in loving memory of, that's a Victorian tradition,' I say. 'It didn't exist before. So you can date headstones by that.'

'I didn't know that.'

'I had to research it for my last book.'

We stare out at the sea and listen to the gulls scream.

'She was five years with the Robinsons, wasn't she? And this was their holiday home.' I say.

'Yeah, and she bloody loved it here.'

'It was also her most sustained period of employment.'

'Yeah, and I wonder with Anne, whether being apart from that family, and away from the claustrophobic parsonage, away from the dictated roles of the family, if that was something she was seeking.  I wonder whether coming here, all this space, a different sort of wildness to the moors she'd left behind, but something familiar too – distance, big skies, a sense of freedom. It was just so entirely different. It must have had a profound effect on her.'

'She would have written some of both *Agnes Grey* and *The Tenant of Wildfell Hall* while she was here.'

'I think there's mention of this is one of her diary pages?'

We talk about a poem Wendy has written about Scarborough which describes 'hat stealing wind'. It's a very exposed peninsula. There's also a good line about the funicular railway.

'I like the word "funicular",' I say.

'Yeah. I think in Latin it means something like rope.'

'So the idea is a vertical method of being pulled up or down?'

'I think so. It comes up in pub quizzes round here all the time. In the summer when you pass the tramway you always overhear someone saying, "you do know it's called funicular, don't you?", it's just one of those things. Everyone thinks of it as a unique piece of knowledge.'

'You mention the suicides over Valley Bridge. What's been done about that?'

'They've put this spiked railing that curves round. You can't climb over them. It used to be a very regular occurrence, to have people jump there. People do still, determined people. My dad was a bus driver and he was driving under the bridge one day.  At first he thought it was a person. But it was a Guy Fawkes effigy.'

'Well, we all did it. As kids I mean.'

'I never did it.'

'Maybe it was just me. The poem mentions the statue of Richard III.'

'Richard III was the last king to occupy the castle. They poked a cannon through the church tower and fired at the castle.'

'Who did?'

'This was much later. During the civil war. The bombardment was so intense it destroyed half the building.'

We look at the church tower and imagine the cannons firing up and over to the castle. The castle stands above us, on a massive promontory of rock that rises above the sea. It's not hard to see why it was a favoured location for a fortress.

'There was a statue outside The King Richard III. On the foreshore, a very old statue. But it got stolen.' Wendy says.

'Isn't that a difficult thing to do? I mean, didn't anyone notice?'

'That's Scarborough. At the Spa there used to be a massive golden lion's head sculpture, where the spa waters themselves would come out of the lion's mouth, and someone pinched that as well.'

'And that's what the empty cage in the poem refers to then? Where Richard III was?'

'We keep thinking in Scarborough that we've got something. Like we own our history, but history in seaside towns is transient, it gets whisked away, it gets replaced with something supposedly better. Like the Futurist Theatre, which has just been demolished to build a roller coaster. It was a beautiful building, a hugely historical building, and now it's gone. It's difficult to have an identity when the identity you think you have is changed so easily. We don't have an identity here. We have nostalgia. We need to write new stories, and we need to honour our old stories.'

She explains that she has just finished writing a poem that deals with Anne Brontë in a more direct way. A tribute to her time here in Scarborough.  I thank her and tell her I will read it later. I fold it up and put it in my coat pocket.

We head back into town to meet Wendy's husband, Chris, for a drink. Just at the bottom of the slope below Anne's grave, Wendy points to a sign, 'Paradise'. It's the name of the street. Next to it is a brick wall with a gap in it. We look through the gap and see the whole of the bay and it's beautiful. Wendy points out that Anne's grave is on Paradise Hill.

We pass a pub with a Shirley Bassey tribute act and a sign outside saying, 'Dogs Welcome. Sorry, No Children.' It's the day of the Grand National and all the pubs are packed with punters. We start at The Merchant, opposite The Golden Last. The Golden Last is a notorious drinking hole. Headquarters to the Scarborough EDL, and known locally as the Stabbers' Arms. Outside there are six St. George flags and two Loyalist flags flapping in the breeze. The landlord was arrested a few years ago on suspicion of supporting a banned paramilitary organisation. The white and black exterior is badly in need of a lick of paint. I want to go inside to get a better look, but I'm told this would not be a good move.

We have a few drinks in the Merchant, then we move on to The Turks Head, then The Waterhouse. Huge plasma screens show the race and people stand and watch, cheering on the horses. A horse called 'Up For Review' falls at the first hurdle. It writhes in pain. It is taken away and shot. As I watch, I think about what Anne would make of it. She wouldn't be a fan. We go to another bar with men dressed as nuns. We encounter those drinking their winnings and others who are sinking that losing feeling with vodka shots.

We end up in the Sub-Aqua Club. We are surrounded by the flotsam and jetsam of wreck salvage: old bells, brass lamps and ship wheels. We're all a bit worse for wear. I'm standing at the bar, surrounded by Sub-Aqua Club members doing a pub quiz. There are wreck divers, underwater photographers and marine biologists. I think the drink in front of me is mine. The quiz master says, 'Question three, what does the word "funicular" mean?' I smile. I look around for Wendy and Chris, but they've gone. I can't remember saying goodbye to them. I see that a group of women have kidnapped my dog, and I go over to rescue him.

I'm woken early the next morning by the shrieking gulls and crashing waves. Wolfie is still fast asleep. My van is parked close to the coast beneath a sign that says, 'no campervans to be parked here overnight'. I get up, make some breakfast, and brew a big mug of tea. I sit by the sea, watching the sun rise through the fret, and the tide go out, wave by wave. I think about what Wendy said, about Anne not being able to see the sunset from her window. Did she instead, sit and watch it rise over the waves?

Once the tide has subsided and the remnants of tea have gone cold, I take my dog on the beach. It's more or less deserted. Just two women clad in neoprene wetsuits, attempting a bit of wild swimming. I think about the 'solitary ramble' Agnes takes, breathing in the 'freshness of the air'. It is here, towards the end of the book, that Agnes and Weston are reunited. There's a sci-fi conference at the Spa, and people dressed as their favourite sci-fi characters are parking up and congregating in the café. A Spock look-a-like tumbles out of a Mitsubishi Mirage.   The sea fret covers everything like a milky shroud.

 I climb up by the funicular tram to the Royal Hotel where a statue of Queen Victoria overlooks the bay. She would have still been a young queen in Anne's day. Born just eight months before Anne, they were more or less the same age. Outside the Grand Hotel a coach pulls up and I watch as hotel staff help the elderly clients onto the coach. Some shuffle, others hobble, using sticks and zimmer frames. Mostly old women, but a few old men too. A disfigured pigeon pecks at a pool of lumpy sick.

I wander round town. Above the bookies and the pawn shops with signs outside saying, '£10 per gram of gold', there are still signs of Victorian grandeur. Splendid carved edifices and high windows. But the general atmosphere is heavy. As I walk up Castle Road again, towards Anne's grave, I think, it's not just the sea fret that makes this place oppressive, it's more pervasive than that. It goes beyond the weather. It comes from the bricks and the concrete. It spills out onto the street from the £2 a pint lager bars. It comes dressed in a JD Sports tracksuit, coughing up phlegm.

The high bay windows are netted. I want to take in Anne's grave without the tourists and the Brontë fans. I just want a moment alone with her. But when I get there the bells of St. Mary's are pealing. It's only nine in the morning but the clanging cacophony of the bells is deafening. I imagine they are no comfort to those sleeping off a hangover.

Last night I walked from the more popular South Bay to the North Bay. There isn't much between the two bays, and not much at North Bay when you arrive. One bar and one shop. Boy racers were congregating in souped-up coupes. Their one litre cars modified with body kits and noisy exhausts. Imitation alloy wheels, spoilers and bonnet scoops. They bounced from the bass coming from the woofers that take up the entire boot. Girls in their teens gathered at street corners sipping from brightly coloured bottles of alcoholic pop.

Scarborough now is no different to so many English towns: inwardlooking, dejected, unsure of its own identity. Scarborough still wears the edifices of a once opulent and prosperous society, but it is crumbling into the ocean, turning into sand and dust, leaving a lurid scene of poverty and desperation. Plastic bottles and plastic bags are all that will remain of us. We are all like Wendy's gulls now, huddled up against the oncoming storm.

I sit on the bench staring at Anne's grave, but there is no peace for the dead. I climb up to the castle where the air is clean. The branches of the trees are alive with green buds. I put my ear to a trunk and listen to the sap rise. It's a great vantage point, and looking back at the town, all the rude features that were brought into close range yesterday have now shrunken back and the effect, along with the sea fret that still clings to the edges of buildings and softens them, is that Scarborough looks beautiful. Scarborough is beautiful. And I can feel for the first time, that elevation of spirit, that Anne must have felt, as she stood here, looking over the bay at the grey seals and lobster pots, white-washed walls and terracotta roofs. The sun rising over golden sands and silver waves. The little houses clustered together, kittiwakes cutting into a gusty breeze. I sit at the edge of the promontory. I feel in my back pocket for Wendy's poem. I take it out, unfold it, and read it.

## **For Anne, a Stone's Throw from Paradise**

At *Paradise* I saw the way the treadle-footed tourists came, how they'd worn the words to nothing on the faces of the other stones. Yet still, no votive offerings; no pens, no letters for this girl of sea frets and horizons.

We've both been here before. Sometimes we up and leave, I drag you with me; bones and silk and boots, and walk you back to Wood's.  I puppet you along your death, add details and embellishments. But it can't last.

You're mute; a doll thing, thin as air. We breathe your mizzle-ghost and pray to know you. The obsession's there, we search for likenesses in photographs and diagnose your mental health, we scour the *Antiques Roadshow* for a locket of your hair.

On *Castle Road* I walk your coffin path to sit beside the other beloveds, to see you fed into the ground. And I'm surprised each time: you're peaceful here, above the sounds of paradise: the seagulls, kittiwakes, the penny slots and Kiss-Me-Quicks.

On the Monday morning, not physically capable of descending the stairs herself, Ellen carried Anne in her arms to the room downstairs. She had a breakfast of boiled milk. She died at about 2pm on Monday 28th May 1849. Shortly after, dinner was announced. The last words she said were, 'Take courage, Charlotte, take courage.' A week later, Charlotte wrote to William Smith Williams, 'Papa has now me only – the weakest, puniest, least promising of his six children. Consumption has taken the whole five.'

*Anne's final view. Drawing from August 1849. Artist unknown*

# acknowledgements

Throughout my journey, *The Brontës* by Juliet Barker, has been my constant companion (its dog-eared pages are stained with tea, wine and mustard), as well as *The Oxford Companion to The Brontës*. Early on I found inspiration in Ann Dinsdale and Mark Davis's *In The Footsteps of the Brontës*. I've read many other books along the way. These include, *Anne Brontë Reimagined* by Adelle Hay, *Crave the Rose* by Nick Holland, *A Life of Anne Brontë* by Edward Chitham, *Take Courage: Anne Brontë and the Art of Life* by Samantha Ellis, and *Anne Brontë: The Other One* by Elizabeth Langland.

I'd like to thank poet and playwright Wendy Pratt for agreeing to show me around Scarborough and also for kind permission to print her poem here. I'd also like to thank Dr Claire O'Callaghan, lecturer at Loughborough University, Victorian and neo-Victorian literature specialist, who has written a great deal about the Brontës, and was kind enough to read this chapter and give me feedback. With that in mind, I'd also like to thank Simon Crump, Steve Ely and Jim Greenhalf, writers and friends, who have all extended the offer to provide feedback.

A special thanks goes to everyone at the Brontë Society and the Brontë Parsonage Museum, who have helped this chapter and the forthcoming book at every stage. In particular, I'd like to thank: Danielle Cadamarteri, Ann Dinsdale, Diane Fare, Harry Jelley, Sarah Laycock, Lauren Livesey, Sue Newby, Kitty Wright and Rebecca Yorke.

I have been given permission to use the illustrations in this book by Scarborough Library, whose archive is a wonderful resource. I'd like to thank the librarian, Angela Kale, for going out of her way to source and scan these images.

The map of Scarborough was devised and designed by cartographer, Chris Goddard, who also devised and designed the Brontë Stones maps. A big thanks to Chris for these exquisite drawings.

The journal, *Brontë Studies*, has been a great resource and source of expertise and scholarship, and I would like to thank the editorial panel for all their help.

Thanks and gratitude also go to everyone at HarperCollins and HQ Stories, especially Lisa Milton, who spotted the book's potential, and Kate Fox who has offered her time and editorial expertise.

Finally, a special thanks go to the publishers of this limited edition book and everyone at the University of Huddersfield Press, who have been so instrumental. Many thanks to Megan Taylor for all her editorial input, and Monty Adkins for his valuable support of the project. Thanks also to Martin Gill, Alison McNab, Catherine Parker, Zoe Johnson and Dawn Cockcroft, who have all worked under great pressure, during the lockdown, to ensure it was ready in time.


**1. Introduction**

Until recently, central nervous system (CNS) malformations were the second most common congenital abnormalities after congenital cardiac defects [1, 2]. Recent reports have documented CNS malformations to be the most common anomalies among all systems, with a prevalence of 3 to 6% in stillbirth and 0.14 to 0.16% in live births [3]. There is limited information about the precise etiology of congenital CNS anomalies, and most of the cases are idiopathic. It is speculated that a combination of genetic and environmental factors plays a major role in the pathogenesis of these defects [4]. Management and diagnosis of these conditions are challenging and require a proper understanding of their etiology and categories.

CNS anomalies (CNSA) include those of the spinal cord (such as meningocele, myelomeningocele, and encephalocele) and brain (including growth disorders of the cerebrum, cerebellum, and brain stem) [5]. CNSA may be associated with other anomalies pertaining to other systems as well, such as those of the heart [6]. These malformations need complex surgeries along with long-term intensive care and impose a significant financial impact on the families and healthcare system. In this chapter, we review the classification, epidemiology, and the newest modalities of treatment of congenital CNS anomalies with respect to NTDs.

**104**

*Congenital Anomalies in Newborn Infants - Clinical and Etiopathological Perspectives*

[1] Abdellatif M, Ghozy S, Kamel MG, et al. Association between exposure to macrolides and the development of infantile hypertrophic pyloric stenosis: A systematic review and meta-analysis.

European Journal of Pediatrics. 2019;**178**:301-314. DOI: 10.1007/

[2] Greenfield Lazar J, Mulholland Michael W, editors. Greenfield's Surgery: Scientific Principles and Practice. 5th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams &

[3] Soares KC, Goldstein SD,

[5] Dimick JB, Upchruch JR, Sonnenday CJ, editors. Clinical

Williams & Wilkins; 2012

sdigestive/mitteldarm01.html

Ghaseb MA, et al. Pediatric Surgery International. 2017;**33**:637. DOI: 10.1007/

[4] Fiser SM. The ABSITE Review: Practice Questions. 2nd ed. Richmond, VA: Hancock Surgical Consultants; 2015

Scenarios in Surgery: Decision Making and Operative Technique. Philadelphia: Wolters Kluwer Health/Lippincott

[6] http://www.embryology.ch/anglais/

[7] https://discovery.lifemapsc.com/ library/review-of-medical-embryology/ chapter-84-development-of-themidgut-general-introduction https:// www.pedsurglibrary.com/apsa/view/ Pediatric-Surgery-NaT/829186/all/ Posterior\_Sagittal\_Anorectoplasty#0

[8] https://coloproctol.org/journal/view.

[9] Morris G, Kennedy A, Cochran W. Current Gastroenterology Reports.

php?number=1558

2016;**18**:16. DOI: 10.1007/ s11894-016-0490-4

s00431-018-3287-7

**References**

Wilkins; 2011

s00383-017-4083-6


**5.2 Immunophenotypic features of ovarian mucinous carcinomas**

Immunohistochemistry of OMCs is characterised by diffuse intense positivity for CK7 and variable positivity for CA19-9, CEA, CK20 and CDX2 focal weak positivity for PAX8 can be seen in a subset of tumours [111, 112], whereas WT1, Napsin A, Vimentin, CA125, ER and PgR are mostly negative. SATB2 is rarely expressed in ovarian mucinous tumours (5–7%) and its expression is associated with the presence of mature teratoma [113] p53 may demonstrate wild-type or mutation-type immunoreactivity and p16 is usually negative or focally positive [114, 115].


# **Celebrating Applied Sciences Reaches 20,000 Articles Milestone Feature Papers in Applied Biosciences and Bioengineering Section**

Edited by Francesco Cappello and Magdalena Gorska-Ponikowska Printed Edition of the Special Issue published in *Applied Science*

www.mdpi.com/journal/applsci


**3.4 Epigenetics of OPRM1 gene and its impact on cell function**

Human genome has about 45,000-C-phosphate-G-(CpG) islands, many in the promoter regions of genes. The CpG islands are located upstream of the transcription start site to within the first exon [57]. Nielsen et al*.* [58] and Chorbov et al*.* [59] reported that in DNA obtained from peripheral lymphocytes, two of 16 CpG sites in a region of *OPRM1* gene promoter had significantly higher methylation in former heroin addicts than in controls. These two CpG sites are located in binding sites for the potential Sp1 transcription factor. Oertel et al*.* [60] showed that substitution of an A with a G at gene position +118 introduces a new CpG-methylation site at position +117, which leads to enhanced methylation of *OPRM1* gene resulting in decreased expression. Using m-fold software, Johnson et al*.* [61] showed that 118G variant demonstrated an altered folding that could affect mRNA stability. The epigenetic mechanism reported by Oertel et al*.* [60] impedes μ-OR upregulation in brain tissue, and they concluded that while in wild-type subjects, a reduced signaling efficiency associated with chronic heroin exposure was compensated for by an increased receptor density; this upregulation was absent in carriers of the 118G receptor variant due to diminished *OPRM1* mRNA transcription. The *OPRM1* 118A > G SNP variant not only reduces μ-OR signaling efficiency, but by a genetic-epigenetic interaction, also reduces OR expression and therefore, depletes the opioid system of a compensatory reaction to chronic exposure, providing evidence that a change in the genotype can cause a change in the epigenotype with major functional consequences.