**5. Conclusions**

Zoonotic trematodes cause a number of diseases some of which have major public health or animal health consequences or have huge financial implications. A key element in the parasites' life cycle are the first intermediate host which depending on the parasitic species particular species of gastropod mollusks. Control of these snails could be an important element in an integrated approach to control these diseases following the "One-Health" approach.


**Abstract**

The management of municipal solid waste (MSW) is a major obstacle for the majority of municipalities in developing countries because of the impacts related to the landfilling of waste. Garbage is an energy-rich material. As a result, energy recovery is considered to be a sustainable waste management method. In Morocco, 7.4 million tons are produced annually; most of the waste is landfilled without any recovery despite the impacts related to this method of disposal. The objective of this chapter is to characterize combustible fractions (RDF) from household waste in Morocco and to study the economic and environmental benefits of their use as alternative fuels in cement kilns. The results of this research show that the combustible fractions contained in household waste in Morocco constitute a potential sustainable energy source with a high lower calorific value (4454 kcal/kg). The study of the advantages of co-incineration shows that the substitution of pet coke by 15% RDF reduces the pollution linked to gaseous emissions. In addition, the cement plant can make financial savings 389 USD/h by minimizing the use of fossil fuels.

**Keywords:** municipal solid waste, energy recovery, RDF, cement plant, Morocco

## **1. Introduction**

With population growth and improved living standards, the generation of household waste continues to increase throughout the world [1, 2]. Despite its health and environmental impacts, landfilling remains the most common method of waste disposal in developing countries [3, 4]. Several treatments can be used to recover the material and energy contained in the waste. The main energy recovery methods that can be used are incineration, pyrolysis, gasification, bio-methanation and RDF production [5]. The choice of treatment method depends on the qualitative and quantitative characteristics of the waste. A study conducted by Ouigmane et al. [6], showed that household waste in Morocco is a potential source of combustible fractions [6]. These fractions are the basic element of RDF production. Indeed, studies have been carried out in developed countries related to the use of RDF in cement kilns as an alternative fuel for petroleum coke have shown several economic and environmental advantages of this line of recovery [7–18].

**Figure 1.**

*World cement and fossil energy production until 2016 [21, 22].*

**Figure 2.**

The cement sector is a major energy consumer [19], a simple change in the management of energy consuming sources can have a positive effect on the plant's energy consumption [20]. Indeed, the cement sector is largely linked to the consumption of fossil fuels (**Figure 1**) [21, 22].

Improving the energy efficiency of energy-intensive industries (e.g. cement plants) will contribute significantly to improving their economic competitiveness in the global market [23, 24]. In order to improve their image, cement plants are trying to replace petroleum coke, which is a polluting fossil fuel, with less polluting alternatives such as RDF or renewable energies. To achieve the 2030 sustainable development goals, the share of fossil fuels must decrease as shown in **Figure 2** [25].

The objective of this chapter is to assess the economic and environmental benefits of using RDF as a substitute for fossil fuels in a cement plant for a region in Morocco.

#### **2. Waste management in Morocco**

#### **2.1 Legislative framework for waste management in Morocco**

The management of municipal solid waste (MSW) requires knowledge of the legal and institutional contexts that concern this sector. In Morocco, the main

*Overall thermal energy consumption for cement production by fuel [25].*

*Sustainable Solid Waste Management in Morocco: Co-Incineration of RDF as an Alternative… DOI: http://dx.doi.org/10.5772/intechopen.93936*

problem of the MSW management sector is manifested in the legal and institutional context, in particular the weakness of the means available to the competent local authorities in terms of solid sanitation and the inadequacy of legal and regulatory texts. Despite all these problems, Morocco has made several efforts and launched Law 28-00 on waste management in 2006 and the national plan for household waste management. Thus, the new Organic Law of Territorial Communes 113-14 relating to communal organization, specifies in its Article 83 that solid sanitation, household waste collection and waste treatment fall within the competence of the communities [26].

### **2.2 The production of household waste in Morocco**

The production of waste in Morocco, as in the rest of the world, does not stop increasing due to the intervention of several factors (population, standard of living, urbanism etc.). The waste production rate in urban areas is 0.76 kg per capita per day and 0.28–0.3 kg per capita per day in rural areas [6–28]. Waste production varies from one region to another, in the region of Rabat the production ratio is 0.96 kg per capita per day [29] and in the city of Beni Mellal the rate is 0.89 kg per capita per day [30].

#### **2.3 The composition of household waste in Morocco**

The qualitative aspect of waste is a key parameter in the choice of treatment method. The general composition of waste in Morocco is characterized by a high content of biodegradable material with a percentage that varies according to urban and rural areas and decreases over time as a result of the improvement in living standards. The average composition of MSW in Morocco is shown in **Figure 3** [28].

#### **2.4 Waste disposal in Morocco**

Prior to the implementation of the National Plan for Household Waste, almost all of the waste generated in Morocco is disposed of in uncontrolled landfills without any control. The country has become increasingly sensitive to the impacts of uncontrolled dumping and the costs of environmental degradation that can result. As a result, the Ministry of the Environment has launched the National Plan for Household Waste to rehabilitate all uncontrolled landfills and replace them with controlled landfills or landfill and recovery centers (LRC) with a 20% recovery by 2022.

**Figure 3.** *Average composition of household waste in Morocco [28].*

#### **Table 1.**

*Costs related to environmental degradation by the waste sector [31].*

### **2.5 The cost of environmental degradation through waste management in Morocco**

Environmental deterioration is not limited to damage to health and ecosystems, but can affect a country's economy. Morocco's efforts in recent years have led to an improvement in the situation of the environmental sector, which is proven by the results of the studies of the cost of environmental degradation in Morocco in 2000 and 2014. These studies are carried out by the World Bank and have shown a 20% reduction in this cost in 14 years. Indeed, the cost of environmental degradation has gone from 590 MAD per inhabitant in 2000 to 450 MAD per inhabitant in 2014 [31]. The costs of environmental degradation related to waste management are presented in **Table 1** [31].

#### **3. Cement sector in Morocco**

#### **3.1 Data on the cement sector in Morocco**

Cement is one of the oldest industries developed in Morocco, with a first plant in Casablanca at the beginning of the 20th century. Today, the activity is structured and covers the whole territory. The Moroccan cement sector is made up of four companies that operate a total of 12 production plants distributed throughout the country (**Figure 4**) with an annual production capacity of almost 21 million tons [32]. All the plants benefit from modern technologies and an efficient management system. In addition, the professionalism and expertise of the staff and the support of three of the world's largest cement groups guarantee product quality.

The development of the cement industry is linked to the economic development of the country and the upgrading of the construction and housing sectors. In 2011, cement consumption peaked at 16 million tons. Since 2012, the construction materials market has been in decline, mainly due to the drop in construction activity [32].

#### **4. Case study**

The objective of this part is to make a case study on a region in Morocco in order to assess the economic and environmental benefits of the co-incineration of RDF in a cement kiln.

*Sustainable Solid Waste Management in Morocco: Co-Incineration of RDF as an Alternative… DOI: http://dx.doi.org/10.5772/intechopen.93936*

**Figure 4.** *Distribution of cement production plants in Morocco.*

### **4.1 Materials and method**

#### *4.1.1 Waste management in the study area*

The province of Khenifra is located in the center of Morocco and is made up of 22 communities (**Figure 5**) with an estimated population of 379,639 inhabitants in 2019 [33]. The management of household waste is a major obstacle for the territorial communes, especially in the disposal phase. Since 2017, an evolution in the mode of waste management in the province of Khenifra has been noticed, with the organization of burial by changing the mode of uncontrolled dumping to a mode of controlled landfill. Despite the efforts made by the communities and the Ministry of the Environment, current waste management is encountering problems and does not meet expectations, since the energy and matter contained in the waste is lost underground. In addition, the controlled landfill is poorly located, as its geographical location can cause several impacts on health and the environment (**Figure 5**). Therefore, it is necessary to look for sustainable and cost-effective solutions to take advantage of the material and energy without causing any impact on the environment.

#### *4.1.2 Presentation of the cement plant in the study area*

The closest cement plant to the Study Area is located in the province of Beni Mellal and is 81 km from the Study Area landfill (**Figure 5**). The potential for

**Figure 5.** *Location of the study area and cement plant.*

cement production at the plant is 1,600,000 million tonnes per year. The dry process is used to produce clinker. Calcination is done in a rotary kiln using fossil fuels (petroleum coke) (**Figure 6**).

#### *4.1.3 Dry fractions potential in the study area*

The scenario taken for this work is the recovery of all dry fractions that can be extracted from household waste following a sorting into two fractions (**Figure 7**). The percentage of dry fractions in household waste in Morocco is 30% of the total flow of household waste [30]. The lower caloric value (LCV) of these fractions has been determined in the laboratory according to the EN 15400 standard [34] the value found is 4454 kcal/kg.

The annual RDF potential in the study area is estimated based on the population of the municipalities in the province and the national average waste generation in urban and rural areas. The results of the RDF production estimated are presented in **Table 2**.

#### *4.1.4 Calculation method*

In order to demonstrate the economic and environmental benefits of coincineration of RDF in cement kilns, several aspects will be evaluated in this case study. The economic gain of the cement plant and the carbon dioxide reduction will be determined. Several data have been collected in order to complete this study, including information on the cement plant in the study area (petroleum coke consumption, LCV of petroleum coke, clinker production, etc.), data on waste management in Khenifra province (potential in combustible fraction, PCI of RDF waste, etc.).

*Sustainable Solid Waste Management in Morocco: Co-Incineration of RDF as an Alternative… DOI: http://dx.doi.org/10.5772/intechopen.93936*

**Figure 6.** *Clinker firing stage in the cement plant.*

**Figure 7.** *RDF production scenario.*

An economic model has been proposed taking into account the quantity of RDF produced, cost savings of fossil fuels, CO2 emissions. Formula (1) is used to calculate the net savings of the cement plant.

$$\begin{aligned} \text{Net savings} &= ((\text{Savings on traditional fuel} + \text{CO}\_2 \text{ emission cost reduction}) \times \text{(100 - Cargy loss)})/100 \\ &- \text{RDF production cost reduction}) \times \text{(100 - Cargy loss)})/100 \end{aligned} \tag{1}$$

#### *<IP_ADDRESS> The substitution rate*

The substitution rate of RDF in cement plants can reach very high percentages up to 45% depending on the process. In the case of the present study, a substitution

#### **Table 2.**

*Estimated RDF production in the Study Area.*

rate of 15% was chosen based on the results of the study by Kara [8] and Hemidat et al. [9] who found that 15% is the most optimal substitution rate from a technical and environmental point of view [8, 9].

### *<IP_ADDRESS> RDF requirement in cement plant*

In order to calculate the amount of RDF required achieving 15% substitution percentage of pet coke, the energy content of RDF was taken into account based on the LCV value determined for the waste in the study area. In fact, a ton of pet coke does not have the same calorific value as a ton of RDF. The data used for the calculation of RDF required to substitute 15% of the pet coke are presented in **Table 3**.

#### *<IP_ADDRESS> Energy requirement*

The amount of energy required to produce one kilogram of clinker is 730 kcal/ kg. Therefore the amount of energy to be extracted from RDF with 15% substitution is given in Eqs. (2) and (3).

$$\text{ERDF} = \frac{730 \times 15}{100} = 109.5 \text{ kcal} \tag{2}$$

$$\text{EPC} = \frac{730 \times 85}{100} = 620.5 \text{ kcal} \tag{3}$$

where ERDF is the energy required from RDF with a 15% substitution rate to produce one kilogram of clinker; EPC is the energy required from pet coke with a substitution rate of 85% to produce one kilogram of clinker.

**Table 3.** *RDF requirement calculation data.* *Sustainable Solid Waste Management in Morocco: Co-Incineration of RDF as an Alternative… DOI: http://dx.doi.org/10.5772/intechopen.93936*

#### *<IP_ADDRESS> Requirement in mass quantity*

The NCV of the RDF in the study area is 4453 kcal/kg, hence, the amount of RDF required to produce one kilogram of clinker in the case of 15% substitution is given by Eq. (4).

$$\text{Mass of RDF corresponding to 109.5 kcal} = 109,5 \,\text{kcal} \times \frac{1}{\text{LCV RDF}} = 0.024 \text{kg} \tag{4}$$

where LCV RDF is the lower calorific value of RDF (for the case of this study, it equals 4454 kcal/kg).

The quantity of pet coke required is given according to Eq. (5).

Mass of pet coke corresponding to 620*:*5 kcal ¼ 620*:*5 kcal � 1 LCV PC ¼ 0*:*083 kg (5)

where LCV PC is the lower calorific value of Pet coke (for the case of this study it is equal to 7500 kcal/kg).

The production of one ton of clinker requires the mixture of 0.024 ton of RDF and 0.083 ton of pet coke.

#### *<IP_ADDRESS> Fuel consumption as a function of time*

The production capacity of the cement plant close to the study area is 1,600,000 tons of cement per year, the calculation to be made in this study will concern the economic cost of using RDF to produce 1 million tons of clinker per year. **Table 4** shows the daily and hourly production of clinker. The cement plant is shut down for a period of one month in order to maintain the combustion furnace.

On the basis of clinker production as a function of time, the tonnage of fuel used will be deducted according to Eq. (6).

fuel consumption per hour ¼ Production of clinker hour

� The amount of RDF needed to produce one tonne of clinker

(6)

RDF consumption per hour ¼ 124*:*37 � 0*:*024 ¼ 2*:*99 tons per hour (7)

The same formula (6) is used to calculate pet coke consumption per hour:

pet coke consumption per hour ¼ 124*:*37 � 0*:*083 ¼ 10*:*32 tons per hour (8)

*<IP_ADDRESS> CO2 emissions*

The amount of carbon dioxide (CO2) saved has been estimated by assuming that 70% of one kilogram of petroleum coke is emitted as CO2 [8]. It is then converted

**Table 4.**

*Clinker production per year, per day and per hour.*

into CO2 savings in the form of an emission of 1 kg of CO2, which is estimated at USD 0.015 [36].

### **5. Results and discussion**

As shown in the experimental study by Hemidat et al. [9] and Kara [8], the use of 15% RDF as a substitute for pet coke for the generation of thermal energy for clinker production does not cause any quality problems [8, 9]. The consumption of pet coke in kg/hour is 10,320 if the substitution rate is 15% and 2990 kg/hour of RDF which will save 1782 kg/hour of petroleum coke (**Table 5**).

Hence, the use of 15% RDF will save 1782 kg/h of pet coke, so the annual saving of pet coke is:

$$1.782 \frac{\text{tons}}{\text{hour}} \times 24 \frac{\text{hour}}{\text{day}} \times 335 \frac{\text{days}}{\text{year}} = 14327.28 \frac{\text{tons}}{\text{year}} \tag{9}$$

As a result, 15,610. 32 tons of pet coke will be saved annually if 15% RDF is used in the cement plant kiln.

**Table 6** shows the results of the calculation of the CO2 emission parameters as well as the net savings in USD per hour.

The price of petroleum coke is variable and depends on the price of oil. In this study a price of 150 USD/ton was taken as the annual average selling price of petroleum coke, so the annual saving in terms of petroleum coke if 15% RDF is used as a substitute is:

$$14 \text{ } 327.28 \frac{\text{tons}}{\text{ans}} \times \frac{150 \text{USD}}{\text{ton}} = 2,149,092 \text{ USD/year} \tag{10}$$

Since the consumption of RDF is 2.99 ton/h, then the annual consumption is:

$$\text{annual consumption of RDF} = \frac{2.99 \text{tons}}{\text{h}} \times \frac{24 \text{h}}{\text{day}} \times \frac{335 \text{day}}{\text{year}} = 24.039.6 \text{ tons/year (11)}$$

#### **Table 5.**

*Calculation results for the pet coke saving.*

#### **Table 6.**

*The relation between emission and saving in the view of CO2.*

*Sustainable Solid Waste Management in Morocco: Co-Incineration of RDF as an Alternative… DOI: http://dx.doi.org/10.5772/intechopen.93936*

The market price of one ton of RDF is 24 USD/ton, hence, the annual purchase price of RDF is:

$$\frac{24\text{ 039.6tons}}{\text{year}} \times 24\text{USD} = 576,950.4\text{ USD/year} \tag{12}$$

The real financial economy is:

$$12,149,092 \frac{\text{USD}}{\text{year}} - 576,950.4 \frac{\text{USD}}{\text{Year}} = \frac{1,572,141.6 \text{USD}}{\text{year}} \tag{13}$$

The results obtained show the real savings achieved through the use of RDF in the cement plant.

In addition, savings related to the substitution of petroleum coke by the more expensive RDF, indirect savings related to CO2 emissions can be achieved.

Given that the price of a ton of pet coke is USD 150, with 15% substitution by RDF. Therefore, the financial saving on pet coke is:

$$\frac{1.782 \text{kg}}{\text{h}} \times \frac{0.15 \text{USD}}{\text{Kg}} = 267.3 \text{ USD/h} \tag{14}$$

The amount of CO2 emissions to be saved is:

$$\frac{1.782 \text{kg}}{\text{h}} \times 0.7 = 1.247.7 \text{ kg/h} \tag{15}$$

The saved cost related to CO2 emissions is:

$$\frac{1247.7\text{ kg}}{\text{h}} \times \frac{0.015 \text{USD}}{\text{kg}} = 18.72 \frac{\text{USD}}{\text{h}} \tag{16}$$

The cost of RDF is:

$$\frac{10 \text{ 320kg}}{\text{h}} \times \frac{0.024 \text{USD}}{\text{kg}} = 247.68 \text{ USD/h} \tag{17}$$

The loss of efficiency is given as follows:

$$\mathbf{20\%} \times \text{RDF\%} \text{conumption} \times \mathbf{100} \tag{18}$$

$$(\mathbf{0.2} \times \mathbf{0.15}) \times \mathbf{100} = \mathbf{3\%}$$

The net saving per hour is:

Net Savings ¼ ððSavings on Conventional Fuels þ CO2 Emission Cost Reduction � RDF Production Cost ReductionÞ ∗ 100 � Loss of Energy Þ*=*100 (19)

$$\frac{[(267.3 + 18.72 - 247.68) \times (100 - 3)]}{100} = 37.19 \text{ USD/h}$$

The energy recovery of RDF in the cement plant has shown the economic and environmental advantage of substituting fossil fuels (Petroleum Coke) by alternative fuels (RDF). This case study shows that the cement plant can save its pet coke consumption as well as CO2 emissions with a saving of 37 USD/hour. Such a study on a cement plant in Turkey showed that it can save 19 USD/h [8]. In Jordan,

Hemdiat et al. found that the savings that a cement plant can make by substituting 15% RDF for petroleum coke can be as high as 389 USD/h [9]. The results found and the potential for the combustible fraction in Moroccan waste [6, 30] are encouraging in order to develop an RDF production chain in all the regions where there are cement plants in Morocco. In addition to the advantage of sustainable disposal of household waste, other types of waste such as medical waste, pandemic waste such as COVID-19, and sludge from wastewater treatment plants can be thermally treated in the kilns of cement plants [37, 38].


**8. Conclusion**

408 Biochemistry

inflammation, it is now well accepted that ceramides are key elements in the inflammatory response (Lamour & Chalfant, 2005; Wijesinghe et al., 2008; Gomez-Munoz et al., 2010). For instance, it was reported that activation of A-SMase and the subsequent formation of ceramides play an important role in pulmonary infections as it facilitates internalization of bacteria into lung epithelial cells (Gulbins & Kolesnick, 2003). In this context, inhibition of

Inflammatory mediators include chemokines, cytokines, vasoactive amines, products of proteolytic cascades, phospholipases, or lipids such as eicosanoids and sphingolipids. A major mediator of inflammation is PLA2 activity. In particular, group IV cytosolic cPLA2 ( or cPLA2-alpha) has been involved in receptor-dependent and independent production of eicosanoids, which are major components of inflammatory responses. Sphingolipids, including ceramides, have also been described as key mediators of inflammation (Hayakawa et al., 1996; Serhan et al., 1996; Manna & Aggarwal, 1998; Newton et al., 2000). More recently a role for ceramide in the development of allergic asthmatic responses and airway inflammation was established (Masini et al., 2008), and exogenous addition of C2 ceramide to cultured astrocytes induced 12-lipoxigenase leading to generation of reactive oxygen species (ROS) and inflammation (Prasad et al., 2008). Also, A-SMase-derived ceramide was involved in platelet activating factor (PAF)-mediated pulmonary edema (Goggel et al., 2004). Subsequently, it was proposed that at least some of the proinflammatory effects of ceramides might in fact be mediated by its further metabolite C1P. The first report on the regulation of arachidonic acid (AA) release and the production of prostaglandins by C1P was from the laboratory of Charles Chalfant (Pettus et al., 2003b). This group demonstrated that C1P was able to stimulate AA release and prostanoid synthesis in A549 lung adenocarcinoma cells. In a follow up report, the same group showed that the mechanism whereby C1P stimulates AA release occurs through direct activation of cPLA2 (Pettus et al., 2004). Subsequently, it was found that C1P is a positive allosteric activator of cPLA2-alpha, and that it enhances the interaction of the enzyme with PC (Subramanian et al., 2005). In further work, the same group demonstrated that activation of cPLA2-alpha by C1P is chain length-specific; in particular, C1P bearing acyl chains equal or higher than six carbons were able to efficiently activate cPLA2-alpha in vitro, whereas shorter acyl chains (in particular C2-C1P) were unable to activate the enzyme. It was concluded that the biological activity of C2-C1P does not occur via eicosanoid synthesis (Wijesinghe et al., 2008). Also, C1P was shown to act in coordination with S1P to ensure maximal production of prostaglandins. Specifically, S1P was shown to induce cyclooxigenase-2 (COX-2) activity, which then uses cPLA2-derived AA as substrate to synthesize prostaglandins (Pettus et al., 2005). Further details on the role of C1P in inflammatory response can be found in different reviews (Chalfant & Spiegel, 2005; Lamour et al., 2007; Wijesinghe et al., 2007), Wijesinghe et al., and recent work by Murayama and co-

A-SMase by C1P could be important to reduce or prevent infection in the lung.

workers (Nakamura et al., 2011).

**6. Ceramide 1-phosphate and the control of cell migration**

Macrophage populations in tissues are determined by the rates of recruitment of monocytes from the bloodstream into the tissue, the rates of macrophage proliferation and apoptosis, and the rate of macrophage migration or efflux. Recently, our group demonstrated that exogenous addition of C1P to cultured Raw 264.7 macrophages stimulated cell migration The implication of simple sphingolipids in the regulation of cell activation and metabolism has acquired special relevance in the last two decades. Most attention was first paid to the effects elicited by ceramide because this sphingolipid turned out to be essential in the regulation of cell death, differentiation, senescence, and various metabolic disorders and diseases. However, its phosphorylated form, C1P, was thought not to be so important. However, C1P has emerged as a crucial bioactive sphingolipid, and this chapter highlights the relevance of C1P in cell biology. Specifically, C1P has now been established as key regulator of cell growth and survival, and its relevance in the regulation of cell migration is beginning to emerge. Also importantly, the discovery that C1P can act both intracellularly or as receptor ligand opens a broad avenue to investigate its implication in controlling cell metabolism. In addition to this, C1P has been postulated to be a potent proinflammatory agent, acting directly on cPLA2 to trigger eicosanoid production. Therefore, C1P and CerK, the major enzyme responsible for its biosynthesis, may be key targets for developing new pharmacological strategies for treatment of illnesses associated to cell growth and death, and cell migration , such as chronic inflammation, cardiovascular diseases, neurodegeneration, or cancer.


**[Oligonucleotides]**

420 Salmonella – A Dangerous Foodborne Pathogen

Individual samples (1 ml) were centrifuged at 10,000 X g for 3 min. The cell pellets were resuspended in RNase free water (100 l) and placed in a 100°C heating block for 20 min. The samples were cooled for 2 min at room temperature and centrifuged at 16,000 X g for 5 min. The supernatant fluids (5 l) were used to make 25 l of a multiplex PCR reaction mixture, which included 5 l of 5 X reaction buffer (2.5 mM MgCl2 and 0.8 mM concentration of each dNTP), 4 l of the primer mixtures of the five food-borne bacteria, 1 l of Super Taq plus polymerase (Rexgene Biotech., Cheongwon, South Korea), and 10 l of DNase free water in a single tube. The multiplex PCR was run for 35 cycles on a Tpersonal cycler (Whatman Biometra, Goettingen, Germany) under the following conditions : denaturation at 94°C for 30 sec, primer annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. The final cycle included an additional 5 min of extension time at 72°C. A 5 l aliquot of the reaction mixture was then electrophoresis on a 2% agarose gel electrophoresis in 0.5 X Tris-borate buffer at 100 V for 25 min. The amplification products were stained with

**[Extraction and preparation of DNA templates for PCR assay]**

ethidium bromide and visualized by UV trans-illumination.

Fig. 4. Flow diagram of experimental protocols for PCR template preparation

The oligonucleotide primers designed with Primer 3.0 software (Whitehead Institute, Cambridge, Mass.) were based on sequences obtained from Genbank and were used to amplify chromosomal DNA for the five food-borne pathogens (Table 2). The oligonucleotides and all reagents for PCRs were synthesized and purchased from Incorporation Bioneer (Daejeon, South Korea) and KoGene BioTech. (Seoul, South Korea).

Table 2. Oligonucleotide primers used in this study


**2. The relationship between walking efficiency and life‐space assessment in elderly people**

#### **2.1. Life‐space assessment**

The increased metabolic cost of walking can detract from the activity and quality of life of elderly people as a decline in physical activity rapidly degrades physical and psychological functions [8, 9]. Life space has been reported to have good construct and criterion validity for measuring the severity of mobility limitations, and achieving effi‐ cient walking plays a crucial role in the extension of life space. Life‐space assessment (LSA) is a tool that measures mobility and reflects participation in society based on the distance through which a person reports moving during the 4 weeks prior to the assessment [10, 11]. Life space, a relatively understudied concept in gerontology, can be defined as the size of the spatial area a person intentionally moves through in daily life, as well as the frequency of travel within a specific time frame [10]. Also, a reduced frequency of going outdoors could represent reduced life space, which has been hypoth‐ esized to be a risk factor in future physical disability. Murata et al. reported that life space was related not only to age or health status but also to environmental or psychosocial factors [12]. Shimada et al.'s reports confirm that LSA was more strongly associated with gait speed than the other gait variables [13] and also show that these declines in physical performances were apparent at age 80 years and over in women and at age 90 years and over in men [13].

LSA may reflect the physical activity status indirectly in elderly people because the LSA score is associated with physical performance, Activities of Daily Living (ADL), and sociodemo‐ graphic factors [11].

Life‐space mobility was measured using a Japanese translation of the LSA [14]. The LSA provided a score based on the distance a person reported moving during the 4 weeks before the assessment [10, 13].

The LSA scores range from 0 ("completely room‐bound") to 120 ("travel out of town every day without assistance") [13].

#### **2.2. Walking efficiency in elderly people**

of walking [1, 2]. In addition, a defect or slowing of this mechanism has been suggested to explain the difficulties experienced by older persons when trying to control their posture [3, 4]. Moreover, both muscle strength and lower postural stability decline with aging [5, 6]. Therefore, aging is characterized by changes in the neuromuscular system that decreases

Meanwhile, this progressive decline in physical capacities reduces the ability of elderly peo‐ ple to perform complex motor tasks and is associated with impaired mobility and a reduction in the ability [7]. Assessment of motor function contributes to the identification of factors that generate impairments to the performance of daily activities and an increase in the risk of falls for elderly people. Moreover, assessment of motor function by physical therapist provides important information about age‐related progressive reduction in muscle strength, ability to

This chapter provides an overview of the assessment method on motor function in elderly

**2. The relationship between walking efficiency and life‐space assessment**

The increased metabolic cost of walking can detract from the activity and quality of life of elderly people as a decline in physical activity rapidly degrades physical and psychological functions [8, 9]. Life space has been reported to have good construct and criterion validity for measuring the severity of mobility limitations, and achieving effi‐ cient walking plays a crucial role in the extension of life space. Life‐space assessment (LSA) is a tool that measures mobility and reflects participation in society based on the distance through which a person reports moving during the 4 weeks prior to the assessment [10, 11]. Life space, a relatively understudied concept in gerontology, can be defined as the size of the spatial area a person intentionally moves through in daily life, as well as the frequency of travel within a specific time frame [10]. Also, a reduced frequency of going outdoors could represent reduced life space, which has been hypoth‐ esized to be a risk factor in future physical disability. Murata et al. reported that life space was related not only to age or health status but also to environmental or psychosocial factors [12]. Shimada et al.'s reports confirm that LSA was more strongly associated with gait speed than the other gait variables [13] and also show that these declines in physical performances were apparent at age 80 years and over in women and at age 90 years and

LSA may reflect the physical activity status indirectly in elderly people because the LSA score is associated with physical performance, Activities of Daily Living (ADL), and sociodemo‐

muscle strength, balance, and proprioception.

walk, and postural control.

**in elderly people**

over in men [13].

graphic factors [11].

**2.1. Life‐space assessment**

people.

26 Clinical Physical Therapy

It may be that the decline in walking efficiency is a common result of the decrease of many bodily functions that capture the overall impact of life space and is an important indica‐ tor of life space. However, the mechanism by which older people's life space and walking efficiency decline is not well investigated and remains poorly understood. Also, the specific mechanisms that explain the difference between the decline in walking efficiency and life space in early elderly and late elderly are not yet clear. Additionally, little is known about the relationship between the walking efficiency decline and confined life space to influence on aging.

In older adults with mobility limitations, abnormalities in posture and gait contribute to the greater energy cost of inefficient gait, with adjustments for age and gait speed [15]. Limitations in life space, as measured by the University of Alabama at Birmingham (UAB) life‐space assessment, reflect lifestyle as well as walking efficiency and may be a valuable measure of functional decrease for community‐dwelling elderly people, especially since life space specifi‐ cally relates to mobility and a person's participation in society.

#### *2.2.1. Walking efficiency assessment*

Walking efficiency during the 6MD trials was measured using the Cosmed K4b2 (Rome, Italy), an indirect calorimetry system specifically designed to measure energy expenditure in nonlaboratory settings (**Figure 1**).

  Flexible facemask is attached to a digital turbine flowmeter to measure the volume of expired air and inspired. Sampling line from the turbine to analyzer unit system delivers expired air for the measurement (O2 , CO2 content). Before test, the K4b2 was calibrated according to the manufacturer's guidelines. After warming up the unit for 60 min, the CO2 and O2 analyzers were calibrated against room air as well as a reference gas of known composition (4.94% CO2 , 16.07% O2 ). Before walking efficiency assess‐ ment, each participant was required to sit quietly for 10 min as a rest period. The speed and distance of the 6‐min walking distance test (6MD) was recorded using standardized procedures.

**Figure 1.** Expiration gas analyser.

Walking efficiency was calculated based on net efficiency as follows: Walking efficiency = (walking VO2  ml/kg ‐ resting VO2  ml/kg)/6MD average walking speed (m/s) (**Figure 2**) [16]. The Cosmed K4b2 system uses the increased VO2 /kg ratio after noise processing to predict walking effi‐ ciency from O2 consumption.

Breath‐by‐breath values from the Excel spreadsheet were calculated from the increased ratio over 1‐breath intervals. Outliers from the increased ratio were calculated from exploratory data analysis of participants. We defined outliers (noise) as values beyond an increased ratio of 100%. Data that were more than 100% of the increased ratio were removed, and a below‐100% value was calculated from the remaining test data. This method contributes to noise processing of the VO2 /kg signal. For each subject, walking efficiency was calculated by recording the noise‐processed VO2 /kg values.

#### **2.3. Relationship between walking efficiency and life‐space assessment**

Ito et al. reported the significant relationship between walking efficiency and LSA in late elderly [16]. Moreover, the data from this study suggest that as walking efficiency declines with age, life space increasingly declines (**Figure 3**). This suggests that the age‐related decline of walking efficiency is caused by physiological changes of the late elderly. Other studies have suggested that as aerobic capacity declines with age, walking at a habitual speed becomes an increasingly more intense and therefore difficult activity, resulting in a slowing of walking speed in an effort to reduce fatigue [17]. Also, the previous study has shown that the walking energy cost for a comparable speed is generally higher for healthy

**Figure 2.** Walking efficiency = (walking VO2  ml/kg ‐ resting VO2  ml/kg)/6MD average walking speed (m/s). Horizontal axis of blue shows the meaning of 10 min of the rest period and 6‐min walking trial. Vertical axis of red shows the meaning of last 3 min of the 6‐min walking trial. Following the transfer of data from the instrument, an Excel spreadsheet was used to calculate steady‐state VO2 /kg and mean counts per minute during the last 3 min of the 6MD trial and 10 min of the rest period.

Walking efficiency was calculated based on net efficiency as follows: Walking efficiency = (walking

Breath‐by‐breath values from the Excel spreadsheet were calculated from the increased ratio over 1‐breath intervals. Outliers from the increased ratio were calculated from exploratory data analysis of participants. We defined outliers (noise) as values beyond an increased ratio of 100%. Data that were more than 100% of the increased ratio were removed, and a below‐100% value was calculated from the remaining test data. This method contributes to

Ito et al. reported the significant relationship between walking efficiency and LSA in late elderly [16]. Moreover, the data from this study suggest that as walking efficiency declines with age, life space increasingly declines (**Figure 3**). This suggests that the age‐related decline of walking efficiency is caused by physiological changes of the late elderly. Other studies have suggested that as aerobic capacity declines with age, walking at a habitual speed becomes an increasingly more intense and therefore difficult activity, resulting in a slowing of walking speed in an effort to reduce fatigue [17]. Also, the previous study has shown that the walking energy cost for a comparable speed is generally higher for healthy

/kg values.

**2.3. Relationship between walking efficiency and life‐space assessment**

 ml/kg)/6MD average walking speed (m/s) (**Figure 2**) [16]. The Cosmed

/kg signal. For each subject, walking efficiency was calculated by

/kg ratio after noise processing to predict walking effi‐

VO2

ciency from O2

28 Clinical Physical Therapy

 ml/kg ‐ resting VO2

**Figure 1.** Expiration gas analyser.

noise processing of the VO2

recording the noise‐processed VO2

K4b2 system uses the increased VO2

consumption.

**Figure 3.** Schematic diagram of the incidence of walking efficiency decline in late elderly people.

elderly people, particularly those above 65 years, compared with younger people [18, 19]. Mechanisms related to the initiation and stepping patterns of gait, such as hip extension, step width, and cadence, have previously been reported to be related to the energy cost of walking in older adults with slow and variable gait [15]. Abe et al. reported that women of advanced age (75 years or older) have diminished pulmonary function, physical function, and mobility, and that diminished pulmonary function is associated with declining physical function [20]. Malatesta et al. reported that healthy octogenarians exhibited higher walking cost and greater stride time variability [21] and also reported that these declines in physi‐ cal performances were apparent at age 80 years and over in women and at age 90 years and over in men [13]. Shimada reported that increased VO2 in older adults manifests as walking becomes inefficient and reduced endurance capacity occurs [22].

This suggests that going activity to extension of LSA may better impact walking efficiency than efforts to improve gait speed and muscle strength.


**1. Introduction**

In the last decades, because of the huge advances of recombinant DNA technology, recombinant antibodies have found increasing applications in the therapy of many diseases, whether of genetic, infectious, or tumour origin. Several antibodies and antibody-based products are either approved or under investigation in clinical trials and, particularly for tumours, many of them have revolutionized classical chemotherapy based on drugs [1]. Through recombinant antibodies, it is possible to interfere with specific protein functions at DNA, RNA, or protein level. Direct targeting of pathogenic proteins can even be advantageous over the targeting of genomic sequences with an on/off mode, because it allows modulating and tailoring protein activity without affecting genomic sequences.

Currently, thanks to the ability of the mammalian immune system to produce antibodies against virtually any antigen, and to over 30 years of molecular technology studies on antibody manipulation, well-established methods allow the selection of ligands for specific protein epitopes in either intra- or extra-cellular environment. Antibody selection can be performed from recombinant antibody libraries of different kinds, even originating from animals immunized with antigens of interest. Specific antibodies can be delivered directly to the cells as purified proteins or expressed as intracellular antibodies (intrabodies) by recombinant DNA technology. Different antibody formats representing more or less extended regions of an immunoglobulin (Ig) are presently available. The small size formats, i.e., antibodies in single-chain format (scFvs) and single domain antibody or nanobodies

**Citation:** Donà, M.G.; Di Bonito, P.; Chiantore, M.V.; Amici, C.; Accardi, L. Targeting Human Papillomavirus-Associated Cancer by Oncoprotein-Specific Recombinant Antibodies. *Int. J. Mol. Sci.* **2021**, *22*,

9143. https://doi.org/10.3390/ ijms22179143

Academic Editor: Yong-Seok Heo

Received: 25 May 2021 Accepted: 20 August 2021 Published: 24 August 2021

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

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(sdAb or Nbs) [2], are the most suitable for expression as intrabodies because they are easily engineerable.

Several monoclonal antibodies (mAbs) in different formats reached the clinical stage or are in different clinical trial stages for the treatment of numerous pathologies including tumours [1,3]. We are principally interested in tumours associated to Human Papillomaviruses (HPVs), which represent a global health problem in terms of morbidity and mortality and for which many therapeutic strategies are under study. Among these, the approach based on recombinant antibodies deserves particular attention because of its potentialities related to safety, precision, and feasibility [3–5].

Here we describe, to the best of our knowledge, the different formats of recombinant antibodies against the HPV oncoproteins of Human Papillomaviruses characterized to date or currently under study and discuss whether and why they show promise for the treatment of pre-neoplastic and neoplastic lesions caused by these viruses.

#### **2. Different Antibody Formats: mAbs, scFvs and Nanobodies**

Recombinant antibody technology has undergone tremendous development in recent decades, so to prompt much progress in disease diagnosis and therapy. The use of display technologies allows in vitro selection from non-animal-derived recombinant (naïve or synthetic) repertoires (libraries) of peptides and antibody fragments in different formats such as Fab fragments (Fabs), scFvs, and Nbs. Different platforms are available such as phage display, yeast display, ribosome display, bacterial display, mammalian cell surface display, mRNA display, and DNA display. All of them mimic what occurs in vivo during antibody generation by the immune system as they rely on (1) genotypic diversity, which can be obtained by immune stimulation of a competent organism or by cloning; (2) the link existing between the genotype and phenotype; (3) selective pressure for increasing antibody specificity; and (4) amplification of specific clones originated by selective pressure. The coding sequences of binders specific for a given antigen, identified by the display technology of choice, can be expressed in prokaryotic or eukaryotic systems and tested both in vitro and in vivo for their ability to counteract the target antigen activity.

The possibility to engineer the originally identified antibody sequence represents an added value, since affinity, stability, and expression level can be improved while maintaining the desired antigen-binding properties. Furthermore, it is possible to modify the format so that the antibody could acquire new kinetic properties. Importantly, it is feasible to bypass the risk of immune reactions during clinical use by constructing antibodies from human scaffolds.

A whole IgG molecule (150 kDa) comprises heavy (H) and light (L) chains each consisting of a variable (VH and VL) and a constant (CH and CL) region covalently linked to each other and to oligosaccharides necessary for antibody effector functions and for long serum half-life. The antigen-binding regions responsible for diversity among antibodies are the complementary determining regions (CDRs), three for each VH and VL. The VH and VL joined by a disulphide bond and covalently linked to the first CH domain are obtainable by IgG proteolysis, resulting in a Fab monovalent antibody fragment (55 kDa) (Figure 1).

A few decades ago, it was observed that the N-terminal IgG fragment including the VH and VL retains the same antigen-binding capacity as the whole IgG molecule. The so-called scFvs (27 kDa) lack the constant regions and include only the VH and VL linked by a short peptide consisting of a sequence of glycine and serine residues such as (Gly4Ser)3. This arrangement provides flexibility, hydrophilicity, and resistance to proteases digestion. The linker length can be modified to favour or not the formation of multimers. In fact, shortening the linker to 3–12 amino acids prevents the formation of monomeric forms supporting inter-molecular VH-VL combinations also in different orientations, with spontaneous formation of a scFv dimer called "diabody" (60 kDa), where each of the two antigen-binding sites are formed by the VH of one scFv and the VL of the other one (Figure 1). The linkage of two scFvs in a unique molecule forms a tandem scFv. Both diabodies and tandem scFvs can have two different binding specificities, and in this case, they are called bispecific (bs). Interestingly, even the VH and VL arrangement in the scFv fusion protein can influence the binding activity, and it is currently possible to predict the best functional structure so as to design scFvs that meet all requirements by molecular modelling using a computer-aided homology method [6,7].

**Figure 1.** Schematic representation of the structure of conventional and camelidae monoclonal antibodies and of different antibody fragments. On the left, the whole monoclonal antibody (**top**) and camelid antibody (**bottom**) structures are represented. The variable light (VL) and heavy (VH) chains, as well as the constant light (CL) and heavy (CH1, CH2, CH3) chains are indicated. The complementarity determining regions responsible for antigen binding, three for each VL and each VH, are represented by stripes highlighted in different colours according to the different antigen specificity. Arrows indicate different monospecific and bispecific antibody fragments derived from the original antibody molecules with their nomenclature and molecular weight. The VH and VL in the different arrangements are connected by peptide linkers of 3–12 amino acids represented by black curved lines.

The CDRs of a scFv are embedded in an amino acid scaffold of either human or animal origin, according to the library utilized for selection. Of note, CDRs with specific binding activities can be isolated and grafted onto different scaffolds suitable for the purposes of interest. Both scFv and Fab fragments can be engineered into stable oligomers to increase binding avidity and widen antigen specificity. Specific applications of these formats are the recruitment of T-cells to tumours in immunotherapy, viral retargeting in gene therapy, and targeting of multiple antigens for a synergic/additive effect. All the mentioned antibody formats, having a size of 15–80 kDa (Figure 1), show an easy tumour penetration and are cleared from the bloodstream flow more quickly with respect to the full size IgGs (150 kDa, Figure 1). Furthermore, their genes can be easily manipulated to modify their stability, specificity, and affinity for the antigens.

Antibody engineering also allows cloning of scFv sequences into eukaryotic vectors equipped with intracellular localization signals, for the scFv expression in specific cell compartments as intrabodies. These can reach and recognize target antigens in the cellular compartments where they are located, with outcomes ranging from direct antigen blockade to indirect impairment of its activity through delocalization, to its targeted degradation [8,9].

The discovery of the smallest format of antibody fragments, the Nbs, expanded the possibilities of targeting intracellular antigens through biotechnology. Nbs derive from Camelidae species (e.g., llamas, dromedaries and camels), which in their antibody repertoire have IgG lacking both light chains and CH1 domains (heavy-chain-only antibodies: HCAbs) [10]. The variable domains of these HCAbs are named VHHs and can be isolated as single-domain antibodies (sdAbs), which are small-sized (~15 kDa) but retain the antigen-binding capability of the full-size antibody. Thanks to the small size, VHHs can easily penetrate tissues and access cryptic epitopes [11,12]. They are more soluble and capable of efficient folding with respect to conventional mAbs, which renders them suitable for high-yield production in *E. coli* and even for delivery to or expression in infected cells as intrabodies. Nbs can resist a wide pH range and high temperatures, and some of them tolerate the presence of organic compounds. Despite the non-human origin, VHHs are rarely immunogenic due to the small size and high sequence homology to the human VH3 gene family, which avoids the necessity of humanization for translation into clinic [13]. The small size also favours rapid renal clearance and facilitates the in vivo application in diagnosis rather than therapy, as the latter use requires prolongation of their half-life, which is approximately 2 h. Nevertheless, VHHs targeting haematological, oncological, infectious, inflammatory/auto-immune, bone and neurological diseases are already being evaluated in clinical trials, while the humanized VHH Caplacizumab (CabliviTM) was recently approved in Europe and USA for the treatment of acquired thrombotic thrombocytopenic purpura [14–16].


**About the Editors**

**Ryszard Gołdyn** (Prof. Dr.) is a hydrobiologist who deals with the functioning of aquatic ecosystems. He obtained his PhD based on phytoplankton research in dam reservoirs; he obtained his postdoctoral degree on the influence of preliminary reservoirs on the main reservoir, used for recreation and water sports. He is interested in the relationships between various groups of organisms, as well as the mutual relations between abiotic conditions and organisms, especially in the role of internal nutrient loading from bottom sediments and the influence of external loading from the catchment area. Recently, he has been working on the possibility of using nature-based solutions for lake restoration. His research covers several lakes that are subject to sustainable restoration. According to the Web of Science database, he is an author of 65 published papers and many more published in non-indexed journals and book chapters. He belongs to many scientific societies; he is the vice president of the Polish Hydrobiological Society and the national representative of SIL.

**Piotr Klimaszyk** (Dr.) obtained a PhD degree in freshwater ecology from the Adam Mickiewicz University of Poznan, Poland. His dissertation concerned the functioning of small water bodies in ´ diverse landscapes. He also conducted research on the influence of colonies of water birds on the transfer of nitrogen and phosphorus between aquatic and terrestrial ecosystems. He analyzed the evolution of soft-water lakes with isoetides and problems related to the reaction of water ecosystems to human impact. He is interested in environmental toxicology. According to ResearchGate he is an author of over 70 research articles and 12 chapters. Currently, he is an associate professor and the head of the Department of Water Protection at the Faculty of Biology of Adam Mickiewicz University in Poznan, Poland. ´


*Article* **Evaluation and Numerical Investigations of the Cyclic Behavior of Smart Composite Steel–Concrete Shear Wall: Comprehensive Study of Finite Element Model**

**Hadee Mohammed Najm 1,\* , Amer M. Ibrahim 2,\*, Mohanad Muayad Sabri Sabri 3 , Amer Hassan 1 , Samadhan Morkhade 4 , Nuha S. Mashaan 5 , Moutaz Mustafa A. Eldirderi 6 and Khaled Mohamed Khedher 7,8**

**Abstract:** The composite shear wall has various merits over the traditional reinforced concrete walls. Thus, several experimental studies have been reported in the literature in order to study the seismic behavior of composite shear walls. However, few numerical investigations were found in the previous literature because of difficulties in the interaction behavior of steel and concrete. This study aimed to present a numerical analysis of smart composite shear walls, which use an infilled steel plate and concrete. The study was carried out using the ANSYS software. The mechanical mechanisms between the web plate and concrete were investigated thoroughly. The results obtained from the finite element (FE) analysis show excellent agreement with the experimental test results in terms of the hysteresis curves, failure behavior, ultimate strength, initial stiffness, and ductility. The present numerical investigations were focused on the effects of the gap, thickness of infill steel plate, thickness of the concrete wall, and distance between shear studs on the composite steel plate shear wall (CSPSW) behavior. The results indicate that increasing the gap between steel plate and concrete wall from 0 mm to 40 mm improved the stiffness by 18% as compared to the reference model, which led to delay failures of this model. Expanding the infill steel plate thickness to 12 mm enhanced the stiffness and energy absorption with a ratio of 95% and 58%, respectively. This resulted in a gradual drop in the strength capacity of this model. Meanwhile, increasing concrete wall thickness to 150 mm enhanced the ductility and energy absorption with a ratio of 52% and 32%, respectively, which led to restricting the model and reduced lateral offset. Changing the distance between shear studs from 20% to 25% enhanced the ductility and energy absorption by about 66% and 32%, respectively.

**Keywords:** composite steel plate shear wall; hysteresis curves; ductility; energy absorption; finite element model