**2. The Study Areas**

As shown in Figure 1, Beijing is located in the north of China and north of the BTH region. It borders Tianjin in the east and some cities of Hebei in the rest. It is the capital of the People's Republic of China, a municipality directly under China's central government, a national central city, and a megacity in the world. The total area of Beijing is 16,410 square kilometers, and it has 16 districts under its jurisdiction. By the end of 2019, there were 21.536 million permanent residents and 18.5 million urban residents, representing an urbanization rate of 86.6 percent. The GDP of Beijing in 2019 was RMB 3537.13 billion yuan, and the per capita GDP was RMB 164,000 yuan. The proportions of primary, secondary, and tertiary industries are 0.3%, 16.2%, and 83.5%, respectively. Besides, the energy consumption per unit of GDP in 2019 was about 0.25 tons of standard coal per RMB 10,000 yuan, which was the lowest in China.

**Figure 1.** Locations of the Beijing–Tianjin–Hebei (BTH) region and Henan Province.

Tianjin, a municipality directly under the central government of China, is a national central city, a megacity, and the largest port city in north China. It borders Beijing in the west and some cities of Hebei in the rest. The total area of Tianjin is 11,966 square kilometers with 16 districts under its jurisdiction. By the end of 2019, there were 15.618 million permanent residents and 13.038 million urban residents, representing an urbanization rate of 83.5 percent. The GDP of Tianjin in 2019 was RMB 1410.43 billion yuan, and the per capita GDP was RMB 90,000 yuan. The proportions of primary, secondary, and tertiary industries were 1.3%, 35.2%, and 63.5%, respectively. Besides, the energy consumption per unit of GDP in 2019 was about 0.41 tons of standard coal per RMB 10,000 yuan. Now, the ecological civilization has also become an important element for the urban development of Tianjin under the coordinated development strategy of the BTH region [11].

Hebei province is located in the north of China, bordering the Bohai Sea in the east and Beijing and Tianjin in the inner ring. The total area of Hebei province is 188,800 km2, and it has 11 prefecture-level cities under its jurisdiction, including Shijiazhuang, Tangshan, Qinhuangdao, Baoding, Handan, Xingtai, Zhangjiakou, Chengde, Cangzhou, Hengshui, and Langfang, with Shijiazhuang as its provincial capital. By the end of 2019, there were 75.920 million permanent residents and the permanent resident urbanization rate was 57.6 percent. The GDP of Hebei in 2019 was RMB 3510.45 billion yuan, and the per capita GDP was RMB 46,000 yuan. The proportions of primary, secondary, and tertiary industries were 10.3%, 39.7%, and 50.0%, respectively. Besides, the energy consumption per unit of GDP in 2019 was about 0.84 tons of standard coal per RMB 10,000 yuan, which was much higher than the national average of 0.51 tons of standard coal equivalent per RMB 10,000 yuan. Hebei province is rich in mineral resources because 156 kinds of mineral resources have been discovered, 39 of which are among the top 5 in China. It has formed a mining economic system with metallurgy, coal, building materials, and petrochemicals. In April 2017, the central government decided to establish Xiong'an New Area in Hebei province. In August 2019, the state council established a new China (Hebei) Pilot Free Trade Zone.

The control group, Henan Province, is located in central China, south of Hebei province, with a total area of 167,000 km2. It has jurisdiction over 17 prefecture-level cities, including Zhengzhou, Kaifeng, Luoyang, Pingdingshan, Anyang, Hebi, Xinxiang, Jiaozuo, Puyang, Xuchang, Luohe, Sanmenxia, Shangqiu, Zhoukou, Zhumadian, Nanyang, and Xinyang, with Zhengzhou as the provincial capital and Jiyuan as a county-level city directly under the jurisdiction of Henan Province. By the end of 2019, there were 109.52 million permanent residents and the permanent resident urbanization rate was 53.2 percent. The GDP of Henan in 2019 was RMB 5425.92 billion yuan, and the per capita GDP was RMB 56,000 yuan. The proportions of primary, secondary, and tertiary industries were 8.5%, 43.5%, and 48.0%, respectively. Energy consumption per unit of GDP in 2019 was about 0.48 tons of standard coal per RMB 10,000 yuan. Henan province is also rich in mineral resources because 142 kinds of mineral resources have been discovered, 58 of which are among the top 5 in China.

In general, Beijing is dominated by the service industry, Tianjin is dominated by the processing and manufacturing industry and by the port service industry, and Hebei is dominated by the resource-intensive industry and agriculture because of the rich mineral resources and rural labor force. Therefore, we can also conclude the following socioeconomic characteristics in the BTH region. First, the issues of industrial pollution are very serious. The heavy industries in Hebei and Tianjin lead to great pressure on the environment, especially serious air pollution. Second, economic development is very unbalanced. Hebei is geographically advantaged with three port cities including Tangshan, Cangzhou, and Qinhuangdao, and most of the cities are very close to Beijing and Tianjin. However, there is a huge gap in economic development as the above statistics show. Third, as the capital city, Beijing has strong political, economic, cultural, scientific, and technological strength. However, there has always been a "siphon effect" of talents, funds, and other resources transferring from Hebei and even Tianjin to Beijing. Therefore, Beijing has a very weak radiation effect on the surrounding cities. Fourth, under the BTH coordinated development strategy, Tianjin has changed to a more sustainable urban development model. For example, the Sino-Singaporean Eco-city replaced Hanggu to become one subcenter of the Binhai New Area of Tianjin, as it is a national project that retains a high level of support from the central government for environment protection in the BTH region.

Based on the outline of the coordinated development plan, Beijing is the national center for political, cultural, and international exchanges and for scientific and technological innovation; Tianjin is the national advanced manufacturing research and development base, the northern international shipping core zone, and the financial innovation operation and reform pilot demonstration zone, and Hebei is an important base for modern trade and logistics in China, a pilot area for industrial transformation and upgrading, a demonstration area for new urbanization and urban-rural integration, and a supporting area for the BTH ecological environment. The overall orientation of the BTH region is "a world-class city cluster with the capital as the core, a leading area for coordinated regional development and reform, a new national engine for innovation-driven economic growth, and a demonstration area for ecological restoration and environmental improvement". The overall development plan can be concluded as "one core, two cities, three axes, four areas, and multiple nodes". "One core" means Beijing, and "two cities" refer to Beijing and Tianjin. They are the main engines for the coordinated development of the BTH region so the linkage between Beijing and Tianjin needs to be further strengthened. "Three axes" refer to the three industrial development and urban agglomeration axes, including Beijing–Tianjin, Beijing–Baoding–Shijiazhuang, and Beijing–Tangshan–Qinhuangdao. "Four areas" refer to the central core functional area, the eastern coastal development area, the southern functional extension area, and the northwest ecological conservation area. "Multiple nodes" include regional central cities such as Shijiazhuang, Tangshan, Baoding, and Handan and node cities such as Zhangjiakou, Chengde, Langfang, Qinhuangdao, Cangzhou, Xingtai, and Hengshui, with the focus on improving these cities' comprehensive carrying capacity and service capacity and on promoting industry and population aggregation in an orderly manner.

There are some reasons why the Chinese government made great effort to develop this region. First, the coordinated development of this region is conducive to solving Beijing's "big-city diseases" such as population expansion, traffic congestion, housing difficulties, environmental degradation, and resource shortage. Second, the BTH region lags behind China's Yangtze River Delta and Pearl River Delta but it has a huge development potential. Third, the economic capabilities are extremely uneven among cities. Beijing and Tianjin lead the country in urbanization rate and per capita GDP, while those of Hebei cities are below the national averages. For example, Beijing's pillar industries are finance, information technology, and science and technology research, but Tianjin and Hebei are dominated by medium- and low-end manufacturing industries with lots of pollution. The milestones are as follows. By 2017, remarkable progress was made in the orderly relocation of noncapital functions of Beijing and some breakthroughs occurred in key areas such as transportation integration, environmental protection, and industrial upgrading and relocation. By 2020, the population of Beijing should be controlled within 23 million, an integrated regional transport network will be shaped, the environment

will effectively improved, major progress will be made in the coordinated development of industries, and the development gaps within the region should be narrowed. By 2030, the core functions of Beijing will be improved, the regional economic structure will become more reasonable, the environmental quality will improve, and the levels of public services should be balanced. In the end, the BTH region will become a region with strong international competitiveness and influence, thus playing a greater role in guiding and supporting China's economic and social sustainable development.


**1. Introduction**

In the United States, 150,000 new cases of colorectal cancer will be diagnosed in 2022 with an estimated 55,000 deaths caused by the disease [1]. Cisplatin, a commonly used chemotherapeutic agent, is used to treat colorectal cancer, albeit with negative side effects, including chemotherapeutic-induced peripheral neuropathy (CIPN), for which there is a lack of treatment options [2–4]. The prevalence of CIPN is agent-dependent, with reported rates varying from 19% to more than 85% [5] and is the highest in the case of platinumbased drugs (70–100%), including cisplatin [6]. This outcome results in an impaired quality of life for affected patients. Therefore, there is a need to identify alternative treatments for colorectal cancer, as well as identify anti-nociceptive agents capable of treating CIPN in patients undergoing cisplatin-based chemotherapy for colorectal cancer.

A number of cannabinoids have been found to reduce cancer cell growth with Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD)—the two most often studied cannabinoids for anti-cancer activity [7–12]. In addition to anti-cancer effects, evidence suggests that cannabinoids (e.g., cannabigerol (CBG), THC, and CBD) have anti-nociceptive properties, resulting in ongoing clinical investigations related to cannabinoid effects on pain [13–18]. The mechanisms by which cannabinoids reduce cancer cell growth as well as mitigate pain are currently under investigation. However, it is known that cannabinoids

**Citation:** Raup-Konsavage, W.M.; Sepulveda, D.E.; Morris, D.P.; Amin, S.; Vrana, K.E.; Graziane, N.M.; Desai, D. Efficient Synthesis for Altering Side Chain Length on Cannabinoid Molecules and Their Effects in Chemotherapy and Chemotherapeutic Induced Neuropathic Pain. *Biomolecules* **2022**, *12*, 1869. https://doi.org/10.3390/ biom12121869

Academic Editors: Marialuigia Fantacuzzi, Barbara De Filippis and Alessandra Ammazzalorso

Received: 3 October 2022 Accepted: 9 December 2022 Published: 13 December 2022

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

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

are promiscuous molecules with the ability to bind to a number of receptors involved in pain processing and inflammation (e.g., CB1, CB2, 5-HT1A, α2-adrenergic receptor) as well as induce immunogenic cell death [15,18–20].

Cannabis produces a number of bioactive compounds that are of pharmaceutical interest. Broadly, these molecules fall into one of three classes: terpenes, flavonoids, and cannabinoids (which are unique to the genus *Cannabis*). Over 100 different types of cannabinoids are produced by cannabis, with CBD and THC being the two most abundant and most well studied. The predominant cannabinoids in *Cannabis* cultivars have a 5-carbon side chain off of the aromatic ring; however, shortly after the discovery of the major 5-carbon phytocannabinoids, CBD and THC, two additional variations of these molecules were described with a shorter, 3-carbon, side chain, cannabidivarin (CBDV) and tetrahydrocannabivarin (THCV) [21]. More recently, CBD and THC variants with carbon side chains of 4, 6, and 7 carbons have been isolated from cannabis, albeit in much lower concentrations [22–24].

The biosynthesis of all cannabinoids begins with geranyl pyrophosphate and a benzoic acid, olivetolic acid in the case of the 5-carbon cannabinoids, by the enzyme geranylpyrophosphate-olivetolic acid geranyltransferase (GOT) [25]. This reaction produces CBG, and the respective variations, which serve as the precursor molecules to all other cannabinoids produced in the plant. However, in most cultivars, CBG is found at low levels because this precursor is efficiently converted to downstream products, which means that the variant molecules will therefore be present in trace amounts. Novel and trace side chain length variants of CBD and THC have previously been isolated from plant material [22]. Here, we set out to develop a novel synthetic mechanism to produce side-chain variants of CBG. Additionally, we tested CBG and CBG variants for potential anti-cancer activity in colorectal cancer cell lines, as well as anti-nociceptive properties in a model of chemotherapy-induced neuropathic pain (CIPN).

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

Thin-layer chromatography (TLC) was performed on aluminum-supported, precoated silica gel plates (EM Industries, Gibbstown, NJ, USA). Flash column chromatography was performed using silica gel SI 60. 1H NMR spectra were recorded on a 500 MHz Bruker mass spectrometer (Billerica, MA, USA). Proton chemical shifts are reported in parts per million (δ). The following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, dd = double doublet, t = triplet, dt = doublet of triplet, m = multiplet. Mass spectrometry analysis was performed on a 4000 Q-trap hybrid triple quadrupole/linear ion trap instrument (Applied Biosystems/MDS Sciex, Waltham, MA, USA) at the proteomic facility of the Penn State College of Medicine, Hershey, PA. High Resolution Mass Spectrometry (HRMS) was performed on AB Sciex TripleTOF 5600 mass spectrometer (Farmington, MA, USA) with electrospray ionization (ESI) in positive-ion mode at the metabolomics core at Penn State University, University Park, PA. The sample was analyzed by flow infusion with a Prominence UFLC system (Shimadzu, Kyoto, Japan) at flow 300 μL/min rates of 0.1% formic acid in a mixture of methanol and water 60:40. MS1 and MS2 data were acquired using a declustering potential (DP) of 80 V. MS2 data were collected in IDA mode with collision energy (CE) of 40 V and collision energy spread (CES) 20 V. During the analysis, an ion spray voltage (IS) of 5500 V, curtain gas (CUR) of 35 psi, nebulizer gas (GS1) of 50 psi, heater gas 2 (GS2) of 55, and heater temperature of 550 C were applied. CBG was purchased from a commercial source (Cayman Chemical, Ann Arbor, MI, USA). 3,5-dimethoxybenzyltriphenylphosphonium bromide (**1**) was prepared as reported in the literature [26]. Briefly, 3,5-dimethoxybenzylbromide (10 g, 43.2 mmol) was heated under reflux with triphenyl phosphine (12.6 g, 47.6 mmol) in toluene (60 mL) for 6 h to give a quantitative yield of compound **1**. All starting materials were obtained from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. Synthesis of 5-alkyl substituted-1,3-dihydroxybenzene precursors was conducted as described below (and illustrated in Scheme 1).

**Scheme 1.** Synthesis of 5-alkyl substitute-1,3-dihydroxybenezene.


**6. Vestibular system and proprioception in nomophobia kids**

The central and peripheral vestibular organs, in conjunction with the visual and somatosensory systems, are responsible for balance, equilibrium, and orientation in space. Vestibular system as major role in subjective awareness of body position and movements in space, postural tone and equilibrium and stabilization of the eyes in space during head movement along with visual system and proprioception continuous use of smart phones may lead the child hyper expressive and also lead to gravitational insecurity, which is an sensory integration issue which may cause to react to movements in an extreme way. This is due to the impact of vestibular system by which the child's gravity receptors become extra sensitive.


**9. Strategies for containment of an infectious disease**

Barlett postulated that any infectious disease cannot be maintained if the population size is below the critical community size unless there is supply of susceptible or regular migration of the infected class from the adjoining places. This will ultimately lead to disease extinction. Disease is said to fade-out if the duration of disease extinction is more than the disease generation length [6]. Although, theoretically, it may be possible to estimate and predict the time for disease eradication from the population, practically it is not feasible. Therefore, in practice, attempts are made to prevent spreading of the infection.

**10. Vaccination**

Implementation of proper and effective vaccination protocol is of primary concern to the epidemiologists and public health decision makers. Vaccination program is usually a govern‐ ment initiative applied on large spatial and temporal scales to reduce the level of complexity of disease. The ultimate goal of any vaccination program is to keep the value of basic repro‐ duction ratio below unity by altering the various control parameters. Mathematical modeling of vaccination recognizes that linear transfer occurs between the susceptible and the removed compartments. For modeling purpose, important variables related to vaccination that should be included are vaccination rate, i.e. the rate at which the susceptible individuals are vaccinated and efficacy or the proportion of susceptible left unprotected even, after immunization [26].

Unearthing the Complexities of Mathematical Modeling of Infectious Disease Transmission Dynamics

http://dx.doi.org/10.5772/57387

195

Two schemes are being widely employed worldwide for prevention and eradication of vaccine-preventable infections-mass vaccination and pulse vaccination. Mass vaccination is usually carried out in infants before the mean age at infection. But, it may not always be practically the most effective approach for global disease eradication. Pulse vaccination, on the other hand, is a cheaper and a better alternative where periodic vaccination of a certain proportion of the population renders sufficient and enough protection against further spread of the disease as the percentage of susceptible is always maintained below the threshold level required for an epidemic to start. But, pulse vaccination suffers from the side effect of resonance. Impact of single- and multiple-dose vaccination on rubella eradication has been studied extensively in an age-structured model with constant transmission rate using Floquet analysis. It has been established that eradication likelihood is governed by the effective duration of immunity. Booster vaccination has also been studied, but, in less details. Permanent immunity is conferred by booster dose in contrast to partial immunity imparted by primary vaccines. A new threshold quantity, known as re-infection thresh‐ old in case of vaccination-induced partial immunity, indicates the condition when vaccina‐ tion will not succeed thereby producing high levels of infection. Prevention or reduction in epidemicity is more readily achievable by booster vaccination, depending on the level of primary vaccination program, because it has the capacity to increase the vaccination coverage and herd immunity of the population as a whole. Outcome of a booster dose depends highly on precise timing of the additional vaccine doses as well as the propor‐ tion of individuals receiving the second-dose. During vaccination, decision of the individu‐ al or mass to undergo program greatly affects the efficacy of the program. Therefore, human

behavior plays a significant role in ensuring success of the strategy [3], [6], [27].

An interesting thing to note while conducting mass scale immunization is that it is possible to protect the whole population from an outbreak even if there are some susceptibles in the population, at less than 100% immunization. This effect is called herd immunity. When R0 < 1, the DFE is globally asymptotically stable resulting in disease eradication as observed in case of measles. Vaccination reduces the force of infection and increases the mean age at infection when the infection may be acquired. Vaccination desynchronizes local dynamics which will prevent migration of susceptible population from neighboring places and thus can facilitate extinction of the disease. However, vaccination of each and every individual in the susceptible

Once the epidemiological data have been modeled into a reliable mathematical model, they can be used to identify population subgroups at high risk of disease and develop preventive interventions or measures according to time, place and person. These measures include education, immunization, quarantine regulations or social distancing to restrict interaction with others and treatment options. Mathematical modeling of intervention strategies can be done in two ways. In the first method, the goal is to assess the effect on the disease dynamics by changing the value of a constant parameter associated with the disease. This indicates the best parameter value for a given performance measure. In the second method, intervention measures are varied as a function of time and the objective is to determine the best parameter value for a given performance measure. Pontryagin's Maximum Principle (PMP) is applied for comparison of a wide range of time varying functions. The best strategy for mitigation of spreading of a contagious disease would be the one which evolves with time during different phases of infection and thus focuses on progressively changing classes of populations. Optimal control theory suggests the most effective mitigation strategy to minimize the risk of individ‐ uals being infected by applying and balancing vaccination and administering drug in a costeffective manner. Based on this, vaccination has been found to be the most common strategy in reversing the epidemic growth of an infection within the population in the initial stages of an outbreak. But in absence of the strategy, due to some reason or other, medical practitioners either isolate symptomatic individuals or trace and quarantine contacts of symptomatic cases. These strategies require proper diagnosis of the symptoms of the disease in each and every individual. Efficacy of any implementation strategy is decided by the efficacy at which the infected person is isolated and the efficacy at which the persons with whom the infectious person came in contact can be quarantined. An infection can be sufficiently controlled if the values of basic reproduction ratio and the proportion of transmission occurring asymptotically can be reduced below a critical line. Since SARS is characterized by low R0 and low infectious‐ ness prior to clinical symptoms, effective isolation of symptomatic patients can sufficiently control an epidemic outbreak. For those diseases where the proportion of asymptomatic transmission is more than 1/ R0, contact tracing should be added to the set of control measures used. This proportion can be determined from the longitudinal data on clinical symptoms and pathogen load within the patient. Mathematically, it has been deduced that influenza is more difficult to control than small pox. Moreover, quarantining and contact tracing would not provide any extra benefit in case of influenza because of very short incubation and infectious periods. AIDS has taken an alarming proportion because of high risk of pre-symptomatic transmission and under such circumstance, self-isolation and contact tracing would provide little respite [10], [22], [24], [25].


**2. Classification of emerging pollutants**

Emerging pollutants are classified and categorized as follows [15–17]:

The personal care products and pharmaceuticals as ECs contain active substances in illicit, prescribed and non-prescribed drugs for human and animal consumption. Active ingredients are also found in disinfectants, biocides, preservatives and personal hygiene and beautification products [18]. PPCPs are classified (**Table 1**) base on the consumer product and their uses [19].

**5**

hydrate)

*Classification, Potential Routes and Risk of Emerging Pollutants/Contaminant*

ATII (traseolide) Boisvelone/Iso-E super

AHDI (phantolide) Boisvelone/Iso-E super

2,4-Dihydroxybenzophenone Dihydromethyljasmonate

ADBI (celestolide) Drometrizole

Galaxolide Methylsalicylate *g*-Methylionone Musk ambrette Hexamethyldisiloxane (HM or HMDS) Musk ketone Hexylcinnamaldehyde Musk xylene

Isobutyl paraben Octocrylene Methylparaben Oxybenzone

Methyl-iso-propylcyclohexenone Propyl paraben *p*-*t*-Bucinal (Lilial) Tonalide

*N*,*N*-Diethyltoluamide D-Limonene

2,6-Di-*tert*-butylphenol Triethylcitrate

1-Hydroxy ibuprofen Cyclophosphamide

17-alpha-Estradiol Danofloxacin 17-alpha-Ethinylestradiol Dantrolene 17-beta-Estradiol Dapsone

2-Hydroxy ibuprofen Desmethylnaproxen (metabolite of naproxene)

Daunorubicin

Butylated hydroxyanisole Butylated hydroxytoluene 2-Ethylthioacetic acid ethylester Dipropyltrisulfide 2-Methylthioacetic acid ethylester Ethylene brassylate 3-Methylthiopropionic acid Habanolide

Category III: Personal care products/food additive compounds

Methyldihydrojasmonate (methyl 3-oxo-2-

Category II: Personal care products/biocides

**Category I: Pharmaceuticals compounds**

1,1,1-Trichloro-2,2-dihydroxyethane (chloral

pentylcyclopentaneacetate)

compounds

Benzaldehyde, (phenylmethylene) Cineole hydrazone (Eusolex) Damascone

Bayrepel Butyl methoxydibenzoylmethane

Benzophenone Decamethylcyclopentasiloxane (D5)

alpha-Terpineol Decamethyltetrasiloxane (MD2M)

4-Methylbenzylidene camphor Dodecamethylcyclohexasiloxane(D6)

Acetylcedrene Dodecamethylpentasiloxane(MD3M)

Ethylhexyl methoxycinnamate Drometrizole trisiloxane (INCI)

Homosalate Octamethylcyclotetrasiloxane (D4) Isobornylacetate Octamethyltrisiloxane (MDM)

Carvone

Triclosan

*DOI: http://dx.doi.org/10.5772/intechopen.94447*

**Category I: Personal care products compound**

4-Oxoisophorone

**Table 1.**

*Classification of pharmaceuticals and personal care products [16].*

#### *Classification, Potential Routes and Risk of Emerging Pollutants/Contaminant DOI: http://dx.doi.org/10.5772/intechopen.94447*

*Emerging Contaminants*

**2. Classification of emerging pollutants**

biocides, food additives).

[pesticides])

1.Pharmaceuticals (illicit and prescribed drugs).

flame retardant, antimicrobial substances)

(**Table 1**) base on the consumer product and their uses [19].

**Personal care products Pharmaceuticals**

*Classification of pharmaceuticals and personal care products [16].*

Fragrances, cosmetics, repellents, food supplements and their metabolites, and transformation products personal hygiene products, sunscreen agents, domestic insect,

treatment plants (WTP) [9]. Furthermore, unregulated chemicals such as nitrosamines (NAs), dioxane, nanomaterials, pharmaceuticals and personal care products (cosmetics, disinfectant, antiseptic, deodorant stick, soap, fragrances, insect repellent, sunscreen, surfactants and toothpaste) as well as perfluorinated alkyl acids (PFAA) are EPs [8, 10, 11]. For instance, N-Nitroso-dimethylamine (NDMA) an emerging contaminant produced as byproducts of chloramines in drinking water treatment plants [12]. Nanomaterials (NMs) are substances produced with a dimension in nanoscale range from 1 to 100 nanometer as to improve the chemical strength and reactivity [10]. However, some NMs introduced contaminants when used for water treatment. Polybrominated biphenyl ethers (PBDEs) PBDEs are brominated hydrocarbons used as flame retardants in the production of furniture, plastics, upholstery, electrical equipment, electronic devices and many other household products [10]. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) fluorinated organic compounds with many industrial applications; such as surfactants in fluoropolymers and as grease, soil and water resistant in fluorinated polymer [13, 14].

Emerging pollutants are classified and categorized as follows [15–17]:

2.Personal care products (cosmetic, surfactants, disinfectants, domestic

methanes, haloacetonitriles, trihalomethanes, halo acetic acids).

5.Algal toxins (toxic released from some algae: Cyanotoxins, microcystin)

6.Biocides and their metabolites (plants and agricultural preventive agents

7.Bioterrorism and disruption devices (Biological and Chemical weapons).

The personal care products and pharmaceuticals as ECs contain active substances in illicit, prescribed and non-prescribed drugs for human and animal consumption. Active ingredients are also found in disinfectants, biocides, preservatives and personal hygiene and beautification products [18].

More than 1036 EC and secondary metabolites as well as biotransformation products are identified on NORMAN List in **Table 2** (NORMAN [20]). The most common classes/categories of ECs include; industrial chemicals, disinfection byproducts, pesticides, sweeteners endocrine disrupting compounds, nanoparticles, sunscreens, UV filters, pharmaceuticals and personal care products among others [21–23].

#### **3. Potential routes emerging pollutants**

Series of anthropogenic activities such as agricultural, domestic and industrial activities leads to the discharged of the pollutants into our environments (**Figure 1**). Water and sediments serve as a sink to these contaminants [25, 26]. Constant consumption of drugs due to less attention given to traditional medicine for the treatment aliments and frequent use of personal care products as vogue has led to the release of different by-products at low concentrations into our environments [27]. The concentration of these pollutants are based on the production processes of some products (drugs) applied in various countries. For example, the concentration of bisphenol A in European water and North American were 43 and 12 ppb correspondingly [28, 29]. Their presence in water has affected the water physicochemical parameter and required urgent attention to drinking issues. Essentially however, many wastewater treatment plant (WWTP) are not considered or designed for the removal of emerging contaminants as shown **Figure 1**. As a result of their hydrophobic nature PPCPs and other EC metabolites as well as associated particular mater settled below water surface when discharged in water. Treated and untreated urban water as well as WWTP (**Figure 1**) are the main sources and path route of EC.

**9**

*Classification, Potential Routes and Risk of Emerging Pollutants/Contaminant*

PPCPs are present in our environment at very low concentrations ranging from ng/L to μg/L but there is paucity of information about the various secondary metabolites generated during the biotransformation of parent compounds [16] The impact of the metabolites could be more toxic than the main compounds and their adverse effect on non-target organism is less understood [30]. The longtime exposure to PPCPs pose severe threat to aquatic biota and human life. Also, many studies on environmental toxicology on the exposure of non-target organs to PPCP are results from acute toxicity data [31–33]. Many PPCPs contained bioactive ingredient that could have a chronic effect non-target biota. Goldfish shows bio-centration factor of 113 when it is exposed to high concentration of PPCPs for 14 days [34]. Chronic effect could lead to gene mutation and decline in fish population. For instance, diclofenac may cause ill-effect on fish organs. Fish are susceptible to PPPCPs contaminants since they live in the environment where these contaminants are present [35]. The presence of carbamazepine and diclofenac in aquatic environs damaged algal chloroplasts [36]. Long time exposure to Sulfamethoxazole cause severe toxicity and inhibit photosynthesis process [37]. Also, ciprofloxacin is toxic to green algae [38]. Natural bacteria developed antibiotic tolerance in PPCP antibiotic contaminated environments [39]. Endocrine disrupting chemicals are also referred to as hormones in aquatic or aqueous environments. Some endocrine chemicals reduce the proper functioning of endocrine system (ES) that is physiological activities [40]. For example, retardation in reproductive processes such as sex variation, poor metabolic and embryonic developments [41]. These environmental hormones could cause serious effects on both aquatic and terrestrials animals through inhibition and modification of hormonal growth in ES

*DOI: http://dx.doi.org/10.5772/intechopen.94447*

**4. Risk of emerging pollutants**

*Potential sources of PPCPs/EDCs in water cycle modified [24].*

**Figure 1.**

and hormones in the cell correspondingly [42].

*Classification, Potential Routes and Risk of Emerging Pollutants/Contaminant DOI: http://dx.doi.org/10.5772/intechopen.94447*

**Figure 1.**

*Emerging Contaminants*

Methicillin Sulfamethazine Nandrolone Sulfamethoxazole Nafcillin Sulfapyridine Nadolol Tetracycline Naproxen Temazepam *N*-Methylphenacetine Terbutaline Neomycin B Taloxa

Norfloxacin Tolfenamic acid Nordiazepam Tilmicosin Novobiocin Timolol Ofloxacin Tiamulin Oxacillin Tramadol Omeprazole Trimethoprim Oleandomycin Tylosin Oxazepam Verapamil Oxytetracycline Valnemulin Oxprenolol Zolpidem

*Identified pharmaceuticals and personal care products (NORMAN [16, 20]).*

More than 1036 EC and secondary metabolites as well as biotransformation products are identified on NORMAN List in **Table 2** (NORMAN [20]). The most common classes/categories of ECs include; industrial chemicals, disinfection byproducts, pesticides, sweeteners endocrine disrupting compounds, nanoparticles, sunscreens, UV filters, pharmaceuticals and personal care products among

Series of anthropogenic activities such as agricultural, domestic and industrial activities leads to the discharged of the pollutants into our environments (**Figure 1**). Water and sediments serve as a sink to these contaminants [25, 26]. Constant consumption of drugs due to less attention given to traditional medicine for the treatment aliments and frequent use of personal care products as vogue has led to the release of different by-products at low concentrations into our environments [27]. The concentration of these pollutants are based on the production processes of some products (drugs) applied in various countries. For example, the concentration of bisphenol A in European water and North American were 43 and 12 ppb correspondingly [28, 29]. Their presence in water has affected the water physicochemical parameter and required urgent attention to drinking issues. Essentially however, many wastewater treatment plant (WWTP) are not considered or designed for the removal of emerging contaminants as shown **Figure 1**. As a result of their hydrophobic nature PPCPs and other EC metabolites as well as associated particular mater settled below water surface when discharged in water. Treated and untreated urban water as well as

**8**

others [21–23].

**Table 2.**

**3. Potential routes emerging pollutants**

WWTP (**Figure 1**) are the main sources and path route of EC.

*Potential sources of PPCPs/EDCs in water cycle modified [24].*


**Corrosion in the Food Industry and Its Control**

Benjamín Valdez Salas, Michael Schorr Wiener, Margarita Stoytcheva, Roumen Zlatev and Monica Carrillo Beltran *Universidad Autonoma de Baja California. Instituto de Ingeniería, Mexicali, Baja California, Mexico*

### **1. Introduction**

362 Food Industrial Processes – Methods and Equipment

356

Wium; H.; Pedersen, P.S. & Qvist, K.B. (2003). Effect of coagulation conditions on the

from ultrafiltered milk. Food Hydrocolloids, Vol.17, No.3, pp. 287-296

microstructure and the large deformation properties of fat-free Feta cheese made

From ancient times human beings have survived on a diet consisting on a relatively few species of plants and animals, domesticated and then cultivated and grown. Three cereals, wheat, rice and corn, supply the need of human energy, protein and vitamins requirements for the network of metabolic processes to maintain normal body function and temperature. In a prehistoric era, indigenous peoples all over the world were moving in inhospitable grounds obtaining their daily sustenance by hunting and gathering fruits, seeds and roots.

Actually, the three largest markets worldwide, according to their production extent, the number of consumers and their economic and social significance are the food, energy and water markets. Furthermore, their increasing scarcity and soaring prices lead to a global critical situation. The demand for increased food supply is related both, to population increase and personal and family income. Consequently, the food market is the largest one, including all the inhabitants of this planet, about seven billion, since everyone eats!

The organized food production and supply starts with the agricultural revolution, developed and implemented in the fertile valleys of the rivers Tigris-Euphrates in Mesopotamia and the Nile in ancient Egypt. Afterwards the food industry expanded, avoided widespread famine and ensured that sufficient food is supplied for all people to stay healthy. Current food research had been largely stimulated by rapidly growing world demand but technological advances in food processing, equipment and production plants have also contributed. A most significant aspect in the search of new nutritional food is the requirement for adequate protein in regions where meat and fish are not available. Additionally, advances in the food industry (FI) such as preservation, packaging and storage facilitate food delivery and minimized health hazards. Space flight conditions have stimulated the creation of space food which meets highly demanding standards for conservation and to be ready for easy digestion e.g. solid dehydrated food easily converted into liquid or paste food.

Techniques for preserving food from natural deterioration following harvest or slaughter dated to prehistoric times applying drying, salting, fermentation of milk and fruits and pickling of vegetables. Modern techniques include canning, freezing, dehydration, cooking under vacuum and addition of chemicals. The principal causes of food spoilage are growth

1. Alkaline: such as caustic soda (NaOH), alkali phosphates (Na3PO4), sodium carbonate

This great variety of corrosive environments and aggressive chemical agents require the use of Corrosion Resistant Alloys (CRAs). SS are the obvious choice of the food industry to

Food main constituents.- Foods are constituted by two essential groups of substances: those with nutritional values: proteins, carbohydrates and fats and the components imparting particular tastes: salt (NaCl) for saltiness, acids for sourness or acidity and sugars for sweetness. Few peculiars foods have a bitter taste e.g. almonds. A central component of food

**Water.-** The vegetables and ripe fruits contain a considerable amount of water between 70% and 90%. Since water is used as a medium for food preparation e.g. cooking , confectionery, hot and cold beverages, even cooked foods contain water. The water behaviour and its interaction with food depends on its physicochemical properties, its molecular and ionized structure and its chemical bonding. Water is considered as the universal solvent since all chemical substances have a finite solubility in water ranging from acids, bases, salts, sugars,

H3O+ a bonding between an hydrogen ion H+ and a water molecule, these are the ions involved in corrosion of metallic materials used for the construction of food processing

2FeCl3 + 3H2O → 6HCl + Fe2O3, (1)

Water is the medium for countless chemical and biological reactions. With the exception of petrochemical and combustion reaction, most chemical processes occur in aqueous systems, including vegetal and animal respiratory and metabolic functions. Food preparation involving dehydration, softening, takes places in water. The operation requires some energy to break hydrogen bond and form new bonds between the solute: food components

**Salt.-** Table salt (NaCl) is encountered in many natural environments: seawater, natural brines, enclosed seas, e.g The Dead Sea, Israel and the Salt Lake, Utah, USA, with a salt concentration of about 300 g/L; in aboveground and underground mines found as rock salt. An aqueous salt solution is decomposed by electrolysis into several useful products: metallic sodium, hydrogen, sodium hydroxide and chlorine, all important raw materials for the chemical processing industry (CPI). The meat packing, sausage making, fish curing and food conservation employ salt as a preservative or seasoning or for both purposes. Up to the 19th Century, during the invention of industrial food freezing and cannery; commercial and navy ships conserved meat in wood barrels full of solid, granular salt for their long voyages since putrefaction bacteria cannot live in salt.  Salt is part of mankind history and tradition. Modern and ancient words, in many languages, are derived from salt: salary,

forming the hydronium ion

359

3. Oxidizers: chlorine, nitric acid, ozone, hypochlorite, hydrogen peroxide (H2O2)

and bicarbonate (Na2CO3, NaHCO3). 2. Acidic: phosphoric, citric and sulphamic acids.

prevent equipment damage and food contamination.

is water as in fruits, vegetables, meat, eggs, milk, etc.

Acidic metallic salts undergo hydrolysis in water:

forming an acidic and corrosive environment.

equipment.

and water: the solvent.

alcohols, etc. Water is dissociate into ions: H2O → H+ + OH-

of microorganism, enzyme action, oxidation and dehydration. The economic and social relevance of the FI is evident by the diverse international and national professional associations, R & D institutions, regulation and standards agencies and potent multinational industrial enterprises involved in all aspects of food science, engineering and technology (S.O. Jekayinfa et al, 2005). It includes authorities from government, industry and academia that address progress crucial for national and global prosperity. Lately, the threats of bioterrorism by poisoning food have pushed the FI to the front of efforts by safety and security organizations to prevent these hazards events.

Among others, aluminium, tin, cooper, titanium and mainly stainless steel (SS) are widely used in FI for the manufacture of processing, production, storage and transportation equipment and machinery. Modern science and technology have developed an extended range of materials with increased corrosion resistance, improved mechanical strength, easier forming and fabrication, weldability and health friendly features. The workhorses of food processing industry are UNS S30400 and S31600, but other austenitic and duplex SS are in useful service in food plants.

Food consists mainly of proteins, carbohydrates and fats. Processed foods contain diverse aqueous solutions, syrups and additives, used to improve food appearance, quality and preservation. They have a wide pH range and salt, water and vinegar content, that impact on food corrosivity.

Many cleaning and sanitation agents are employed to remove bacteria, scale, fouling and corrosive biological and mineral deposits. They include alkaline, acidic, strong or weak oxidizing and reducing chemicals to ensure a high hygiene level. This great variety of corrosive environments and aggressive chemical agents require the use of Corrosion Resistant Alloys (CRAs). SS are the obvious choice of the FI to prevent equipment damage and food contamination.


**3.2 Systemic contact dermatitis**

*Trace Metals in the Environment - New Approaches and Recent Advances*

and oxygen reactive species (ROS). Cr(III) as the real allergen is bound to certain proteins to form the hapten, while ROS induces the releasing of interleukin-1β (IL-1β) which then activates antigen-presenting cells (Langerhans cells (LC)). Activated antigen-presenting cells bind with the hapten, mature, travel to the regional lymph nodes, and stay in paracortical T-cell areas. After that, activated antigen presenting cells-hapten complex activates naïve T cells by helping in vigorous blast formation and proliferation to become chromium-specific T cells. Activated chromium-specific T cells then travel through blood stream and recirculate to give hypersensitivity effect detecting a lower concentration of hapten in different parts of the skin. Treatment of chromium-induced contact dermatitis could be conducted in several approaches including avoiding direct contact to chromium source and topical application of chelating agent and barrier creams to prevent any cutaneous permeation, corticosteroid to relieve inflammation, and antioxidant to reduce oxidative stress [36, 50]. Various antioxidants have been tested in treating chromium-induced contact dermatitis such as *N*-acetylcysteine [51], ascorbic acid [52], pine bark extract (pycnogenol®) [53], and pterostilbene [54]. Two chelating agents, ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), also have been examined for preventing contact dermatitis, but both of these ligands showed a low effectiveness as a protecting agent [55, 56]. The developments in

*Simplified elicitation mechanism of chromium-induced contact dermatitis adapted from several references [48, 49].*

**126**

**Figure 2.**

Systemic contact dermatitis is a kind of dermatitis elicited by systemic administration of certain allergen that previously sensitizes the skin through direct dermal contact [57]. Several groups of allergens have been identified to cause this inflammatory disease such as metals, medications, food, plants and herbals, and certain chemicals [57, 58]. Systemic administration of these allergens is also described in various routes including oral, subconjunctival, intramuscular, pulmonary inhalation, intranasal, intrauterine, endocardial, arthroplastic, intravenous, intraarticular, subcutaneous, intradermal, dental, intratubal, and endovascular [59]. The pathophysiology of this disease still remains unclear, but several mechanisms have been proposed [60–65]. Moreover, the theory suggesting type 3 immune response (antigen-antibody complexes) involved in systemic contact dermatitis (SCD) has not fully been proven [58].

Chromium as a metal allergen has been found to cause systemic contact dermatitis either through oral, dental, or arthroplastic routes [13, 66, 67]. Consumption of Cr(III)-based supplements in the form of chromium picolinate [68] and chromium chloride [69] has been shown to cause SCD. Oral ingestion of potassium dichromate previously used as a homeopathic drug also induces dermatitis as clinical manifestation of SCD [70, 71]. In certain case, SCD is also induced by various metal alloys applied in orthopaedical, cardiac, neurological, and abdominal associated devices [72]. In these cases, chromium-containing alloys such as stainless steel SAE 316 L, cobalt-chromium-molybdenum steel, and Vitalium™ release metal ion [73] are reported to cause SCD [74–79]. These reported SCD cases are identified with the presence of several manifestations including erythroderma [74], and localized/generalized eczema or urticarial [76–78]. These findings suggested that any chromium sources applied in systemic routes could elicit SCD with certain clinical manifestations and degrees of severity.

Treatment of chromium-induced systemic contact dermatitis may be conducted in several approaches including managing diets and lifestyles by gut remediation and avoiding the food and sources that contain chromium; systemic or topical treatment using immune-suppressants such as corticosteroid; phototherapy; and hyposensitization therapy [80, 81]. Sharma developed a guideline for the preparation of low chromate diet that could help in controlling daily chromium consumption from food and ameliorating skin condition [82]. In case of SCD from arthroplastic routes, revision or removal of implant needs to be conducted by considering the time of hypersensitivity incidence after surgery and degree of severity [74]. Revision of implant could be conducted by using less allergenic implant such as titanium-based implant or chromium-based implant coated with certain biocompatible materials such as polytetrafluoroethylene, ZrN multilayers, diamond-like carbon, titanium nitride, graphite-like carbon, and tantalum [74, 79, 83]. Revision or removal of implants may not produce rapid disappearance [76] probably due to the presence of soluble or particulate debris of implant that produce inflammations [84, 85].

#### **3.3 Skin cancer**

Chromium, especially Cr(VI), is a potent human carcinogen. In a human cell, carcinogenesis of chromium(VI) (**Figure 3**) occurs through the penetration of chromium(VI) species into the cell via sulphate/phosphate anion transport system, reduction of chromium(VI) by endogenous antioxidant to produce ROS and chromium(III), and alteration of DNA directly by chromium(VI) or indirectly by ROS [86]. The alteration of DNA then produces different types of products including Cr-DNA adducts, DNA–protein and DNA interstrand cross-links, DNA breaks, and DNA base damage [87]. Carcinogenicity of chromium(VI) has induced lung cancer in workers from various chromium-based industries [88–90] and has been associated to incidence of other cancers [91]. However, the meta-analysis study showed that the correlation between exposure of chromium(VI) and the high mortality in skin cancer is not significant [91]. This study indicated that there is no supporting data confirming the chromium species as carcinogen in inducing skin cancer in human.

Several studies, however, showed that chromium species could induce skin cancer in rats and mice either as single carcinogen or cocarcinogen. Oral administrations of drinking water containing sodium dichromate dehydrate to male F344/N rats for 2 years showed that the sample developed various types of skin cancer [92]. Two other studies using hairless SK1-hrBR mice also exhibited that chromium(VI) could act as cocarcinogen in promoting UV-induced skin tumor [93, 94]. Davidson and co-workers [93] showed that oral administration of chromium(VI)-containing drinking water and UV irradiation to hairless mice have synergistic effect in promoting skin tumor. Exposure of chromium(VI) or UV radiation alone did not induce skin tumors [93]. Uddin and co-worker also conducted the same experiment and found that systemic administration of exogenous antioxidant (vitamin E and selenomethionine) did not improve skin condition [94]. It indicated that chromium(VI) cocarcinogenicity may be occurred in different mechanisms without involving ROS [94]. These three studies indicate that acute or chronic oral administration of chromium(VI) species has a great potency in promoting skin cancer in mammals including humans.

**129**

*Dermatologic Toxicities and Biological Activities of Chromium*

Irritation and chemical burn are caused by dermal exposure of chromium(VI) particle, solution, or mist in large quantities. Solid deposition of chromium(VI) would develop to "chromium ulcers" or "chrome holes" [67], while high concentration of chromium(VI) solution would lead to chromium burn. A mechanism for this ulcer formation is still unclear, but it may be related to the disruption of actin cytoskeleton by chromium(VI) leading to mitochondria-dependent apoptosis in skin fibroblasts cells [96]. Several reports exhibited these irritation and burning effects from different chromium species such as solid CrO3 [97], chromic acid solution [98–100], hot chromium(III) sulphate solution [101, 102], and chromium acid

Management of irritation is conducted by considering preventive and treatment approaches. Prevention of irritation is conducted by using barrier creams, moisturizers, etc., while treatment could be done by using moisturizers and corticosteroids [50]. For chromium burn, treatment is conducted by combining mechanical excision, hemofiltration, and systemic administration of chelating agent and antioxi-

Human hair is naturally exposed to a certain amount of chromium [104] that come from various sources [105–107]. Excessive and repeated exposures of chromium in certain environmental condition cause discoloration of blond, dyed-blond, and white hair (to become green) [108–110] and cause rapid hair fall [111, 112]. The mechanisms of these two effects are still unclear. Hair discoloration is probably the result of interaction between chromium ions (and also copper and nickel) and protein in hair (keratin) [113], while rapid hair fall may be related to several mechanisms such as promoting premature end of hair cycle [114] or disruption of

Acne vulgaris is a common dermatological condition that affects physical and psychological aspects of patients [116]. Several diseases that show the presence of a certain degree of acne also relate with depression and emotional stress such as type-2 diabetes, rheumatoid arthritis, and polycystic ovarian syndrome (PCOS) [117–119]. Pathophysiology of this disease involves several key mechanisms including excessive sebum production due to hormonal and environmental conditions, alteration of fatty acids composition due to sebum metabolism by *Cutibacterium acnes*, hyperkeratinization within the follicle that clogs up the pore in the form of whitehead or blackhead comedones, inflammation induced by bacterial colonization, and malfunction of locale innate and adaptive immune system [120]. The presence of acne vulgaris is also correlated to the clinical depression in patients [116]. In this case, depression or stress can influence the regulation of sebaceous gland as the main part in sebum production [121]. Catecholamines (epinephrine and norepinephrine) as the main stress hormones also affect the growth of certain *Cutibacterium acnes* strains [122–124]. Catecholamine-treated *C. acnes* strain also can stimulate a limited but significant increase of lipid production in sebaceous

*DOI: http://dx.doi.org/10.5772/intechopen.90347*

**3.4 Irritation and chromium burn**

mist [103].

dant [100].

**3.5 Hair disorders**

hair shaft formation [115].

**4.1 Acne vulgaris (antiacne)**

**4. Biological activities of chromium**


**2. Two types of virus progeny serve at different steps of host invasion**

Alphabaculoviruses and betabaculoviruses produce virions of two phenotypic classes: occlusion-derived virus (ODV) and budded-virus (BV). Nucleocapsids of both types of virus particles are assembled in the nucleus of the infected cell. During the late phase of infection, when proteins of the nucleocapsid are expressed, BVs are produced as the newly formed nucleocapsids exit the cell, acquiring their envelopes from the cell membrane during the budding process. BVs disseminate the infection within the host by entering other cells via a mechanism of receptor-mediated endocytosis. ODVs arise at very late times, when nucleocapsids are enveloped in membrane units derived from the nuclear envelope to finally be embedded in the OBs. OBs persist in the environment after liquefaction of the insect cadaver and are responsible for the horizontal transmission of the virus between hosts. Upon ingestion by a susceptible insect larva, OBs are dissolved in the alkaline environment of the midgut and ODVs are released. The ODVs move through the peritrophic membrane and nucleocapsids are delivered into midgut epithelial cells through a mechanism of membrane fusion mediated by specific viral proteins known as *per os* infectivity factors (PIFs). This primary infection in the midgut is followed by a secondary infection, consisting in the dissemination of BVs to other tissues. In contrast to this infection cycle, which is typical of lepidopteran-specific baculoviruses, infections of gammabaculovi‐ ruses and deltabaculoviruses are restricted to the midgut of their hosts.

## **3. Nuclear events associated with infection**

Baculovirus infection causes cells to enlarge and stop dividing; the nucleus swells and forms the virogenic stroma (VS), which is the nuclear compartment where the viral DNA is replicated and the nucleocapsids of virus progeny are assembled. The host chromatin adopts a marginal distribution at 24 hours post infection (hpi), when the VS becomes evident. At this time, it was shown that histone H4 fused to fluorescent protein markers colocalizes with the chromatin in the periphery but not with the VS [5]. The new distribu‐ tion of the chromatin is determined by the replication of the virus and may have effects in changes operated in the expression of host genes and the progression of the cell cycle. At late times, ODVs become occluded into OBs in the periphery of the VS.

Apparently, the nucleocapsids of NPVs enter the nucleus through the nuclear pores, whereas the genome of GVs is probably injected [6]. Virions of baculoviruses are devoid of histones, in turn the DNA in the nucleocapsid is packed in association with viral protein P6.9, a basic DNA-binding protein. This small polypeptide is rich in arginine, serine and threonine residues, a feature similar to proteins called protamines present in the nuclei of spermatids in many animals and plants. The positive arginine residues in protamines neutralize the negative charges in the DNA backbone while serine and threonine medi‐ ate interaction between protamine molecules, resulting in a high condensation of genom‐ ic DNA. Once it is uncoated into the nucleus, the DNA dissociates from P6.9 through the phosphorylation of the protein. During infection the viral DNA appears to be organized in the form of nucleosomal-like structures in association with P6.9, as suggested by experiments of micrococcal nuclease digestion of isolated nuclei [7].


14

**Figure 5.** Success factors of implementation of PED and To-PED projects.

PED Towards PED **Figure 6.** Challenges and barriers for implementation of PED and To-PED projects.

A big challenge for successful implementation processes are legislation and regulations—an in-depth analysis of the particular legal barriers (on either local, national, or European levels) therefore seems to be highly relevant in particular for PED. Within the +CityxChange project, possible strategies to enable regulatory mechanisms have been studied [13].

#### **4. Discussion and Conclusions**

The analysis presented in this report was performed on data included in the first update of the PED Booklet (February 2020). The aim of this study is to evaluate trends, features, successes, or critical factors that contribute to development of PED projects in Europe.

There is a broad variety of projects addressing energy transition. While only 29 of 61 projects are planning to implement PEDs, all of them provide valuable approaches and strategies regarding actions towards the energy transition in the urban context. Most of the projects classified as Towards PEDs seem to include the possibility to develop into PEDs in a further step. However, the actual feasibility of many project designs is not yet assessable since a large share of the projects listed are in planning or early implementation stages. The PED Reference Framework, with its focus on energy functions and overall sustainability goals, provides an open framework—the results of the comparison between PED projects and Towards PEDs projects indicate that differences between the two groups are not categorical and the main issues and challenges of project implementation are similar. This therefore supports the assumption that projects with no self-declared PED ambition may be further developed to achieve PED status according to the PED Reference Framework. A further operationalization of the PED Reference Framework [5], which is currently under development, aims at a common PED definition that includes local differences and diverse approaches.

For this analysis it must be considered that not all countries have equally contributed to the Booklet yet. The Booklet is based on voluntary contributions from individual PED-related projects. Therefore, the results do not allow any interpretation about the level of PED efforts across Europe in general.

Nevertheless, these preliminary results highlight the importance of national programs and strong R&I involvement in the replication of PED strategies, as for example in Norway where ZEN Research Center on Zero Emission and Neighborhoods in Smart Cities is helping the spreading of pilot projects in line with the PED framework. Future investigations on the impact of climatic zones on PED mainstreaming will be also considered.

Projects that declared a PED ambition are mainly in their planning or implementation stage (93% versus 57% of To-PED). This suggests that most of PED strategies are still to be tested and optimized and a regular update of the Booklet cases and data analysis is needed. Efforts will continue to extend the data base, improve data quality, and assess these projects to identify good practice as well as critical issues at further stages.

Most projects with PED ambitions (66%) combine newly built neighborhoods with existing neighborhoods. This result is in good agreement with the framework that suggests the combination of new urban development areas with existing buildings. Only 7% of PED are developed in an existing neighborhood. The high complexity of a PED project requires deep interventions to fit with the new paradigm and might inhibit ambitions to apply PED ambitions to existing structures. The need for dealing with complex ownership structures or technological limitations, for instance regarding PV-instalment, in connection with other aspects of revitalization, for example, building protections and preservation of cityscape, are just two factors that need to be mentioned. Regarding the implementation of a PED ambition, it does not seem to be a common strategy to count only on existing neighborhood. However, for most European cities, transformation of the existing building stock is a key aspect for achieving the energy transition. Therefore, exploring, integrating and applying PED solutions and approaches to the existing urban structure seems to be a crucial challenge in the long run. An 8-step process to develop a PED is proposed by the JRC report [14].

A significant amount of PED projects involves a highly mixed land use (39%) while only 18% of Towards PED projects do. Multifunctional urban structures can be seen as a basic requirement for successful PED implementation since they support efficient use of energy and more opportunities for energy flexibility. A high percentage of PED mentioned commercial and social use of the land. Multifunctionality is also a crucial requirement for embedding PED strategies in integrated urban planning strategies aiming at high quality of life and sustainable local development. This is in line with the framework definition that requires social and economic sustainability.

Another feature of PED is their highly mixed combination of energy typologies. Fifty percent of PED projects integrate four to five energy sources, whereas 60% of To-PED projects use two or three. This is a reasonable result given the fact that PED projects have the ambition to have an annual positive energy balance produced from renewable sources. To satisfy this ambitious aim, the combination of multiple energy typologies is key to exploit changing energies' availabilities, consumptions with different weather conditions, or with the alternation of day and night and seasons. Photovoltaics is used in 71% of PED projects versus 30% of To-PED projects. Photovoltaics is a mature and suitable technology for decentralized and renewable energy production. Its profitability is already reached in some European countries and increasing diffusion in building rooftop is expected [15]. This explains why a high percentage of PEDs rely on photovoltaics solar energy as local production of renewable energy.

Stakeholders, citizens involvement strategies, integrated technology, and political support are considered main success factors for PED development. A successful PED implementation requires collaboration with citizens and final users right from initial planning stages to avoid their opposition to the change of paradigm that a PED aims to do in terms of social, economic and energy sustainability. Political support is a necessary factor to activate national programs and new funding opportunities. Furthermore, the technical complexity of a PED is the reason why integrated technology was mentioned as a main feature to its success.

The main barrier for a PED is the access to adequate funding and business models (Figure 6). PEDs are more expensive than traditional projects and they require multiple sources of financing and advanced business models. Innovations and changes introduced by PED projects require ad hoc regulations and a dedicated legal framework. This explains why legislation and regulations, together with existing governance structures within city administrations and a lack of cross-sectoral approaches, are often mentioned as main reasons for inhibited implementation processes (Figure 6).

Considering the issues discussed, key areas of action to be further explored can be summarized as follows:

Based on these preliminary observations, the next activities will focus on the following aspects:

**Author Contributions:** Conceptualization: C.G. and S.B.; methodology: S.B. and C.G.; software: S.B., S.T., and C.G.; validation: S.B. and C.G.; formal analysis: S.B. and C.G.; investigation: S.B. and C.G.; resources, S.B. and C.G.; data curation, S.B. and S.T.; writing—original draft preparation, S.B.; writing—review and editing, C.G.; visualization, S.B.; supervision, C.G.; project administration, C.G.; funding acquisition, C.G. All authors have read and agreed to the published version of the manuscript.

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

**Acknowledgments:** The authors would like to thank all project representatives who provided data on the projects presented in the PED Booklet, which served as basis for this analysis. Furthermore, the authors would also like to acknowledge the support of SET Plan Action 3.2 Italian National Delegate Paola Clerici and the work of the PED Program Management, specifically Margit Noll, Hans-Günther Schwarz, Robert Hinterberger, and Susanne Meyer, and the contributions of the PED Program Stakeholders Group, specifically EERA JPSC, regarding overall conceptualization of the PED Program and the elaboration of the PED Framework, which is a fundamental basis for the analysis of the data provided.

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

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