**Advances and Challenges in Organic Electronics**

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**Fr ´ed ´eric Dumur Fabrice Goubard**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Fred´ eric Dumur ´ Aix-Marseille University France

Fabrice Goubard Cergy-Pontoise University France

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**Abstract**

Survival and quality of life of cancer patients have improved in the last decades. However, some forms of cancer still escape current treatment options and continue to have an ominous prognosis. A plausible strategy to change this situation is to identify unexploited pathways in the cancer cell that open genuinely new therapeutic pathways. Ion channels are among such targets since they participate in all steps in the cancer process, from initiation through growth and metastasis to drug resistance. In some cases, ion channels can thus serve as therapeutic targets. Kv10.1 is particularly well suited for this purpose because the channel appears outside of the brain almost exclusively in cancer cells. Recent research showed that besides its functions as a canonical ion channel, Kv10.1 is required by dividing cells to complete division. For this, healthy cells express the channel only during a short period in the cell division cycle. Cancer cells, rather than increasing the channel's expression, maintain relatively constant levels throughout their lives, which confers a selective advantage and favors tumor progression. The mechanisms leading to abnormal expression and its consequences, and how we can take advantage of this knowledge to improve current cancer treatments will be discussed.

**Keywords:** Kv10.1, ion channels, cell cycle, cancer target


*Review* **Neuroprotective Effect of Vascular Endothelial Growth Factor on Motoneurons of the Oculomotor System**

**Silvia Silva-Hucha, Angel M. Pastor and Sara Morcuende \***

Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla, 41012 Sevilla, Spain<EMAIL_ADDRESS>(S.S.-H<EMAIL_ADDRESS>(A.M.P.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +34-954-55-95-49

**Abstract:** Vascular endothelial growth factor (VEGF) was initially characterized as a potent angiogenic factor based on its activity on the vascular system. However, it is now well established that VEGF also plays a crucial role as a neuroprotective factor in the nervous system. A deficit of VEGF has been related to motoneuronal degeneration, such as that occurring in amyotrophic lateral sclerosis (ALS). Strikingly, motoneurons of the oculomotor system show lesser vulnerability to neurodegeneration in ALS compared to other motoneurons. These motoneurons presented higher amounts of VEGF and its receptor Flk-1 than other brainstem pools. That higher VEGF level could be due to an enhanced retrograde input from their target muscles, but it can also be produced by the motoneurons themselves and act in an autocrine way. By contrast, VEGF's paracrine supply from the vicinity cells, such as glial cells, seems to represent a minor source of VEGF for brainstem motoneurons. In addition, ocular motoneurons experiment an increase in VEGF and Flk-1 level in response to axotomy, not observed in facial or hypoglossal motoneurons. Therefore, in this review, we summarize the differences in VEGF availability that could contribute to the higher resistance of extraocular motoneurons to injury and neurodegenerative diseases.

**Keywords:** VEGF; oculomotor system; trophic factors; motoneurons; neurodegeneration; axotomy; amyotrophic lateral sclerosis

**1. Vascular Endothelial Growth Factor (VEGF)**

*1.1. History*

VEGF was initially described as an angiogenic factor and, consequently, it was named vascular permeability factor (VPF) for its role in inducing vascular permeability in tumor cells [1]. It was not until 1989 when the VEGF protein, whose molecular weight is approximately 45 kDa, was purified and sequenced, and it was definitively assigned the name of vascular endothelial growth factor [2,3].

It is well known that this factor is a highly specific mitogen for vascular endothelial cells, whose family consists of multiple cell signaling proteins involved in angiogenesis, lymphangiogenesis, vasodilation, and vascular leakage, among other functions [4]. In 2001, his essential role in motoneuronal protection was revealed for the first time [5], as will be discussed later in detail.


**10.1. Establishing a general approach**

Decisions regarding revascularization are complex and have been founded on decades of evidence. This body of evidence has evolved in parallel with advances in treatment but also a patient population with increasing medical complexity. Therefore, a contemporary approach to MVD and revascularization must be founded on an understanding of the wide spectrum of disease severity, advances in medical/surgical therapy, diversity in patient populations, patient preference and social circumstances. Optimal treatment strategies must apply the most current evidence in an appropriate clinical context. Furthermore, guiding principles of management with a multidisciplinary 'Heart Team' approach should be the cornerstone of state of the art treatment of multivessel coronary artery disease as supported by recent revascularization guidelines [14]. The basic approach should address a number of basic clinical questions which address the (1) therapeutic goals of the case, (2) the presence or absence of clinical evidence to support revascularization, (3) the presence/absence of prognostic factors that may make surgical revascularization more favorable, (4) whether the anatomy favor PCI or CABG, (5) is the patient a good surgical candidate should prognostic disease be present, (6) does the patient have any particular preferences and (7) are there ambiguities that would benefit from further discussion by a Heart Team [Table 4].

**Table 4.** Key clinical questions forming the basis of the therapeutic decision for management of multivessel coronary artery disease.

The fundamental basis of our decisions rest on what therapeutic goals can be achieved: *improvement of survival, improvement of symptoms or both*. It is important to make this distinction because although the goal would naturally to improve on both; consider the following two clinical scenarios:

If the intent is to improve survival, there is good evidence that supports revascularization in certain patient populations: significant left main (>50%) or 3VD, 2VD with proximal LAD with diabetes LV dysfunction and/or high burden of functional ischemia).

If the intent is primarily to *improve symptoms (eg. The clinical profile undermines the prognostic benefit of surgical revascularization)*, there is good evidence that revascularization with PCI is of benefit in those who are symptomatic despite optimal medical therapy if technically feasible. But with advances in medical therapy, it is reasonable to maximize medical treatment before considering revascularization [56].


**Acknowledgements**

I wish to thank people involved in the studies described here, including Kyoji Urayama, Célia Guilini, Gulen Turkeri, Monia Boulberdaa, Mojdeh Dormishian, Rehana Qureshi, Himanshu Arora and Adeline Gasser. The publication was supported in part by grants from Fondation pour la Recherche Médicale (Equipe Labellisée), Centre National de la Recherche Scientifique and Université de Strasbourg. This work has also been published within the LABEX ANR‐ 10‐LABX‐ 0034\_Medalis and received a financial support from the French government managed by Agence Nationale de la Recherche (ANR) under "Programme d׳investissement d׳avenir."


*2.4. Leaf Relative Water Content (RWC) Determination*

Leaf RWC was measured following the method of Barrs and Weatherley [43]. Fresh leaves were collected, weighed instantly by a standardized weighing balance, and then dipped into water in separate Petri-dishes for 12 h. The turgid weight was calculated by weighing the wet leaves. Afterwards, the leaf samples were oven-dried at 80 ◦C for 48 h, and the dry weight was recorded. The RWC was calculated using the following formula:

RWC (%) = [(Fresh weight − Dry weight)/(Turgid weight − Dry weight)] × 100.


4.1.1. Health Risk Assessment

The concentrations of ions in water were used to assess the possible health risks from human exposure to drinking water through both oral intake and skin contact. This study considered two groups, namely adults and children [41], and evaluated the carcinogenic risk of eight ions and the non-carcinogenic risk of two heavy metal ions [42].


**2. Materials and Methods**

To study the process of preparing forage from grain, a laboratory-scale plant has been developed consisting of a frame on wheels, a tank with a built-on passive grinder, a 1SM65-50-160/2 centrifugal pump and a NGD-1.1 disperser, material pipelines, and a control cabinet (Figure 1). The measuring station operates as follows. Water is poured into the tank based on the required volume of prepared forage at the outlet, which is 2:1 with respect to the grain. The amount of water poured was recorded by a water meter SVK 15-3-8 (Figure 2). Next, the centrifugal pump is turned on, and the heating process to 30 ◦C takes place. When this temperature is reached, the tank is uniformly filled with pre-processed and cleaned grain at the given water:grain ratio. Next, enzymes are added. Through special inspection ports in the built-in grinder (chopper), the uniformity and homogeneity of the working mass are visually monitored, and, upon reaching a temperature of 60 ◦C, the pump is switched off. The prepared mass is allowed to infuse for one hour for the more efficient operation of enzymes. The result is a high-carbohydrate forage from grain recommended for feeding farm animals as a single feed or as a part of feed mix.

**Figure 1.** The measuring station for preparing forage from grain.

**Figure 2.** Water meter SVK 15-3-8 used for recording the amount of water poured into the tank of the measuring station.

While the plant pump is operating, the grain is crushed by its blades. To accelerate the process of grain grinding and to obtain a more even forage composition, it was proposed to install a passive grinder in the form of a grid-work (grill) or a plate in the upper part of the tank (Figure 3). During the experiments, the influence of the angle α of setting the grid-work (plate) and the distance S from the nozzle to the grid-work (Figure 4) on the quality of forage have been studied. It was modified by moving the grid-work (plate) between the holes of the fixture of the laboratory-scale plant (Figure 5). The distance S between the grid-work (plate) and the nozzle was adjusted by moving the grid-work (plate) along the slots in the cover (Figure 6).

**Figure 3.** Passive grain grinder made in the form of (**a**) grid-work and (**b**) plate.

a b

**Figure 4.** A device for adjusting the inclination angle of the grid-work (plate) and its distance to the nozzle. (**a**) Diagram; (**b**) General vie: (1) tank with a built-in passive grinder, (2) case of the passive grinder, (3) cover of the passive grinder, (4) grid-work (plate), (5) nozzle, (6) nozzle flange.

**Figure 5.** The system for adjusting the inclination angle of the grid-work (plate).

**Figure 6.** Cover with slots for adjusting the clearance S between the grid-work (plate) and the nozzle.

To determine the performance of the proposed plant under scientific laboratory conditions, a technique was developed to assess the influence of design and operating parameters on the work process of preparing forage from grain. Wheat was used as the source raw material. To carry out the fermentation process, the multi-enzyme composition MEC-AC-3 produced by Vostok LLC, Kirov Region (Russian Federation), was used. The volume of forage prepared was 50 L.

Routine of the experiment was as follows. First, 17 kg of grain was put into 33 L of water pre-heated to a temperature of 30 ◦C, and 80 g of enzyme was added, previously weighed with the accuracy of up to 0.01 g using the Biomer VK-300.01 laboratory balance (Novosibirsk) (Figure 7). From that moment, the process timing began using the HUAWEI P20 lite stopwatch (China). Every 7 min 30 s before the working mass temperature reached 60 ◦C, a test batch was taken from the tank: the tank cover was opened, anda1L volume bucket was placed under a stream and filled up. Within the same time intervals, in addition to recording the temperature values, the power consumed by the pump electric motor was recorded using the Mastech MS2203 clamp meter (Pittsburg U.S.) (Figure 8). After that, the feed sample was poured onto a sieve with a sieve size of 0.5 mm. Free water was removed by vigorous stirring with a brush. The resulting mass was poured into a plastic bag, which was assigned an information tag (Figure 9). Three to five packets with samples were obtained before the temperature reached 60 ◦C. After that, the pump was turned off, and one hour was timed until the forage from grain was completely prepared.

**Figure 7.** Weighing the multi-enzyme composition MEC-AC-3 using the VK-300.01 laboratory balance.

**Figure 8.** Measuring the power consumption of the electric motor using the Mastech MS2203 clamp meter.

**Figure 9.** Bags with samples of forage from grain.

Temperature of the forage from grain (and water) was recorded using the TST81 sensor, the data from which was transmitted to the TL-11-250 temperature controller (Figure 10). During the experiment, the dynamics of temperature changes was recorded from the digital display of the temperature controller (Figure 10).

**Figure 10.** Temperature controller TL-11-250 with a temperature sensor TST81 (**a**) and recording the temperature of the forage from grain (**b**).

a b

Next, a 100 g sample weight was selected from each bag and distributed on the sieve with a sieve size of 3 mm. After that, the sample was rinsed out in a bath with water. Next, the sieve residue was weighed, and the number of whole grains was counted.

An hour later, a control sample was taken from the premixed forage from grain. To determine the uniformity, screen sizing was collected using sieves of the following sizes: 2; 1.4, 1; 0.5 mm and the bottom. Then, 100 g of the sample weight was fed to the upper sieve, and then the plansifter (RL brand) was turned on for 5 min (Figure 11). First, the dry sieves were weighed, and their mass was determined. Then, they were weighed with the residue, and the difference was determined. For best data reliability, this analysis was performed in triplicate.

**Figure 11.** Laboratory plansifter RL brand with a set of sieves for determining the granulometric composition of the forage.

For energy estimation of the plant operation, specific energy consumption was used referred to a unit volume of the working mixture, w1:

$$\mathbf{w1} = \frac{\mathbf{W} \cdot \mathbf{r}}{V},\tag{1}$$

where

W is the average power consumption, kW;

τ is the process time, s;

*V* is the volume of water and working mixture, l.

#### **3. Results**

After the experiments, tables and graphs were constructed that characterize the change in the quality of forage from grain depending on the installation parameters (α and S) of the grid-work and the time t of the fermentation process (Tables 1 and 2, Figures 12–15). According to the results of experiments and calculations according to the formula (1), histogram 16 was constructed. Histogram 16a shows what full electric motor power is spent on preparing the forage. Figure 16b presents the change in the specific costs of electricity calculated using the expression (1). The constructed histograms show how the power consumption of the electric motor changes when the angle α of setting the grid-work (plate) and the distance S between the grid-work (plate) and the nozzle are modified.

Regardless of the angle α of setting the grid-work and the distance S, the number of whole grains decreases to 0 after 1350 s (22.5 min) of the plant operation (Figure 12). After 900 s (15 min) of the plant operation, the minimum number of whole grains is observed when the grid-work is installed according to options 1 and 3 (Figure 12). That is, according to the indicator m1 "the number of whole grains in the forage," we can recommend the grid-work installation scheme using options 1 and 3.

The number of whole grains reaches the value of 0 in 1350 s (in 22.5 min) when the plate is installed at an angle α = 90◦ at S = 140 mm (Figure 13). In other cases, in order to avoid the presence of whole grains in the forage, the operation time of the plant should be at least 30 min. The graph also shows that with decreasing angle α, the number of whole grains in the forage increases. This is explained by the fact that, when the angle α is decreased, the impact force of the grain on the plate goes down. From

the analysis of the graphs presented in Figure 13, we can conclude that the best results of preparing forage using a plate are obtained when it is installed according to options 3, 4, and 5 (Figure 13).

The best performance in terms of the indicator "residue on the sieve with a sieve size of 3 mm" is also achieved when the plate is installed according to options 3, 4, and 5 (Figure 14). In these cases, the m2 indicator is close to zero after 1800–1850 sec of the plant operation. When the plate is installed at an angle of 30 and 45◦ for a time t = 1800 s, m2 equals 0.1% and 0.4%, respectively, and a zero value is reached only after 2080 and 1989 s, respectively. That is, in terms of the indicator "residue on a sieve with a diameter of 3 mm," it can be recommended to prepare forage when the plate is installed according to options 3 and 5.

The average power consumed by the engine while preparing forage did not exceed 5 kW. The lowest value was recorded when the grid-work was installed at an angle of 30◦ and at a distance of 205 mm from the nozzle—it was 4 kW. In general, the average engine power during the preparation of forage using a grid-work is lower than when using a plate. When using the grid-work, it did not exceed 4.5 kW, and when using the plate, it varied from 4.51 to 5 kW (Figure 16a).

Change in specific energy consumption required to prepare 1 L of forage from grain is shown in Figure 16b. In general, it can be seen that when a grid-work is used, the specific energy consumption is lower than when a plate is used. Based on the findings presented, we can recommend the use of a grid-work installed at an angle of 30◦ at a distance of 140 and 205 mm from the nozzle, or a plate installed at an angle of 90◦ at a distance of 140 mm from the nozzle. With this combination of parameters, the minimum specific energy consumption is observed, which amounts to 41.5–48.4 W·h/L. In other cases, there is an increase in this indicator to 50–52 W·h/L (Figure 15c).

**Table 1.** The change in the quality of forage from grain depending on the installation parameters (α and S) of the grid-work (where M is average, SD is standard deviation, and SKE is skewness factor).

**Table 2.** The change in the quality of forage from grain depending on the installation parameters (α and S) of the plate (where: M—average, SD—standard deviation, SKE—skewness factor).

**Figure 12.** Number of whole grains in molasses when prepared using a grid-work: 1—the distance from the grate to the nozzle is 140 m the angle of inclination of the grate is 30 degrees; 2—the distance from the grate to the nozzle is 140 mm, the angle of inclination of the grate is 45 degrees; 3—distance between the grate and nozzle 205 mm, angle of inclination of the grate 45 degrees; 4—distance between the grate and nozzle 205 mm, angle of inclination 30 degrees.

**Figure 13.** The residue on the sieve with a sieve size of 3 mm when preparing forage using a grid-work: 1—distance from the grid-work to the nozzle is 140 mm, grid-work inclination angle is 30 degrees; 2—distance from the grid-work to the nozzle is 140 mm, grid-work inclination angle is 45 degrees; 3—distance from the plate to the nozzle is 205 mm, grid-work inclination angle is 45 degrees; 4—distance from the grid-work to the nozzle is 205 mm, grid-work inclination angle is 30 degrees.

**Figure 14.** The number of whole grains in forage when it is prepared using a plate: 1—distance from the plate to the nozzle is 140 mm, plate inclination angle is 30 degrees; 2—distance from the plate to the nozzle is 140 mm, plate inclination angle is 45 degrees; 3—distance from the plate to the nozzle is 140 mm, plate inclination angle is 90 degrees; 4—distance from the plate to the nozzle is 205 mm, plate inclination angle is 90 degrees; 5—distance from the plate to the nozzle is 305 mm, plate inclination angle is 90 degrees.

**Figure 15.** Residue on the sieve with a sieve size of 3 mm when preparing forage using a plate: 1—distance from the plate to the nozzle is 140 mm, plate inclination angle is 30 degrees; 2—distance from the plate to the nozzle is 140 mm, plate inclination angle is 45 degrees; 3—distance from the plate to the nozzle is 140 mm, plate inclination angle is 90 degrees; 4—distance from the plate to the nozzle is 205 mm, plate inclination angle is 90 degrees; 5—distance from the plate to the nozzle is 305 mm, plate inclination angle is 90 degrees.

**Figure 16.** The influence of the studied parameters on: a—power consumption of the electric motor; b—specific energy consumption; 1—distance from the grid-work to the nozzle is 140 mm, grid-work inclination angle is 30 degrees; 2—distance from the grid-work to the nozzle is 140 mm, grid-work inclination angle is 45 degrees; 3—distance from the plate to the nozzle is 205 mm, grid-work inclination angle is 45 degrees; 4—distance from the grid-work to the nozzle is 205 mm, grid-work inclination angle is 30 degrees; 5—distance from the plate to the nozzle is 140 mm, plate inclination angle is 30 degrees; 6—distance from the plate to the nozzle is 140 mm, plate inclination angle is 45 degrees; 7—distance from the plate to the nozzle is 140 mm, plate inclination angle is 90 degrees; 8—distance from the plate to the nozzle is 205 mm, plate inclination angle is 90 degrees; 9—distance from the plate to the nozzle is 305 mm, plate inclination angle is 90 degrees.


**1. Introduction**

130 Electropolymerization

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The electronically conducting polymers (ECPs), such as polypyrrole (PPy), polythiophene (PT) and polyaniline (PANI) are known to possess unusually high electrical conductivity in the doped state. Due to this, these materials have been of great interests for chemists as well as physicists since their electrical properties were reported (Diaz et al., 1979). The ECP films behave like a redox polymer and have potential applications in electrocatalysis, solar energy conversion, corrosion, electronics, etc. The redox polymer reaction is accompanied by a change in the electrical properties of the film from an insulator to an electrical conductor involving both electron and ion transport within the film (Kaplin & Qutubuddin, 1995).

Conducting polymers can be synthesized either chemically or electrochemically. Electrochemical synthesis is the most common method as it is simpler, quick and perfectly controllable. PPy is one of the most interesting conducting polymers since it is easily deposited from aqueous and non-aqueous media, very adherent to many types of substrates, and is well-conducting and stable. Electrochemical polymerization produces thin films with a thickness of few micrometers on an electrode surface (Diaz et al., 1979), while a chemical oxidation yields a fine-grained material. However,the yield and quality of the resulting polymer films are influenced by several factors, such as nature and concentration of monomer and the counter ion, solvent, cell conditions (e.g. electrode and applied potential), temperature and pH (Sadki et al. 2000; Ansari, 2006 & Pina et al. 2011).

ECPs can be modified in several ways (Juttner et al., 2004) to obtain tailored materials with special functions: (i) derivatization of the monomer by introducing aliphatic chains with functional groups; (ii) variation of the counterion, incorporated for charge compensation during the polymerization process; (iii) inclusion of neutral molecules with special chemical functions and (iv) formation of compounds with noble metal nanoparticles as catalyst for electrochemical oxidation and reduction processes.

The electropolymerization reaction is a complex process and its mechanism is still not fully understood. A number of mechanisms have been proposed (Genies et al., 1983; Kim et al., 1988; Asavapiriyanont et al., 1984; Qui & Reynolds, 1992) and are comprehensively reviewed (Sadki et al. 2000; Ansari, 2006 & Pina et al. 2011). Among these, Diaz's mechanism is the most accepted one (Genies et al., 1983) and supported by Waltman and Bargon (1984 & 1985) also. In this mechanism, the pyrrole (Py) activation occurs through electron transfer

Polypyrrole Composites: Electrochemical Synthesis, Characterizations and Applications 133

Electrochemical film formation is often followed by stoichiometric determination of the number of electrons donated by each molecule. This value is generally found to be in between 2 and 2.7, where 2 electrons serve in the film formation and the excess charge is

The final step in the polymerization is not clear and different hypotheses have been proposed. Funt and Diaz (1991) believe that the reaction with water could be responsible for the polymerization quench. While, Street (1986) believes that the growth of the chain stops because the radical cation becomes relatively unreactive towards the chain propagation or

This article presents an overview of electrochemical synthesis of PPy nanocomposites and their structural and electrochemical properties and applications in electrochemical devices. Applications of nanocomposites described include solar cells, fuel cells, batteries, corrosion protection coatings and super capacitors. Nanocomposites consists of PPy and one or more

PPy films are obtained through electropolymerization of Py on suitable substrates/working electrodes by using different electrochemical techniques such as cyclic voltammetry (CV), potentiodynamic, galvanostatic, potentiostatic, reversal potential pulsing technique, etc. To carry out the electropolymerization of Py, a three-electrode one-compartment cell (50 or 100 ml capacity) with provision of passing an inert gas (N2/Ar) into the electrolyte before the start of the oxidation and above the surface of the electrolyte during the polymerization process is employed. Saturated calomel electrode (SCE) or Ag/AgCl (saturated KCl) is used as the reference electrode. PPy composites prepared were either in the form of sandwich-

Generally, the electroplymerization is carried out on noble metals or inert materials such as Pt, Au, glassy carbon (GC), or indium tin oxide (ITO). It is because of the fact that the standard oxidation potential of pyrrole is fairly high (Eox = 0.70 V vs. SCE) and so, when scanning the potential the dissolution of most of metal supports takes place prior to the oxidation of monomer is reached and, thus, the electropolymerization reaction is inhibited. However, when we are using supports which are oxidizable, for example, Fe, Zn, Al, etc, as the working electrode, it is necessary to find new electrochemical conditions for slowing the dissolution of the working electrode without preventing electropolymerization. Recently, a new electrosynthesis process of PPy films on oxidizable metals Al and Fe has been reported (Bazzaoui et al. 2005). In this process, the formation of homogeneous and strongly adherent

components, which can be carbon nanotubes, metals, oxide nanomaterials, etc.

(a) (b)

because the reactive chain ends become sterically blocked.

consumed by the polymer oxidation.

Fig. 1.

**2. Synthesis**

type or simple films.

**2.1 Polypyrrole (PPy) films**

from the monomer forming a radical cation-rich solution near the electrode in several steps. Details of steps involved are given in Scheme 1.

Scheme 1.

The propagation continues via the same sequences: oxidation, coupling, deprotonation until the final polymer product is formed (Fig.1a). The electropolymerization does not give the neutral nonconducting polymer but its oxidized conducting form (doped). The final polymer chain, in fact, carries a positive charge every 3-4 Py units, which is counter balanced by an anion. The structure of the doped polymer can be given as shown in Fig.1b.

#### Fig. 1.

132 Electropolymerization

from the monomer forming a radical cation-rich solution near the electrode in several steps.

The propagation continues via the same sequences: oxidation, coupling, deprotonation until the final polymer product is formed (Fig.1a). The electropolymerization does not give the neutral nonconducting polymer but its oxidized conducting form (doped). The final polymer chain, in fact, carries a positive charge every 3-4 Py units, which is counter balanced by an anion. The structure of the doped polymer can be given as shown in Fig.1b.

Details of steps involved are given in Scheme 1.

Scheme 1.

Electrochemical film formation is often followed by stoichiometric determination of the number of electrons donated by each molecule. This value is generally found to be in between 2 and 2.7, where 2 electrons serve in the film formation and the excess charge is consumed by the polymer oxidation.

The final step in the polymerization is not clear and different hypotheses have been proposed. Funt and Diaz (1991) believe that the reaction with water could be responsible for the polymerization quench. While, Street (1986) believes that the growth of the chain stops because the radical cation becomes relatively unreactive towards the chain propagation or because the reactive chain ends become sterically blocked.

This article presents an overview of electrochemical synthesis of PPy nanocomposites and their structural and electrochemical properties and applications in electrochemical devices. Applications of nanocomposites described include solar cells, fuel cells, batteries, corrosion protection coatings and super capacitors. Nanocomposites consists of PPy and one or more components, which can be carbon nanotubes, metals, oxide nanomaterials, etc.


**Author details**

Sandip Mitra and Kunaal Kharbanda\*

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Nephrology, Central Manchester Foundation Trust, Manchester Academy of Health Sciences Centre (MAHSC), Manchester, United Kingdom

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**3. Propesticides**

A biologically inactive compound requires structural transformation(s) after application to become pesticidally active. Derivatives of known active ingredients that are converted to parent compound for activity. These are various process require for the activation of propesticide to the pesticidally active one [6]. Ideally these activation processes takes place only in the target organism. Even though it can also takes place in the environment, including soil and atmosphere. These can be one of or combination of the following four types:

#### **3.1. Activation by primary biochemical target**

This activation process is carried out in presence of certain enzyme or enzyme system. Enzymatic conversion of a proinsecticide to the active toxophore at the target tissue results in disruption of activating enzyme [7]. The activating enzymes, present in the tissue of target organism, are carrying out certain biochemical function in the body of target organism. When active toxophore are released from the proinsecticide their normal functions are discontinued resulting in killing of organisms. This is also known as suicide inactivation as their natural process is being inactivated [8]. Likewise, there also takes place the following:

#### **3.2. Activation by detoxification system**

to animals for the control of insects, arachnids or other pests in or on their bodies [1]. The term includes substances intended for use as a plant growth regulator, defoliant, desiccant or agent for thinning fruit or preventing the premature fall of fruit, and substances applied to crops either before or after harvest to protect the commodity from deterioration during storage and transport. Present day pesticides which are common and widely used have number of adverse affects on non-target organisms. The consequences lead to environment toxicity affecting ecosystem [2]. Therefore safer insecticide deserves attention. A propesticide is a biologically inactive compound requiring structural transformation(s) after application to become pesticidally active. Activation process for propesticides can be one of or a combination of the three following types: (a) chemical (nonenzymatic); (b) biochemical (enzymatic); or (c) physical, e.g., photochemical [3]. In practice, however, propesticidal substances are sometimes active without chemical modification when measured *in vitro*; nevertheless, their metabolites contribute sig-

Safer pesticides are those chemicals which have no or minimum acute or chronic toxicity to mammals and harmless to non-target organisms as well as non-persistent in the environment. The harmful effects of pesticides to non-target organisms can be overcome to certain extend by increasing the selectivity pf pesticide. Pesticide selectivity as such can be attained by either physical or biochemical means that is often combined in practice. In the former, only the target species is exposed to the control agent, and this can be accomplished by special formulation or precise application techniques [4]. In the later, selectivity is based on differences of the biochemical processes or target receptors of the pest and non-pest species. Furthermore, physiochemical factor such as differential uptake by and translocation within target and nontarget organisms can contribute to disparate biological activities [5]. Nevertheless, differences in metabolic pathways, which convert toxic xenobiotics into less harmful and readily excretable product, and in metabolic rates of various organisms are frequently the basis of selectivity.

A biologically inactive compound requires structural transformation(s) after application to become pesticidally active. Derivatives of known active ingredients that are converted to parent compound for activity. These are various process require for the activation of propesticide to the pesticidally active one [6]. Ideally these activation processes takes place only in the target organism. Even though it can also takes place in the environment, including soil and

This activation process is carried out in presence of certain enzyme or enzyme system. Enzymatic conversion of a proinsecticide to the active toxophore at the target tissue results

atmosphere. These can be one of or combination of the following four types:

**3.1. Activation by primary biochemical target**

nificantly to the overall biological activity of the applied material.

**2. Safer pesticides**

108 Insecticides - Agriculture and Toxicology

**3. Propesticides**

By detoxification system we meant the various processes carried out by the target organisms to detoxify any xenobiotics compound [9]. These are usually degradation processes and it includes oxidation, reduction, hydrolysis or conjugation reactions. Here, the xenobiotic compounds, i.e., propesticides are acted upon by these processes resulting in production of a more toxic material than the original one.

#### **3.3. Activation by symbiont metabolism**

Most of the insects harbor symbiotic or parasitic microorganisms in their guts or hemolymph which posses enzymes lacking in them [10]. The various endogenous xenobiotic processes can be brought under control to activate proinsecticidal agents in such organism specific fashion.

#### **3.4. Activation by symbiotic routes**

In this path enzymes are not involved. Here, the activation process is not metabolic but results in toxicity because a change in the propesticide occurs in biological milieu.


**2.3 Color Doppler imaging (CDI) of orbital (retro-bulbar) vessels**

Approximately 25% of patients with temporal artery biopsy (TAB) - proven GCA have ophthalmologic complications: usually unilateral visual loss (due to the vasculitic involvement of orbital vessels:

Schmidt compared the results of TAs-US examinations with the occurrence of visual ischemic complications (A-AION, CRAO, branch retinal artery occlusion, diplopia, or amaurosis fugax) in 222 consecutive patients with newly diagnosed, active GCA [21–24].

However, findings of TAs US did not correlate with eye complications [21–24].

This is the reason why we always have to exam the orbital (retrobulbar) vessels in GCA patients or in patients with unilateral abrupt visual loss [9–16] (**Figure 8A,B**).


*3.1.1 Tensile strength*

Tensile strength is the ability of a material to resist a force that tends to pull it apart. It is a basic provision for classifying the properties of given polymer materials at a specified loading rate and temperature. The ultimate tensile strength of PE at 0-70°C is 11 MPa–25 MPa, while its yield strength at the same temperature range is 6 MPa-30 MPa. This polymer property depends on percentage crystallinity, the thermodynamic stability of the primary PE chain, chain orientation, and packing density of the crystalline chain [17]. Polyethylene's strength, rigidity, friction, and hardness are low but have high impact strength and ductility. Three main tensile strengths are essential in the study of the properties of a polymer. The yield strength is the stress the PE can withstand without permanent deformation. The ultimate tensile strength is the maximum stress the PE can withstand, while the breaking strength is the stress coordinate on the strain–stress curve at the point of rupture.

Polyethylene shows excellent creep under an applied force. Elastic strain is the strain in the region of stress/strain curve of material under deformation, which recovers its shape on the release of applied stress. Elastic strain is reversible. Young's modulus is the slope of the graph ab covering from a being the origin of the stress–strain graph to point b, representing the reversible region of the stress–strain in **Figure 1**. After point b, strain is no longer proportional to stress, and the slope of the stress–strain graph changes at an increasing rate, and the strain is irreversible. The materials continue to deform after point c until it breaks at point d [18]. The percentage elongation we get during a tensile experiment is significant because it provides information on the ductility of the polyethylene under investigation. Materials with a high degree of elongation will exhibit high ductility. This is because the force necessary to sustain sample elongation and finally break the specimen changes very little at the yield point, and this makes the value of the yield strength and the breaking strength quite close [14].


**2. Screening and evaluation for action on protein folding**

view will not be undertaken here. The canonical neurodegenerative diseases are Alzheim‐ er's disease (AD), Parkinson's disease (PD), Huntington's disease, amyotrophic lateral sclerosis and the prion diseases. Although each involves distinct proteins, misfolding has a common role. Huntington's disease is caused by an excessive number of sequential gluta‐ mines near the N terminus of the protein huntingtin. An expansion of the natural glutamine stretch exceeding approximately 33 residues results in Huntington's symptoms and the age of onset of the disease declines with increasing residue number [10-12]. A likely reason for this is the increased propensity for aggregation with increasing polyglutamine length [13]. Amyotrophic lateral sclerosis is a disease associated with the death of the upper and lower motor neurons in the spinal cord, brain stem and motor cortex. The affected neurons accu‐ mulate aggregate protein inclusions that may be causing the cells to die. Mutations in genes encoding superoxide dismutase-1, TAR DNA binding protein 43 and fused in sarcoma/ translation in liposarcoma (FUS/TLS) are implicated in ALS (reviewed in [14]). Prion diseas‐ es display several interesting commonalities with these other neurodegenerative diseases. Prion diseases emerge from misfolded prion protein, which serves as a template for subse‐ quent misfolding of native prion protein with ensuing aggregation and neuron loss. The propagation of pathogenic misfolding from one protein molecule to its healthy neighbours by templating was long considered to be unique to prions; however, recent studies have suggested that molecular templating of misfolded proteins occurs with other neurodegener‐ ative diseases as well. Furthermore, templating is inferred by observation of their spatial spread from select foci in the brain [5, 15, 16]. It is notable that prion diseases, which are in‐ fectious, may occur in animals or humans of all ages, whereas the apparently non-transmis‐ sible protein folding diseases occur mainly with ageing. This implies that a key step in all protein folding disease may be the initial establishment of a misfolded protein in a neuron, whether it is a misfolded protein taken in at any age by infection or misfolding that emerges in a susceptible endogenous protein due to faltering proteostasis with ageing. Therefore, the prevention of misfolding and/or the promotion of disaggregation and refolding of misfold‐ ed proteins *in vivo* appear to be the most direct means of dealing with all these diseases.

96 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

In this chapter, the focus is on AD and PD, as these are the two most prevalent neurodege‐ nerative diseases, and the proteins that misfold in these diseases are well studied. AD ac‐ counts for approximately two thirds of all cases of dementia [17]; it leads to cognitive deficits in reasoning, memory, abstraction and motor skills, with eventual death. It is charac‐ terized at the molecular level by extracellular aggregates of a short peptide called amyloid beta (Aβ, and also called beta amyloid), which can form oligomers and larger fibrils with amyloid structure and later emerging intracellular neurofibrillary tangles that are composed of hyperphosphorylated tau protein (reviewed in [18]). In this chapter, Aβ normally refers to Aβ42, which is the 42-residue peptide and also appears to be most toxic form. There is in‐ creasing evidence that the Aβ oligomers have a key and likely causative role in AD. Most compelling are studies showing mutations in the regulatory region or encoded protein se‐ quence in amyloid precursor gene in families predisposed to AD [19-21]. In contrast to AD, PD has been found to involve the misfolding of α–synuclein (αS) into aggregates found in Lewy bodies and Lewy neurites.  These have relied upon the measurement of protein misfolding modulation as an early step in screening and evaluation of new active molecules or as a follow-up step in the evaluation of known and promising candidates because of their suspected effects on the diseases. Identification of a molecule as a folding modulator raises the possibility that it could address the cause of AD or PD, rather than only addressing their symptoms, which makes products identified in this way potentially valuable in prevention as well as in amelioration of existing disease.

#### **2.1.** *In vitro* **analysis of protein misfolding modulation**

For both Aβ and αS, the transition from the native state to an amyloid conformation is not direct, but involves steps or stages. These may include unfolding, oligomerization and further aggregation into amyloid fibrils, with the possibility of off-pathway aggregation or reversal of these processes [25, 26]. This is summarized in Fig. 1. Compounds may prevent misfolding and/or templating by inhibiting the unfolding of a natively folded protein, by promoting the folding of an unfolded form, by preventing amyloid formation or by diverting the protein toward an alternative aggregation pathway leading to a less harmful aggregate. A selection of assay approaches to misfolding evaluation will be compared here in terms of information value, throughput and versatility.

**Figure 1.** Overview of the general trajectory of protein misfolding with amyloid formation. Solid black arrows show pathway toward amyloid formation and stippled arrows show pathways of prevention or reversal of amyloid and re‐ lated oligomers. This is an original diagram based upon information in reviews [2, 23].

#### *2.1.1. Protein unfolding*

For the discovery and development of novel agents against diseases such as AD and PD, a fuller picture of the action of a compound can be obtained by investigating the distinct steps of unfolding, aggregation, misfolding and amyloid formation. Proteins may remain largely folded and still produce amyloid or similar aggregates, provided that a local aggregationprone sequence is exposed [27]. Nonetheless, for most proteins that readily form amyloid, it appears that that the loss of native folding is a required step [27]. Both Aβ and αS appear to have some unfolded or disordered character, but with regions that can become helical under appropriate conditions [28-30]. Inducing or preserving this helical conformation is a rational approach to preventing amyloid formation [29, 31, 32].

Folded and unfolded proteins differ in several characteristics that can be easily measured and that would allow folding modulators to be detected and evaluated. Some of the most common methods are summarized here. For example, proteins absorb right and left-circu‐ larly polarized light differently; this circular dichroism (CD) can be measured over a series of wavelengths to give informative spectra. The CD spectra of unfolded and folded proteins differ and those of proteins with predominantly α-helix and β-sheet secondary structure can be easily distinguished. In more detailed studies, the proportion of each major secondary structure in a protein can be calculated from the spectrum using software such as DICHRO‐ WEB [33]. Moreover, diagnostic features such as minima at wavelengths of 208 and 222 nm for α-helical proteins can be monitored in order to quantify changes in fold in response to temperature, solution components or other factors. To date, CD has been used for smallscale studies on protein folding and protein-ligand interaction because throughput is limited to a single sample at a time. The recent development of a high-speed automated CD spec‐ trometer (ACD, Applied Photophysics) may allow this approach to be employed more broadly in sample screening. An advantage of CD measurement is that it is an intrinsic property of a protein; thus, the spectra require no additive and the protein can be measured directly without accessory molecules or modifications. Another intrinsic feature of proteins that can be measured directly is fluorescence. Proteins with aromatic side chains fluoresce when exposed to UV light and this can undergo a shift in intensity or wavelength of intensi‐ ty maximum with folding. Fluorescence can also be measured using new devices that allow miniaturized measurements in a parallel manner using a similar rapid-throughput approach (e.g. the Optim 1000, Avacta Innovative Analysis). Likewise, differential binding of dyes such as SYPRO Orange allow protein forms with different exposure of hydrophobic resi‐ dues to solvent to be distinguished. In the case of SYPRO Orange, the dye is quenched when free in an aqueous solution, but it becomes unquenched when it interacts with the hydro‐ phobic regions that are exposed upon protein unfolding and its fluorescence increases as a consequence. This measurement can be performed efficiently on large numbers of samples using devices designed for quantitative PCR [34]. Measurements by these approaches allow protein folding to be assessed and changes in fold and/or fold stability to be examined. An informative and frequently employed measure of fold stability is the melting temperature (Tm), as proteins with more stable folds normally melt at higher temperatures. The Tm can be determined by any of the above techniques. A compound that stabilizes the folded con‐ formation of a protein would raise its Tm and this change can be easily quantified by the above approaches. A caveat with amyloid-forming proteins is that stabilized folding, as ex‐ hibited by a higher Tm, may not necessarily represent the native folding that is assumed to preclude amyloid formation. For this reason, CD is particularly valuable, as it also allows the secondary structure to be determined; a spectrum distinct from that of β-sheet would provide a reasonable indication that the stable form is native and unrelated to amyloid.

#### *2.1.2. Amyloid-like structure*

**Figure 1.** Overview of the general trajectory of protein misfolding with amyloid formation. Solid black arrows show pathway toward amyloid formation and stippled arrows show pathways of prevention or reversal of amyloid and re‐

For the discovery and development of novel agents against diseases such as AD and PD, a fuller picture of the action of a compound can be obtained by investigating the distinct steps of unfolding, aggregation, misfolding and amyloid formation. Proteins may remain largely folded and still produce amyloid or similar aggregates, provided that a local aggregationprone sequence is exposed [27]. Nonetheless, for most proteins that readily form amyloid, it appears that that the loss of native folding is a required step [27]. Both Aβ and αS appear to have some unfolded or disordered character, but with regions that can become helical under appropriate conditions [28-30]. Inducing or preserving this helical conformation is a rational

Folded and unfolded proteins differ in several characteristics that can be easily measured and that would allow folding modulators to be detected and evaluated. Some of the most common methods are summarized here. For example, proteins absorb right and left-circu‐ larly polarized light differently; this circular dichroism (CD) can be measured over a series of wavelengths to give informative spectra. The CD spectra of unfolded and folded proteins differ and those of proteins with predominantly α-helix and β-sheet secondary structure can be easily distinguished. In more detailed studies, the proportion of each major secondary

lated oligomers. This is an original diagram based upon information in reviews [2, 23].

98 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

approach to preventing amyloid formation [29, 31, 32].

*2.1.1. Protein unfolding*

It was through dye binding that amyloid plaques were first discovered. Fibrous aggregates in tissues stained blue when treated with acid followed by iodine and the aggregates were termed amyloid because they were believed to be starch based upon the colour develop‐ ment (reviewed in [35]). Although it is a misnomer, the name amyloid has endured and sev‐ eral different dyes have become invaluable in the study of protein misfolding and amyloid formation *in vitro*.

Thioflavin T (ThT), a benzothiazole dye, and Congo red, a diazobenzidine dye, are both commonly used to quantify the amount of amyloid protein present in a solution. ThT was first described to have amyloid-binding ability over 50 years ago [36] and it has since been in widespread use in the study of amyloidosis and the search for possible binding mole‐ cules. Congo red has also been in use for amyloid detection and quantification for several decades since its first description in that role [37]. Both of these dyes undergo a red shift in their fluorescence emission when they interact with amyloid fibrils. They cannot be used to distinguish between amyloid fibrils and smaller aggregates; however, they allow quantita‐ tive analysis and they continue to find wide use in amyloid analysis. Thioflavin S is a mix‐ ture of components derived from ThT and it has no spectral shift in absorbance or emission, which makes it suitable only for imaging and not for quantitation in solution because of high background [35]. Derivatives of N-arylaminonaphtalene including ANS and bis-ANS bind primarily to early stage aggregation in amyloid formation [38, 39]. They have not been as widely used as ThT and Congo Red, but they may find greater use with the increasing focus on aggregation pathways (see section 3 below).

Recently, there has been interest in identifying newer binding fluorophores that allow oligo‐ meric aggregates to be distinguished from fibrils. An example is the indole compound tryp‐ tophanol. It allows specific detection and quantitation of prefibrillar oligomeric Aβ because its fluorescence is quenched only in the presence of that form of Aβ with no similar quench‐ ing in the presence of Aβ fibrils [40]. Another is the carbocyanine dye JC-1 that distinguishes different aggregation states of αS [41]. JC-1 allows real-time tracking of αS aggregation, as different fluorescent signals are emitted for monomer and fibrillar binding and the ratio of the two signals allows for real-time visualization of αS aggregation [41].

Another recent development is the elaboration of antibodies and related agents that recog‐ nize proteins exclusively in their amyloid conformation, allowing the amyloid portion of the protein in question to be detected and quantified. A monoclonal antibody raised against αS was shown to recognize the protein in its amyloid form and, although the anticipated use is mainly *in vivo*, it showed effective recognition in an *in vitro* ELISA format [42]. Novel anti‐ bodies, termed gammabodies, were engineered by grafting a series of peptide sequences from Aβ into immunoglobulin variable regions [43]. These have found use in distinguishing oligomeric and amyloid forms of Aβ and this technology would be expected to be applicable to other aggregating proteins [43]. Since an amyloid-like structure is relevant to many dis‐ eases, a new capture and detection peptoid that behaves in a manner similar to an antibody offers unusual versatility. This peptoid, based upon a 6-amino acid stretch in the human PrP sequence, binds to the amyloid forms of Aβ, αS, amylin and serpin [44].

Model proteins that undergo transition to an amyloid-like conformation may also offer alternatives for amyloid formation-related studies [45]. For example, the 37-residue natively α-helical winter flounder (*Pseudopleuronectes americanus*) antifreeze protein wflAFP-6 (HPLC-6) adopts an amyloid-like structure during the freeze-thaw process [46, 47]. This may offer an interesting model system for the study of the conversion of a protein from its native form into amyloid fibrils [47, 48]. The ability to generate rapid amyloid formation in wflAFP-6 using the freeze-thaw process would offer low background compared with Aβ assays and the consistent α-helical nature of the native AFP may also allow initial unfolding steps to be more clearly evidenced than in proteins with less defined native structure. Nonetheless, a molecule found to be an amyloid-relevant folding modulator using AFP amyloid-like transition assays may be universal or nearly so, as in the case of the peptoid detection system described above, or it may be specific to the wflAFP-6 and have no effect on other amyloid-forming proteins. Therefore, in spite of its potential advantages, further study of the antifreeze protein model would be required before its adoption in an assay platform.

#### *2.1.3. Protein interaction during amyloid formation*

first described to have amyloid-binding ability over 50 years ago [36] and it has since been in widespread use in the study of amyloidosis and the search for possible binding mole‐ cules. Congo red has also been in use for amyloid detection and quantification for several decades since its first description in that role [37]. Both of these dyes undergo a red shift in their fluorescence emission when they interact with amyloid fibrils. They cannot be used to distinguish between amyloid fibrils and smaller aggregates; however, they allow quantita‐ tive analysis and they continue to find wide use in amyloid analysis. Thioflavin S is a mix‐ ture of components derived from ThT and it has no spectral shift in absorbance or emission, which makes it suitable only for imaging and not for quantitation in solution because of high background [35]. Derivatives of N-arylaminonaphtalene including ANS and bis-ANS bind primarily to early stage aggregation in amyloid formation [38, 39]. They have not been as widely used as ThT and Congo Red, but they may find greater use with the increasing

Recently, there has been interest in identifying newer binding fluorophores that allow oligo‐ meric aggregates to be distinguished from fibrils. An example is the indole compound tryp‐ tophanol. It allows specific detection and quantitation of prefibrillar oligomeric Aβ because its fluorescence is quenched only in the presence of that form of Aβ with no similar quench‐ ing in the presence of Aβ fibrils [40]. Another is the carbocyanine dye JC-1 that distinguishes different aggregation states of αS [41]. JC-1 allows real-time tracking of αS aggregation, as different fluorescent signals are emitted for monomer and fibrillar binding and the ratio of

Another recent development is the elaboration of antibodies and related agents that recog‐ nize proteins exclusively in their amyloid conformation, allowing the amyloid portion of the protein in question to be detected and quantified. A monoclonal antibody raised against αS was shown to recognize the protein in its amyloid form and, although the anticipated use is mainly *in vivo*, it showed effective recognition in an *in vitro* ELISA format [42]. Novel anti‐ bodies, termed gammabodies, were engineered by grafting a series of peptide sequences from Aβ into immunoglobulin variable regions [43]. These have found use in distinguishing oligomeric and amyloid forms of Aβ and this technology would be expected to be applicable to other aggregating proteins [43]. Since an amyloid-like structure is relevant to many dis‐ eases, a new capture and detection peptoid that behaves in a manner similar to an antibody offers unusual versatility. This peptoid, based upon a 6-amino acid stretch in the human PrP

Model proteins that undergo transition to an amyloid-like conformation may also offer alternatives for amyloid formation-related studies [45]. For example, the 37-residue natively α-helical winter flounder (*Pseudopleuronectes americanus*) antifreeze protein wflAFP-6 (HPLC-6) adopts an amyloid-like structure during the freeze-thaw process [46, 47]. This may offer an interesting model system for the study of the conversion of a protein from its native form into amyloid fibrils [47, 48]. The ability to generate rapid amyloid formation in wflAFP-6 using the freeze-thaw process would offer low background compared with Aβ assays and the consistent α-helical nature of the native AFP may also allow initial unfolding steps to be more

the two signals allows for real-time visualization of αS aggregation [41].

sequence, binds to the amyloid forms of Aβ, αS, amylin and serpin [44].

focus on aggregation pathways (see section 3 below).

100 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

As the interaction between locally or entirely misfolded monomers appears critical to nonnative protein association, there is interest in identifying molecules that directly interfere with that process. Therefore, assays for binding competition or impedance have been developed. A convenient new microplate assay employs fluorescently labelled Aβ. Unlabelled Aβ is coated onto the plate surface and then the labelled Aβ is applied in solution and allowed to bind [49]. After unbound material is washed off, the fluorescence remaining can be used to determine the level of interaction of Aβ [49]. Although the aggregation state of the Aβ in solution or on the plate is not indicated, the assay indicates the interaction between in-solution and on-plate Aβ that is inhibitable, which can be taken to represent either oligomer formation or assembly into larger fibrils. Thus, molecules that affect any part of this process can be readily identified. Nonetheless, with the aggregation state of the binding units unclear except where Aβ frag‐ ments are used, this assay does not indicate which step(s) in assembly of the monomeroligomer-fibril are being affected.

Aβ oligomerization and fibril formation can be challenging to study *in vitro* using dyes and other methods because of spurious background oligomer formation and the difficulty in determining how much oligomer may already have formed in a starting sample [50]. For these reasons, assays that avoid these problems have garnered interest. In one example, a procedure was developed involving a semi-denaturing detergent-agarose gel electrophoresis followed by blotting and immunological detection with an appropriate antibody recognizing the protein in all its forms [51]. The method allows amyloid aggregate size distributions to be determined and the user can distinguish among the monomeric, oligomeric and large aggregated (likely fibril) forms. A drawback is that it involves a vacuum transfer of proteins from the gel to a PVDF membrane. A derived method employing capillary blotting to a nitrocellulose mem‐ brane, reminiscent of nucleic acid blotting, has made the assay more precise and scaleable for screening purposes [52]. These methods allow the association state of the starting material to be determined and the effect of any product to be evaluated in comparison. Another approach is to eliminate the possibility of aggregates in the starting solution. An example is an assay based upon the expression of the Aβ sequence in tandem with green fluorescent protein (GFP) as a fusion within a bacterial cell [53]. Aggregation of Aβ precludes GFP folding and so there is no fluorescence, whereas addition of a molecule impeding the aggregation of Aβ results in GFP folding to its native structure with consequent fluorescence. A more directly quantifiable *in vitro* form of this assay was developed using the Venus yellow fluorescent protein (vYFP) instead of GFP, which allows denaturation and refolding under precisely controlled conditions to detect molecules that promote or inhibit aggregation [50].

#### **2.2. Cell- and organism-based analyses**

Once an active extract is identified by efficient *in vitro* assay and similar assay-guided frac‐ tionation has revealed the class of molecules or the molecule that is responsible, determina‐ tion of effects *in vivo* is of interest. Using culture models such as the PC12 primary neuronal cells or the SH-SY5Y neuroblastoma cell line, effects of active molecules can be determined. In a first approach, cells can be treated by transfection or other means to harbour high levels of the protein of interest and then the effect of active molecules on cell survival can be meas‐ ured using one of several commercially available assays for dead cells, which measure lac‐ tate dehydrogenase leakage or other indicative parameters. However, more precise information can be gleaned by histological analysis using stains such as Congo red or thio‐ flavin S, described above, that reveal the presence of amyloid accumulation. Immunohisto‐ chemistry using appropriate antibodies against Aβ or αS can be even more informative, as some allow distinction of oligomeric denatured forms from amyloid fibrils.

Cell-based assays can also provide an indication of the possibility of a molecule crossing the blood-brain barrier. Although Aβ circulates in the serum [54] and αS is expressed in red blood cells [55], it is considered preferable for a compound to have direct access to the brain where the pathology takes place. Therefore, blood-brain barrier models in cell culture, such as the MDR-MCCK monolayer [56], are available to predict the permeability for specific substances. Nonetheless, blood brain barrier physiology is more complex than the models or direct plasma/brain ratio determinations would predict [57]. For that reason, results must be interpreted with care.

Animal models provide critical understanding in terms of the distribution, mode of action and effectiveness of an active molecule. Besides the well-known mouse models for many neuro‐ degenerative diseases, there are valuable invertebrate and fish models. An αS-transgenic *Drosophila* fly model is available for PD [58] and an Aβ-transgenic model for AD [59] The nematode worm, *Caenorhabditis elegans,* can be used to study AD and PD [60, 61]. Zebrafish (*Danio rerio*) have an Aβ protein precursor-encoding gene [62] and synuclein genes have been identified in this species [63]. Zebrafish may also become informative for studies of αS once the encoded proteins in this organism are better understood.

## **3. Protein folding modulators from common terrestrial natural products**

A large number of molecules have been shown to influence the aggregation of Aβ [18] and a growing number have shown effects on αS (discussed below). Although no effective thera‐ py for PD or AD has emerged to date, progress on a number of natural products is encour‐ aging. This situation is not surprising, as synthetic and combinatorial chemistry normally rely on a limited number of structural scaffolds and random permutations of structures and synthesizable units [64]. In contrast, natural molecules are highly diverse and they have been subject to evolutionary pressure under distinct conditions in which valuable activities have been selected. Therefore, they may offer a combination of variety and biological preselection that may make a therapeutic activity more likely [64].

#### **3.1. Natural protein folding modulators from terrestrial sources**

Several natural molecules have garnered interest because they modulate amyloid formation by Aβ (reviewed in [18, 65, 66]). A number of salient examples involving Aβ and αS are presented here.

#### *3.1.1. Curcumin*

**2.2. Cell- and organism-based analyses**

102 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

interpreted with care.

Once an active extract is identified by efficient *in vitro* assay and similar assay-guided frac‐ tionation has revealed the class of molecules or the molecule that is responsible, determina‐ tion of effects *in vivo* is of interest. Using culture models such as the PC12 primary neuronal cells or the SH-SY5Y neuroblastoma cell line, effects of active molecules can be determined. In a first approach, cells can be treated by transfection or other means to harbour high levels of the protein of interest and then the effect of active molecules on cell survival can be meas‐ ured using one of several commercially available assays for dead cells, which measure lac‐ tate dehydrogenase leakage or other indicative parameters. However, more precise information can be gleaned by histological analysis using stains such as Congo red or thio‐ flavin S, described above, that reveal the presence of amyloid accumulation. Immunohisto‐ chemistry using appropriate antibodies against Aβ or αS can be even more informative, as

Cell-based assays can also provide an indication of the possibility of a molecule crossing the blood-brain barrier. Although Aβ circulates in the serum [54] and αS is expressed in red blood cells [55], it is considered preferable for a compound to have direct access to the brain where the pathology takes place. Therefore, blood-brain barrier models in cell culture, such as the MDR-MCCK monolayer [56], are available to predict the permeability for specific substances. Nonetheless, blood brain barrier physiology is more complex than the models or direct plasma/brain ratio determinations would predict [57]. For that reason, results must be

Animal models provide critical understanding in terms of the distribution, mode of action and effectiveness of an active molecule. Besides the well-known mouse models for many neuro‐ degenerative diseases, there are valuable invertebrate and fish models. An αS-transgenic *Drosophila* fly model is available for PD [58] and an Aβ-transgenic model for AD [59] The nematode worm, *Caenorhabditis elegans,* can be used to study AD and PD [60, 61]. Zebrafish (*Danio rerio*) have an Aβ protein precursor-encoding gene [62] and synuclein genes have been identified in this species [63]. Zebrafish may also become informative for studies of αS once

**3. Protein folding modulators from common terrestrial natural products**

A large number of molecules have been shown to influence the aggregation of Aβ [18] and a growing number have shown effects on αS (discussed below). Although no effective thera‐ py for PD or AD has emerged to date, progress on a number of natural products is encour‐ aging. This situation is not surprising, as synthetic and combinatorial chemistry normally rely on a limited number of structural scaffolds and random permutations of structures and synthesizable units [64]. In contrast, natural molecules are highly diverse and they have been subject to evolutionary pressure under distinct conditions in which valuable activities

some allow distinction of oligomeric denatured forms from amyloid fibrils.

the encoded proteins in this organism are better understood.

Curcumin is a natural polyphenol found in the traditional Indian spice turmeric. This molecule has been extensively investigated, in part because of its role in traditional Ayurvedic medicine [67]. Numerous studies have shown curcumin to have wide ranging biological activities, including Aβ folding modulation. Curcumin shows promising interaction with Aβ. For example, using an oligomer-specific antibody for immunoblotting, cucurmin was found prevent Aβ oligomerization and toxicity *in vitro*. In the same study, curcumin was shown to bind to amyloid plaques in brain sections of mice transgenic for the human amyloid precursor protein [68]. In addition, mature fibrils of the slightly shorter Aβ<sup>40</sup> peptide were destabilized by curcumin [68]. Curcumin and other polyphenols, including tannic acid, rosmarinic acid, and myricetin, were shown to inhibit fibril formation and destabilized mature fibrils for both Aβ and αS [69]. As a result, curcumin has been a component of interest in clinical trials and, most recently the exclusive molecule in a trial for prevention of early cognitive decline and abnormal Aβ accumulation in the brain [70]. If an effect is observed, it would be interesting to see if it acts directly upon misfolding at the dosage used in subjects.

#### *3.1.2. Epigallocatechin-3-gallate*

In terms of bioactive molecules in traditional medicine, perhaps the most recognized is (-) epigallocatechin 3-gallate (EGCG) that is found in unfermented (green) tea (*Camellia sinensis*). EGCG is a pleiotropic polyphenolic molecule with interesting protein folding modulation activity. In *C. elegans* expressing human Aβ in muscle cells, western blotting analysis using an anti-Aβ antibody showed EGCG to inhibit Aβ oligomerization [71]. Studies of Aβ and αS using ThT fluorescence, electron microscopy and other biophysical methods showed interaction with EGCG and ensuing "off-pathway" aggregation into spherical oligomers, which thereby prevented the formation of typical aggregation intermediates and amyloid fibrils by both proteins [25]. Further investigation revealed EGCG to bind directly to the β-sheet structure in the amyloid fibrils formed by Aβ and by αS and to direct their rearrangement into smaller non-toxic protein aggregates [26]. A clinical trial is planned to examine the effect of EGCG on early stage AD [72] and this may have effects based upon the protein folding modulation described here, other properties of the molecule, or a combination thereof if adequate levels are attained in the brain. Other tea compounds may offer similar protection. Theaflavins are formed by oxidation of EGCG during the fermentation process to produce black tea. These derivatives were shown to have similar activity to EGCG in modulating the polymerization of Aβ and αS [73].

#### *3.1.3. Carotenoids and related molecules*

Carotenoids (pro-vitamin A) and vitamin A are present in many commonly consumed foods. The *in vitro* examination of vitamin A and β-carotene activity on AB fibril formation, as measured using dye binding and electron microscopy suggest protective effects that appear to result from binding to a region in the C-terminal half of the peptide [74, 75]. This is consistent with reports that vitamin A and related molecules inhibit the progression of AD symptoms [76]. Also using dye binding and electron microscopy, vitamin A and β-carotene were shown to inhibit the formation of αS amyloid fibrils and to destabilize existing fibrils [77].

#### *3.1.4. Scyllo-inositol*

Another natural molecule that has garnered interest is scyllo-inositol, which is abundant in the coconut palm (*Cocos nucifera*) [78]. Scyllo-inositol is a stereoisomer of myo-inositol that stabilizes a non-toxic oligomeric aggregate of Aβ [78-80]. This is in contrast to phosphatidyli‐ nositol, which was found to promote fibrillogenesis and membrane insertion of Aβ [78]. Scylloinositol also protected against Aβ oligomer-induced inhibition of long-term potentiation [80]. A clinical trial of scyllo-inositol was inconclusive after the highest doses were discontinued [81]. Nonetheless, it remains a compound of interest provided that drawbacks can be ad‐ dressed.

#### *3.1.5. Emerging sources*

In addition to the well-studied molecules above, there are growing possibilities for other sources of folding modulators. For example, another source of interesting polyphenols is the wolf berry, *Lycium barbarum*, which is used in traditional Chinese medicine and was shown to protect against Aβ-related cell death [82]. Extracts obtained from other species used in traditional Chinese medicine, including cat's claw (*Uncaria rhynchophylla*) and tree peony (*Paeonia suffruticosa*) have shown relevant activity [83-85]. The tree peony contains 1,2,3,4,6 penta-O-galloyl-beta-D-glucopyranose, which prevented Aβ fiber formation and reversed the process in existing fibers [84]. In addition, other polyphenols procyanidins from apple (*Malus domestica*), silymarin from thistle (*Carduus marianus*) and reservatrol from red grapes (*Vitis sp.*) show interesting activities that may warrant further study [86-88].

#### **3.2. Challenges with natural folding modulators**

Although much attention has been brought to the natural folding modulators present in various terrestrial species, none have yet translated into meaningful therapies. The best prospect currently appears to be EGCG [89], which nonetheless presents challenges.

#### *3.2.1. Challenges in delivery of natural products*

EGCG is susceptible to decomposition during storage prior to being consumed, much of it may be destroyed by normal stomach acid during digestion, it is not highly bioavailable and it carries the possibility of hepatotoxicity (reviewed in [90]). Therefore, ideal handling, delivery and dosage would need to be determined before this molecule could be employed successfully to manage disease-related misfolding in animals or humans. Nonetheless, studies have suggested that the use of green tea is negatively correlated to the prevalence of cognitive impairment [91] and that tea shows a dose-dependent effect in protection from PD [92]. Therefore, even with regular storage, preparation and consumption of tea as part of a normal diet, it appears that tea components, and likely EGCG or its derived theaflavins, have a measure of benefit. It is not yet clear whether the reported benefits involve the modulation of protein misfolding, and this would be an area for future study. Curcumin presents challenges similar to those of EGCG and derivatives. Dietary ingestion of curcumin appears sufficient to offer some measure of protection from neurodegenerative diseases in populations that consume it (reviewed in [93]), suggesting its bioavailability. Yet, bioavailability was found to be limiting and work has been undertaken toward improving delivery of curcumin by combining it with phosphatidyl choline, olive oil and stearic acid [94]. Similarly, plasma levels of EGCG following oral consumption were higher when the product was encapsulated in chitosan nanoparticles [95]. The promising suggestion is that formulation and delivery may be optimized to enhance availability of these protein folding modulators *in vivo*. Other compounds such as carotenoids are bioavailable and cross the blood-brain barrier as well, possibly making them convenient for the establishment of protein folding modulators [96].

#### *3.2.2. Requirement for crossing the blood-brain barrier*

*3.1.3. Carotenoids and related molecules*

104 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

*3.1.4. Scyllo-inositol*

dressed.

*3.1.5. Emerging sources*

Carotenoids (pro-vitamin A) and vitamin A are present in many commonly consumed foods. The *in vitro* examination of vitamin A and β-carotene activity on AB fibril formation, as measured using dye binding and electron microscopy suggest protective effects that appear to result from binding to a region in the C-terminal half of the peptide [74, 75]. This is consistent with reports that vitamin A and related molecules inhibit the progression of AD symptoms [76]. Also using dye binding and electron microscopy, vitamin A and β-carotene were shown

Another natural molecule that has garnered interest is scyllo-inositol, which is abundant in the coconut palm (*Cocos nucifera*) [78]. Scyllo-inositol is a stereoisomer of myo-inositol that stabilizes a non-toxic oligomeric aggregate of Aβ [78-80]. This is in contrast to phosphatidyli‐ nositol, which was found to promote fibrillogenesis and membrane insertion of Aβ [78]. Scylloinositol also protected against Aβ oligomer-induced inhibition of long-term potentiation [80]. A clinical trial of scyllo-inositol was inconclusive after the highest doses were discontinued [81]. Nonetheless, it remains a compound of interest provided that drawbacks can be ad‐

In addition to the well-studied molecules above, there are growing possibilities for other sources of folding modulators. For example, another source of interesting polyphenols is the wolf berry, *Lycium barbarum*, which is used in traditional Chinese medicine and was shown to protect against Aβ-related cell death [82]. Extracts obtained from other species used in traditional Chinese medicine, including cat's claw (*Uncaria rhynchophylla*) and tree peony (*Paeonia suffruticosa*) have shown relevant activity [83-85]. The tree peony contains 1,2,3,4,6 penta-O-galloyl-beta-D-glucopyranose, which prevented Aβ fiber formation and reversed the process in existing fibers [84]. In addition, other polyphenols procyanidins from apple (*Malus domestica*), silymarin from thistle (*Carduus marianus*) and reservatrol from red grapes (*Vitis*

Although much attention has been brought to the natural folding modulators present in various terrestrial species, none have yet translated into meaningful therapies. The best

EGCG is susceptible to decomposition during storage prior to being consumed, much of it may be destroyed by normal stomach acid during digestion, it is not highly bioavailable and it carries the possibility of hepatotoxicity (reviewed in [90]). Therefore, ideal handling, delivery and dosage would need to be determined before this molecule could be employed successfully

prospect currently appears to be EGCG [89], which nonetheless presents challenges.

*sp.*) show interesting activities that may warrant further study [86-88].

**3.2. Challenges with natural folding modulators**

*3.2.1. Challenges in delivery of natural products*

to inhibit the formation of αS amyloid fibrils and to destabilize existing fibrils [77].

It is widely accepted that molecules must cross the blood-brain barrier in order to have relevant biological effects on neurodegenerative diseases; however, interesting new findings are suggesting that this may not always be the case. In a study using radiolabeled Aβ40 adminis‐ tered to the brains of rats, elevated peripheral Aβ40 levels reduced protein clearance from the brain [97]. Conversely, a decline of Aβ levels in blood plasma can lead to cognitive improve‐ ment, presumably by reducing Aβ levels in the brain [98, 99]. Furthermore, genetic study of the presenilin gene encoding a secretase with a role in Aβ synthesis revealed a heritable expression level in the liver, suggesting a role of liver-expressed protein in brain Aβ levels [100]. Follow-up investigation using ST571, a drug that lowers peripheral Aβ levels but that does not cross the blood-brain barrier, showed reduction in brain Aβ [100]. Although these were studies of complete Aβ levels rather than the different conformations and aggregates, together they do imply that modulation of peripheral oligomerized and fibrillar forms of Aβ, which would facilitate clearance, could affect the brain ratios of differently associated Aβ forms. Therefore, protein folding modulators that do not cross the blood-brain barrier may still be worth considering in the prevention and treatment of disease. These studies have the drawback of assuming a stable and functional blood-brain barrier over the course of these diseases; however, other findings suggest that functioning of the blood-brain barrier may become compromised over the course of AD and other neurodegenerative diseases (reviewed in [101]), which may result in inappropriate dosages of molecules from plasma. Therefore, the prevention phase may be critical to the benefit of any molecule that acts peripherally to modulate Aβ, as that is the point at which normal transfer of molecules such as Aβ across the barrier can occur. Alternatively, the compromised blood-brain-barrier during the disease phase may allow therapeutic molecules to reach the brain more efficiently. These possibilities require further study.