*2.2. Laser Peening Treatment*

The LPwC treatment was performed in the Department of Industrial Engineering Laboratories of the University of Bologna (Bologna, Italy). The setup used for the laser shock peening is shown in Figure 3.

**Figure 3.** Setup used for the laser shock peening without coating treatment.

The laser source was provided by a Nd:YAG pulsed laser produced by Quanta Systems (Samarate, Italy). Its settings are as follows: wavelength of 1064 nm, nominal pulse energy of 1.8 J, and a pulse duration equal to 8 ns. The nominal laser power density was 7 GW/cm2.

The peening was applied in the central part of the notch on a rectangular area of 40 × 4 mm2, as shown in Figure 4a. A circular laser spot size of 2 mm in diameter was used, with a distance of 0.25 mm between two adjacent spot-centers. A spot overlapping of 75%, as shown in Figure 4b, was chosen so as to attain complete coverage of the treated area on a single layer, as discussed in the literature [13]. This resulted in a pulse density equal to 1600 pulses/cm2.

**Figure 4.** (**a**) Detailed view of the peened zone. (**b**) The treated area is bounded in the red box.


*Article* **Acceleration of Plasma Nitriding at 550** ◦**C with Rare Earth on the Surface of 38CrMoAl Steel**

**Dongjing Liu 1 , Yuan You 1,2,\*, Mufu Yan 2 , Hongtao Chen 3 , Rui Li 1 , Lin Hong 1 and Tingjie Han 1**

1 School of Materials Science and Engineering, Qiqihar University, Qiqihar 161006, China;

**Abstract:** In order to explore the effect of the addition of rare earth (RE) to a steel microstructure and the consequent performance of a nitrided layer, plasma nitriding was carried out on 38CrMoAl steel in an atmosphere of NH3 at 550 ◦C for 4, 8, and 12 h. The modified layers were characterized using an optical microscope (OM), a microhardness tester, X-ray diffraction (XRD), a scanning electron microscope (SEM), a transmission electron microscope (TEM), and an electrochemical workstation. After 12 h of nitriding without RE, the modified layer thickness was 355.90 µm, the weight gain was 3.75 mg/cm2 , and the surface hardness was 882.5 HV0.05. After 12 h of RE nitriding, the thickness of the modified layer was 390.8 µm, the weight gain was 3.87 mg/cm2 , and the surface hardness was 1027 HV0.05. Compared with nitriding without RE, the ε-Fe2-3N diffraction peak was enhanced in the RE nitriding layer. After 12 h of RE nitriding, La, LaFeO3 , and a trace amount of Fe2O3 appeared. The corrosion rate of the modified layer was at its lowest (15.089 × 10−2 mm/a), as was the current density (1.282 × 10−5 A/cm2 ); therefore, the corrosion resistance improved.

**Keywords:** 38CrMoAl steel; plasma nitriding; microhardness; corrosion resistance; modified layer

#### **1. Introduction**

Plasma nitriding is a widely used chemical heat treatment technology. This method uses the active nitrogen atoms generated during the cathode sputtering process at a relatively low temperature (350–570 ◦C) to accumulate on the surface of a workpiece, diffuse into the substrate, and finally form a nitriding-modified layer with excellent performance. For a variety of alloy materials, it can improve surface hardness, fatigue resistance, wear resistance, and corrosion resistance [1–7]. At present, the methods most commonly used to increase the thickness of the nitride layer are to increase the nitriding temperature or to extend the process time [8]. However, these methods have some disadvantages, such as the coarsening of the structure and reduced hardness [9,10]. Therefore, the question of how to increase the thickness of the modified layer in a short time presents a difficult challenge. Researchers have found that adding some catalysts can improve the efficiency of nitriding. Among them, rare earth (RE) elements have been proven to be effective catalysts for chemical heat treatment [11].

In the early 1980s, researchers studied the chemical heat treatment of RE. In the following decades, scholars explored the effects of RE catalysis and microalloying [12]. Many researchers observed that in the process of gas carburizing, gas carbonitriding, gas nitrocarburizing, and plasma nitriding, RE elements can diffuse deeper to the surface [13–15]. At the same time, the involvement of RE elements causes the nitrogen to diffuse deeper into the nitrided layer. Yan et al. [16,17] showed that rare earth elements induced nitriding and observed similar catalytic effects; they also studied the general catalytic effect of rare

**Citation:** Liu, D.; You, Y.; Yan, M.; Chen, H.; Li, R.; Hong, L.; Han, T. Acceleration of Plasma Nitriding at 550 ◦C with Rare Earth on the Surface of 38CrMoAl Steel. *Coatings* **2021**, *11*, 1122. https://doi.org/10.3390/ coatings11091122

Academic Editor: Kyong Yop Rhee

Received: 3 August 2021 Accepted: 10 September 2021 Published: 16 September 2021

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

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

earth elements on the nitriding process of various steels, including stainless steel [18,19] and alloy steel [20–22].

Due to its good wear resistance, high fatigue strength and high strength, 38CrMoAl steel is widely used in many industries [23,24]. However, in some applications, it cannot meet the requirements of high surface hardness and corrosion resistance. Therefore, surface modification approaches are used to improve the structure and properties of 38CrMoAl steel. However, there are few studies on the surface modification of 38CrMoAl steel, especially with the addition of RE. Therefore, it is very important to study the effect of plasma nitriding following the addition of RE on the thickness, surface hardness, phase composition, and corrosion resistance of a modified 38CrMoAl steel layer.

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

The test material was 38CrMoAl steel with a thickness of 5 mm and a diameter of φ 20 mm, and its chemical compositions are shown in Table 1. The steel was solution-treated in an SXL-1400C box-type electric furnace and austenitized at 940 ◦C for 1 h, after which oil quenching treatment was performed. A QG-1 metallographic cutting machine was used to cut the solution-treated sample into 5 mm thin slices. The surface was polished with 120# water sandpaper. Next, absolute ethanol was used for ultrasonic cleaning for 10–15 min. The sample was then blow dried with a hair dryer before being wiped with a dust-free cloth, weighed, and tied together with a thin iron wire. The 1 × 1 × 1 cm3 rare earth (RE) lanthanum was cut into 1/8 cube blocks, and the RE was hung on the cathode platform with the iron wire, in order to maximize the sputtering of RE.

**Table 1.** Chemical composition of 38CrMoAl steel (wt.%).

Before plasma nitriding in the LDMC-30AFZ ion nitriding furnace, the furnace was evacuated to below 10 pa. The nitriding atmosphere was NH3 (100 mL/min) at 550 ◦C for 4, 8, and 12 h. A CPA-225D electronic balance (Sai Dolis scientific instruments Beijing co., Ltd., Beijing, China) was used to measure the mass before and after the nitriding sample. After nitriding, the surface of the sample was polished with 120#-2000# water sandpaper. The polished sample was corroded with 4% nitric acid alcohol solution for 15 s, rinsed with alcohol, and dried. The cross-section structure and thickness of the modified layer was observed with a 9XB-PC metallographic microscope, the surface morphology was observed with a ThermoScientific™Apreo C (TEM, JEOL Ltd, Tokyo, Japan) model field emission scanning electron microscope, and a HV-1000IS microhardness tester (Shanghai Jvjing Precision Instrument Manufacturing Co., Ltd., Shanghai, China) was used to test the hardness of the modified layer. The phase composition of the sample surface after nitrogen treatment was characterized by a D8 Advance X-ray diffractometer (Bruker, Karlsruhe, Germany). Anodic polarization tests were employed to estimate the corrosion resistance of the specimens in a 3.5 wt.% NaCl solution. The reference electrode was Ag/AgCl and a platinum column was used as the auxiliary electrode. A corrosion resistance test was performed in a CHI604E B16276 electrochemical workstation.

#### **3. Results and Discussion**

#### *3.1. Microstructure and Phase Composition of Modified Layer*

Figure 1 shows the metallographic microstructure of the modified layer of 38CrMoAl steel without rare earth (RE) and with rare earth. The nitrided layer without RE was clearly divided from the matrix, and the microstructure of the nitrided layer was uniform. After nitriding for 4, 8, and 12 h, the thickness of the modified layer was 222.2, 345.7, and 355.9 µm, respectively; the thickness of the modified layer increased gradually with the extension of nitride time. However, at the same temperature of 550 ◦C, after the addition

of RE, the nitrided layer thickened with time to 252.4, 358.0, and 391.8 µm (4, 8, 12 h), respectively., The thickened histograms of the nitrided modified layer with RE and without RE after nitriding for 4, 8, and 12 h are shown in Figure 2. The modified layer was thickened by 30.2, 12.3, and 34.9 µm at 4, 8, and 12 h, respectively. Compared with the nitriding layer without the addition of RE, the thickening rates of the RE nitriding layer were 13.6%, 3.50% and 9.80%, respectively, indicating that it was easier to obtain a thicker nitriding layer with the addition of RE at the same nitriding time intervals. The addition of RE in nitrided steel increased the concentration of microscopic crystal defects, such as vacancies and dislocations, in the modified layer and caused lattice distortion of the iron atoms around it, which was beneficial for the adsorption and diffusion of the nitrogen atoms. Therefore, in a short period of time, a higher nitrogen concentration was formed in the matrix, providing a very high nitrogen potential and concentration gradient for the inward diffusion of nitrogen atoms, which also provided good thermodynamic conditions for the diffusion of nitrogen atoms. of RE, the nitrided layer thickened with time to 252.4, 358.0, and 391.8 μm (4, 8, 12 h), respectively., The thickened histograms of the nitrided modified layer with RE and without RE after nitriding for 4, 8, and 12 h are shown in Figure 2. The modified layer was thickened by 30.2, 12.3, and 34.9 μm at 4, 8, and 12 h, respectively. Compared with the nitriding layer without the addition of RE, the thickening rates of the RE nitriding layer were 13.6%, 3.50% and 9.80%, respectively, indicating that it was easier to obtain a thicker nitriding layer with the addition of RE at the same nitriding time intervals. The addition of RE in nitrided steel increased the concentration of microscopic crystal defects, such as vacancies and dislocations, in the modified layer and caused lattice distortion of the iron atoms around it, which was beneficial for the adsorption and diffusion of the nitrogen atoms. Therefore, in a short period of time, a higher nitrogen concentration was formed in the matrix, providing a very high nitrogen potential and concentration gradient for the inward diffusion of nitrogen atoms, which also provided good thermodynamic conditions for the diffusion of nitrogen atoms.

clearly divided from the matrix, and the microstructure of the nitrided layer was uniform. After nitriding for 4, 8, and 12 h, the thickness of the modified layer was 222.2, 345.7, and 355.9 μm, respectively; the thickness of the modified layer increased gradually with the extension of nitride time. However, at the same temperature of 550 °C, after the addition

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As shown in the XRD patterns of Figure 1a–c, which correspond to Figure 3a without RE nitridating, the main phase of the modified layer without RE nitridation was γ'-Fe4N and the secondary phase was ε-Fe2-3N. However, La and LaFeO3 oxides were detected in the XRD patterns shown in Figure 1d–f, corresponding to Figure 3b with the addition of RE nitridation for 12 h. Because LaFeO3 showed a strong adsorption effect on N atoms [25], the thickening rate of the modified layer increased with the addition of RE nitriding within the same timeframe. As shown in the XRD patterns of Figure 1a–c, which correspond to Figure 3a without RE nitridating, the main phase of the modified layer without RE nitridation was γ'-Fe4N and the secondary phase was ε-Fe2-3N. However, La and LaFeO3 oxides were detected in the XRD patterns shown in Figure 1d–f, corresponding to Figure 3b with the addition of RE nitridation for 12 h. Because LaFeO3 showed a strong adsorption effect on N atoms [25], the thickening rate of the modified layer increased with the addition of RE nitriding within the same timeframe.

**Figure 1.** Microstructure of modified layer of 38CrMoAl steel under different nitriding conditions: (**a**) PN, 4 h; (**b**) PN, 8 h; (**c**) PN, 12 h; (**d**) RE, 4 h; (**e**) RE, 8 h; (**f**) RE, 12 h. **Figure 1.** Microstructure of modified layer of 38CrMoAl steel under different nitriding conditions: (**a**) PN, 4 h; (**b**) PN, 8 h; (**c**) PN, 12 h; (**d**) RE, 4 h; (**e**) RE, 8 h; (**f**) RE, 12 h.

**Figure 2.** Cross-section thickening histogram of nitrided layer of 38CrMoAl Steel without and with RE. **Figure 2.** Cross-section thickening histogram of nitrided layer of 38CrMoAl Steel without and with RE. **Figure 2.** Cross-section thickening histogram of nitrided layer of 38CrMoAl Steel without and with RE.

**Figure 3.** XRD pattern of 38CrMoAl steel without and with RE nitriding. (**a**) Without RE (**b**) With RE. **Figure 3.** XRD pattern of 38CrMoAl steel without and with RE nitriding. (**a**) Without RE (**b**) With RE. **Figure 3.** XRD pattern of 38CrMoAl steel without and with RE nitriding. (**a**) Without RE (**b**) With RE.

#### *3.2. Weight Gain and Microhardness of Modified Layer 3.2. Weight Gain and Microhardness of Modified Layer 3.2. Weight Gain and Microhardness of Modified Layer*

Figure 4 shows the histogram (4, 8, and 12 h) of the thickening of the steel without the RE-nitridated layer. The weight gain was 2.42, 3.35 and 3.75 mg/cm2 at each respective time interval. Figure 5 shows the microhardness diagram of the modified layer. After 4 h of nitriding, the surface hardness was 701.3 HV0.05. After nitriding for 8 h, the surface hardness was 975.3 HV0.05. After 12 h of nitriding, the surface hardness of the modified layer was 882.5 HV0.05. Figure 4 shows the histogram (4, 8, and 12 h) of the thickening of the steel without the RE-nitridated layer. The weight gain was 2.42, 3.35 and 3.75 mg/cm2 at each respective time interval. Figure 5 shows the microhardness diagram of the modified layer. After 4 h of nitriding, the surface hardness was 701.3 HV0.05. After nitriding for 8 h, the surface hardness was 975.3 HV0.05. After 12 h of nitriding, the surface hardness of the modified layer was 882.5 HV0.05. Figure 4 shows the histogram (4, 8, and 12 h) of the thickening of the steel without the RE-nitridated layer. The weight gain was 2.42, 3.35 and 3.75 mg/cm2 at each respective time interval. Figure 5 shows the microhardness diagram of the modified layer. After 4 h of nitriding, the surface hardness was 701.3 HV0.05. After nitriding for 8 h, the surface hardness was 975.3 HV0.05. After 12 h of nitriding, the surface hardness of the modified layer was 882.5 HV0.05.

**Figure 4.** Histogram of weight gain of modified layer under nitriding conditions without RE. **Figure 4.** Histogram of weight gain of modified layer under nitriding conditions without RE. **Figure 4.** Histogram of weight gain of modified layer under nitriding conditions without RE.

**Figure 5.** Microhardness of modified layer cross-section under nitriding conditions without RE. **Figure 5.** Microhardness of modified layer cross-section under nitriding conditions without RE. **Figure 5.** Microhardness of modified layer cross-section under nitriding conditions without RE.

Figure 6 shows the histogram of the weight gain after nitriding with the addition of for 4, 8, and 12 h. The weight gain was 2.49, 3.43, and 3.87 mg/cm2, respectively. Figure 7 shows the microhardness distribution under nitriding conditions with RE. After nitriding for 4, 8, and 12 h, the surface hardness was 840.9, 989.6, and 1027.0 HV0.05, respectively, and the surface hardness reached its peak at 12 h. According to the microhardness bar chart of the nitriding surface without and with rare earth in Figure 8, the increase percentage of the surface hardness after nitriding with RE over time was 19.7%, 1.47%, and 16.3%, for each respective time interval. Therefore, the surface hardness improved after the addition of RE nitriding. This was mainly because the addition of RE refines the microstructure of the nitrided layer and adjusts the phase proportion [26]. Moreover, there are dispersion strengthening and solid solution strengthening effects after adding rare earth nitriding. At the same time, the infiltration of rare earth elements leads to serious lattice Figure 6 shows the histogram of the weight gain after nitriding with the addition of for 4, 8, and 12 h. The weight gain was 2.49, 3.43, and 3.87 mg/cm2, respectively. Figure 7 shows the microhardness distribution under nitriding conditions with RE. After nitriding for 4, 8, and 12 h, the surface hardness was 840.9, 989.6, and 1027.0 HV0.05, respectively, and the surface hardness reached its peak at 12 h. According to the microhardness bar chart of the nitriding surface without and with rare earth in Figure 8, the increase percentage of the surface hardness after nitriding with RE over time was 19.7%, 1.47%, and 16.3%, for each respective time interval. Therefore, the surface hardness improved after the addition of RE nitriding. This was mainly because the addition of RE refines the microstructure of the nitrided layer and adjusts the phase proportion [26]. Moreover, there are dispersion strengthening and solid solution strengthening effects after adding rare earth nitriding. At the same time, the infiltration of rare earth elements leads to serious lattice Figure 6 shows the histogram of the weight gain after nitriding with the addition of for 4, 8, and 12 h. The weight gain was 2.49, 3.43, and 3.87 mg/cm2 , respectively. Figure 7 shows the microhardness distribution under nitriding conditions with RE. After nitriding for 4, 8, and 12 h, the surface hardness was 840.9, 989.6, and 1027.0 HV0.05, respectively, and the surface hardness reached its peak at 12 h. According to the microhardness bar chart of the nitriding surface without and with rare earth in Figure 8, the increase percentage of the surface hardness after nitriding with RE over time was 19.7%, 1.47%, and 16.3%, for each respective time interval. Therefore, the surface hardness improved after the addition of RE nitriding. This was mainly because the addition of RE refines the microstructure of the nitrided layer and adjusts the phase proportion [26]. Moreover, there are dispersion strengthening and solid solution strengthening effects after adding rare earth nitriding. At the same time, the infiltration of rare earth elements leads to serious lattice distortion and increases dislocation around the boundary, resulting in grain boundary strengthening.

distortion and increases dislocation around the boundary, resulting in grain boundary

distortion and increases dislocation around the boundary, resulting in grain boundary

strengthening.

strengthening.

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**Figure 6.** Bar graph of weight gain of modified layer under nitriding conditions with RE. **Figure 6.** Bar graph of weight gain of modified layer under nitriding conditions with RE. **Figure 6.** Bar graph of weight gain of modified layer under nitriding conditions with RE. **Figure 6.** Bar graph of weight gain of modified layer under nitriding conditions with RE.

**Figure 7.** Microhardness of modified layer cross-section under nitriding conditions with RE. **Figure 7.** Microhardness of modified layer cross-section under nitriding conditions with RE. **Figure 7.** Microhardness of modified layer cross-section under nitriding conditions with RE. **Figure 7.** Microhardness of modified layer cross-section under nitriding conditions with RE.

with and without RE. **Figure 8.** Histogram of the surface microhardness of the modified layer under nitriding conditions with and without RE. **Figure 8.** Histogram of the surface microhardness of the modified layer under nitriding conditions with and without RE. **Figure 8.** Histogram of the surface microhardness of the modified layer under nitriding conditions with and without RE.

#### *3.3. Element Distribution in Modified Layer and EDS Energy Spectrum Analysis 3.3. Element Distribution in Modified Layer and EDS Energy Spectrum Analysis*

The modified surface layers of the 12 h samples without RE nitriding were magnified by 100. Point scanning and plane scanning were performed for a, b, c, d, and e in Figure 9a by an EDS analyzer. Figure 9b1–b5 and Figure 9c1–c4 show the modified layer EDS spectrum test results. The distance from the surface of the modified layer was gradually transferred to the matrix, and the content distribution of each element was uneven. In combination with the line scan and plane scan in Figure 9a,b1–b5, it is also shown that under nitriding conditions without RE, the content of the element N in the modified layer didnot change significantly, and the distribution of the elements C, Fe, and Cr in the modified layer was relatively uniform. The modified surface layers of the 12 h samples without RE nitriding were magnified by 100. Point scanning and plane scanning were performed for a, b, c, d, and e in Figure 9a by an EDS analyzer. Figure 9b1–b5 and Figure 9c1–c4 show the modified layer EDS spectrum test results. The distance from the surface of the modified layer was gradually transferred to the matrix, and the content distribution of each element was uneven. In combination with the line scan and plane scan in Figure 9a and Figure 9b1–b5, it is also shown that under nitriding conditions without RE, the content of the element N in the modified layer didnot change significantly, and the distribution of the elements C, Fe, and Cr in the modified layer was relatively uniform.

**Figure 9.** Element distribution diagram of the 12 h-nitrided layer without the addition of RE. (**a**) Fe, N, line-scan; (**b1**–**b5**) a.b,c,d,e points correspond to dot energy spectrum; (**c1**–**c4**) C, N, Cr, Fe element surface scanning. **Figure 9.** Element distribution diagram of the 12 h-nitrided layer without the addition of RE. (**a**) Fe, N, line-scan; (**b1**–**b5**) a,b,c,d,e points correspond to dot energy spectrum; (**c1**–**c4**) C, N, Cr, Fe element surface scanning.

Figure 10 shows the surface morphology and elements content after 12 h of RE nitriding. As can be seen from Figure 10b1–b5, the La content in the modified layer was small, while the N content showed a downward trend. At position c in Figure 10a, dot analysis was performed at 130.60 μm of the permeable layer, and the content of La was consistent with the surface content of the nitrided layer, indicating that RE atoms can be diffused into the modified layer. This was confirmed by mid-plane scanning, shown in Figure 10d5, but with the thickening of the nitrided layer, the modified layer contained a trace of La. Comparison between Figures 9b1 and 10b1 shows that the content of N element on the surface without RE nitriding was higher than it was after RE nitriding, although the thickness of the modified layer on the surface without RE nitriding was thinner Figure 10 shows the surface morphology and elements content after 12 h of RE nitriding. As can be seen from Figure 10b1–b5, the La content in the modified layer was small, while the N content showed a downward trend. At position c in Figure 10a, dot analysis was performed at 130.60 µm of the permeable layer, and the content of La was consistent with the surface content of the nitrided layer, indicating that RE atoms can be diffused into the modified layer. This was confirmed by mid-plane scanning, shown in Figure 10d5, but with the thickening of the nitrided layer, the modified layer contained a trace of La. Comparison between Figures 9b1 and 10b1 shows that the content of N element on the surface without RE nitriding was higher than it was after RE nitriding, although the thickness of the modified layer on the surface without RE nitriding was thinner than

that of the layer subjected to nitriding with RE [27]. This indicates that the increase of the nitrogen concentration or nitrogen the potential did not necessarily lead to the thickening of the modified layer. The thickness of the modified layer is not only related to the external N potential, but also to the internal diffusion of N atoms [28]. than that of the layer subjected to nitriding with RE [27]. This indicates that the increase of the nitrogen concentration or nitrogen the potential did not necessarily lead to the thickening of the modified layer. The thickness of the modified layer is not only related to the external N potential, but also to the internal diffusion of N atoms [28].

**Figure 10.** Element distribution diagram in 12 h-nitrided layer with the addition of RE.(**a**) Fe, N, line-scan; (**b1**–**b5**) a.b,c,d,e points correspond to dot energy spectrum; (**c**) The distance between La and the surface of the modified layer; (**d1**–**d5**) C, N, Cr, Fe, La element surface scanning. **Figure 10.** Element distribution diagram in 12 h-nitrided layer with the addition of RE. (**a**) Fe, N, line-scan; (**b1**–**b5**) a.b,c,d,e points correspond to dot energy spectrum; (**c**) The distance between La and the surface of the modified layer; (**d1**–**d5**) C, N, Cr, Fe, La element surface scanning.

transmission electron microscopy (TEM). The bright-field image and the corresponding selected region electron diffraction (SAED) map are shown in Figure 11. A large number of nanocrystals were observed, as shown in Figure 11a. The size of the precipitated phase was extremely small, with the maximum size no greater than 60 nm. The surface structure of the 12 h RE-nitrided samples was further studied by transmission electron microscopy (TEM). The bright-field image and the corresponding selected region electron diffraction (SAED) map are shown in Figure 11. A large number of nanocrystals were observed, as shown in Figure 11a. The size of the precipitated phase was extremely small, with the maximum size no greater than 60 nm. The results showed

The surface structure of the 12 h RE-nitrided samples was further studied by

that the addition of RE refined the grain size and produced a microalloying effect on the modified layer. Figure 12b shows a clear diffraction ring caused by a large number of nanocrystals, indicating that these La, γ'-Fe4N, ε-Fe2-3N, and La phases existed in the outermost layer in the form of nanocrystals. The size of these phases was generally limited to 100 nm, so they can be referred to as nanophases. Yan et al. [29,30] obtained similar results with plasma nitriding, and found that the existence of the nano-phase helps to improve the hardness and wear resistance of the nitriding layer. Furthermore, these phases are consistent with the XRD results in Figure 3b. The results showed that the addition of RE refined the grain size and produced a microalloying effect on the modified layer. Figure 12b shows a clear diffraction ring caused by a large number of nanocrystals, indicating that these La, γ'-Fe4N, ε-Fe2-3N, and La phases existed in the outermost layer in the form of nanocrystals. The size of these phases was generally limited to 100 nm, so they can be referred to as nanophases. Yan et al. [29,30] obtained similar results with plasma nitriding, and found that the existence of the nano-phase helps to improve the hardness and wear resistance of the nitriding layer. Furthermore, these phases are consistent with the XRD results in Figure 3b.

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**Figure 11.** TEM image after rare earth nitriding for 12h (**a**) Bright-field image; (**b**) Corresponding SAED. **Figure 11.** TEM image after rare earth nitriding for 12h. (**a**) Bright-field image; (**b**) Corresponding SAED.

*3.4. Polarization Curve and Fitting Data*

tent of the ε-phase in the samples. Because ε-nitrides are the most important parameters controlling the corrosion resistance of nitrided samples, the corrosion resistance of our **Figure 12.** Corrosion resistance curve of modified layer under different nitriding conditions (4, 8, 12 h): (**a**) PN; (**b**) RE. **Figure 12.** Corrosion resistance curve of modified layer under different nitriding conditions (4, 8, 12 h): (**a**) PN; (**b**) RE.

rare earth-nitrided samples was enhanced at 12 h.

#### **Table 3.** Fitting data of modified 38CrMoAl steel layer under nitriding conditions with RE. *3.4. Polarization Curve and Fitting Data*

for the formation of rare earth nitride [31].

the following rare earth nitriding reactions occur:

in the modified layer [32]. The calculated negative ΔG1 *T*

(H) to form rare earth hydrides.

a stable gas.

**Table 2.** Fitting data of modified 38CrMoAl steel layer under nitriding conditions without RE. **Polarization Curve Fitting Data Unit Untreated 4 h 8 h 12 h**  Corrosion rate (×10−2 mm/a) 17.472 21.405 21.939 25.714 Rp(×103 Ω/cm2) 1.756 1.434 1.400 1.193 Io(×10−5 A/cm2) 1.486 1.820 1.865 2.186 Eo/V −0.674 −0.960 −0.955 −0.967 Passivation zone width/V 0.715 2.219 2.267 2.395 **Polarization Curve Fitting Data Unit Untreated 4 h 8 h 12 h**  Corrosion rate (×10−2 mm/a) 17.472 16.092 20.754 15.089 Rp (×103 Ω/cm2) 1.756 1.906 1.479 2.034 Io (×10−5 A/cm2) 1.486 1.368 1.765 1.282 Eo/V −0.674 −0.953 −0.954 −0.962 Passivation zone width/V 0.715 2.955 2.934 2.458 *3.5. Thermodynamics of the Modified Layer*  Because the nitrided atmosphere was NH3, the main reaction in the furnace was: The polarization curve data in Figure 12a were used for fitting, and the results are shown in Table 2. After nitriding for 4, 8, and 12 h, the corrosion rates of the modified layer were 21.405 × 10−2 mm/a, 21.939 × 10−2 mm/a, and 25.714 × 10−2 mm/a, respectively. The corrosion rates peaked after 12 h of nitriding. The current corrosion density was also increased to 2.186 × 10−5 A/cm2 compared with that of the non-nitrided sample. After fitting the polarization curve data shown in Figure 12b, we obtained the fitting results shown in Table 3. After nitriding with RE for 4, 8, and 12 h, the corrosion rates of themodified layer were 16.092 × 10−2 mm/a, 20.754 × 10−2 mm/a, and 15.089 × 10−2 mm/a,

The decomposition of ammonia produced [N] atoms (Equation (1)), some of which penetrated into the surface of the workpiece, and the other part formed N2, which became

The nitride produced by the decomposition of iron nitride provided the conditions

The RE formed compounds with nitrogen (N), a gap element of chemical heat treatment, and the bond changed from metal to ionic. Rare earths also reacted with hydrogen

Trace RE hydrides floating in the furnace flow improved the adsorption capacity of the workpiece surface diffusion. When RE hydrides are attracted to the workpiece surface,

The thermodynamic driving force of the decomposition of ε-Fe2N (Reaction 2) was greater than that of γ′-Fe4N (Reaction 3). Therefore, it is speculated that γ′-Fe4N was still

Since the hydrogenation of rare earth can occur above 300 °C:

NH3 → 3/2H2 + [N] (1)

2ε-Fe2N → Fe4N + 2[N] (2)

γ′-Fe4N → 4α-Fe + [N] (3)

La+3 + 2Cl− + H2 = LaH2-3 + 3/2Cl2 (4)

LaH2 + [N] → La[N] + H2 (5)

shows that the reaction occurs

respectively; the corrosion rate decreased, increased and then decreased again. The corrosion rates of the RE-nitrided samples reached their minimum at 12 h, and the current density of the RE-nitrided samples decreased to 1.282 × 10−5 A/cm2 compared with the samples without RE, and the width of passivation zone was wider. Tang [25] discussed the main reasons for the enhanced corrosion resistance of ε-nitride compounds on the workpiece surface. The X-ray diffraction analysis in Figure 3b shows that the presence of La and oxide LaFeO3 significantly improved the intensity of the ε-Fe2−3N diffraction peak and increased the content of the ε-phase in the samples. Because ε-nitrides are the most important parameters controlling the corrosion resistance of nitrided samples, the corrosion resistance of our rare earth-nitrided samples was enhanced at 12 h.

**Table 2.** Fitting data of modified 38CrMoAl steel layer under nitriding conditions without RE.

**Table 3.** Fitting data of modified 38CrMoAl steel layer under nitriding conditions with RE.

## *3.5. Thermodynamics of the Modified Layer*

Because the nitrided atmosphere was NH3, the main reaction in the furnace was:

$$\text{NH}\_3 \rightarrow \text{3/2H}\_2 + \text{[N]} \tag{1}$$

The decomposition of ammonia produced [N] atoms (Equation (1)), some of which penetrated into the surface of the workpiece, and the other part formed N2, which became a stable gas.

The nitride produced by the decomposition of iron nitride provided the conditions for the formation of rare earth nitride [31].

$$\text{2}\text{ε-Fe}\_2\text{N} \rightarrow \text{Fe}\_4\text{N} + \text{2[N]}\tag{2}$$

$$
\gamma' \text{-Fe}\_4\text{N} \to 4\alpha \text{-Fe} + \text{[N]}\tag{3}
$$

The RE formed compounds with nitrogen (N), a gap element of chemical heat treatment, and the bond changed from metal to ionic. Rare earths also reacted with hydrogen (H) to form rare earth hydrides.

Since the hydrogenation of rare earth can occur above 300 ◦C:

$$\rm La^{+3} + 2Cl^{-} + H\_{2} = LaH\_{2-3} + 3/2Cl\_{2} \tag{4}$$

Trace RE hydrides floating in the furnace flow improved the adsorption capacity of the workpiece surface diffusion. When RE hydrides are attracted to the workpiece surface, the following rare earth nitriding reactions occur:

$$\text{LaH}\_2 + \text{[N]} \rightarrow \text{La[N]} + \text{H}\_2 \tag{5}$$

The thermodynamic driving force of the decomposition of ε-Fe2N (Reaction 2) was greater than that of γ 0 -Fe4N (Reaction 3). Therefore, it is speculated that γ 0 -Fe4N was still in the modified layer [32]. The calculated negative ∆G1*T* 0 shows that the reaction occurs spontaneously from left to right in the range of 575–700 ◦C, forming active compounds of rare earth and nitrogen.

#### **4. Conclusions**

We studied the effect of the addition of rare earth (RE) to a modified layer of 38CrMoAl steel by plasma nitriding at 550 ◦C for 4, 8, and 12 h, and in an HN3 atmosphere. The following conclusions were drawn:

(1) For nitriding without the addition of RE for 4, 8, and 12 h, the thickness of the modified layers was 222.2, 345.7, 355.9 µm for each respective timeframe, the weight gains were 2.42, 3.35, and 3.75 mg/cm2 , respectively, and the maximum surface hardness was 882.5 HV0.05, at 12 h. After nitriding with the addition of RE for 4, 8, and 12 h, the modified layers were thickened by 30.2, 12.3 and 34.9 µm, respectively, and the weights were 2.49, 3.43 and 3.87 mg/cm2 , respectively. The maximum surface hardness was 1027.0 HV0.05, at 12 h.

(2) After 12 h of nitriding with RE, La, LaFeO3, and trace Fe2O3 appeared in the modified layer. The diffraction peak of γ'-Fe4N in the RE nitriding layer was weakened compared with the layer nitrided without RE, while the diffraction peak of ε-Fe2-3N was enhanced. Meanwhile, La, γ'-Fe4N, ε-Fe2-3N, and La phases existed in the outermost layer in the form of nanocrystals.

(3) After nitriding with the addition of RE for 12 h, the corrosion rate of the modified layer decreased to a minimum of 15.089 × 10−2 mm/a, and the current density also decreased to a minimum of 1.282 × 10−5 A/cm2 ; consequently, the corrosion resistance increased.

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

**Funding:** Supported by the National Natural Science Foundation of China (51401113) and Natural Science Foundation of Heilongjiang Province of China (E2016069), Heilongjiang Postdoctoral Financial Assistance (LBH-Z16061), The Fundamental Research Funds in Heilongjiang Provincial Universities (135309504).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

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

#### **References**

*Article*


*2.2. In Silico*/*Experimental Interaction of Single Tail Self-Assembling Dendrons and siRNA*

Mesoscale simulations were again exploited for predicting the interactions between the amphiphilic dendrons **1**–**8** and siRNA molecules, as illustrated in Figure 3.

**Figure 3.** *Cont*.

**Figure 3.** Morphologies of the systems formed at a dendrimer-to-siRNA charge ratio (N/P) = 10 by the siRNA molecules and the amphiphilic dendrons **1** (**a**), **2** (**b**), **3** (**c**), **4** (**d**), **5** (**e**), **6** (**f**), **7** (**g**) and **8** (**h**) as obtained from molecular simulations. Left panels are zoomed images of the right panels. Colors as in Figure 2, except for the siRNA molecules, shown as red sticks and the water and counterions, portrayed in the left panels as light blue field for clarity. Adapted from [7] with the permission of John Wiley and Sons, 2016.

The computer images showed that, at the same dendrimer-to-siRNA charge ratio (N/P) of 10, the nucleic acid fragments are well encased within the micellar network formed by all self-assembling amphiphilic dendrimers **1**–**6** and so are efficiently protected from the surrounding environment. For the non-self-assembling systems **7** and **8**, the siRNA molecules are interspersed within an unorganized solution of single dendrons and water, and therefore in principle more susceptible to ultimate degradation by e.g., RNAses.

The in silico quantitative characterization of the siRNA/amphiphilic dendron interactions was next carried out by atomistic molecular dynamics (MD) simulations performed in the framework of the so-called molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) methodology [9–15] (the methodology is described in detail in the Supporting Information in the companion paper [9]). The values of free energy of binding (ΔGbind) between the different self-assembling dendrons **1**–**6** towards the siRNA sequence directed against the mRNA coding for the heat shock protein 27 (Hsp27)—a small molecular chaperone which is a vital regulator of cell survival and a major player in drug resistance—were initially calculated, as listed in the first column of Table 2. However, in order to perform a rigorous discussion about these aspects, the concept of effective free energy of binding (ΔGbind,eff) was introduced, as follows. From an extensive analysis of each dendron micelle/siRNA complex MD trajectory, the number Neff of dendron branches (each bearing a positive charge) in permanent contact with the siRNA was determined, leading to the values listed in the second columns of Table 2. The precise identification of each dendrimer branch involved in the siRNA binding, and the corresponding individual contribution afforded to the overall binding energy, was then carried out by a per-residue free energy decomposition technique (described in the Supporting Information in the companion paper [9]). The cumulative results of this analysis are reported in the third column of Table 2, showing the effective contribution to the total free energy of binding (ΔGbind,eff). However, to be able to compare simulation data among themselves (and with experimental data, see below) the effective-charge-normalized values of ΔGbind,eff, i.e., ΔGbind,eff/Neff, were considered, as shown in the fifth column of Table 2.

**Table 2.** The free energy of micellization (ΔGbind, kcal/mol), number of effective charges (Neff), effective free energy of binding (ΔGbind,eff, kcal/mol), and effective-charge-normalized free energy of binding (ΔGbind,eff/Neff) between siRNA and dendrons **1**–**6** as derived from molecular simulations. The last column reports the siRNA/dendron binding data (C50, μM) obtained from ethidium bromide (EB) displacement assays. Adapted from [7] with the permission of John Wiley and Sons, 2016.

From the values reported in Table 2, the effective interaction between the micelles formed by the different dendrons with siRNA (ΔGbind,eff) increases with increasing alkyl chain length, the last three dendrons definitely being the strongest siRNA binders in the order: **4** < **5** < **6**. Limiting the discussion to these three self-assembling systems, the micelles formed by dendron **4** were characterized by a total number of charges equal to +64, the 48% of which (Neff = 31 are involved in productive siRNA binding. This translates into the corresponding ΔGbind,eff/Neff value of −0.41 kcal/mol. In contrast, the other two efficient self-assembled micelles generated by dendrons **5** and **6** were not only both able to exploit 61% of their positive charges (49/80 and 62/102 for **5** and **6**, respectively) but they also did it more efficiently (ΔGbind,eff/Neff = −0.59 kcal/mol for **5** and −0.56 kcal/mol for **6**, Table 1), ultimately characterizing the corresponding micelles as the best siRNA binders of the whole series.

ǂ ǂ

In silico data were experimentally confirmed by fluorescent ethidium bromide (EB) displacement assays (see Figure A1 in Appendix A). According to these tests, the value of C50 (i.e., the dendron concentration at which the fluorescence of siRNA-intercalated EB is reduced by 50%), is an indication of siRNA binding efficiency: The lower the C50 value, the stronger the siRNA/micelle binding. As seen in Figure A1, the negative-control (i.e., non-self-assembling) dendrons **7** and **8** were not able to displace EB from the nucleic acid. In line, also for the self-assembling dendron **1**, bearing the shortest alkyl tail (C14), the C50 value was not determined, due to the high CMC value required for its self-assembly. On the other hand, for the dendron series **2**–**6**, progressively decreasing C50 values were obtained (Figure A1 and Table 2, last column), with dendron **6** being the most effective siRNA binder, in full agreement with computer-based predictions.

#### *2.3. In Vitro siRNA Delivery Performance of Single-Tail Self-Assembling Dendrons*

Based on the results described above, dendrons **4**–**6** were selected for in vitro delivery of siRNA molecules targeting both the Hsp27 and the translationally controlled tumor protein (TCTP, a highly conserved protein present in all eukaryotic organisms that regulate cell survival in human tumors) in human castration-resistant prostate cancer (HCPC) PC-3 and C4-2 cell lines (Figure 4a,b). In these experiments, all other dendrons (**1**, **3**, **7** and **8**) were also tested for comparison and/or negative controls.

**Figure 4.** *Cont*.

**Figure 4.** Self-assembled dendron-mediated siRNA delivery and gene silencing of Hsp27 (**a**) and TCTP proteins (**b**) in human prostate cancer PC-3 (solid bars) and C4-2 (patterned bars) cell lines (N/P = 10). Control: Untreated cells. The results obtained from non-self-assembling dendrons **1**, **7** and **8** are also shown for negative-control purposes. (**c**) Expression of Hsp27 in PC-3 cells treated with 50 nM siRNA delivered by self-assembled dendron **4** (N/P = 10), by Lipofectamine, and by the high-generation covalent triethanolamine (TEA)-core PAMAM-based dendrimer G7. Control: Untreated cells. Negative controls: Self-assembled dendron **4** alone and a scrambled (i.e., non-silencing) siRNA sequence. The expression levels of Hsp27 and TCTP proteins were quantified by western blots 72 h post treatment using vinculin as the control. Adapted from [7] with the permission of John Wiley and Sons, 2016.

As shown in this Figure, the best siRNA delivery capacity was obtained with the nanovector based on the self-assembled form of dendron **4**, whose related gene silencing effect was even superior to that obtained both with the gold standard commercial vector Lipofectamine and with the high-generation (G7) covalent PAMAM-based dendrimer. This featured an extended triethanolamine (TEA) core developed by our group and discussed in detail in the companion paper [9]. As anticipated by in silico predictions, nanocarriers formed by dendrons bearing shorter aliphatic (**1**–**3**), hydrophilic (**7**) or no chains (**8**) fail to elicit meaningful gene silencing in all cases. Indeed, while **7** and **8** are intrinsically unable to self-assembly (negative controls), the former dendron set (**1**–**3**) likely cannot generate stable micelles and/or siRNA complexes at the experimental conditions employed for transfection, due to their high CMC and C50 values (Tables 1 and 2). On the other hand, nanomicelles from dendrons with longer alkyl tails (**5** and **6**) were also able to achieve significant siRNA delivery and silenced the target Hsp27 and TCTP genes, yet with performance lower than that obtained with dendron **4**.

These last results seem to be in slight contradiction with the computer-based prediction reported in Table 2, according to which the best (i.e., most favorable) values of ΔGbind,eff/Neff and C50 are indeed obtained for self-assembled dendrons **5** and **6**. This ranks these nanovectors as the tightest siRNA binders. To investigate whether this disagreement was real or only apparent, cellular uptake experiments based on flow cytometry were performed using siRNA in complex with micelles from **4**, **5**, and **6** (Figure 5a). These data clearly show that this step is not governing the differential behavior of these nanovector/siRNA performance along the in vitro gene silencing pathway, as all three systems are characterized by comparable cellular uptake efficiency.

**Figure 5.** (**a**) The mean fluorescence intensity of PC-3 cells treated with siRNA labeled with the fluorescent dye Cy-3 in complex with self-assembled nanovectors **4**, **5**, and **6** (50 nM siRNA, N/P = 10). Non-treated cells and cells treated with labeled siRNA alone were used as control. (**b**) siRNA release from their complexes with micelles of dendrons **4** (red), **5** (black), and **6** (yellow) as assessed using EB displacement assay performed in the presence of heparin. Adapted from [7] with the permission of John Wiley and Sons, 2016.

With this aspect ruled out, it was reasoned that the siRNA release of its nanovector could be the factor underlying the best performance in gene silencing of the **4**/siRNA complex. Thus, it was determined that it was the dissociation of the nucleic acid fragments from their **4**, **5**, and **6** self-assembled nanovectors via the EB displacement assay in the presence of heparin, a highly negatively charged polysaccharide that can compete with siRNA in nanocarrier binding. As can be seen in Figure 5b, by increasing heparin concentration the siRNA molecules were more efficiently displaced from their complex with **4** than from the two alternative dendron micelles. This led us to the conclusion that the siRNA release process might be more effective from the self-assembled dendron **4** than from the **5** and **6** complexes, respectively.

Collectively, these results led to the conclusion that, in agreement with computer predictions, the supermolecular assemblies formed by **5** and **6** were actually too stable to dissemble in physiological conditions. This enhanced stability negatively affects the siRNA release in the cellular environment, thereby impairing the relevant gene silencing effect. At the same time, the complexes of micelles originated by dendron **4** and siRNA represent the best compromise in term of nucleic acid fragment binding and release efficiency, and these properties ultimately translate into the most potent biological performance.

The final step of the in vitro characterization concerned the investigation of the cellular toxicity of our single-tail self-assembled dendrons. As shown in Figure 6 for the PC-3 cell line as an example, none of the self- and non-self-assembling dendrons were endowed with cytotoxic properties. Analogous results were obtained with the alternative prostate cancer cell line, C4-2.

**Figure 6.** Metabolite toxicity (**a**) and membrane damaging activity (**b**) of self- and non-self-assembling dendrons **1**–**8** and their complexes with a scrambled (i.e., non-silencing) siRNA sequence (50 nM siRNA, N/P = 10) in PC-3 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the lactate dehydrogenase (LDH) assay, respectively. Untreated cells were used for control. Adapted from [7] with the permission of John Wiley and Sons, 2016.

In essence, all in silico and in vitro results concurred to indicate **4** as the amphiphilic dendron endowed with the best features for efficient self-assembly, and easy and stable formation of complexes with the siRNA molecules for delivery. Once inside the cell, there is effective disassembly during endosomal release for subsequent potent gene silencing.

#### *2.4. In Vivo siRNA Delivery Performance of Single-Tail Self-Assembling Dendron 4*

The in vitro best performing nanomicelles generated by the dendron **4** were finally challenged for in vivo gene silencing in a prostate cancer xenografted nude mouse model. The results obtained are gathered in Figure 7.

**Figure 7.** (**a**) In vivo gene silencing achieved by treating nude mice bearing prostate cancer (PC-3) tumors (30–50 mm3) with Hsp27 siRNA delivered by the self-assembling dendron **4**, buffer solution (control), the dendron **4** alone, and a scrambled siRNA/**4** complex (negative controls). Treatments (3 mg/kg siRNA, N/P = 5) were administered for a period of 4 weeks via intraperitoneal injections (twice per week). (**b**) In vivo inhibition of tumor growth quantified by tumor size and (**c**) antiproliferation activity in tumors assessed by immunohistochemistry in mice treated as described for panel (**a**). Panel (**c**), from left to right: control, **4** alone, **4**/siRNA, **4**/scrambled siRNA. Adapted from [7] with the permission of John Wiley and Sons, 2016.

Images in Figure 7 support the effective and specific in vivo gene silencing induced upon administration of the Hsp27-directed siRNA delivered by the self-assembled nanovector **4**. In particular, an impressive reduction of ~80% in Hsp27 expression and of ~65% in tumor size after 4 weeks of treatment were achieved (Figure 7a,b).

These data, coupled with no discernable signs of in vivo toxicity (Figure 8) and the absence of mice weight loss, support the potential utilization of the amphiphilic compound **4** as a siRNA vector for future therapeutic implementations.

(**g**)

**Figure 8.** In vivo levels of inflammatory cytokines interleukine-6 (IL-6, **a**), interferon-gamma (INF-γ, **b**), and tumor necrosis factor-alpha (TNF-α, **c**), observed in male C57BL/6 mice treated with **4** and its siRNA complex (3.0 mg/kg siRNA, N/P = 5) by intravenous injection. Non-treated mice and animals treated with buffer solutions were used for control. Mice were sacrificed 24 h after treatment and the ELISA assay was performed on the collected serum. (**d**) Hepatic enzyme levels (aspartic aminotransferase AST (solid bars), alanine transferase ALT (patterned bars), gamma-glutamine transferase γ-GLT (empty bars), (**e**) kidney functions (creatinine CRE (mmol/L, solid bars), blood urea nitrogen BUN (μmol/L, patterned bars), and (**f**) cholesterol level in the blood measured in the treated mice after sacrifice. (**g**) Pathology of major organs from treated mice (HES staining). Columns (from left to right): Control, **4**, **4**/siRNA, and **4**/scrambled siRNA. Rows (from top to bottom): Heart, kidney, liver, lung, spleen. Adapted from [7] with the permission of John Wiley and Sons, 2016.

#### **3. siRNA Delivery by Double-Tail Self-Assembling Amphiphilic Dendrons**

#### *3.1. Design, Optimization and Chemico-Physical Charactrization of Double-Tail Self-Assembling Amphiphilic Dendrons*

With the haste of further improving the siRNA delivery and related gene silencing performance of the lead self-assembling amphiphilic dendron **4**, it was questioned whether increasing its hydrophobic portion would render this molecule even more efficient and effective. According to the devised strategy, it was ultimately decided to decorate **4** with a second C18-long alkyl chain. In order to do so, a series of computer-based molecular design and optimization studies were carried out, finally leading to the new double-tail amphiphilic dendron structure **AD** (where **AD** stands for Amphiphilic Dendron) shown in Figure 9a [6]. In buffer solution per se, molecular simulations predicted **AD** to self-assemble into large (~200 nm) vesicle-like structures (Figure 9b) called dendrimersomes [16]. After synthesis of **AD** by click-chemistry, dynamic light scattering (DLS) and transmission electron microscopy (TEM) indeed confirmed both the size and the shape of the supermolecular entities formed by **AD** upon self-assembly (Figure 9c). Notably, the vesicle corona structure closely matched a bilayer, characterized by a thickness of 7 nm—approximately equal to twice the molecular length of **AD** (~3.5 nm).

**Figure 9.** (**a**) The structure of the double-tail amphiphilic dendron simulated (**b**) and experimental TEM images (**c**) of the vesicle-like dendrimersomes formed by **AD** alone upon self-assembling in buffer solution. In panel b, the hydrophilic units of **AD** are portrayed in light green, the hydrophobic units are depicted in dark green, while light grey spheres are used to show some representative water molecules. Simulated (**d**) and experimental TEM images (**e**) of the nanosized spherical micelles formed by **AD** in the presence of siRNA molecules in solution. In panel (**d**), colors as in panel (**b**), while the nucleic acid fragments are portrayed as orange sticks. Adapted from [6] with permission of John Wiley and Sons, 2014.

Quite surprisingly, however, when simulations of the self-assembly of **AD** were performed in the presence of siRNA many highly ordered smaller spherical micelles of 6–8 nm diameter were obtained, with the siRNA fragments nicely interspersed within them (Figure 9d). The explanation for this vesicular to micellar structural transition likely resides in the greater positively charged surface area exposed by the micelles with respect to the dendrimersomes, which provides more efficient electrostatic interactions with the negatively charged siRNA. This, in turn, leads to the formation of more stable and compact complexes between the self-assembled nanocarriers and the nucleic acids molecules. The computer predictions were supported by TEM imaging (Figure 9e), confirming that **AD** is able to dynamically self-assemble into responsive and adaptive supramolecular assemblies in the presence of external stimuli, i.e., negatively-charged small polyanions.

#### *3.2. In Vitro siRNA Delivery Performance of the Double-Tail Self-Assembling Dendron AD*

The nanosized micelles formed by self-assembly of the double-tail amphiphilic dendron **AD** were able to effectively protect siRNA from degradation (e.g., by RNAses) and to promote fast and quantitative uptake by castrate-resistant prostate cancer PC-3 cells—almost 100% internalization of the nanovector/siRNA complexes was reached within 30 min. As concerns the mechanism of cell internalization of the **AD**/siRNA complexes, macropinocytosis was found to be the leading cellular entry mechanism for this nanovector/siRNA system, in utter analogy with the G5 covalent PAMAM dendrimer counterpart discussed in the companion paper (Figure 15a in [9]). Indeed, neither chlorpromazine (an inhibitor of clathrin-mediated endocytosis) nor genistein (a caveolae-mediated uptake specific blocker) were effective while cytochalasin D (a specific macropinocytosis inhibitor that, by binding to actin filaments, interferes with actin polymerization and assembly) inhibited cellular uptake in a dose-dependent manner, as presented in Figure 10b. Again in analogy with what observed for the G5 TEA-core PAMAM covalent dendrimer (Figure 15b in [9]), confocal microscopy provided images showing significant co-localization of the siRNA/**AD** complexes with the macropinocytosis biomarker dextran. Whereas, negligible co-localization was observed in cells treated with the two alternative biomarkers transferrin (clathrin-mediated endocytosis) and CTX-B (caveolae-mediated endocytosis). Thus, these experiments further corroborate the concept that these self-assembling PAMAM-based amphiphilic dendrons behave very similarly to their covalent high generation counterpart.

The gene silencing effect induced by Hsp-27 targeting siRNA delivered by **AD** nanomicelles was next investigated both at the mRNA and protein levels using prostate (PC-3) and breast (MCF-7 and MDA-MB231) cancer cell lines. As seen in Figure 10, a remarkable attenuation of the Hsp27 expression was achieved in all cases. This effect was comparable, if not greater, than that achieved with Lipofectamine, the reference standard reagent for siRNA/DNA transfection (e.g., **AD** was 6 times more efficient than Lipofectamine in downregulating Hsp27 protein expression in PC-3 cells, Figure 10b).

**Figure 10.** *Cont*.

**Figure 10.** Downregulation of Hsp27 at the mRNA (**a**) and protein (b) level in prostate cancer PC-3 cells treated with siRNA (50 nM) delivered by the self-assembled double-tail dendron **AD** (N/P = 5). Inhibition of Hsp27 expression by siRNA/**AD** complexes (50 nM siRNA, N/P = 5) in breast cancer MCF-7 (**c**) and MDA-MB231 (**d**). Adapted from [6] with permission of John Wiley and Sons, 2014.

The knockdown of Hsp27 in these cancer cells by siRNA delivered by **AD** micelles was paralleled by significant anti-proliferative effects (~75% of cancer cell growth inhibition), in agreement with the evidence that Hsp27 silencing negatively affects PC-3 cell survival [9,17]. Similarly to what was observed for the high generation covalent PAMAM dendrimers [9], the mechanism leading to cell death relied on caspase-dependent induced apoptosis, that is, the programmed cell death was promoted by apoptotic caspases, a family of endoproteases that provide critical links in cell regulatory networks controlling cell death. Accordingly, a 3-fold increase in caspase-3/7 activation was observed on average, confirming that siRNA delivered by the nanovectors based on self-assembled **AD** is very effective in silencing Hsp27 and thereby inducing caspase-dependent anticancer activity in vitro. A remarkable additional feature of this biological outcome is that the silencing effect was completely maintained for one week, and was also effective when transfection was performed in the presence of serum, a prototypical Achilles' heel of cationic nanovectors in siRNA delivery. In fact, the negatively charged serum proteins can not only compete with siRNA binding to the nanocarrier, but also may, by virtue of strong electrostatic forces, lead to the the disintegration of the nanoassemblies with consequent premature nucleic acid release and degradation [9].

From another perspective, when confronted with its single-tail analogue **4,** the efficiency of in vitro silencing induced by the double-tailed **AD** dendron/siRNA complexes was practically identical (e.g., 97% for **AD** and 96% for **4** in PC-3 cells, Figure 10b,c, respectively). Most importantly, however, remarkably good results were obtained with **AD** when challenged against cancer stem cells (notoriously the cancer cell population most refractory to treatments), and also stem and primary cells that constitute the target of deadly viruses such as HIV-1. As shown in Figure 11, **AD** was indeed able to elicit significant RNAi interference (RNAi) in glioblastoma stem cells (PBT003), and in HIV-1 infected human primary peripheral blood mononuclear cells (PBMC-CD4+), and hematopoietic stem cells (HSC-CD34+). Specifically, upon treatment of the hardly tractable PBT003 cells with **AD** carrying the siRNA was directed against the signal transducer and activator of transcription 3 (STAT3), which is a key player protein in glioma-initiating cells, thought to be responsible for glioblastoma induction, progression and recurrence. A 35% reduction in the corresponding mRNA level was observed, while only 24% mRNA reduction was achieved upon transfection with Lipofectamine (Figure 11a). The data relative to the two HIV-infected cell lines (PBMC-CD4+ and HSC-CD34+) transfected with the siRNA targeting the HIV-1 Tat/Rev gene are more striking. The Tat and Rev HIV-1 proteins are essential positive regulators of gene expression through interaction with RNA target elements present within the 5 translated leader sequence and envelope gene, respectively. Importantly, the genes encode the Rev and Tat overlap, with each being produced from a different reading frame. As seen from Figure 11b,c, in both cases a remarkable 50% reduction in the Tat/Rev mRNA expression was observed, while the prototypical delivery vector Lipofectamine (aka RNAiMAX) failed to induce any gene silencing in these cells

infected by the acquired immunodeficiency syndrome (AIDS) virus. Fundamentally, this translated into a 50% reduction of the viral infection (Figure 11d,e) and, to the best of the authors' knowledge, constitutes the first report on the successful and safe delivery of siRNA into primary and stem cells.

**Figure 11.** (**a**) STAT3 knockdown in glioblastoma stem cells (PBT003) treated with siRNA/**AD** complexes (50 nM siRNA, N/P = 5). The knockdown of Tat/Rev in primary human peripheral blood mononuclear cells (PBMC-CD4+) (**b**) and hematopoietic CD34+ stem cells (HSC-CD4+) (**c**) treated as in (**a**). Effective inhibition of HIV replication in PBMC-CD4+ (**d**) and HSC-CD4+ (**e**) after treatment with the anti-Tat/Rev siRNA delivered by **AD** (50 nM siRNA, N/P = 5). Viral loading was assessed using HIV-1 p24 antigen ELISA 3 days post-treatment. PBMC-CD4+ cells and HSC-CD34+ cells were infected by NL4-3 virus at a multiplicity of infection (MOI) = 0.001 and by JR-FL virus (MOI = 0.005), respectively, for 5 days before transfection. (**f**) Proton-sponge effect in PC-3 cells transfected with siRNA/**AD** in the absence (solid bars) and presence (patterned bars) of the proton pump inhibitor Bafilomycin A1. Untreated cells were used as the control. Adapted from [6] with permission of John Wiley and Sons, 2014.

As for the high generation covalent PAMAM dendrimer analogues presented in the companion paper [9], it was reasoned that also in the case of the **AD** micelles, the so-called proton sponge effect [18] could be invoked to explain nucleic acid release from the self-assembled double-tail nanovectors and, therefore, potent siRNA delivery within the cell cytoplasm. According to the debated proton sponge concept, when cationic nanoparticles such as those formed by **AD** and its siRNA cargo enter acidic endosomal (and lysosomal) vesicles, unsaturated amino groups sequester protons supplied by the proton pump v-ATPase. These sequestered protons cause the pump to continue functioning, leading to the retention of chloride ions and water molecules. Eventually, osmotic swelling causes the rupture of the vesicle, allowing the cationic nanoparticles to enter the cytoplasm. As the **AD** amphiphiles feature tertiary amines in the interior of their PAMAM heads which become protonated at the endosomal acidic pH (5.5), the authors verified whether the proton sponge effect was effectively at play also during their mediated siRNA delivery. Indeed, as illustrated in Figure 11f, Hsp27 silencing in PC-3 cells was significantly reduced in the presence of Bafilomycin A1 (a proton pump inhibitor that prevents endosome acidification). This implies that the **AD**-mediated siRNA delivery was indeed dependent on the endosomal acidification process and that the proton sponge effect played a role in the endosomal escape and cytoplasmic release of the nucleic acid.

Finally, the same protocol adopted for investigating the cellular toxicity of the single-tail self-assembled dendrons described in Section 2.3 was applied to **AD**. As expected, no discernible toxicity was observed in all cell lines (PC-3, PBMC-CD4+, HSC-CD34+, and PBT003 transfected with 50 nM siRNA and **AD** at N/P = 5 and 10) using both the MTT and LDH assays, supporting further in vivo gene silencing experiments with this nanocarrier.


*2.2. Methods*

The main research program was to test 28 wall masonry panels. The specimens were made under laboratory conditions. The preparation of the panels, their curing, testing, and processing of the test results were carried out in accordance with the EN 1052-1 standard [49]. The angle of the bed joint changed: θ = 0◦, 22.5◦, 45◦, 67.5◦, 90◦ (Figures 4 and 5). The samples had standard dimensions of 50 × 50 × 12 cm3, except for those with bed joints parallel to the load direction (θ = 90◦) of dimensions 27 × 75 × 12 cm3. The dimensions of the panels were selected in accordance with the RILEM guidelines used in the other discussed works in the field of this research [35,50].

**Figure 4.** Description of θ angle measurements of tested samples.

**Figure 5.** Load application diagram for compressed wall samples at different angles of bed joints (**a**) θ = 0◦, (**b**) θ = 22.5◦, (**c**) θ = 45◦, (**d**) θ = 67.5◦, (**e**) θ = 90◦.

Five specimens were prepared for each joint angle, except the θ = 0◦, where eight specimens were used.

The samples were built on the flat surface of the compressive strength test stand plate on a thin sand bed. Until the test, the elements were stored at a temperature of 20 ◦C and an air humidity <65%. The tests were carried out 28 days from the date of preparation of the samples.

The models were loaded with a hydraulic actuator (Pneumat P3000, Minsk, Belarus) with a steadily increasing rate on the stand of own production, with the use of a 1250 kN hydraulic press. The samples were loaded with an increase in force equal to 12 kN per minute up to the value at which their collapse occurred. The force was measured with a dynamometer (Pneumat M, Minsk, Belarus). Dial gauges were installed on both surfaces of each sample to measure horizontal and vertical displacements (Figure 6).

**Figure 6.** Compressive strength test with dial indicator installation, hs—height of the sample, ls—length of the sample (**a**) θ = 45◦, (**b**) θ = 90◦.

Young's modulus and Poisson's ratio were determined in terms of the elastic work of the samples, in accordance with the requirements of the standard [49].

During the experimental tests, the destruction processes were also recorded with high-resolution cameras.

#### **3. Results and Discussion**

*3.1. Results of Compressive Strength Tests*

The main results obtained during the compressive strength tests are presented in Table 2.

There are visible changes in the average strength of the masonry with a change in the load angle in relation to the bed joints. The highest compressive strength of 15.1 MPa was obtained for samples loaded in the direction perpendicular to the bed joints (θ = 0◦). The lowest results (3.6 MPa) were obtained for the angle θ = 67.5◦. The strength of the element was 4.2 times lower than the strength of the model with force acting in the direction perpendicular to the bed joints. Figure 7 presents the changes in the wall strength in relation to the reference model (θ = 0◦).

As the results obtained show, minimal compressive strength should be expected for the load acting on the samples with the angle of the bed joints θ = 57.5◦. Its value is limited to 21% of the compressive strength for the load acting parallel to the bed joints (θ = 0◦). For the samples with angle θ = 90◦, the compressive strength was limited to 75% of the strength of samples with θ = 0◦.


**Part 4**

**Hysterectomy Post-Operative Care**

248 Hysterectomy

Wang F, Shen X, Xu S, Liu Y, Ma L, Zhao Q, Fu D, Feng S, Li X. Negative words on surgical

Wodlin NB, Nilsson L, Arestedt K, Kjølhede P. Mode of anesthesia and postoperative symp-

Wodlin NB, Nilsson L, Kjølhede P. Health-related quality of life and postoperative recovery

Wodlin NB, Nilsson L, Kjølhede P. The impact of mode of anaesthesia on postoperative

Yardeni IZ, Beilin B, Mayburd E, Levinson Y, Bessler H. The effect of perioperative intra-

Yeh YC, Lin TF, Chang HC, Chan WS, Wang YP, Lin CJ, Sun WZ. Combination of low-dose

Zhang XW, Fan Y, Manyande A, Tian YK, Yin P. Effects of music on target-controlled

Zhao LY, Ye TH,Zhang YZ, Zhao H. Combination of morphine with low-dose naloxone for

opioid-related side effects. *J Formos Med Assoc* 2009; 108: 548-53

in fast-track hysterectomy. *Acta Obstet Gynecol Scand* 2011; 90: 362-8

domized, controlled trial. *Pharmacol Rep* 2009; 61: 1198-205

*Scand* 2011; 90: 369-79

2011; 118: 299-308

*Anaesthesia* 2005; 60: 990-4

2005; 27: 228-31

109: 1464-9

wards result in therapeutic failure of patient-controlled analgesia and further release of cortisol after abdominal surgeries. *Minerva Anestesiol* 2008; 74: 353-65 Wang F, Shen X, Xu S, Liu Y. Preoperative tramadol combined with postoperative small-

dose tramadol infusion after total abdominal hysterectomy: a double-blind, ran-

toms following abdominal hysterectomy in a fast-track setting. *Acta Obstet Gynecol*

recovery from fast-track abdominal hysterectomy: a randomised clinical trial. *BJOG*

venous lidocaine on postoperative pain and immune function. *Anesth Analg* 2009;

nalbuphine and morphine in patient-controlled analgesia decreases incidence of

infusion of propofol requirements during combined spinal-epidural anaesthesia.

intravenous patient-controlled analgesia. *Zhongguo Yi Xue Ke Xue Yuan Xue Bao*

**16**

*United Kingdom*

**Innovations in the Care of**

Sepeedeh Saleh and Amitabha Majumdar

*East Lancashire NHS Trust, Lancashire,*

**Postoperative Hysterectomy Patients**

Hysterectomy, removal of the uterus, has traditionally been regarded as the definitive surgical treatment for heavy menstrual bleeding. And is one of the most commonly performed operations in the UK. (Marjoribanks, 2006) Whilst menstrual disorders are the most frequent reasons for performing hysterectomy other common indications include chronic pelvic pain, fibroids, malignancy of the uterus, cervix or ovaries and genital prolapse. Hysterectomy is a major surgical procedure with potential for significant physical and emotional complications. It also carries additional social and economic costs.

Hysterectomy rates have been decreasing in recent years, but there remain large variations in population-based rates of hysterectomy across primary care organisations in England, from fewer than 10 per 100,000 to 100 per 100,000 in female populations. (Information for hospital episode statistics) Although hysterectomies were originally all performed via the abdominal route, alternative types of hysterectomy (for example vaginal and laparoscopic hysterectomies) are increasingly popular, particularly when carried out for benign disease. Vaginal hysterectomy is the procedure of choice for uterovaginal prolapse amongst practitioners in the UK. (Jha, 2007) This move away from abdominal surgery is in part due to the less invasive nature of the vaginal and laparoscopic procedures which, as a result, produce arguably better outcomes: an effect most marked when considering post-operative recovery. Current evidence on the safety and efficacy of laparoscopic techniques for hysterectomy (including laparoscopic-assisted vaginal hysterectomy, laparoscopic hysterectomy, laparoscopic supracervical hysterectomy and total laparoscopic hysterectomy) appears adequate to support their use, provided arrangements are in place

Although hysterectomy is generally considered safe several possible complications are associated with the procedure. These complications can result in mild to severe morbidity and even (in rare cases) mortality. Although their incidence is low, it is important to be aware of the immediate and longer-term complications that may arise from hysterectomy. Immediate complications include haemorrhage, bladder and ureteric injury, bowel injury, infection and venous thromboembolism. Long-term complications include the psychological and emotional aspects of such surgery in addition to complications arising from the surgical

**1. Introduction**

(Lethaby, 1999)

for consent, audit and clinical governance.

menopause and the use of hormone replacement therapies.


*3.6. Ankle Manifold Representation*

In this section, we show the results in the software SageMath Manifolds. We load the model and visualize it as a manifold, we show the axis and the sagittal plane intersection. With the model parameters loaded, *r*1, *r*2, *ω*1, *ω*2, and the origin established in the center of the base modules. We apply the equation:

$$
\hat{r}\_1 = \overline{c}\_0 + \hat{n}\_p \cdot d \tag{62}
$$

where *c*0 is the median center computed from trajectories A, B, and C center fitting, and *n* ˆ *p* is the median planes' normal vectors containing the circles. Table 8 shows values for the TC axis in the PM chart. In Table 9, we show the Plucker coordinates for the TC and ST axes.

Finally, Figure 38a shows the ankle manifold, and Figure 38b, the chart representing the range of movement and angle coordinates.

**Table 8.** Axis estimation data.

**Table 9.** Plucker line coordinates.

**Figure 38.** Ankle joint manifold. (**a**) Manifold for PM, (**b**) chart with ankle axis coordinates.

#### **4. Discussion**

In this work, we addressed the human ankle joint model from an alternative approach. We used statistical measurements for the development of a new device, specially designed to capture the human ankle joint movements. In animal joints, it is difficult to place encoders and linear sensors to measure the range of movement of complex joints in each internal living tissue reference frame. The product of exponential formulas uses only two frames, and it is useful in this case. Furthermore, in our work, we used a trilateration method for finding the device's platform position, which is an analytic method. Therefore we avoid numerical approximations that can diverge and reduce rounding errors. We proposed the ankle joint model as a Riemannian manifold. We can define a chart as a subset of such a manifold with angle coordinates for measuring the range of movement. Our presented device is lightweight, non-invasive, and can be used in remote places, on beds, or on the floor. By characterizing the ankle parameters, we can conduct symmetry studies by

correlating the left and right ankle joints. We can enhance the device configuration in future versions by replacing the draw-wire sensors used from potentiometers to digital encoders connected by a CAN bus, reducing wiring, space, weight, and energy consumption. We will use the model for the synthesis and reconfiguration of an ankle parallel rehabilitation robot, programmed by symmetrical movements at the opposite ankle. By employing the axis location and the screw theory, forces, and torques, we will study the ankle dynamics by using reciprocal screws to the axis location in a re-configurable platform. The robot will be lightweight because of the use of cable-driven actuators, inspired by antagonistic muscles that work with reciprocal inhibition for energy optimization. The robot will reconfigure the structure, considering the ankle joint as a central mast, and referenced it with MMP and MLP markers.

Figure 39 shows a schematic of the re-configurable approach.

**Figure 39.** Re-configurable cable-driven robot concept.

Other applications are, for example, by visualizing the platform trajectories one can explain how the calcaneal Achilles insertion is near to the platform's A point. The platform's normal vector changes abruptly near this region, as was depicted in Figures 24b and 26a,b. Furthermore, Riemannian models have different properties. We will explore diagnosis and treatments based on the model and metrics by employing machine learning algorithms. This approach can be applied to other joints in humans and other animals, by designing specialized re-configurable hardware and software. Tracking the parameters in different ages and weight conditions, and comparing the ankle models in healthy and injured people.

#### **5. Conclusions**

Computer tomography (CT) and magnetic resonance (MR) images have greater precision and accuracy. Measurements in medical imaging will help us compare the errors (RMS) in the HAJ. In biomechanics, we have not found an ideal model for error comparison. Then, we will compare the error with an accurate measurement. The device has limitations regarding mechanical precision and deformation of its parts. We face up to the error through the electronic design system. The calibration process is imperative for enhancing accuracy.

The calibration process is human-dependent. We read the digital measurement and compare it with caliper measurements directly in the sensor. Then, we register the data in a table to find the equivalence. An electronic board with trimmers avoids saturation, bias,

and calibration; a 10 bit ADC and an exponentially weighted moving average (EWMA) filter the noise signals. We have implemented a processing (Software) calibration interface. We avoid adding more specific technical data, such as CMRR, ADC speed, mechanical tolerance, and other issues inherent to the measuring devices.

Digital sensors, communications, and POE function fitting use machine learning techniques.

The ankle is the most commonly injured joint of the lower limb, fundamental to the human body's balance; it is necessary to measure the range of motion by in vivo methods for patients in lying positions in reduced or remote places. The device's development considers ankle anatomy and anthropometry. We propose a Riemannian manifold model based on the device's data readings. Performing simulations enabled us to design the size of the device and the maximal length of the wires. We present a trilateration algorithm, projecting the tetrahedron's sides on the base plane. The sensors are modular and part of the device's lightweight and portable structure. The electronic system is modular, replaced by other single-board computers (SBC) and microcontroller unities. We will also use the TM for ankle characterization and diagnosis for rehabilitation robotics, prosthesis, and orthosis design. The prototype is not a finished product (the TRL is 2). The work's scope is to validate the use of a modern alternative biomechanic representation of the human ankle joint. It is a platform for testing an alternative trilateration method that employs draw-wire sensors (DWS). Such sensors have a constant tension, coiled on a drum attached to a potentiometer, and a flat spiral spring. We also attempted to develop a flexible device design for several foot sizes. We are working on a newer device version with an enhanced attachment system, a more compact design, and digital DWS compatible with a configurable robot. Machine learning and edge computing will assist in disease diagnosis and rehabilitation of patients.

**Author Contributions:** Conceptualization, J.V.-R., Á.V. and Ó.A.-V.; methodology, Á.V. and Ó.A.-V.; software, J.V.-R.; validation, Á.V. and Ó.A.-V.; formal analysis, J.V.-R.; investigation, J.V.-R.; resources, Á.V. and Ó.A.-V.; data curation, Á.V. and Ó.A.-V.; writing—original draft preparation, J.V.-R.; writing—review and editing, Á.V. and Ó.A.-V.; visualization, J.V.-R.; supervision, Á.V.; project administration, Á.V. ; funding acquisition, J.V.-R., Á.V. and Ó.A.-V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by Colciencias-Colfuturo PhD Scholarships Program Educational Credit Forgivable grant number 568, and by Vicerrectorado de Investigación de la Universitat Politècnica de València (PAID-11-21).

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

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The code and CAD electronics and mechanical designs are available.

**Acknowledgments:** The authors thank the Colfuturo Colciencias Collaboration for supporting this work, as well as the Universitat Politècnica de València and the Universidad de los Llanos.

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

#### **Abbreviations**

The following abbreviations are used in this manuscript:


**Acknowledgements**

The authors wish to thank National Council of Scientific and Technical Research (CONICET, Argentina).

**55**

**Author details**

(FCEQyN – UNaM), Argentina

provided the original work is properly cited.

María Eugenia Barrandeguy1,2\* and María Victoria García1,2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Population and Quantitative Genetics, Department of Genetics, Faculty of Exact, Chemicals and Natural Sciences, National University of Misiones

2 Institute of Subtropical Biology – Posadas Node (UNaM – CONICET), National Council of Scientific and Technical Research, Argentina

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*The Sensitiveness of Expected Heterozygosity and Allelic Richness Estimates for Analyzing…*

*DOI: http://dx.doi.org/10.5772/intechopen.95585*


*3.2. Secondary Screening for the Antifungal Activity of Actinobacterial Isolates and Scanning Electron Microscopic Assay*

The six isolates (Figure 4) which showed the strongest antagonism against the three pathogens were further subjected to secondary screening by paired culture antibiosis to further confirm their antagonistic ability against *C. scovillei*, *C. truncatum* and *F. oxysporum*. The results of this assay indicated that all six isolates were capable of inhibiting the growth of *C. scovillei*, *C. truncatum* and *F. oxysporum*. The isolate AR26 was found to be significantly superior to other isolates in inhibiting mycelial growth of *C. scovillei* (59.63%), *C. truncatum* (61.18%) and *F. oxysporum* (63.58%), respectively followed by the isolate AR10 (Supplementary Figure S1). The isolate AFE2 recorded the lowest percentage of mycelial growth inhibition of the tested pathogens, relative to the other five isolates. Scanning Electron Microscopy (SEM) observations indicated a clear evidence for antifungal activity of isolate AR26 against *C. scovillei*, *C. truncatum* and *F. oxysporum*. The antifungal activity was observed as distinct morphological deformities in pathogen hyphae in the presence of antagonist and hyphae were found to be twisted and shrunk in *C. scovillei*, disintegrated in *C. truncatum*, aggregated into clusters in *F. oxysporum* with reduction in mycelial mat (Figure 5). In contrast, the hyphae in the control plate were dense, intact with regular structure.

**Figure 1.** Heat map showing the in vitro antagonism of actinobacterial isolates against chilli fruit rot pathogens as represented by zone of inhibition.

**Figure 2.** Venn diagram representing the actinobacterial isolates exhibiting strong antagonism against fruit rot pathogens.

**Figure 3.** Antifungal activity of actinobacterial isolates against chilli fruit rot pathogens. (**A**) Control *C. scovillei* (**D**) Control *C. truncatum* (**G**) Control *F. oxysporum.* Antifungal activity of actinobacterial isolate AR26 against (**B**) *C. scovillei* (**E**) *C. truncatum* (**H**) *F. oxysporum*. Antifungal activity of actinobacterial isolate AR10 against (**C**) *C. scovillei* (**F**) *C. truncatum* (**I**) *F. oxysporum*.

**Figure 4.** Actinobacterial isolates with potent antagonistic activity against fruit rot pathogens.

**Figure 5.** Scanning Electron Micrographs showing the interaction of antagonist. *S. tuirus* AR26 with *C. scovillei*, *C. truncatum* and *F. oxysporum*; *(***A**) intact mycelium of *C. scovillei* in the absence of antagonist; (**B**) twisted and shrunken hyphae of *C. scovillei* in the presence of antagonist; (**C**) mycelium of *C. truncatum* in absence of antagonist; (**D**) distorted hyphae of *C. truncatum* in the presence of antagonist; (**E**) dense and intact mycelium of *F. oxysporum* in absence of antagonist; (**F**) aggregated hyphae of *F. oxysporum* in presence of antagonist.


**Abstract**

Cardiovascular diseases (CVDs) are still a major cause of mortality worldwide and are a serious health problem. Various factors that contribute toward CVDs include hypertension, tobacco use, physical inactivity, diabetes mellitus, obesity and overweight, alcohol, dietary factors and psychosocial aspects such as stress, anxiety and depression. Nutraceuticals and diet are very important for prevention of CVDs. The potential of nutraceuticals or functional food in mitigating risk of CVDs is discussed. Functional food with health related properties include fruit and vegetable, fish, legumes, nuts, soya protein, coffee, tea, chocolate, animal based functional food. In addition, some diet plans have shown the potential of reducing the incidence of CVDs. This includes the Mediterranean, Dietary Approaches to Stop Hypertension (DASH), Okinawan and vegetarian diets. This chapter examines the risk factors of CVDs, including hypertension, tobacco usage, physical inactivity, diabetes mellitus, overweight and obesity. The chapter also brings to the fore, functional foods with properties related to health and effect of dietary patterns in the treatment and prevention of CVDs.

**Keywords:** non-communicable disease, cardiovascular diseases, nutraceutical, functional foods, dietary patterns


**5.2 Ethical implications**

One of the causes of all such disputes is provoked by the disagreements that may arise concerning the authorship of the works in publications.

Does one invite the other fellow researcher who works with the same group as me? Should I or must I? Should I include the name of my fellow (invite him to participate in my paper) for ethical reasons even if he didn't collaborate with it or only collaborated eventually? Should I invite him only for interest in future partnership?

What if I publish my work all by myself? Will this occasion discomfort? Would the other researcher try to harm me at my institution?

Which decisions are ethical?

All these questions may pass throw the researcher´s mind. Who should decide how to answer them? Each collection has its own policies or habits. There are those who adopt the following criterion: if one did the work alone then one publishes alone and therefore receives all the prestige. Others opt for publication in groups, with partnerships, and therefore for each new publication the whole group will participate again. This is particularly profitable, considering scientific evaluation nowadays.

These questions are quite difficult for the scientist to answer considering the nature of ethics. The individual and society have a double nature. The individual has the powerful principle of egocentrism that stimulates him to be selfish while the society has rivalry, competition, and struggle between the selfish. Society doesn't manage to impose its ethical norms to all individuals. These don't have an ethical behavior that overcomes selfish behavior at all times. This problem becomes worse in very complex societies, where the integration of traditional sympathy bonds is inseparable from the development of individuality (Morin, 2004). Remember that individuality is produced by power (Foucault, 1979).

Ethical sources are also natural because they are older than humanity. The principle of inclusion is prescript at the self-socio-biological organization of the individual, which is transmitted through a genetic path. Mammal societies are communal and rival; they feel at the same time an egocentric conflict and sympathy for the enemies outside. Sympathy for

7 We do not agree with such a simplistic evalution system inside the sciences, because it leads to an artificial partitioning of a scientific work into as many articles as possible, reflecting the greeds of the professional, who, seduced by this system, aims to take maximal advantages of it. This is also a consequence of manufacturing sciences, who see all the professionals as employees (see Callon & Foray, 1997; Oliveira, 2009).

this power is reflected in prestige, which is assigned to the number of publications7 or, in an indirect way, to the possession of the animals-objects that will provide data which will result in publications. So, all this behavior (try to continue being the only one to work with a group), aggravated by the anguish of feeling threatened by our neighbor (since all scientists are clients and competitors at the same time, as Bourdieu affirms), would be similar to the territorial defense of social animals. This could be justified by the pressure that all researchers suffer as a consequence of the scientific system, especially those scientists that

One of the causes of all such disputes is provoked by the disagreements that may arise

Does one invite the other fellow researcher who works with the same group as me?

Should I include the name of my fellow (invite him to participate in my paper) for ethical reasons even if he didn't collaborate with it or only collaborated eventually?

What if I publish my work all by myself? Will this occasion discomfort? Would the

All these questions may pass throw the researcher´s mind. Who should decide how to answer them? Each collection has its own policies or habits. There are those who adopt the following criterion: if one did the work alone then one publishes alone and therefore receives all the prestige. Others opt for publication in groups, with partnerships, and therefore for each new publication the whole group will participate again. This is

These questions are quite difficult for the scientist to answer considering the nature of ethics. The individual and society have a double nature. The individual has the powerful principle of egocentrism that stimulates him to be selfish while the society has rivalry, competition, and struggle between the selfish. Society doesn't manage to impose its ethical norms to all individuals. These don't have an ethical behavior that overcomes selfish behavior at all times. This problem becomes worse in very complex societies, where the integration of traditional sympathy bonds is inseparable from the development of individuality (Morin,

Ethical sources are also natural because they are older than humanity. The principle of inclusion is prescript at the self-socio-biological organization of the individual, which is transmitted through a genetic path. Mammal societies are communal and rival; they feel at the same time an egocentric conflict and sympathy for the enemies outside. Sympathy for

7 We do not agree with such a simplistic evalution system inside the sciences, because it leads to an artificial partitioning of a scientific work into as many articles as possible, reflecting the greeds of the professional, who, seduced by this system, aims to take maximal advantages of it. This is also a consequence of manufacturing sciences, who see all the professionals as employees (see Callon & Foray,

are inserted in a small, local, regional, peripheral and marginal collection.

Should I invite him only for interest in future partnership?

particularly profitable, considering scientific evaluation nowadays.

2004). Remember that individuality is produced by power (Foucault, 1979).

other researcher try to harm me at my institution?

concerning the authorship of the works in publications.

**5.2 Ethical implications**

Should I or must I?

1997; Oliveira, 2009).

Which decisions are ethical?

the struggle against a prey or predator; rivals for alpha males, in fights for primacy, for domination, for conquest of females (Morin, 2004).

Human societies developed and incorporated this double sociological character: the interestrivalry relation and the community bond. The feeling of community is and will be a source of responsibility and sympathy, and these are sources of ethics. Individuality is the source of personal responsibility by your life conduct, and it is also the source of strength of egocentrism. It grows in all fields and tends to inhibit the altruistic and sympathy potentialities, and this contributes to the disintegration of several groups (Morin, 2004).

The bases of ethical crisis belong to a general crisis of the certainty foundations: philosophical knowledge foundation crises and scientific knowledge foundation crises. This is emergence without knowing what emerges. Ethics, as emergence, depends on the social and historical conditions that make it emerge. But it is the individual that makes ethical decisions. It is his duty to choose his values and finalities (Morin, 2004).

So ethics is a relative aspect and that's why some institutions and museums, that usually have a large structure, utilize committees of morals or councils of ethics to decide about authorship, among other things. Usually these places have a previously established policy to follow. In this way, an uncomfortable situation may be avoided. In smaller collections these boards do not exist and the decisions of possible conflict are solved by the curator or even by the professionals themselves. In these cases the problems usually turn into personal problems, where the scientist does not differentiate professional from personal life, since he feels like the owner of the taxonomic groups.

This is one of the reasons why all zoological collections should implant an ethical committee, no matter how small they are. The optimal would be to standardize some protocols to be followed, for example:

	- a. This material is already being studied at that specific moment in that same collection, and, therefore, there is no reason for two persons to develop the same project with the same group.
	- b. The researcher is not connected with any institution or university program.

This protocol should take into account the particular needs of each collection. For example, consider the differences as well as the similarities between an entomological and a mastozoological collection. The elaboration of such a protocol should be accompanied by a juridical foundation, despite their particularities.


**2. Guidelines for selecting potent siRNAs**

As discussed earlier, potency of siRNAs varies greatly with selection of target region. The parameters responsible for effectiveness of siRNAs being target accessibility, uniqueness of target region, absence of SNP sites etc. Other parameters worth considering while selecting the target site is the consideration of alternatively spliced isoforms of the target gene [5].

A parameter which greatly influences siRNA potency is RISC loading of the intended antisense strand [6]. Only one strand of the siRNA duplex enters into RISC and guides the complex to the target mRNA for its silencing. So, the choice of strand that enters into RISC complex plays the vital role in the whole silencing process. Generally RISC complex chooses the siRNA strand which has weaker 5' end binding energy. So this thermodynamic property should be ensured while selecting siRNAs. Also there are a number of sequence compositions that are said to be important for recognition of the intended strand by RISC and some parameters for enhancing the stability of siRNA duplex are also worth considering while selecting siRNAs. These rules can be divided into three categories 1) Selection of target region, 2) Structural and Thermodynamic Consideration and 3) Sequence Characteristics.


**About the Editors**

#### **Charlotte Vermehren**

Charlotte Vermehren has many years of experience within pharmaceutical research, development and use of medicines from both the pharmaceutical industry, biotech, the Danish healthcare system, and academia. For the past 10 years, Charlotte has had an intensive focus on rational pharmacotherapy and polypharmacy. She has published approximately 50 scientific papers and holds a PhD in pharmacology from the University of Copenhagen.

#### **Niels Westergaard**

Niels Westergaard has more than 25 years of experience from both academia and the pharmaceutical industry within pharmaceutical disciplines, which has provided him with a deep insight into the value chain from early drug discovery to development and marketing of new drugs. He has published more than 60 peer reviewed papers and holds a PhD and DSc –degree from the University of Copenhagen.