**1. Introduction**

Diabetic retinopathy (DR) is a neurovascular complication of diabetes mellitus and the leading cause of blindness in working age adults [1]. Diabetic retinal microangiopathy significantly contributes to DR pathogenesis and strategies targeting its occurrence and progression are important for preventing vision loss in affected patients [2,3].

Hyperglycemia-induced retinal vascular pathology is a multi-step process characterized by retinal endothelial cell dysfunction and death, increased vascular permeability leading to diabetic macular edema (DME), and abnormal retinal neovascularization, as seen in proliferative diabetic retinopathy (PDR) [2,3]. Prolonged hyperglycemia affects the retinal microvasculature by altering multiple molecular pathways involving redox imbalance and induction of pro-inflammatory responses [3].

Previously, we showed that hyperglycemia-induced oxidative/nitrative stress accelerates retinal endothelial cell senescence and that this is an important early pathogenic event during the development of diabetic retinal microangiopathy [4]. Retinal oxidative/nitrative stress results from increased production of reactive oxygen and nitrogen species (ROS and RNS, respectively) from cellular and mitochondrial oxidases [5,6] as well as from loss of endogenous antioxidant activities [7,8].

Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase known to exert important biological functions due to its ability to regulate the acetylation state of nuclear and cytoplasmic proteins [9]. Most known targets of HDAC6 are cytoskeletal proteins, transcription factors [9] and endogenous antioxidants such as peroxiredoxin 1 (Prx1) [10,11]. As a consequence of its multiple biological targets, altered HDAC6 expression and activity is linked to inflammation, oxidative stress and the pathogenesis of a number of neurodegenerative [12] and cardiovascular disorders [13] as well as cancer [14].

The potential impact of HDAC6 on diabetic microvascular complications is understudied. Previous studies have shown beneficial e ffects of HDAC6 inhibition in diabetic kidney disease [15] and diabetic heart disease [16]. Moreover, while little is known on HDAC6 contribution to retinal diabetic disorders, studies have implicated pro-oxidative e ffects of HDAC6 in animal models of retinal neurodegenerative diseases [17,18], potentially suggesting a role for this histone deacetylase in retinal pathologies involving oxidative stress, such as DR.

Based on this evidence, we assessed the e ffects of diabetes on HDAC6 retinal expression and activity in human and experimental DR and we investigated the molecular mechanisms involved in this process in a rat model of type 1 diabetes (streptozotocin-induced diabetic rat = STZ-rat) and in cultures of human retinal microvascular endothelial cells exposed to glucidic stress.

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

#### *2.1. Human Postmortem Samples*

De-identified human postmortem retinal samples were obtained from Georgia Eye Bank, Inc. (Alanta, GA, USA). Retinas were obtained from a total of 8 diabetic and 8 non-diabetic donors that were selected based on DR history or lack of reported ocular pathologies (control). Supplementary Table S1 summarizes the demographic and clinical history information available for all donors.

#### *2.2. Animals and Treatment*

All animal experiments strictly adhered to the Statement of the Association for Research in Vision and Ophthalmology (ARVO) for the humane use of laboratory animals for ophthalmological research and to Augusta University approved protocols (#2009-0181). All animals were housed in the vivarium of Augusta University with a 12-h day/night light cycle with light intensity in the room maintained at 130 lux at cage level, and fed ad libitum. Adult male Sprague–Dawley (SD) rats (250−300 g) obtained from Envigo (Indianapolis, IN, USA) were made diabetic by a single intraperitoneal injection of streptozotocin (STZ) (55 mg/kg dissolved in 0.1 mol/L sodium citrate, pH 4.5) (Sigma-Aldrich, St. Louis, MO, USA). Control rats received injections of vehicle alone. Rats with fasting blood glucose ≥250 mg/dL were considered diabetic. A group of STZ-rats was injected intraperitoneally with 10 mg/kg Tubastatin A (TS) (MedChemExpress, Monmouth Junction, NJ, USA) starting two weeks after the STZ injections and continuing every other day for the next six weeks. Control rats received vehicle phosphate-bu ffered saline (PBS) injection. The diabetic rats were sacrificed by an overdose of anesthesia (ketamine 200 mg/kg and xylazine 60 mg/kg) followed by thoracotomy. Blood glucose levels were measured by ReliOn prime blood glucose monitoring system (Bentonville, AR, USA) and glycated hemoglobin A1c (HbA1c) was measured using A1c Now+ System (PTS Diagnostic, Winter Park, FL, USA). A number of other metabolites were also monitored in diabetic and control rat plasma using a biochemistry panel analyzer (Piccolo Xpress analyzer, Princeton, NJ, USA). Rats metabolic profiles in response to the di fferent treatment protocols are reported in Supplementary Table S2.

#### *2.3. Cells and Treatment*

Human retinal endothelial cells (HuREC) were purchased from Cell Systems Corporation (Kirkland, WA, USA) and maintained using complete medium with normal glucose formulation (Cell Systems) at 37 ◦C and 5% CO2 in a humidified atmosphere. All the experiments were carried out using HuREC between passages 3 to 7 and all the tissue culture flasks/plates were pre-coated with attachment factor (Cell Systems). The cells were switched to serum-free medium (Cell Systems) 10 to 12 h before the experiments. To mimic the e ffects of the hyperglycemia (glucidic stress), HuREC were cultured for 48 h in serum-free medium containing 25 mM d-glucose (high glucose, HG). Similarly, control cells were cultured in serum-free and normal glucose medium (5.5 mM d-glucose, NG). Cells grown in normal glucose media with the addition of l-glucose (5.5 mM d-glucose + 19.5 mM l-glucose, LG) served as an osmotic control. Some of the HuREC were treated with 5 μM TS. To determine TS toxicity towards HuREC, a dose-response curve using MTT cell viability assay (Abcam, Cambridge, MA, country) was conducted following the manufacturer's instructions. MTT dye absorbance was read using a microplate reader at 540 nm (Supplementary Figure S1).


1. Introduction

Typical flexible object manipulation by robots has been performed in a static state or a quasi-static state [1–7]. In these methods, dynamical deformation of the flexible object is not considered, making it easier to manipulate the flexible object. However, the robots have to wait until the deformation converges to the steady state, which results in the manipulation time being very long, and the working efficiency is not good. Moreover, although it is desired to manipulate the flexible object while observing its deformation with cameras, the working efficiency becomes even worse because of the low speed of the cameras and image processing for recognizing the deformation. In addition, a deformation that changes momentarily cannot be recognized in real time by general-purpose cameras and image processing, and appropriate feedback control may not be carried out because of the latency from the recognition to the robot motion.

#### Becoming Human with Humanoid - From Physical Interaction to Social Intelligence

Recently, dynamic manipulations and nonprehensile manipulations of objects have been actively investigated with the goal of developing new manipulation techniques [8]. Our goal has been to achieve high-speed flexible object manipulation by handling the flexible object in a "dynamic," nonprehensile state. As a result, we succeeded in simplifying the deformation model of flexible objects by using the high-speed motion of a high-speed robot, and we were able to perform appropriate real-time visual feedback control with a high-speed vision system. This dynamic manipulation technique may enable rapid realization of flexible object manipulation tasks.

In this chapter, we review the proposed method based on high-speed motion and visual feedback, and we demonstrate several tasks carried out by a high-speed robot system using the proposed method.


*Article* **Genome-Wide Identification of the Rose** *SWEET* **Gene Family and Their Different Expression Profiles in Cold Response between Two Rose Species**

**Xiangshang Song, Yaping Kou, Mingao Duan, Bo Feng, Xiaoyun Yu, Ruidong Jia, Xin Zhao, Hong Ge \* and Shuhua Yang \***

> State Key Laboratory of Vegetable Biobreeding, Key Laboratory of Biology and Genetic Improvement of Flower Crops (North China), Ministry of Agriculture and Rural Affairs, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China<EMAIL_ADDRESS>(B.F.)

**\*** Correspondence<EMAIL_ADDRESS>(H.G<EMAIL_ADDRESS>(S.Y.); Tel.: +86-10-8210-9542 (S.Y.)

**Abstract:** Sugars Will Eventually be Exported Transporter (SWEET) gene family plays indispensable roles in plant physiological activities, development processes, and responses to biotic and abiotic stresses, but no information is known for roses. In this study, a total of 25 *RcSWEET* genes were identified in *Rosa chinensis* 'Old Blush' by genome-wide analysis and clustered into four subgroups based on their phylogenetic relationships. The genomic features, including gene structures, conserved motifs, and gene duplication among the chromosomes of *RcSWEET* genes, were characterized. Seventeen types of *cis*-acting elements among the *RcSWEET* genes were predicted to exhibit their potential regulatory roles during biotic and abiotic stress and hormone responses. Tissue-specific and cold-response expression profiles based on transcriptome data showed that *SWEETs* play widely varying roles in development and stress tolerance in two rose species. Moreover, the different expression patterns of cold-response *SWEET* genes were verified by qRT-PCR between the moderately cold-resistant species *R. chinensis* 'Old Blush' and the extremely cold-resistant species *R. beggeriana*. Especially, *SWEET2a* and *SWEET10c* exhibited species differences after cold treatment and were sharply upregulated in the leaves of *R. beggeriana* but not *R. chinensis* 'Old Blush', indicating that these two genes may be the crucial candidates that participate in cold tolerance in *R. beggeriana*. Our results provide the foundation for function analysis of the *SWEET* gene family in roses, and will contribute to the breeding of cold-tolerant varieties of roses.

**Keywords:** rose; *SWEET* gene family; sugar transport; gene expression; cold response


1. Introduction

Proposals for a momentum transfer based launch system are not new. Konstantin Tsiolkovsky, credited with the concept of multi-stage rocket vehicles, also proposed the orbital tower. Much later Yuri Artsutanov inverted this idea to suggest a geostationary satellite with a counterweight and a tether extending to the Earth's surface. This so called 'space elevator' was first published in 1960 in Komsomolskaya Pravda and later discovered independently in the US when the term 'skyhook' was coined [1]. The structure was shown to be stable against the effects of lunar tidal forces and payload motions, and functions by extracting energy from Earth rotation [2]. The problem is that no known material has sufficient strength to construct a space elevator in Earth orbit.

Difficulties with the space elevator led to the proposal of the asynchronous orbital skyhook [3]. (The original concept was credited to John McCarthy at Stanford.) This is an extended orbital structure that rotates so that each end periodically comes to a low altitude and velocity, at which instants the system is easy to access. Initial studies advocated configurations that place a low demand on the tether material properties, as this was thought to be the principal challenge. To replace energy lost during launch it was proposed that the skyhook be used to return a similar quantity of material from orbit to Earth.

Detailed studies of the asynchronous skyhook [4, 5] addressed engineering aspects of the tether and docking mechanism. They proposed a set of configurations in which access is provided by a hypersonic vehicle operating at a speed of at least

3.1 km/s (Mach 10). This high speed of the access vehicle reduces the skyhook rotation rate and so places less stress on the tether material.

vM ¼ RΩ � Lω � 465 (1)

h i þ Lω2 (2)

aM ¼ RΩ2 ð Þ 1 � L=R �2 � 1

Space Access for Future Planetary Science Missions DOI: http://dx.doi.org/10.5772/intechopen.88530

rotation.

access vehicles.

negligible.

3. Mass properties

solved for the tether cross-section:

Here χ2 ¼ ρω2L2

55

Here L and ω are the skyhook half-length and rotation frequency, and Eq. 2 includes the acceleration components due to gravity, orbital velocity and skyhook

Specifying the endpoint velocity and acceleration yields two implicit equations for the skyhook parameters. With a nominal orbital radius of 8000 km the skyhook length is small enough to apply the limit L ≪ R: Then for a minimum energy state at zero velocity and 40 m/s2 acceleration, the skyhook parameters are L ¼ 1090 km and ω ¼ 0:006s�1. This system can be accessed at zero velocity by a vehicle capable of ascending to an altitude of 532 km. Moreover, the maximum acceleration experienced during launch is similar to that of a conventional launch vehicle. One of the skyhook endpoints is at minimum energy when the structure is oriented radially. This state occurs with a period τ ¼ π=ð Þ ω � Ω corresponding to a ground track distance of 3176 km around the equator. The orbital parameters could be adjusted so this distance is an exact fraction of the equatorial circumference, in which case the minimum energy states occur above fixed points on the equator. These locations are natural sites at which to establish bases to operate the suborbital

The skyhook configurations of interest here have an endpoint speed near orbital

Consider a symmetric skyhook comprising two equal masses m connected by a massive tether of length 2L and define the origin at the center. The tether crosssection is a rð Þ and the tether material has uniform density ρ and ultimate tensile strength T. For a skyhook with rotation frequency ω the tension σ at radius r obeys:

ð Þ¼� r ρω2

Substituting a rð Þ¼ σð Þr =T and noting that a Lð Þ¼ mLω2=T this equation can be

T exp χ2 1 � r

By symmetry the mass centroid is at the origin. This structure may be generalized to describe a set of asymmetric configurations with unequal end masses at different distances from the centroid. The symmetric configuration has the benefit of offering two opportunities to access the skyhook in each rotation cycle, but asymmetric

> ðL 0

L

=2T is a dimensionless parameter characterizing the skyhook.

ra rð Þ (3)

� �2 � � � � (4)

a rð Þdr (5)

velocity to allow access at low energy. The high rotation rate means tension is mainly due to centripetal force, with the field gradient contribution being

σ0

configurations allow access to a greater variety of launch trajectories. The tether mass MT and moment of inertia IT are given by:

MT ¼ 2ρ

a rð Þ¼ mLω2

Hypersonic flight technology is not yet capable of providing routine access to the high Mach number regime. By contrast, several reusable vehicles are available that provide access to suborbital flight trajectories using combinations of air-breathing and rocket propulsion [6, 7]. High strength fiber technology has also made substantial progress with the incorporation of carbon nanotubes into the molecular structure [8]. This suggests a need to review the orbital skyhook concept with a focus on configurations that allow low speed access. It is also necessary to explore different approaches to energy replenishment that do not require access to a repository of orbiting material.

Section 2 reviews the skyhook concept and estimates the parameters of a practical launch system. Expressions for the skyhook mass properties are obtained in Section 3 for the case where centripetal force is the dominant source of tension. The dynamics is modeled in Section 4 assuming the structure remains linear, with the tether mass properties represented by a compact object at the mass centroid. Electric propulsion is proposed as a mechanism for energy replenishment in Section 5, and the feasibility of supplying propellant for the thrusters is explored. Section 6 describes the advantages of a skyhook launch system for future planetary science missions, and Section 7 summarizes the main results.

## 2. Concept description

An orbital skyhook launch system involves three phases, each exploiting a different physical process. It begins with the delivery of a payload by suborbital vehicle. Docking occurs at one of the skyhook endpoints when it is near minimum altitude and velocity. The suborbital vehicle is required to attain only a small fraction of the energy needed for orbit, and does not need to operate in a hypersonic flight regime. It can therefore employ mature airframe and propulsion technologies, making it easier to design for efficiency and reusability.

The second phase is momentum transfer from the skyhook to the payload [9]. After docking the payload gains energy as the skyhook rotates, reaching a maximum after half a cycle. If the payload is not released energy transfers back to the skyhook in the second half of the cycle as it returns to minimum energy. By selecting when the payload is released, it can be placed into an elliptical orbit or on an escape trajectory. Note that if the payload is released at a subsequent minimum energy point, the skyhook energy and orbit are left unaffected. This means the vehicle is transported around the Earth at orbital velocity, with the only energy cost being that of gaining access to the skyhook.

In the third phase energy drawn from the skyhook during launch must be replenished. If the payload mass is small relative to the total system mass, the orbital perturbation is also small. In this case the structure remains above the atmosphere through subsequent rotations, and energy replenishment may occur over an extended period. Electric propulsion is proposed for this purpose. It provides a small thrust with a large specific impulse, and therefore high propellant efficiency. Propellant can be delivered with the payload to supply thrusters at the skyhook endpoints, but it will be shown that a better approach is to apply thrust at the skyhook mass centroid.

Of interest here are skyhook configurations that offer low speed access. Ideally the endpoint speed should match the orbital velocity relative to Earth's surface. In addition, acceleration during launch must not be excessive. For a skyhook in a circular equatorial orbit with radius R and orbital frequency Ω the endpoint ground track speed and acceleration are given by:

Space Access for Future Planetary Science Missions DOI: http://dx.doi.org/10.5772/intechopen.88530

3.1 km/s (Mach 10). This high speed of the access vehicle reduces the skyhook

Hypersonic flight technology is not yet capable of providing routine access to the high Mach number regime. By contrast, several reusable vehicles are available that provide access to suborbital flight trajectories using combinations of air-breathing and rocket propulsion [6, 7]. High strength fiber technology has also made substantial progress with the incorporation of carbon nanotubes into the molecular structure [8]. This suggests a need to review the orbital skyhook concept with a focus on configurations that allow low speed access. It is also necessary to explore different approaches to energy replenishment that do not require access to a repos-

Section 2 reviews the skyhook concept and estimates the parameters of a practi-

An orbital skyhook launch system involves three phases, each exploiting a dif-

The second phase is momentum transfer from the skyhook to the payload [9]. After docking the payload gains energy as the skyhook rotates, reaching a maximum after half a cycle. If the payload is not released energy transfers back to the skyhook in the second half of the cycle as it returns to minimum energy. By selecting when the payload is released, it can be placed into an elliptical orbit or on an escape trajectory. Note that if the payload is released at a subsequent minimum energy point, the skyhook energy and orbit are left unaffected. This means the vehicle is transported around the Earth at orbital velocity, with the only energy cost

In the third phase energy drawn from the skyhook during launch must be replenished. If the payload mass is small relative to the total system mass, the orbital perturbation is also small. In this case the structure remains above the atmosphere through subsequent rotations, and energy replenishment may occur over an extended period. Electric propulsion is proposed for this purpose. It provides a small thrust with a large specific impulse, and therefore high propellant efficiency. Propellant can be delivered with the payload to supply thrusters at the skyhook endpoints, but it will be shown that a better approach is to apply thrust at the skyhook mass centroid. Of interest here are skyhook configurations that offer low speed access. Ideally the endpoint speed should match the orbital velocity relative to Earth's surface. In addition, acceleration during launch must not be excessive. For a skyhook in a circular equatorial orbit with radius R and orbital frequency Ω the endpoint ground

ferent physical process. It begins with the delivery of a payload by suborbital vehicle. Docking occurs at one of the skyhook endpoints when it is near minimum altitude and velocity. The suborbital vehicle is required to attain only a small fraction of the energy needed for orbit, and does not need to operate in a hypersonic flight regime. It can therefore employ mature airframe and propulsion technologies,

cal launch system. Expressions for the skyhook mass properties are obtained in Section 3 for the case where centripetal force is the dominant source of tension. The dynamics is modeled in Section 4 assuming the structure remains linear, with the tether mass properties represented by a compact object at the mass centroid. Electric propulsion is proposed as a mechanism for energy replenishment in Section 5, and the feasibility of supplying propellant for the thrusters is explored. Section 6 describes the advantages of a skyhook launch system for future planetary science

rotation rate and so places less stress on the tether material.

missions, and Section 7 summarizes the main results.

making it easier to design for efficiency and reusability.

being that of gaining access to the skyhook.

track speed and acceleration are given by:

54

itory of orbiting material.

Planetology - Future Explorations

2. Concept description

$$v\_M = R\Omega - L\alpha - 4\mathfrak{G} \tag{1}$$

$$a\_M = R\Omega^2 \left[ \left( \mathbf{1} - L/R \right)^{-2} - \mathbf{1} \right] + L\alpha^2 \tag{2}$$

Here L and ω are the skyhook half-length and rotation frequency, and Eq. 2 includes the acceleration components due to gravity, orbital velocity and skyhook rotation.

Specifying the endpoint velocity and acceleration yields two implicit equations for the skyhook parameters. With a nominal orbital radius of 8000 km the skyhook length is small enough to apply the limit L ≪ R: Then for a minimum energy state at zero velocity and 40 m/s2 acceleration, the skyhook parameters are L ¼ 1090 km and ω ¼ 0:006s�1. This system can be accessed at zero velocity by a vehicle capable of ascending to an altitude of 532 km. Moreover, the maximum acceleration experienced during launch is similar to that of a conventional launch vehicle.

One of the skyhook endpoints is at minimum energy when the structure is oriented radially. This state occurs with a period τ ¼ π=ð Þ ω � Ω corresponding to a ground track distance of 3176 km around the equator. The orbital parameters could be adjusted so this distance is an exact fraction of the equatorial circumference, in which case the minimum energy states occur above fixed points on the equator. These locations are natural sites at which to establish bases to operate the suborbital access vehicles.

#### 3. Mass properties

The skyhook configurations of interest here have an endpoint speed near orbital velocity to allow access at low energy. The high rotation rate means tension is mainly due to centripetal force, with the field gradient contribution being negligible.

Consider a symmetric skyhook comprising two equal masses m connected by a massive tether of length 2L and define the origin at the center. The tether crosssection is a rð Þ and the tether material has uniform density ρ and ultimate tensile strength T. For a skyhook with rotation frequency ω the tension σ at radius r obeys:

$$
\sigma'(r) = -\rho a^2 r a(r) \tag{3}
$$

Substituting a rð Þ¼ σð Þr =T and noting that a Lð Þ¼ mLω2=T this equation can be solved for the tether cross-section:

$$a(r) = \frac{mL\alpha^2}{T} \cdot \exp\left[\chi^2 \left\{1 - \left(\frac{r}{L}\right)^2\right\}\right] \tag{4}$$

Here χ2 ¼ ρω2L2 =2T is a dimensionless parameter characterizing the skyhook. By symmetry the mass centroid is at the origin. This structure may be generalized to describe a set of asymmetric configurations with unequal end masses at different distances from the centroid. The symmetric configuration has the benefit of offering two opportunities to access the skyhook in each rotation cycle, but asymmetric configurations allow access to a greater variety of launch trajectories.

The tether mass MT and moment of inertia IT are given by:

$$M\_T = 2\rho \int\_0^L a(r) dr \tag{5}$$

$$I\_T = 2\rho \int\_0^L a(r)r^2 dr\tag{6}$$

'worst case' in the sense that skyhook rotation is specified to allow access at zero velocity relative to the Earth. If the access vehicle provides a horizontal velocity component the rotation rate is smaller, in which case the taper factor and skyhook

Skyhook length is a significant factor in the dynamics because field strength is not uniform across the structure. This differs from most problems in astrodynamics where the object of interest is small compared to the field gradient length scale, or

Here the skyhook is assumed to behave as a rigid body, kept in tension by the rotation and experiencing no stretching or bending. The validity of these assumptions depends on the tether material properties, but they are sufficient for the present purpose. The structure is expected to remain linear due to the large

The equations of motion of a rigid body are typically obtained by a Lagrangian

The skyhook system is modeled here as three objects connected by tethers of fixed length L as illustrated in Figure 1. The central object has the mass properties of the tethers as calculated above. This formulation represents the physical extent of the skyhook in a non-uniform field. It is also a good approximation for the mass distribution of the tether if it has a significant taper factor, in which case much of the mass is concentrated near the centroid. Based on these considerations a Newto-

The system state is described by a six element vector comprising the skyhook centroid location r ¼ ð Þ r; θ and orientation angle φ and their derivatives. The endpoint locations are specified by the vectors r1 and r2 which are functions of the state

Skyhook geometry with the tether mass and moment of inertia represented by a compact object at the mass

method using the mass properties. This formulation ignores the field gradient effect, which is important for skyhook dynamics. To see this note that the skyhook structure experiences a moment due to the two arms being subject to different field strengths according to their proximity to Earth. If the skyhook were treated as a

the system can be simplified by assuming spherical symmetry.

single compact object this behavior would not be represented.

centripetal restoring force that counters any bending.

nian formulation is used for the analysis.

Figure 1.

centroid.

57

mass are also decreased.

Space Access for Future Planetary Science Missions DOI: http://dx.doi.org/10.5772/intechopen.88530

4. Equations of motion

Evaluating the integrals and simplifying:

$$\mathbf{M}\_{\mathrm{T}}/\mathfrak{m} = 2\sqrt{\pi} \,\,\chi\,\exp\left[\chi^2\right] \mathbf{erf}(\chi) \tag{7}$$

$$\mathrm{I}\_{T}/\mathfrak{mL}^{2} = \sqrt{\pi} \chi^{-1} \exp\left[\chi^{2}\right] \mathrm{erf}(\chi) - 2 \tag{8}$$

The limit χ ! 0 represents a material of infinite strength, in which case the tether mass and moment of inertia vanish. Adding the contributions of the two end masses leads to expressions for the mass properties of the entire skyhook:

$$M/m = 2\sqrt{\pi} \,\chi\exp\left[\chi^2\right] \,\text{erf}(\chi) + 2\tag{9}$$

$$\text{I/mL}^2 = \sqrt{\pi} \,\chi^{-1} \exp\left[\chi^2\right] \text{erf}(\chi) \tag{10}$$

These expressions for the skyhook mass properties indicate the dependence on tether material properties, and provide key parameters for dynamical modeling.

An important feature of a tether is the taper factor, the ratio of maximum to minimum cross-section area. A tether constructed from low strength material has a large taper factor, indicating its impracticality. The nominal skyhook described above with a carbon fiber tether has a taper factor of 237, in which case the diameter at the centroid is about 15 times that the end points. If the tether had the properties of carbon nanotubes the taper factor reduces to 3.3. The properties of any future tether material are likely to fall within these bounds.

Table 1 indicates the mass properties of the nominal skyhook for several tether materials. Notionally high strength materials like steel and diamond are excluded by the very large taper factor. Aramid fibers like Kevlar are possible but the total mass is large. The strongest carbon fiber offers a solution with a skyhook mass about 4600 times the endpoint mass. If materials with still greater tensile strength become available, such as by incorporating carbon nanotubes or colossal carbon tubes into the tether material, the taper factor and skyhook mass can be much smaller.

For the skyhook configuration described here the endpoint mass is regarded as the maximum payload capability. This assumes the endpoint mass may be replaced by a docking mechanism of negligible mass to capture the payload. Engineering margins have not been included in this analysis, but the nominal configuration is a

#### Table 1.

Tether mass properties for various materials (from Eqs. (4), (7) and (8)).

'worst case' in the sense that skyhook rotation is specified to allow access at zero velocity relative to the Earth. If the access vehicle provides a horizontal velocity component the rotation rate is smaller, in which case the taper factor and skyhook mass are also decreased.


2.2.6. Static Standing Insole-Type Plantar Pressure Measurement

Based on the methods proposed by Cavanagh in 1987, the movements were designed to test whether the self-made insole-type plantar was able to determine the subject's foot type. The subjects stood evenly on the insole-type plantar pressure instrument, and the designed insole-type plantar pressure instruments were placed in a normal shoe that served as the test shoe in the current study. Before using this normal shoe, all the additional support for the arch had already been removed to ensure that there was no other arch support, which may bias the result. On this statically standing

position, the subjects had to look straight ahead with their hands at their sides for five seconds with the sample rate of 100 Hz (as shown in Figure 7).

**Figure 7.** Static standing plantar pressure measurement position for the subject, with the insole-plantar pressure device inserted in the shoes.


*4.8. Statistics*

Statistical analyses were performed with Prism version 8.4.1 (GraphPad, La Jolla, CA, USA). We analyzed these data to identify outliers and removed all outliers prior to performing statistical comparisons. Normality was determined using the Shapiro–Wilk test. Significant differences between medians were determined by unpaired Mann–Whitney U test. For normally distributed data, significant differences between means were determined by unpaired *t* test and ANOVA. Binary data were compared using Fisher's exact test. Data are presented as median + IQR data or mean ± standard deviation (SD). Statistical significance was defined as *p* < 0.05.

**Supplementary Materials:** Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/18/ 6477/s1.

**Author Contributions:** Conception, C.J.S., J.K.K., N.C.D., N.M.M.; methodology, A.M.B., C.J.S., N.C.D., N.M.M., S.B.; validation, C.J.S., C.C.Y., N.C.D., N.M.M., T.W., V.O.; formal analysis, C.C.Y., N.C.D., N.M.M.; investigation, C.C.Y., N.C.D., N.M.M., N.V.-P., S.B., T.W., V.O.; resources, A.M.B., J.K.K., N.C.D.; writing—original draft preparation C.J.S., N.C.D., N.M.M., N.V.-P.; writing—review and editing C.J.S., J.K.K., N.C.D., N.M.M.; visualization, N.C.D., N.M.M.; supervision, A.M.B., C.J.S., J.K.K., N.C.D.; funding acquisition, N.C.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by National Institutes of Health, grant number: R01HL127013.

**Acknowledgments:** We thank Jason Butler and Ralf Adams for their donations of the experimental mice for generation of our colony. We thank Naiche Adler for her oversight on the development and layout of the project and figures. We thank Sara Morelli and Pranela Rameshwar who provided insightful comments and assistance with design of the project. All schematic representations were created with BioRender.com.

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


**7.3 Dam foundations**

A site investigation shall be carried out prior to construction to verify the nature of the foundation. By knowing the actual foundation condition at the site, the earth dam can then be designed accordingly. An embankment foundation is said to be suitable if it is able to provide stable support for the bridge under all conditions of

saturation and loading and that it provides adequate leakage resistance to avoid excessive water loss [42, 43].

The foundation of the dam may be broadly classified into three types which are rock foundations, coarse-grained material foundations and fine-grained material foundation [43].

These foundations may need to be treated to stabilize any weakness and also to reduce leakage. On the other hand, rock foundation must be inspected for erosive leakage and excessive uplift pressure. If such conditions exist, the foundation must be considered grouting [9, 43].

## **7.4 Dam seismicity**

Induced earthquakes refer to the earthquakes and slight tremors that result from human activity that alter the stresses and stresses on the Earth's crust. Most of the induced earthquakes are of low magnitude [44].

Seismic hazard from induced seismic activity can be assessed using techniques similar to natural earthquakes, although one account for unstable earthquakes. Earthquakes vibrating from induced earthquakes appear to be similar to those observed in natural tectonic earthquakes, although differences in rupture depth need to be taken into account. This means that ground motion models derived from natural seismic recordings can be used, which are often more numerous in robust motion databases compared to induced earthquake data. Then, a risk assessment can be performed, taking into account earthquake risk and the vulnerability of vulnerable items (such as local residents and building stock). Finally, risk can, in theory at least, be mitigated, either through modifications of the risk or reduced exposure or vulnerability [44].


**1. Introduction**

In recent years, the emergence of antibiotic-resistant bacteria has become a global problem. One of the main drivers for the emergence of antibiotic-resistant bacteria is the excessive or inappropriate use of antibiotics [1,2].

Clinical isolates of antibiotic-resistant *Escherichia coli* were first isolated in the 1970s [3], soon followed by strains with extended-spectrum β-lactamase (ESBL) genes, which confer resistance to third generation cephalosporins [4], which are the most commonly prescribed classes of antibiotics with bactericidal activity [5]. Multiple resistant bacteria are now a major problem in human and veterinary medicine, especially for nosocomial infections [3,6]. These antibiotic resistance genes are found on plasmids that also carry genes conferring resistance to other non-β-lactam antibiotics such as aminoglycosides and trimethoprim-sulfamethoxazole [4]. *E. coli* harboring these plasmids are only susceptible to carbapenems and colistin.

Antibiotic-resistant bacteria are commonly found in agricultural settings, hospital environments, factory farms, and contaminated foodstuffs [7]. Regardless of the source of antibiotic-resistant bacteria, development of efficient cleaning and disinfection methods are urgently needed to prevent their spread [8].

Several technologies to eliminate antibiotic-resistant bacteria and/or antibiotic resistance genes have been employed including ultraviolet (UV) light [9], solar photo-Fenton process [10], photocatalysis [11], photoelectrocatalysis [12], and nanoparticle treatment [13]. However, some of these technologies have given disappointing levels of disinfection [14,15].

Recently, plasma technologies have been shown to effectively inactivate various bacteria including antibiotic-resistant bacteria [16,17]. Currently, however, it is not known whether the sensitivity against plasma differs between antibiotic-resistant and non-resistant bacteria. Previous studies were conducted in a way that may have masked possible differences arising from sensitivity due to antibiotic resistance. Here, we compared antibiotic-resistant bacteria harboring a plasmid that confers antibiotic resistance with its non-transformed counterpart in terms of susceptibility against plasma generated by a dielectric barrier discharge (DBD) plasma torch. In addition, we analyzed biochemical changes in the bacteria after exposure to the plasma to better understand the mechanism of inactivation.

#### **2. Results**

To obtain antibiotic-resistant bacteria, *Escherichia coli* DH5α was transformed with pPRO-EX-HT -CAT, which contains an ampicillin resistance gene and chloramphenicol acetyltransferase (CAT) gene. Colonies were picked after overnight selection on Luria-Bertani (LB) agar medium containing ampicillin. The selected colonies were subsequently cultured in LB liquid medium with or without ampicillin for 24 h. Aliquots of the resultant *E. coli* were then treated with a dielectric barrier discharge (DBD) plasma torch (Figure 1). As a control, untransformed *E. coli* were also analyzed. The viable cell counts of all *E. coli* samples decreased after plasma treatment. The viable cell number for *E. coli* transformed/selected with pPRO-EX-HT-CAT and cultured in LB liquid medium supplemented with ampicillin was lower than the control (2.40 × 106 ± 1.38 × 106 CFU/mL, 0 min) (Figure 2). Plasma treatment for 1 min reduced viable cell count to 1.38 × 105 ± 0.80 × 105 CFU/mL. However, cultures of the transformed and selected *E. coli* in ampicillin-free LB liquid medium were 2.27 × 106 ± 1.31 × 106 CFU/mL (0 min) and 1.06 × 105 ± 0.61 × 105 CFU/mL (1 min). In summary, no significant difference in viable cell count was observed between the ampicillin-containing culture and ampicillin-free culture. This result suggests that selection pressure with ampicillin does not change the susceptibility of the bacteria to plasma treatment.

Similarly, *E. coli* transformed/selected with pPRO-EX-HT-CAT and non-transformed *E. coli* were cultured in ampicillin-free LB liquid medium (Figure 3) and then subjected to plasma treatment. Viable cell number was 2.13 × 107 ± 0.20 × 107 CFU/mL (0 min) and 1.80 × 104 ± 1.80 × 104 CFU/mL (1 min) for the transformed *E. coli*, and 2.59 × 107 ± 0.47 × 107 CFU/mL (0 min) and 7.83 × 104 ± 5.17 × 104 CFU/mL (1 min) for the non-transformed *E. coli,* indicating no significant difference between the two groups. In all cases, the number of viable bacteria was below the detection limit after 2 min treatment (Figures 2 and 3).

Next, we aimed to examine the effect of plasma treatment on components of *E. coli* using the DBD plasma torch. To this end, we performed DNA analysis by polymerase chain reaction (PCR), CAT quantification by ELISA and CAT activity using an acetyltransferase activity assay as well as lipopolysaccharide (LPS) analysis by the Limulus test. PCR amplification using sequence-specific primers for bacterial 16S rDNA followed by agarose gel electrophoresis gave a discrete band in the untreated *E. coli* sample (0 min). DNA sequencing of the amplified band verified that it corresponded to *E. coli* DH5α 16S rDNA (i.e., 99.25 ± 0.35% [50→3 0 direction, *N* = 4] and 99.48 ± 0.34% [30→5 0 direction, *N* = 4] identical to Genbank accession number NZ\_CP026085.1) (Figure 4). A band was detected in *E. coli* samples (0 min) in DH5α Amp(−) (non-transformed *E. coli* proliferated in an ampicillin-free LB liquid medium) and pPRO-EX-CAT DH5α Amp(−) (*E. coli* transformed with pPRO-EX-CAT, selected in

of inactivation.

**2. Results**

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW

prevent their spread [8].

ampicillin-containing LB agar, and proliferated in ampicillin-free LB liquid medium), although the levels of intact DNA was low at 1 min and undetectable at 2 min for both *E. coli* samples. viable cell count was observed between the ampicillin-containing culture and ampicillin-free culture. This result suggests that selection pressure with ampicillin does not change the susceptibility of the bacteria to plasma treatment.

CFU/mL (0 min) and 1.06 × 105 ± 0.61 × 105 CFU/mL (1 min). In summary, no significant difference in

Antibiotic-resistant bacteria are commonly found in agricultural settings, hospital environments, factory farms, and contaminated foodstuffs [7]. Regardless of the source of antibioticresistant bacteria, development of efficient cleaning and disinfection methods are urgently needed to

Several technologies to eliminate antibiotic-resistant bacteria and/or antibiotic resistance genes have been employed including ultraviolet (UV) light [9], solar photo-Fenton process [10], photocatalysis [11], photoelectrocatalysis [12], and nanoparticle treatment [13]. However, some of

Recently, plasma technologies have been shown to effectively inactivate various bacteria including antibiotic-resistant bacteria [16,17]. Currently, however, it is not known whether the sensitivity against plasma differs between antibiotic-resistant and non-resistant bacteria. Previous studies were conducted in a way that may have masked possible differences arising from sensitivity due to antibiotic resistance. Here, we compared antibiotic-resistant bacteria harboring a plasmid that confers antibiotic resistance with its non-transformed counterpart in terms of susceptibility against plasma generated by a dielectric barrier discharge (DBD) plasma torch. In addition, we analyzed biochemical changes in the bacteria after exposure to the plasma to better understand the mechanism

To obtain antibiotic-resistant bacteria, *Escherichia coli* DH5α was transformed with pPRO-EX-HT-CAT, which contains an ampicillin resistance gene and chloramphenicol acetyltransferase (CAT) gene. Colonies were picked after overnight selection on Luria-Bertani (LB) agar medium containing ampicillin. The selected colonies were subsequently cultured in LB liquid medium with or without ampicillin for 24 h. Aliquots of the resultant *E. coli* were then treated with a dielectric barrier discharge (DBD) plasma torch (Figure 1). As a control, untransformed *E. coli* were also analyzed. The viable cell counts of all *E. coli* samples decreased after plasma treatment. The viable cell number for *E. coli* transformed/selected with pPRO-EX-HT-CAT and cultured in LB liquid medium supplemented with ampicillin was lower than the control (2.40 × 106 ± 1.38 × 106 CFU/mL, 0 min) (Figure 2). Plasma treatment for 1 min reduced viable cell count to 1.38 × 105 ± 0.80 × 105 CFU/mL. However, cultures of

these technologies have given disappointing levels of disinfection [14,15].

**Figure 1.** Schematic of a dielectric barrier discharge (DBD) plasma torch. (**a**) The DBD plasma torch comprises a ceramic tube (Al2O3) (length, 100 mm) containing a stainless-steel mesh (SUS304) and covered with copper tape (thickness, 80 μm) on the outside. The copper tape and stainless steel mesh with stainless steel wire were connected to a power supply (10 kV, 10 kHz); (**b**) Cross-sectional view **Figure 1.** Schematic of a dielectric barrier discharge (DBD) plasma torch. (**a**) The DBD plasma torch comprises a ceramic tube (Al2O3 ) (length, 100 mm) containing a stainless-steel mesh (SUS304) and covered with copper tape (thickness, 80 µm) on the outside. The copper tape and stainless steel mesh with stainless steel wire were connected to a power supply (10 kV, 10 kHz); (**b**) Cross-sectional view of the torch, which is made up of a ceramic tube (inner diameter, 4 mm; outer diameter, 6 mm). During gas plasma generation, air flow was maintained at 3.5 L/min using an air pump (Suishin SSPP-2S; Suisaku Co., Sakai, Japan). A suspension (20 µL) of *Escherichia coli* was dropped onto a watch glass. The distance from the top of the plasma torch to the liquid surface on the watch glass was fixed at 20 mm. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW of the torch, which is made up of a ceramic tube (inner diameter, 4 mm; outer diameter, 6 mm). During gas plasma generation, air flow was maintained at 3.5 L/min using an air pump (Suishin SSPP-2S; Suisaku Co., Sakai, Japan). A suspension (20 μL) of *Escherichia coli* was dropped onto a watch glass. The distance from the top of the plasma torch to the liquid surface on the watch glass was fixed at 20 mm.

**Figure 2.** Treatment with a DBD plasma torch reduced the viable cell number of *Escherichia coli* transformed with a plasmid conferring ampicillin resistance, which was selected by spreading on ampicillin-containing agar medium and proliferated in ampicillin-containing liquid medium (■) by a similar amount as plasmid-transformed *E. coli* proliferated in ampicillin-free liquid medium (□). A suspension of *E. coli* transformed with an ampicillin resistance gene-containing plasmid pPRO-EX-HT-CAT, selected in ampicillin-containing agar medium, and proliferated either in ampicillincontaining liquid medium (■) or ampicillin-free liquid medium (□) was exposed to a DBD plasma torch for the indicated time (min). Viable cell count (colony forming units (CFU)/mL) was then determined before and after treatment. NS means no significant difference between ■ and □ at each time when verified by the non-repeated measured ANOVA (analysis of variance) followed by the Bonferroni correction. ANOVA for non-repeated measures was used for comparing the intragroup, **Figure 2.** Treatment with a DBD plasma torch reduced the viable cell number of *Escherichia coli* transformed with a plasmid conferring ampicillin resistance, which was selected by spreading on ampicillin-containing agar medium and proliferated in ampicillin-containing liquid medium () by a similar amount as plasmid-transformed *E. coli* proliferated in ampicillin-free liquid medium (). A suspension of *E. coli* transformed with an ampicillin resistance gene-containing plasmid pPRO-EX-HT-CAT, selected in ampicillin-containing agar medium, and proliferated either in ampicillin-containing liquid medium () or ampicillin-free liquid medium () was exposed to a DBD plasma torch for the indicated time (min). Viable cell count (colony forming units (CFU)/mL) was then determined before and after treatment. NS means no significant difference between and at each time when verified by the non-repeated measured ANOVA (analysis of variance) followed by the Bonferroni correction. ANOVA for non-repeated measures was used for comparing the intragroup, while the Bonferroni correction was used as post-hoc analysis.

min) for the transformed *E. coli*, and 2.59 × 107 ± 0.47 × 107 CFU/mL (0 min) and 7.83 × 104 ± 5.17 × 104 CFU/mL (1 min) for the non-transformed *E. coli,* indicating no significant difference between the two groups. In all cases, the number of viable bacteria was below the detection limit after 2 min treatment

Similarly, *E. coli* transformed/selected with pPRO-EX-HT-CAT and non-transformed *E. coli* were

while the Bonferroni correction was used as post-hoc analysis.

(Figures 2 and 3).

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW

(Figures 2 and 3).

while the Bonferroni correction was used as post-hoc analysis.

mm.

time when verified by the non-repeated measured ANOVA followed by the Bonferroni correction.

of the torch, which is made up of a ceramic tube (inner diameter, 4 mm; outer diameter, 6 mm). During gas plasma generation, air flow was maintained at 3.5 L/min using an air pump (Suishin SSPP-2S; Suisaku Co., Sakai, Japan). A suspension (20 μL) of *Escherichia coli* was dropped onto a watch glass. The distance from the top of the plasma torch to the liquid surface on the watch glass was fixed at 20

**Figure 2.** Treatment with a DBD plasma torch reduced the viable cell number of *Escherichia coli* transformed with a plasmid conferring ampicillin resistance, which was selected by spreading on ampicillin-containing agar medium and proliferated in ampicillin-containing liquid medium (■) by a similar amount as plasmid-transformed *E. coli* proliferated in ampicillin-free liquid medium (□). A suspension of *E. coli* transformed with an ampicillin resistance gene-containing plasmid pPRO-EX-HT-CAT, selected in ampicillin-containing agar medium, and proliferated either in ampicillincontaining liquid medium (■) or ampicillin-free liquid medium (□) was exposed to a DBD plasma torch for the indicated time (min). Viable cell count (colony forming units (CFU)/mL) was then determined before and after treatment. NS means no significant difference between ■ and □ at each time when verified by the non-repeated measured ANOVA (analysis of variance) followed by the Bonferroni correction. ANOVA for non-repeated measures was used for comparing the intragroup,

Similarly, *E. coli* transformed/selected with pPRO-EX-HT-CAT and non-transformed *E. coli* were cultured in ampicillin-free LB liquid medium (Figure 3) and then subjected to plasma treatment. Viable cell number was 2.13 × 107 ± 0.20 × 107 CFU/mL (0 min) and 1.80 × 104 ± 1.80 × 104 CFU/mL (1 min) for the transformed *E. coli*, and 2.59 × 107 ± 0.47 × 107 CFU/mL (0 min) and 7.83 × 104 ± 5.17 × 104 CFU/mL (1 min) for the non-transformed *E. coli,* indicating no significant difference between the two

**Figure 3.** Treatment with a DBD plasma torch reduced the viable cell number of *E. coli* transformed with a plasmid conferring ampicillin resistance, which was selected by spreading on ampicillincontaining agar medium and proliferated in ampicillin-free medium (■), compared with nontransformed *E. coli* proliferated in ampicillin-free medium (□). A suspension of *E. coli* transformed with an ampicillin resistance gene-containing plasmid pPRO-EX-HT-CAT and selected in ampicillincontaining agar medium and proliferated in ampicillin-free liquid medium (■) as well as nontransformed *E. coli* proliferated in ampicillin-free liquid medium (□) was exposed to a DBD plasma torch for the indicated time (min). Viable cell count (colony forming units (CFU)/mL) was then

**Figure 3.** Treatment with a DBD plasma torch reduced the viable cell number of *E. coli* transformed with a plasmid conferring ampicillin resistance, which was selected by spreading on ampicillin-containing agar medium and proliferated in ampicillin-free medium (), compared with non-transformed *E. coli* proliferated in ampicillin-free medium (). A suspension of *E. coli* transformed with an ampicillin resistance gene-containing plasmid pPRO-EX-HT-CAT and selected in ampicillin-containing agar medium and proliferated in ampicillin-free liquid medium () as well as non-transformed *E. coli* proliferated in ampicillin-free liquid medium () was exposed to a DBD plasma torch for the indicated time (min). Viable cell count (colony forming units (CFU)/mL) was then determined before and after treatment. NS means no significant difference between and at each time when verified by the non-repeated measured ANOVA followed by the Bonferroni correction. EX-HT-CAT containing CAT gene was treated with the DBD plasma torch and analyzed by CAT ELISA (Figure 5). After exposure to the plasma torch, CAT was reduced in *E. coli* for both transformed and selected *E. coli* cultured in LB medium with or without ampicillin. For *E. coli* transformed with the plasmid and proliferated in the ampicillin-containing medium, CAT concentration before gas plasma treatment was 3147.7302 ± 224.9762 ng/mL (0 min), but was significantly reduced to 0.0325 ± 0.0001 ng/mL and 0.0335 ± 0.0009 ng/mL after plasma treatment for 1 min and 2 min, respectively. In addition, significant reduction of CAT concentration to 0.0344 ± 0.0004 ng/mL at 1 min and 0.0339 ± 0.0004 ng/mL at 2 min was also observed in transformed *E. coli* cultured in ampicillin-free medium compared to the control of 0.3961 ± 0.0182 ng/mL at 0 min. In summary, a significant decrease in the

amount of CAT for the transformed *E. coli* was observed at each time point compared to 0 min.

**Figure 4.** Genomic DNA (16S rDNA) of non-transformed *E. coli* proliferated in ampicillin-free medium (DH5α Amp(−)) and *E. coli* transformed with a plasmid conferring ampicillin resistance, selected in ampicillin-containing agar medium and proliferated in ampicillin-free medium (pPRO-EX-CAT DH5α Amp(−)), was degraded by DBD plasma torch treatment. A suspension of nontransformed *E. coli* proliferated in ampicillin-free medium (DH5α Amp(−)) and *E. coli* transformed with an ampicillin resistance gene-containing pPRO-EX-CAT plasmid, selected in ampicillincontaining agar medium and proliferated in ampicillin-free liquid medium (pPRO-EX-CAT DH5α **Figure 4.** Genomic DNA (16S rDNA) of non-transformed *E. coli* proliferated in ampicillin-free medium (DH5α Amp(−)) and *E. coli* transformed with a plasmid conferring ampicillin resistance, selected in ampicillin-containing agar medium and proliferated in ampicillin-free medium (pPRO-EX-CAT DH5α Amp(−)), was degraded by DBD plasma torch treatment. A suspension of non-transformed *E. coli* proliferated in ampicillin-free medium (DH5α Amp(−)) and *E. coli* transformed with an ampicillin resistance gene-containing pPRO-EX-CAT plasmid, selected in ampicillin-containing agar medium and proliferated in ampicillin-free liquid medium (pPRO-EX-CAT DH5α Amp(−)), was exposed to a DBD plasma torch for the indicated time (min). Viable cell count (colony forming units (CFU)/mL) was then determined before and after treatment. Bands corresponding to amplified *E. coli* 16S rDNA are indicated by arrowheads. Bands corresponding to a DNA size ladder (M) are labelled on the right-hand side of the gel.

Next, to examine the effect of plasma on the quantity of protein, *E. coli* transformed with pPRO-EX-HT-CAT containing CAT gene was treated with the DBD plasma torch and analyzed

by CAT ELISA (Figure 5). After exposure to the plasma torch, CAT was reduced in *E. coli* for both transformed and selected *E. coli* cultured in LB medium with or without ampicillin. For *E. coli* transformed with the plasmid and proliferated in the ampicillin-containing medium, CAT concentration before gas plasma treatment was 3147.7302 ± 224.9762 ng/mL (0 min), but was significantly reduced to 0.0325 ± 0.0001 ng/mL and 0.0335 ± 0.0009 ng/mL after plasma treatment for 1 min and 2 min, respectively. In addition, significant reduction of CAT concentration to 0.0344 ± 0.0004 ng/mL at 1 min and 0.0339 ± 0.0004 ng/mL at 2 min was also observed in transformed *E. coli* cultured in ampicillin-free medium compared to the control of 0.3961 ± 0.0182 ng/mL at 0 min. In summary, a significant decrease in the amount of CAT for the transformed *E. coli* was observed at each time point compared to 0 min. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW Amp(−)), was exposed to a DBD plasma torch for the indicated time (min). Viable cell count (colony forming units (CFU)/mL) was then determined before and after treatment. Bands corresponding to amplified *E. coli* 16S rDNA are indicated by arrowheads. Bands corresponding to a DNA size ladder (M) are labelled on the right-hand side of the gel.

**Figure 5.** The amount of chloramphenicol acetyltransferase (CAT) in *E. coli* transformed with a plasmid conferring ampicillin resistance, selected on ampicillin-containing agar medium and then proliferated in liquid medium with ampicillin (■) or without ampicillin (□), was reduced by DBD plasma torch treatment. *E. coli* transformed with an ampicillin resistance gene- and CAT genecontaining plasmid pPRO-EX-HT-CAT and selected on ampicillin-containing LB agar were proliferated in ampicillin-containing liquid LB medium (Amp(+)) (■) (**a**) or ampicillin-free liquid LB medium (Amp(−)) (□) (**b**) and then subjected to DBD plasma torch treatment for 0–2 min. The concentration of CAT (ng/mL) was subsequently measured by enzyme-linked immunosorbent assay (ELISA). Differences where *p* < 0.01 (\*\*) versus control (0 min) were significant when verified by the **Figure 5.** The amount of chloramphenicol acetyltransferase (CAT) in *E. coli* transformed with a plasmid conferring ampicillin resistance, selected on ampicillin-containing agar medium and then proliferated in liquid medium with ampicillin () or without ampicillin (), was reduced by DBD plasma torch treatment. *E. coli* transformed with an ampicillin resistance gene- and CAT gene-containing plasmid pPRO-EX-HT-CAT and selected on ampicillin-containing LB agar were proliferated in ampicillin-containing liquid LB medium (Amp(+)) () (**a**) or ampicillin-free liquid LB medium (Amp(−)) () (**b**) and then subjected to DBD plasma torch treatment for 0–2 min. The concentration of CAT (ng/mL) was subsequently measured by enzyme-linked immunosorbent assay (ELISA). Differences where *p* < 0.01 (\*\*) versus control (0 min) were significant when verified by the non-repeated measured ANOVA followed by the Bonferroni correction.

non-repeated measured ANOVA followed by the Bonferroni correction. Next, to examine the effect of plasma on protein function, the activity of CAT in transformed *E. coli* was measured by an acetyltransferase activity assay (Figure 6). We speculated that plasma might interfere with the activity of CAT. Specifically, the activity of CAT in *E. coli* transformed with pPR-EX-HT-CAT, selected in ampicillin-containing LB agar, and then proliferated in ampicillincontaining LB liquid medium was 5617.49 ± 92.17 RFU (0 min), but decreased to 322.22 ± 1.07 RFU and 345.11 ± 10.88 RFU after DBD plasma torch treatment for 1 min and 2 min, respectively. In addition, 1991.12 ± 27.76 RFU (0 min) and 343.10 ± 7.47 RFU (1 min) and 343.27 ± 5.74 RFU (2 min) were also observed in cultured *E. coli* after transformation/selection of pPRO-EX-HT-CAT in the Next, to examine the effect of plasma on protein function, the activity of CAT in transformed *E. coli* was measured by an acetyltransferase activity assay (Figure 6). We speculated that plasma might interfere with the activity of CAT. Specifically, the activity of CAT in *E. coli* transformed with pPR-EX-HT-CAT, selected in ampicillin-containing LB agar, and then proliferated in ampicillin-containing LB liquid medium was 5617.49 ± 92.17 RFU (0 min), but decreased to 322.22 ± 1.07 RFU and 345.11 ± 10.88 RFU after DBD plasma torch treatment for 1 min and 2 min, respectively. In addition, 1991.12 ± 27.76 RFU (0 min) and 343.10 ± 7.47 RFU (1 min) and 343.27 ± 5.74 RFU (2 min) were also observed in cultured *E. coli* after transformation/selection of pPRO-EX-HT-CAT in the ampicillin-free LB liquid medium. Thus, a significant decrease in CAT activity of the transformed *E. coli* was observed at each time compared to 0 min.

ampicillin-free LB liquid medium. Thus, a significant decrease in CAT activity of the transformed *E. coli* was observed at each time compared to 0 min. Finally, a chromogenic Limulus test was performed to measure the amount of intact LPS in non-transformed *E. coli* following DBD plasma torch treatment (Figure 7). The results showed that intact LPS in *E. coli* after plasma treatment significantly decreased from 218.94 ± 16.38 EU/mL at 0 min to 4.14 ± 0.22 EU/mL at 1 min and 1.53 ± 0.24 EU/mL at 2 min. These findings indicate that *E. coli* LPS lipid A, which is located on the outer surface of the bacteria, is degraded after plasma treatment. In conclusion, plasma treatment may degrade the integrity of the cell wall of *E. coli*.

**Figure 6.** CAT activity in *E. coli* transformed with an ampicillin resistance gene- and CAT genecontaining plasmid, selected on ampicillin-containing agar medium, and proliferated in liquid medium with ampicillin (■) or without ampicillin (□) was decreased by DBD plasma torch treatment.

*coli* was observed at each time compared to 0 min.

Amp(−)), was exposed to a DBD plasma torch for the indicated time (min). Viable cell count (colony forming units (CFU)/mL) was then determined before and after treatment. Bands corresponding to amplified *E. coli* 16S rDNA are indicated by arrowheads. Bands corresponding to a DNA size ladder

**Figure 5.** The amount of chloramphenicol acetyltransferase (CAT) in *E. coli* transformed with a plasmid conferring ampicillin resistance, selected on ampicillin-containing agar medium and then proliferated in liquid medium with ampicillin (■) or without ampicillin (□), was reduced by DBD plasma torch treatment. *E. coli* transformed with an ampicillin resistance gene- and CAT genecontaining plasmid pPRO-EX-HT-CAT and selected on ampicillin-containing LB agar were proliferated in ampicillin-containing liquid LB medium (Amp(+)) (■) (**a**) or ampicillin-free liquid LB medium (Amp(−)) (□) (**b**) and then subjected to DBD plasma torch treatment for 0–2 min. The concentration of CAT (ng/mL) was subsequently measured by enzyme-linked immunosorbent assay (ELISA). Differences where *p* < 0.01 (\*\*) versus control (0 min) were significant when verified by the

Next, to examine the effect of plasma on protein function, the activity of CAT in transformed *E. coli* was measured by an acetyltransferase activity assay (Figure 6). We speculated that plasma might interfere with the activity of CAT. Specifically, the activity of CAT in *E. coli* transformed with pPR-EX-HT-CAT, selected in ampicillin-containing LB agar, and then proliferated in ampicillincontaining LB liquid medium was 5617.49 ± 92.17 RFU (0 min), but decreased to 322.22 ± 1.07 RFU and 345.11 ± 10.88 RFU after DBD plasma torch treatment for 1 min and 2 min, respectively. In addition, 1991.12 ± 27.76 RFU (0 min) and 343.10 ± 7.47 RFU (1 min) and 343.27 ± 5.74 RFU (2 min) were also observed in cultured *E. coli* after transformation/selection of pPRO-EX-HT-CAT in the

non-repeated measured ANOVA followed by the Bonferroni correction.

(M) are labelled on the right-hand side of the gel.

**Figure 6.** CAT activity in *E. coli* transformed with an ampicillin resistance gene- and CAT genecontaining plasmid, selected on ampicillin-containing agar medium, and proliferated in liquid medium with ampicillin (■) or without ampicillin (□) was decreased by DBD plasma torch treatment. **Figure 6.** CAT activity in *E. coli*transformed with an ampicillin resistance gene- and CAT gene-containing plasmid, selected on ampicillin-containing agar medium, and proliferated in liquid medium with ampicillin () or without ampicillin () was decreased by DBD plasma torch treatment. *E. coli* transformed with an ampicillin resistance gene- and CAT gene-containing plasmid pPRO-EX-HT-CAT and selected on ampicillin-containing medium were proliferated in ampicillin-containing liquid medium (Amp(+)) () or ampicillin-free liquid medium (Amp(−)) () and then subjected to DBD plasma torch treatment for 0–2 min. The activity of CAT in the bacteria was subsequently measured using an acetyltransferase activity assay kit (Enzo Life Sciences, Inc.) with an excitation wavelength of 400 nm and emission wavelength of 490 nm. Differences where *p* < 0.01 (\*\*) versus control (0 min) were significant when verified by the non-repeated measured ANOVA followed by the Bonferroni correction. wavelength of 400 nm and emission wavelength of 490 nm. Differences where *p* < 0.01 (\*\*) versus control (0 min) were significant when verified by the non-repeated measured ANOVA followed by the Bonferroni correction. Finally, a chromogenic Limulus test was performed to measure the amount of intact LPS in nontransformed *E. coli* following DBD plasma torch treatment (Figure 7). The results showed that intact LPS in *E. coli* after plasma treatment significantly decreased from 218.94 ± 16.38 EU/mL at 0 min to 4.14 ± 0.22 EU/mL at 1 min and 1.53 ± 0.24 EU/mL at 2 min. These findings indicate that *E. coli* LPS lipid A, which is located on the outer surface of the bacteria, is degraded after plasma treatment. In conclusion, plasma treatment may degrade the integrity of the cell wall of *E. coli*.

**Figure 7.** Changes to the level of lipopolysaccharides (LPS) in *E. coli* following DBD plasma torch treatment. The concentration of LPS measured by the Limulus test (Limulus-color KY test, Wako Pure Chemical Industries Ltd., Osaka, Japan) is expressed as endotoxin units (EU) per mL. LPS level was reduced after plasma treatment for 0–2 min. Differences where *p* < 0.01 (\*\*) versus control (0 min) were considered significant when verified by the non-repeated measured ANOVA followed by the Bonferroni correction. **Figure 7.** Changes to the level of lipopolysaccharides (LPS) in *E. coli* following DBD plasma torch treatment. The concentration of LPS measured by the Limulus test (Limulus-color KY test, Wako Pure Chemical Industries Ltd., Osaka, Japan) is expressed as endotoxin units (EU) per mL. LPS level was reduced after plasma treatment for 0–2 min. Differences where *p* < 0.01 (\*\*) versus control (0 min) were considered significant when verified by the non-repeated measured ANOVA followed by the Bonferroni correction.

#### **3. Discussion**

**3. Discussion**  The growing risk of disease from antibiotic-resistant bacteria is now a global public health concern [18]. The spread of antibiotic resistance is caused, in part, from the extensive and indiscriminate use of antibiotics, which leads to the emergence of antibiotic-resistant bacteria. To The growing risk of disease from antibiotic-resistant bacteria is now a global public health concern [18]. The spread of antibiotic resistance is caused, in part, from the extensive and indiscriminate use of antibiotics, which leads to the emergence of antibiotic-resistant bacteria. To restrict the increase of antibiotic-resistant bacteria, efficient inactivation methods that do not rely on antibiotics are required.

restrict the increase of antibiotic-resistant bacteria, efficient inactivation methods that do not rely on antibiotics are required. Previous studies have shown that exposure to plasma, which is a state of matter predominantly composed of ions and electrons, can inactivate a variety of antibiotic-resistant bacteria. For example, Previous studies have shown that exposure to plasma, which is a state of matter predominantly composed of ions and electrons, can inactivate a variety of antibiotic-resistant bacteria. For example, glow discharge plasma has been shown to inactivate antibiotic resistant *E. coli* [16].

the same or different level of sensitivity to plasma treatment.

glow discharge plasma has been shown to inactivate antibiotic resistant *E. coli* [16]. Surface micro-

including *Yersinia enterocolitica*, *Staphylococcus aureus*, *Klebsiella*, *E. coli*, *Acinetobacter*, and *Enterococcus faecium* [17]. Moreover, a floating-electrode DBD plasma device has successfully inactivated methicillin-resistant *S. aureus* (MRSA) [19]. In addition, suspensions and biofilms of MRSA were inactivated by RF (radiofrequency) plasma [20] and floating-electrode DBD [21]. Therefore, plasma treatment provides a robust and efficient way of eliminating antibiotic-resistant bacteria. However, to date, there are no reports showing whether antibiotic-resistant and non-resistant bacteria display Surface micro-discharge (SMD) plasma treatment was demonstrated to inactivate antibiotic-resistant bacteria including *Yersinia enterocolitica*, *Staphylococcus aureus*, *Klebsiella*, *E. coli*, *Acinetobacter*, and *Enterococcus faecium* [17]. Moreover, a floating-electrode DBD plasma device has successfully inactivated methicillin-resistant *S. aureus* (MRSA) [19]. In addition, suspensions and biofilms of MRSA were inactivated by RF (radiofrequency) plasma [20] and floating-electrode DBD [21]. Therefore, plasma treatment provides a robust and efficient way of eliminating antibiotic-resistant bacteria. However, to date, there are no reports showing whether antibiotic-resistant and non-resistant bacteria display the same or different level of sensitivity to plasma treatment.

In this study, we have clarified that antibiotic-resistant and non-resistant bacteria show no significant difference in susceptibility to treatment with plasma. Furthermore, this study suggests that the emergence of plasma-resistant bacteria following plasma treatment is low.

Our analysis showed a reduction in the levels of CAT, DNA, and LPS in *E. coli* after plasma treatment. Previous studies have analyzed gas plasma [22–25], which led to oxidation/modification of DNA and protein as well as their degradation. LPS, also termed as endotoxin, is a component of the outer membrane of Gram-negative bacteria and is known to be highly resistant to both physical and chemical treatment [26]. However, a previous study has shown that the level of endotoxin can be reduced by exposure of bacteria to nitrogen gas plasma [27]. Here, we found that LPS is degraded by DBD plasma torch treatment. These findings are consistent with a previous study, in which the authors proposed that cell surface components are important in the mechanism of inactivation because the thickness of the polysaccharide membrane influences sensitivity to plasma [17]. Furthermore, as previously determined [28], the temperature of the liquid surface was 38.40 ± 0.12 ◦C at the 2 min treatment time using the same DBD plasma torch under identical conditions. *E. coli* can survive at around 38 ◦C [29]. Thus, heating during operation of the plasma torch cannot have been the cause of *E. coli* inactivation.

To clarify the likely inactivation mechanism, analysis of plasma components by electron spin resonance (ESR) was also performed (Supplementary Figure S1). These experiments showed that OH radicals (OH·) and H radicals (H·) were present in the plasma. Thus, reactive chemical species such as OH and H radicals may act to eliminate bacteria by oxidizing biomolecules. Indeed, these findings are consistent with a previous study that showed that the OH radical is the most important factor for bacterial inactivation during plasma treatment [16,30]. Thus, oxidation and degradation of biomolecules may be caused by reactive chemical species such as reactive nitrogen species (RNS) and reactive oxygen species (ROS) generated by the plasma. Nevertheless, it is possible that the reactive chemical species may act on other biomolecules. In addition, as antioxidants have antimicrobial activity against antibiotic-resistant bacteria [31], modulation of the oxidative conditions might change the antibacterial activity. Thus, further studies on the potential relationship between oxidative modulation by plasma and its antibacterial activity would be necessary to perform.

Regardless of the underlying mechanism of inactivation, the DBD plasma torch was found to be highly effective at eliminating antibiotic-resistant bacteria. Presumably, this was because changes to cell components induced by the plasma treatment such as modifications to the proteins and DNA were independent of antibiotic resistance. Further studies are also needed to clarify the inactivation mechanism of the DBD plasma torch including detailed analysis of bactericidal factors generated by the plasma that induce damage to DNA and proteins as well as cell surface degradation including LPS, which is a component of the outer membrane of Gram-negative bacteria. Antibiotic resistant bacteria employ multiple mechanisms to confer resistance including antibiotic inactivation, target-site modification, and reduction of cytoplasmic antibiotic concentration [32]. The specific type of antibiotic resistance mechanism might be related to plasma susceptibility. Therefore, further studies on the relationship between the various mechanisms of antibiotic resistance and plasma susceptibility are required.

Finally, the development of process compatible technology design is required to convert the DBD plasma device into a commercial and practical technology for disinfection in agricultural settings. To approach this issue, we have recently designed a new type of DBD plasma device (a roller conveyer plasma device) that is well suited to the disinfection of agricultural products such as vegetables and fruits during the sorting process on rollers [33]. To achieve broad applicability for the DBD plasma device across a range of agricultural food products, further development and improvement such as scale-up and better cost-performance will be needed. Thus, further optimization of plasma generation to maximize disinfection efficiency will be required before the device can be widely applied in a commercial setting.

#### **4. Materials and Methods**

#### *4.1. DBD Plasma Torch*

The DBD plasma torch used in the present study was the same as described in a previous report [34]. Briefly, a ceramic tube (Al2O3) (inner diameter, 4 mm; outer diameter, 6 mm; length, 100 mm) was wound around the tube by a copper tape (thickness, 80 µm; length, 60 mm) that was used as an earth electrode (Figure 1). A stainless steel mesh (SUS304) with stainless steel wires was placed inside the tube to act as a high voltage electrode. Next, a low frequency and high-voltage power supply (10 kV peak-to-peak, 10 kHz) was connected to the two electrodes. During plasma generation, an air pump (Suishin SSPP-2S; Suisaku Co., Sakai, Japan) was used at an air flow rate of 3.5 L/min. The distance from the torch top to the liquid surface was set at 20 mm. Aliquots (20 µL) of *E. coli* suspension were dropped on a watch glass and subjected to plasma treatment.

#### *4.2. Plasma Treatment of Transformed Bacteria and Colony Counting*

*E. coli* (DH5α Competent Cells, Takara Bio, Shiga, Japan) was used for the plasma treatment. Transformation of pPRO-EX-HT-CAT (Thermo Fisher Scientific, Waltham, MA, USA) with a plasmid carrying an ampicillin-resistance gene and chloramphenicol acetyltransferase (CAT) gene was performed according to a conventional methodology [35]. *E. coli* obtained after selection on LB (Luria-Bertani) agar medium supplemented with ampicillin (50 mg/L) were picked and then cultured in LB liquid medium with or without ampicillin (50 mg/L) at 37 ◦C for 24 h. The resultant *E. coli* were then subjected to plasma treatment. As a control, untransformed *E. coli* were also exposed to plasma. Viable cell count after plasma treatment was performed by spreading onto LB agar medium with or without ampicillin (50 mg/L). After overnight culture at 37 ◦C, colony counting was performed to determine the colony forming units per mL (CFU/mL).

#### *4.3. Enzyme-Linked Immunosorbent Assay (ELISA)*

CAT was quantified by ELISA using a CAT ELISA Kit (cat no. #11 363 727 001; Roche, Rotkreuz, Switzerland) in the accordance with the manufacturer's instructions.

#### *4.4. Acetyltransferase Activity Assay*

CAT activity was measured using an acetyltransferase activity assay kit (Enzo Life Sciences Inc., Farmingdale, NY, USA) as the index of RFU (relative fluorescence units) with an excitation wavelength of 400 nm and an emission wavelength of 490 nm.

## *4.5. PCR*

The level of intact *E. coli* genomic DNA was assessed using a Bacterial 16S rDNA PCR Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. Bacterial DNA samples were subjected to 30 cycles of PCR amplification conditions (94 ◦C for 0.5 min, 55 ◦C for 0.5 min, 72 ◦C for 1 min) using a PC320 thermal cycler (Astec Co. Ltd., Fukuoka, Japan). Amplified DNA was analyzed by agarose gel electrophoresis (1% gel) and the DNA was visualized using a WSE-5200 Printgraph 2M device (ATTO Corporation, Tokyo, Japan). The amplified products were extracted using a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany) and directly subjected to DNA sequencing using primers F1 and R1 (*bacterial*) of the Bacterial 16S rDNA PCR Kit with an ABI 373 OXL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

## *4.6. Measurement of LPS*

DBD plasma-treated or untreated bacterial suspensions were resuspended in distilled water (Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan) and subjected to the chromogenic Limulus test (Limulus-color KY test; Wako Pure Chemical Industries Ltd., Osaka, Japan) for quantifying LPS. Absorbance at 415 nm by reference to that at 655 nm was compared with a standard curve obtained using a LPS solution (Wako Pure Chemical Industries Ltd., Osaka, Japan). LPS concentrations (Endotoxin Units (EU)/mL) were then estimated from the index of absorbance.

#### *4.7. Statistical Analysis*

GraphPad Prism 7.02 software (GraphPad Prism Software Inc., La Jolla, CA, USA) was used for the statistical analysis. Mean ± standard deviation of experiments carried out at least in triplicate are shown. Non-repeated analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test was applied to the statistical analysis of significant difference.

#### **5. Conclusions**

The present study established that there was no difference in susceptibility between antibiotic-resistant and non-resistant bacteria to plasma treatment using an DBD plasma torch. In addition, ESR has shown that the plasma torch generates reactive chemical species including OH and H radicals, which may contribute to the inactivation of DNA, proteins, and LPS. However, OH and H radicals may react with various other bacterial components to generate oxidation products. Therefore, the bactericidal mechanism needs to be further clarified by the detailed analysis of changes to the components of the bacteria induced by radicals generated by the plasma.

Nonetheless, the mechanism of inactivation is unlikely to differ between antibiotic-resistant and non-resistant bacteria. Therefore, the plasma method may be especially useful for eliminating antibiotic-resistant bacteria from the environment. Data on the likely inactivation mechanism may contribute to improving the efficiency of the plasma system by fine-tuning plasma generation to maximize bactericidal action. Indeed, future optimization of the plasma system based on knowledge gained concerning the inactivation mechanism(s) may facilitate increased inactivation efficiency.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/21/17/6326/s1.

**Author Contributions:** Conceptualization, A.S. and T.M.; Resources, T.M.; Data curation, A.S.; Writing—original draft, A.S.; Writing—review and editing, A.S. and T.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by Grant-in-Aid from The Ito Foundation. This work was also supported by the JSPS (Japan Society for the Promotion of Science) KAKENHI grant numbers JP20K03919, JP16K04997, JP22110514, and JP24110717, and the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry (Grant number 26015A) as well as the Promotion of Basic Research Activities for Innovative Biosciences from Bio-oriented Technology Research Advancement Institution (BRAIN).

**Acknowledgments:** The authors would like to acknowledge Risa Yamashiro (University of the Ryukyus, Japan) and Kunihiko Tajima (Kyoto Institute of Technology) for their technical assistance.

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