*Communication* **Induction of Micronuclei in Cervical Cancer Treated with Radiotherapy**

**Daijiro Kobayashi 1 , Takahiro Oike 2,\*, Kazutoshi Murata 3 , Daisuke Irie 2 , Yuka Hirota 2 , Hiro Sato 2 , Atsushi Shibata 4 and Tatsuya Ohno 2,5**

Received: 11 August 2020; Accepted: 1 September 2020; Published: 3 September 2020

**Abstract:** Micronuclei (MN) trigger antitumor immune responses via the cyclic GMP-AMP synthase-signaling effector stimulator of interferon genes (cGAS-STING) pathway. Radiotherapy induces MN in peripheral blood lymphocytes. However, data for solid tumors are lacking. Here, we analyzed MN post-radiotherapy in solid tumor samples. Tumor biopsy specimens were obtained from seven prospectively recruited patients with cervical cancer, before treatment and after receiving radiotherapy at a dose of 10 Gy (in five fractions). The samples were stained with 4 0 ,6-diamidino-2-phenylindole dihydrochloride, and 200 nuclei per sample were randomly identified and assessed for the presence of MN or apoptosis, based on nuclear morphology. The median number of MN-harboring nuclei was significantly greater in samples from patients treated with radiotherapy than in pre-treatment samples (151 (range, 16–327) versus 28 (range, 0–61); *p* = 0.015). No significant differences in the number of apoptotic nuclei were observed between pre-treatment and 10 Gy samples (5 (range, 0–30) versus 12 (range, 2–30); *p* = 0.30). This is the first report to demonstrate MN induction by radiotherapy in solid tumors. The results provide clinical evidence of the activation of antitumor immune responses by radiotherapy.

**Keywords:** micronuclei; uterine cervical cancer; radiotherapy; cGAS; STING; abscopal effect; immunotherapy

#### **1. Introduction**

Immunotherapy is rapidly becoming a promising strategy for cancer treatment. Recent reports on the combination use of immunotherapy with radiotherapy (RT) are overwhelming [1,2]. However, this kind of treatment strategy is hampered by the fact that its efficacy is unpredictable [1]. The cost for cancer immunotherapy is unsustainably high [1], therefore, a stratification of the patients who benefit from the combination treatment is needed. To this end, at the present, identification of reliable biomarkers of response to cancer immunotherapy combined with radiotherapy represents one of the mainstream immune-oncology research lines [1].

Micronuclei (MN) are induced following aberrant mitotic events in response to ionizing radiation, and are identified as one or a few smaller nuclei independent from the main nucleus [3,4]. After irradiation, cyclic GMP-AMP (cGAMP) synthase (cGAS) localizes to MN and binds to double-stranded DNAs (dsDNAs) that activate the stimulator of interferon genes (STING), an endoplasmic reticulum adaptor protein [5,6]. This induces GAMP-driven proinflammatory response [7,8], leading to type-1 interferon secretion [9]. As such, MN formation is directly related to the activation of cGAS-STING pathway that is the key axis of antitumor immune responses following radiation [7,8,10,11].

Taken together, RT-induced intratumoral MN may be used as a biomarker of the response to cancer immunotherapy combined with radiotherapy. Induction of MN by RT has been demonstrated in peripheral blood lymphocytes obtained from individuals treated with RT [12–15]. However, the induction of MN by RT in solid tumors has not been reported. This study aimed to obtain proof of principle for the induction of MN by RT in solid tumors. To this end, we evaluated tumor specimens obtained at pre-treatment and intra-RT time points from the same patients with cervical cancer.

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

#### *2.1. Patients*

Patients with cervical cancer were prospectively enrolled in the study if they met the following criteria: (a) pathologically-confirmed newly diagnosed cervical cancer; (b) treated with definitive RT at Gunma University Hospital between November 2017 and November 2018; (c) staged as IB1–IVA according to the 2009 International Federation of Gynecology and Obstetrics staging system; and (d) no previous exposure to radiotherapy or cytotoxic agents.

This study was approved by the Ethical Review Board Committee of Gunma University Hospital (approval number, 1109). Written consent was obtained from all participants.

#### *2.2. Treatment*

Patients were treated with concurrent chemoradiotherapy using standardized protocols [16]. Briefly, RT consisted of EBRT using 10 MV X-rays and computed tomography-based high dose-rate image-guided brachytherapy (IGBT). For EBRT, whole pelvic irradiation was delivered at a dose of 50 Gy in 25 fractions, and a central shielding technique was used for the latter 10–30 Gy. IGBT was performed with a 192Ir Remote-After-Loading System (microSelectron, Elekta, Stockholm, Sweden), using tandem and ovoid applicators. A total of 24 Gy was delivered in four fractions, one fraction per week. Cisplatin (40 mg/m2 ) was administered weekly during the RT treatment period, with the first course administered on day 1. Patients ≥ 75 years old did not receive chemotherapy.

#### *2.3. Sample Collection and Nuclear Morphology Assessment*

Specimens were obtained by punch biopsy from the center of tumors of the uterine cervix before the initiation of RT and after RT at 10 Gy. The nuclei contained in the tumor specimens were stained with DAPI (Sigma-Aldrich, St. Louis, MO, USA), as described previously [17]. Briefly, the biopsy specimens were kept in ice-cold phosphate buffered saline (PBS) and transported immediately from the hospital to the laboratory. Within 10 min from the biopsy, the tumor was minced using a surgical knife and incubated in a solution containing 0.25% trypsin (Thermo Scientific, Waltham, MA, USA) and 1 mM ethylenediaminetetraacetic acid (Sigma-Aldrich) at 37 ◦C for 5 min. The samples were then passed through a 70-micron nylon mesh cell strainer (Falcon, New York, NY, USA) in 5 mL PBS. Strained cells were collected by centrifugation at 1500× *g* rpm for 5 min and suspended in 0.4 mL PBS. A Cytospin™ 4 Cytocentrifuge (Thermo Scientific) was used to spread 0.1 mL of the cell suspension in a monolayer on slide glasses. The cells were then fixed with 3% paraformaldehyde-2% sucrose solution for 10 min. Fixed cells were incubated with 0.7% Triton X-100 (Sigma-Aldrich) and stained with DAPI. Coverslips were mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA).

Microscopic images were obtained under a fluorescence microscope (Eclipse Ni, Nikon, Tokyo, Japan), using the settings for conventional image capture with a 40× objective lens. In continuous

fields, 200 nuclei were identified and the nuclear events (i.e., MN and apoptosis) for each nucleus were recorded. MN were determined by the presence of small fragments separate from the main nuclei (Figure 1b) [8]. Apoptosis was determined by the presence of apoptotic bodies, nuclear condensation, or nuclear fragmentation (Figure 1c) [18]. *J. Pers. Med.* **2020**, *10*, x FOR PEER REVIEW 3 of 8 were recorded. MN were determined by the presence of small fragments separate from the main nuclei (Figure 1b) [8]. Apoptosis was determined by the presence of apoptotic bodies, nuclear condensation, or nuclear fragmentation (Figure 1c) [18].

. **Figure 1.** Representative images of nuclei stained with 4′,6-diamidino-2-phenylindole **Figure 1.** Representative images of nuclei stained with 40 ,6-diamidino-2-phenylindole dihydrochloride. (**a**) Normal nucleus. (**b**) Nucleus harboring micronuclei (arrow). (**c**) Apoptosis. Scale bar = 10 µm.

#### dihydrochloride. (**a**) Normal nucleus. (**b**) Nucleus harboring micronuclei (arrow). (**c**) Apoptosis. Scale *2.4. Statistical Analysis*

bar = 10 μm. *2.4. Statistical Analysis*  Differences in the prevalence of nuclear events between pre-RT and post-RT samples in the same patient were examined by paired non-parametric Wilcoxon test. Differences in the induction rate of nuclear events between the CCRT group and the RT group alone were examined by unpaired nonparametric Mann–Whitney test. Correlations of the interval between two biopsies with the induction rate of nuclear events were examined by Spearman's rank order test. Differences were considered Differences in the prevalence of nuclear events between pre-RT and post-RT samples in the same patient were examined by paired non-parametric Wilcoxon test. Differences in the induction rate of nuclear events between the CCRT group and the RT group alone were examined by unpaired non-parametric Mann–Whitney test. Correlations of the interval between two biopsies with the induction rate of nuclear events were examined by Spearman's rank order test. Differences were considered statistically significant at *p* < 0.05. Statistical analysis was performed using Prism8 (GraphPad, San Diego, CA, USA).

#### statistically significant at *p* < 0.05. Statistical analysis was performed using Prism8 (GraphPad, San **3. Results**

Diego, CA, USA). **3. Results**  Seven patients were enrolled in this study (Table 1). The median age was 57 (42–82) years. Four patients received concurrent chemoradiotherapy (CCRT) with weekly cisplatin (40 mg/m2), and the Seven patients were enrolled in this study (Table 1). The median age was 57 (42–82) years. Four patients received concurrent chemoradiotherapy (CCRT) with weekly cisplatin (40 mg/m2 ), and the remaining three patients received RT alone because of older age (i.e., ≥ 75 years). The median total dose of external beam radiotherapy (EBRT) was 55.6 (50.0–58.0) Gy. We confirmed that more than 95% of the planned doses were appropriately delivered to tumors (Figure 2).

remaining three patients received RT alone because of older age (i.e., ≥ 75 years). The median total dose of external beam radiotherapy (EBRT) was 55.6 (50.0–58.0) Gy. We confirmed that more than 95% of the planned doses were appropriately delivered to tumors (Figure 2). **Table 1.** Patient characteristics. **Characteristics Median (Range)**  Tumor biopsy was performed before the initiation of RT and after exposure to 10 Gy in the same patients. The median interval between first biopsy and second biopsy was 7 days (range, 4–10 days). Two patients with a 4-day interval underwent biopsies on Monday and Friday of the same week. The patient with a 10-day interval underwent biopsy before and after the year-end holiday. The other four patients underwent biopsies on Monday of the first week and Monday or Tuesday of the following week (i.e., 7- and 8-day intervals, respectively).

Age (years) 57 (42–82) Age ≤ 50 vs. > 50 3 vs. 4 Menstrual status (premenopausal vs. postmenopausal) 2 vs. 5 MN and apoptosis in each sample were determined by morphological assessment of nuclei stained with 40 ,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (see Section 2.3 for detailed methods). Representative images of MN and apoptosis are shown in Figure 1.

Follow-up (months) 11 (1–21) FIGO stage IB 0 II 4 III 2 A random 200 nuclei were evaluated for each experimental setting; the median number of nuclei harboring MN was 28 (range, 0–61) pre-RT and 151 (range, 16–327) after RT, at a dose of 10 Gy. The number of MN-harboring nuclei was significantly greater in 10 Gy samples than in pre-RT samples (*p* = 0.015) (Figure 3). In contrast to MN, the number of apoptotic nuclei did not differ significantly between the pre-RT samples and the 10 Gy samples (5 (range, 0–30) versus 12 (range, 2–30); *p* = 0.30).

IVA 1

Squamous cell carcinoma 7

Histology

**Table 1.** Patient characteristics.

FIGO, International Federation of Gynecology and Obstetrics; CEA, carcinoembryonic antigen; SCC, squamous cell carcinoma antigen; EBRT, external beam radiotherapy; IGBT, image-guided brachytherapy; fr., fraction. brachytherapy; fr., fraction.

**Figure 2.** Representative images of dose distribution of external beam radiotherapy. Dashed line **Figure 2.** Representative images of dose distribution of external beam radiotherapy. Dashed line indicates tumor (yellow). (**a**) Axial plane; (**b**) sagittal plane.

indicates tumor (yellow). (**a**) Axial plane; (**b**) sagittal plane.

Tumor biopsy was performed before the initiation of RT and after exposure to 10 Gy in the same patients. The median interval between first biopsy and second biopsy was 7 days (range, 4–10 days). Two patients with a 4-day interval underwent biopsies on Monday and Friday of the same week. The patient with a 10-day interval underwent biopsy before and after the year-end holiday. The other four patients underwent biopsies on Monday of the first week and Monday or Tuesday of the following week (i.e., 7- and 8-day intervals, respectively). To analyze the influence of chemotherapy administered concurrently with RT on MN induction, we compared the rate of MN-harboring nuclei at 10 Gy relative to that for pre-RT settings (i.e., MN induction rate) between the CCRT group (*n* = 4) and the RT alone group (*n* = 3) (Figure 4a). The median MN induction rate was 21.5% (4.0–46.5%) in patients treated with RT alone and 58.5% (11.5–163.5%) in those treated with CCRT; there was no significant difference in MN induction rate between the two groups (*p* = 0.22). The interval between the first and second biopsies was not significantly correlated with the MN induction rate (*p* = 0.29, R2 = 0.22) (Figure 4b).

MN and apoptosis in each sample were determined by morphological assessment of nuclei stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (see Section 2.3 for detailed

A random 200 nuclei were evaluated for each experimental setting; the median number of nuclei harboring MN was 28 (range, 0–61) pre-RT and 151 (range, 16–327) after RT, at a dose of 10 Gy. The

between the pre-RT samples and the 10 Gy samples (5 (range, 0–30) versus 12 (range, 2–30); *p* = 0.30).

methods). Representative images of MN and apoptosis are shown in Figure 1.

test.

*J. Pers. Med.* **2020**, *10*, x FOR PEER REVIEW 5 of 8

**Figure 3.** The number of nuclei harboring micronuclei (MN) (**a**) and those undergoing apoptosis (Ap) (**b**) pre-radiotherapy or after 10 Gy RT. *p* values were assessed by the paired non-parametric Wilcoxon **Figure 3.** The number of nuclei harboring micronuclei (MN) (**a**) and those undergoing apoptosis (Ap) (**b**) pre-radiotherapy or after 10 Gy RT. *p* values were assessed by the paired non-parametric Wilcoxon test. 163.5%) in those treated with CCRT; there was no significant difference in MN induction rate between the two groups (*p* = 0.22). The interval between the first and second biopsies was not significantly correlated with the MN induction rate (*p* = 0.29, R2 = 0.22) (Figure 4b).

**Figure 4.** Induction rate of micronuclei (MN) at 10 Gy, relative to pre-RT settings. (**a**) Comparison between patients treated with radiotherapy (RT) alone and those receiving concurrent chemoradiotherapy (CCRT). *p* value was assessed by the Mann–Whitney test. (**b**) Correlation between the interval between two biopsies and the induction rate of MN. *p* value and R square value were **Figure 4.** Induction rate of micronuclei (MN) at 10 Gy, relative to pre-RT settings. (**a**) Comparison between patients treated with radiotherapy (RT) alone and those receiving concurrent chemoradiotherapy (CCRT). *p* value was assessed by the Mann–Whitney test. (**b**) Correlation between the interval between two biopsies and the induction rate of MN. *p* value and R square value were assessed by Spearman's Rank Order test.

#### **4. Discussion**

assessed by Spearman's Rank Order test.

**(a) (b) Figure 4.** Induction rate of micronuclei (MN) at 10 Gy, relative to pre-RT settings. (**a**) Comparison between patients treated with radiotherapy (RT) alone and those receiving concurrent chemoradiotherapy (CCRT). *p* value was assessed by the Mann–Whitney test. (**b**) Correlation between the interval between two biopsies and the induction rate of MN. *p* value and R square value were assessed by Spearman's Rank Order test. **4. Discussion**  This study examined the induction of MN by radiotherapy, using cervical biopsy samples obtained before and after radiotherapy. Intratumoral MN showed a 5.4-fold increase after RT for 10 Gy in 5 fractions compared with pre-treatment setting. The increase in MN after irradiation was comparable with that reported previously in peripheral blood lymphocytes [12–15]. The novelty of this study is that, for the first time, we demonstrated the induction of MN in primary tumors treated **4. Discussion**  This study examined the induction of MN by radiotherapy, using cervical biopsy samples obtained before and after radiotherapy. Intratumoral MN showed a 5.4-fold increase after RT for 10 Gy in 5 fractions compared with pre-treatment setting. The increase in MN after irradiation was comparable with that reported previously in peripheral blood lymphocytes [12–15]. The novelty of this study is that, for the first time, we demonstrated the induction of MN in primary tumors treated with clinical radiation beams. In contrast to MN, there was no significant difference in the rate of This study examined the induction of MN by radiotherapy, using cervical biopsy samples obtained before and after radiotherapy. Intratumoral MN showed a 5.4-fold increase after RT for 10 Gy in 5 fractions compared with pre-treatment setting. The increase in MN after irradiation was comparable with that reported previously in peripheral blood lymphocytes [12–15]. The novelty of this study is that, for the first time, we demonstrated the induction of MN in primary tumors treated with clinical radiation beams. In contrast to MN, there was no significant difference in the rate of apoptosis before and after irradiation. In a previous in vitro study, we investigated the nuclear morphology of cell death after X-ray irradiation at 4 Gy [19]. In that study, the most common mode of clonogenic cell death induced by X-rays was mitotic catastrophe, and the median rate of apoptosis was approximately 1%. MN represent an increase in genetic instability, and they are a hallmark of mitotic catastrophe [20–22], which is consistent with the results of this study. In addition, it is worth noting that a previous study showed that excessive expression of Trex1 by high dose irradiation in a single fraction decreases cytosolic dsDNA [11]. These data indicate that the fractionated RT schedule employed in the present study (i.e., 2 Gy per fraction and five fractions per week) might be effective in inducing MN.

with clinical radiation beams. In contrast to MN, there was no significant difference in the rate of

Advances in radiotherapy techniques have improved the outcomes of patients with newly diagnosed uterine cervical cancer [16]. However, the platinum-based combinations used in first-line chemotherapy for metastatic or recurrent tumors rarely provide durable disease control [23]. McLachlan et al. reported the outcome of second-line chemotherapy in the patients with recurrent or metastatic cervical cancer [24]. In that report, they showed the response rate was insufficient as the median overall survival was 9.3 months. As such, better treatments are still needed for patients with recurrent or metastatic cervical cancer, and patients should be considered for clinical trials whenever feasible, including novel targeted agents and immunotherapy.

Almost all cases of uterine cervical cancer are caused by high-risk human papillomavirus (HPV) infection [25] and HPV expresses foreign antigens within host cells. Although immunotherapy has the potential to improve the survival of uterine cervical cancer patients, the response rate of uterine cervical cancer to checkpoint immunotherapy is 10–25% [26]. Previous studies suggest the differences in the immunological microenvironments and escape mechanisms by histological subtypes. Reddy et al. showed that the expression of programmed cell death-ligand 1 (PD-L1) was higher in squamous cell carcinoma than in adenocarcinoma [27]. Boussios et al. showed that diffuse PD-L1 expression in squamous cell carcinoma is correlated with poor disease-free survival and disease-specific survival compared to the cases with marginal PD-L1 expression; in adenocarcinoma, survival benefit was observed for the patients with tumors lacking PD-L1-positive tumor-associated macrophages [28,29]. The cohort of the present study solely consisted of squamous cell carcinoma. From these perspectives, future studies should compare post-RT MN induction between squamous cell carcinoma and adenocarcinoma.

The abscopal effect is an anti-tumor effect observed in the unirradiated sites of RT-treated patients. Abscopal effect "comes by chance, not through seeking", i.e., not observed in all RT-treated cases and the methods for its prediction has not been established, despite a number of clinical trials as summarized by Wang and colleagues [30]. This heterogeneity in the induction of abscopal effect post-RT can be attributable to the variance in the strength of post-RT antitumor immune responses. cGAS-STING pathway may be the mediator of post-RT abscopal effect, and MN may be the predictive biomarker. This possibility warrants further investigation in future.

In this study, we selected the time point corresponding to a dose of 10 Gy irradiation. Gamulin et al. reported that the number of MN increases gradually during RT, reaching the highest value after the administration of the last RT fraction [31]. Nevertheless, we thought, from clinical experience of rapid shrinkage of uterine cervical tumors during RT course, that sufficient samples of viable cells may not be available at later time points; therefore, we chose 10 Gy for sample collection in this study.

#### **5. Conclusions**

MN trigger antitumor immune responses via the cGAS-STING pathway. For the first time, we present clinical evidence of MN induction by RT in solid tumors. The present data provide an important insight into the activation of antitumor immune responses by RT.

**Author Contributions:** Conceptualization, D.K.; data acquisition, D.K., D.I., K.M., and Y.H.; formal analysis, D.K.; writing—original draft preparation, D.K.; writing—review and editing, H.S.; T.O. (Takahiro Oike); supervision, A.S., T.O. (Takahiro Oike), and T.O. (Tatsuya Ohno); funding acquisition, D.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Grants-in-Aid from the Japan Society for the Promotion of Science for Exploratory Research KAKENHI [19K17260]. This work was also supported by Gunma University Heavy Ion Medical Center.

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

#### **Abbreviations**

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

*Article*


**2. Analysis of plant fiber composition**

High-performance anion-exchange chromatography with an integrated pulsed amperometric method (HPAEX-IPAM) is a newly developed method for sugar analysis in recent years. Zheng [23, 24] established HPAEX-IPAM for the

*Study on Pyrolysis Behaviors of Various Plant Fibers DOI: http://dx.doi.org/10.5772/intechopen.109294*

#### **Table 1.**

*Information of plant fiber compositions (%) [27].*

determination of monosaccharides (arabinose, galactose, glucose, xylose, and mannose), glycuronate acid (galacturonic acid and glucuronic acid), and cellobiose in plant fiber hydrolysate. In order to hydrolyze cellulose, National Renewable Energy Laboratory (NREL) method optimized by the heating method is used to hydrolyze cellulose into monosaccharides. Then, monosaccharides are separated by an efficient anion exchange column in a strong alkaline medium, and then, the current generated by the oxidation reaction of hydroxyl groups in the sugar molecular structure on the gold electrode surface is detected [25, 26]. This method is simple in pretreatment, and the pulsed amperometric detector has high sensitivity, selectivity, accuracy, and reproducibility. Calculate the content of fiber components, according to the concentration of hydrolysis products of each fiber component. Zheng [23, 24] used this method to determine the acid hydrolysis monosaccharides of six common plant fibers (coniferous fiber, broadleaf fiber, bamboo fiber, flax fiber, grass fiber, and cotton fiber), and calculated the composition of cellulose and hemicellulose of each plant fiber, according to the corresponding hydrolysis monosaccharides mass fraction of cellulose and hemicellulose (as shown in **Table 1**). The lignin content shown in **Table 1** is the sum of acid-insoluble lignin and acid-soluble lignin, which are determined by weighing the filter residue and detecting acid hydrolysate with UV.


**Author details**

Marta Świerczyńska1,2\*, Agnieszka Tronina3,4 and Ewa Mrukwa-Kominek1,2

1 Faculty of Medical Sciences in Katowice, Department of Ophthalmology, Medical University of Silesia, Katowice, Poland

2 Department of Ophthalmology, Kornel Gibiński University Clinical Center, Medical University of Silesia, Katowice, Poland

3 Faculty of Medical Sciences in Katowice, Department of Pediatric Ophthalmology, Medical University of Silesia, Katowice, Poland

4 Department of Pediatric Ophthalmology, Kornel Gibiński University Clinical Center, Medical University of Silesia, Katowice, Poland

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 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, provided the original work is properly cited.


*Article* **Exploring the Measurement of Regional Forestry Eco-Efficiency and Influencing Factors in China Based on the Super-Efficient DEA-Tobit Two Stage Model**

**Junlan Tan 1, Xiang Su 1 and Rong Wang 2,\***

**Abstract:** This paper adopts the super-efficient DEA (data envelopment analysis) model to measure the forestry eco-efficiency (FECO) of 30 Chinese provinces and cities from 2008 to 2021, and then introduces the Tobit model to explore the influencing factors of FECO to better understand the sustainable development level of forestry. It draws the following conclusions: (1) The average value of FECO in China is 0.504, which is still at a low level, and the FECO of each region has significant regional heterogeneity; the provinces with higher FECO are mainly concentrated in the eastern region, while the FECO of the central and western regions is lower; (2) In terms of the main factors affecting FECO in China, the regression coefficients of market-based environmental regulations are significantly positive in the national, eastern and central regions, while they are significantly negative in the western region. The coefficient of impact of scientific research funding investment on forestry industry eco-efficiency is negative and shows a significant promotion effect in the eastern region, but the elasticity coefficient in the central and western regions is negative but not significant. Economic development has a positive but insignificant effect on FECO, with the eastern region showing a positive correlation, while the central and western regions are insignificant. Industrial structure has a significant negative effect on FECO in the national, eastern and central regions, but the effect of industrial structure on FECO in the western region is not significant. The effect of foreign direct investment on FECO was negative for the national, central and western regions, but the central region did not pass the significance test, while the eastern region reflected a significant promotion effect.

**Keywords:** forestry; eco-efficiency; sustainable development; forestry resources


3.5.2. Cell Treatment

Calu-3 cells were seeded in 96-well plates at a density of 1.5 × 104 cells.well−1 and incubated for 24 h in the culture medium. For assessment of cell viability and cytotoxicity, Calu-3 cell line was incubated with the samples NCUR-2 (200.00–25.00 μmol·L−1), NMTX-1 (100.00–12.50 μmol·L−1 and 4.00–0.50 μmol·L−1) and NCUR/MTX-2 (247.00–61.75 μmol·L−1 and 9.88–2.47 μmol·L−1 to CUR; 100.00–25.00 μmol·L−1 and 4.00–1.00 μmol·L−1 to MTX) for 72 h at 37 ◦C with 5% CO2 atmosphere. For co-loaded formulation, final concentrations were standardized using MTX due to its lower content in NCs and EE compensation was also performed for CUR content, resulting in fractional values. Tests were obtained using serial dilution procedure and were performed as four independent experiments containing *n* = 4 samples per assay.

#### 3.5.3. Cell Viability by Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) Test

After 72 h of the treatments, 200 μL of a solution of MTT at 0.5 mg·mL–1 was added to the wells following a standard method [56]. The cultures were then incubated at 37 ◦C for 2 h, protected from light, until the presence of formazan crystals. The supernatant was then removed. For the solubilization of these crystals, 200 μL of dimethyl sulfoxide was added. The spectrophotometric absorbance reading was performed at a wavelength of 550 nm in a μQuant microplate reader (BioTek, Winooski, VT, USA). To calculate cell viability (%), Equation (2) was used. The concentration that inhibited 50% of cell growth (IC50) was then calculated by Probit regression [57]:

$$\text{Cell viability } (\%) = \frac{\text{absolute of test}}{\text{absorbance of control}} \times 100 \tag{2}$$

#### 3.5.4. Combination Index

To define the drug-drug interaction potential from the use of NCs containing CUR and MTX against Calu-3 cells, the combination index (CI) was calculated by Equation (3) [58] considering the results obtained by the MTT method:

$$\text{Combination index (CI)} = \frac{(D)1}{(Dx)1} + \frac{(D)2}{(Dx)2} \tag{3}$$

where (Dx)1 and (Dx)2 are the concentration of the tested substance 1 (CUR) and the tested substance 2 (MTX) used in the single treatment that was required to decrease the cell viability by 50% and (D)1 and (D)2 are the concentration of the tested substance 1 (CUR) in combination with the concentration of the tested substance 2 (MTX) that together decreased the cell viability by 50%.


*5.2.10 Metallic powders as pigments*

The powdered form of some metals, as well as some alloys, are used as pigments. Finely powdered aluminium and bronze have been used as pigments in lacquers. Pigments containing finely powdered zinc have been used for protective coatings on iron and steel to protect them from atmospheric corrosion [17–30].


*3.3. Method Used to Diagnose Battery Fault*

Figure 9 illustrates the proposed battery fault diagnosis algorithm.

The fault diagnosis algorithm considers two situations. After the battery information is sensed through the BMS and the battery efficiency is evaluated regarding whether the value corresponds to the over range, charging proceeds. If the battery efficiency is not higher than the over range, the charge-discharge process is performed; however, if the battery efficiency is higher than the over range and the battery SoH is 40% or less, the charge-discharge process is terminated.

This over range value changes depending on the battery state, battery type, and other factors, and this value should be set before the operation. In this paper, the fault state was set when the efficiency was below 80% and the SoH was below 40%.

Although the battery's charge-discharge SoC is used correctly at 0–100% for the ESS, in this paper, the SoC was charged at 20–80%, the optimal operation region for lithium-ion batteries from a safety viewpoint. Furthermore, the SoH was subjected to charge-discharge cycles up to the maximum region of the battery. Charging was terminated based on the experimental requirements and safety considerations—when the SoH reached 40% or less—to confirm the disposal of the battery through a signal.

**Figure 9.** Proposed fault diagnosis algorithm.

The BMS senses the final output values of Equations (1) and (9), then a chargedischarge termination signal is transmitted through CAN communication if the value is within the over range. The paper predicted the correct battery state through BMS and diagnosed the fault using the proposed method during the charge-discharge process to propose a BMS algorithm for an ESS that uses a large battery capacity.


**7. Conclusion**

The majority of patients suffering from acute pancreatitis will have a mild, self-limited and uncomplicated course. Local and systemic complications, mild or life-threatening, such as pancreatic and/or peripancreatic fluid collections, walledoff necrosis, infected pancreatic necrosis, disconnected pancreatic duct syndrome and vascular complications can occur.

The successful management of these patients needs a multidisciplinary team composed by gastroenterologists, surgeons, interventional radiologists, and specialists in critical care medicine, infectious disease, and nutrition. However, it must be considered that the requisite technical expertise and judgment for many of these procedures is not widely available in all centres. Intervention is generally required for infected pancreatic necrosis and less commonly in patients with sterile necrosis who are symptomatic. The surgical odyssey in managing necrotizing pancreatitis has been described. Operative approaches to the treatment of acute pancreatitis complications have undergone a dramatic transformation over the past few decades. Prospective, randomized trials have further clarified the value of the latest minimally invasive approaches to the treatment of this disease. This is the notable example of how evidence-based knowledge leads to improvement in patient care.

*Recent Advances in Pancreatitis*


**Author details**

Hideki Nakano Neurorehabilitation laboratory, Graduate School of Health Sciences, Kyoto Tachibana University, Kyoto, Japan

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 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, provided the original work is properly cited.


**A. Input data of the scenario analysis**

#### **Table A1.**

*Locations of suppliers, distribution centers, and suppliers of the scenario analysis.*

#### **Table A2.**

*The relative distances among suppliers and distribution centers in [km].*

#### **Table A3.**

*The relative distances among distribution centers and customers in [km].*

#### **Table A4.**

*The customers' demands in [TEU].*

**Table A5.**

*The specific transportation cost among suppliers and distribution centers in [EUR/TEU/km].*


**1. Introduction**

Over the past years, electro mobility has become an important topic in emission free transportation plans. In combination with renewable energies, it is even possible to keep vehicles carbon neutral. These advantages have triggered the interest in electric vehicles in recent years, but the technology has always been expensive. Significant factors leading to low user acceptance include the large and expensive batteries in short-range vehicles and slow charging solutions. Wireless power transfer (WPT) is focused on solving these current problems, such as the inconvenient plug-in charging process. Modern systems have already demonstrated the potentials of wireless energy transfer in cars [1,2]. However, there are currently no solutions for the industrial production of these components.

Within this paper the results of a feasibility study of contactless charging will be introduced, followed by the production processes for inductive power transfer (IPT) systems as well as automation concepts, which have been developed at the Institute for Factory Automation and Production Systems (FAPS) of the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU).