*2.4.2 Heavy metals in wastewater from the textile industry*

*Perovskite and Piezoelectric Materials*

GeI2 doped FA0.98EDA0.01SnI3 and GeI2 doped EA0.98EDA0.01SnI3 PSCs and shows the power conversion efficiency of 13.24% for lead-free perovskite solar cell has been

To survive on this planet the clean air, water, and foods are essential to all forms of life. The surface and the groundwaters are only the sources of clean water which help to all living systems as well as human activities such as consuming, irrigation of crops, industrial application, etc. [97]. Water pollution is one of the most worldwide common issues as the population outbursts and industrial evolutions are there. Day by day, the heavy metals (maybe in the form of ions) are released into water bodies by various industries [98] and are exceedingly water-soluble, non-decomposable, oncogenic agents and cause adverse health complications on the animals as well as human beings. Wastewaters coming out from various industries contain many heavy metal ions, for example, Cu2+, As5+, Ni2+, Sb5+, Zn2+, Cd2+, and Pb2+ [99]. In addition to heavy metal ions, the different organic and inorganic dyes are alternative pollutant releases from different industries for example papers, textiles, and plastics where the dyes are used for coloring their product and also generate significant volumes of wastewater. Many of these dyes containing heavy metal ions have a tendency to store in the living entities causing a different type of diseases and disorders [100–102]. Hence, it is essential to purify the metal-contaminated water before its discharge to the environment. Among all compare to current methods to remove heavy metal from the contaminated water [100, 101] adsorption method is the most likely one because it low cost-effective, high efficiency, and simple to run.

The group of Zhang et al. synthesized the porous nano-calcium titanate microspheres via a citric acid assisted modified sol-gel method and used for absorption of heavy metals like lead, cadmium, and zinc [103]. Haron et al. reported that the nano-crystalline LaGdO3 perovskite was synthesized by the co-precipitation method could adsorb heavy metal ions (Cd2+ and Pb2+) which should be the attention in an application such as wastewater treatment [104]. Zhang et al. synthesized porous nano-barium-strontium titanate via sol-gel method using sorghum straw as a template and investigate about adsorption mechanism of Pb, Zn, and Cd from contaminated water [105]. LaFeO3 nanoparticles were synthesized by Rao et al. by the sol-gel method in presence of different chelating agents and these nanoparticles utilized for an adsorbent of the removal of heavy metal ions in particular cadmium ion. The LaFeO3 sample prepared with succinic acid (SA) as a chelating agent shows a higher removal efficiency of Cd2+ ions from aqueous systems [106]. Zhang et al. investigated Sr modified LaFeO3 and its structural and catalytic activity. La0.8Sr0.2FeO3 contributed significantly enhanced activity in methane combustion and CO oxidation because the oxygen vacancies accelerated the dissociation of gaseous oxygen on the surface in CO oxidation and facilitated the diffusion of lattice oxygen from the bulk to the surface during CH4 combustion [107]. The perovskite LaAlO3 was manufactured using the co-precipitation method by Haron et al. The structural and efficiency of removal of heavy metal (Cd2+ and Pb2+) were extremely investigated by them. The adsorption performance was studied which fit with the Langmuir isotherm. The results disclosed that LaAlO3 perovskite showed high efficiency as heavy metal ions remover from the contaminated water. This adsorbent could be recycled with an EDTA solution and reprocessed with only slightly less efficient than that of the fresh sample [108]. The group of Chen et al. synthesized ternary photocatalyst ZnTiO3/Zn2Ti3O8/ZnO

demonstrated with mixed cation and surface passivation [96].

**2.4 Removal of heavy metals from wastewater**

*2.4.1 Heavy metals from contaminated water*

**38**

The growing population in our globe, demands to clothe and increase with the taming sense of fashion and lifestyle thus textiles are contrived to meet the growing demands. In several countries such as India and Sri Lanka; the production of textile becomes their source of income that subsidizes their gross domestic product (GDP). However, this has brought both significances to such countries either in a positive way which is an enhancement of the economy or in a negative way indorsed to environmental pollution. The textile industries have been adapted as the worst reprobates of pollution contributors [110]. Especially, in India, according to the Central Pollution Control Board [111], a total of 2324 textile industries are set up. The textile industries employ different types of dyes for the manufacturing of various fabric materials. In reality, about 1 million different dyes are found in the market [112] and roughly 700,000 tons of artificial dyes are produced per year [113]. The disposal of dyes in waters exemplifies a severe environmental issue due to the coinciding presence of various types of pollutants [114–116]. All traditional methods used for the treatment of dyes and/or heavy metals have limitations because of cost, efficiency and operational complications. Among all of them, adsorption was exposed as one of the most effective methods due to its simplicity in operation, adaptability, high-treatment efficiency and low cost, and hence it is extensively applied for wastewaters treatment [117–121].

The perovskite oxide La0.9Sr0.1FeO3, capped with cetyl trimethyl ammonium bromide (CTAB) cationic surfactant, and used as a sorbent for the removal of the anionic Congo red (CR) dye from aqueous solutions was reported by Ali et al. [122]. The group of Chu et al. demonstrated the efficiency of Ag-La0.8Ca0.2Fe0.94O3-δ for the removal of organic and bacterial pollutants by catalytic peroxymonosulfate (PMS) activation. The oxygen vacancies in the B-site of perovskite enhances PMS activation and The SO4• and •OH radicals enhance the biocidal activity [123]. Nanocrystalline LaAlO3:Sm3+:Bi3+ composites are used to adsorb Direct Blue-53 (DB-53) dye was reported by Pratibha et al. [124]. This adsorbent is good and promising in the adsorption capacity and is advantageous in the elimination of toxic and non-biodegradable pollutants from water. The group of Dong et al. hydrothermally synthesized perovskite BaZrO3 in the form of hollow micro- and nano-sphere. This size-tunable BaZrO3 hollow nanospheres exhibited an excellent adsorption performance for reactive dyes in acidic conditions and can be used as excellent circular adsorbents for removing reactive dyes. They show the adsorption capacities are over

#### **Figure 5.**

*Representation of the adsorption process over LaFeO3 nanoparticles surfaces prepared using succinic acid (SA), citric acid (CA), and oxalic acid (OA) [106].*

160 mg g−1 for different investigated dyes at a pH value of 2. The adsorbents were easily recovered by using a basic solution with the adsorption performance persistent and the desorption rate is more than 97 wt% [125]. Siddharth et al. synthesized the perovskite structure of Ti-doped BaMnO3 (BaMn0.85Ti0.15O2.93) and its enhanced photocatalytic degradation (~99%) as compared to BaMnO3 toward toxic water impurities like RhB and MB dyes within 270 and 150 min under sunlight [126].


**6.2 Airworthiness and flight safety in military aviation**

The phrase 'acceptable level of safety' in the definition of airworthiness is a complex consideration as absolute safety is hypothetical and can be achieved only at infinite cost. Therefore, the airworthiness standards have to balance between safety concerns on the one hand and the cost and practicability from design and manufacture point of view on the other.

In aviation, safety may be defined as freedom from death, injury or damage to people on board and loss property and life on ground (accident). Safety of any flying effort or machine would depend primarily upon, whether we are operating below or above a particular level known as 'risk threshold'. The risk threshold is the level of risk beyond which accidents are inevitable. It also must be appreciated that this 'risk threshold' is not a stationary one and it keeps varying based on the role, function and a host of other associated factors. It needs therefore to be reassessed under each changing scenario. Airworthiness control is to minimize the risk and maximize the effectiveness. All the airworthiness standards, military or civil, whether that of the USA, Europe or Russia, have a common point of reference which is that an inverse relation should exist between probability of occurrence of an event and the degree of hazard inherent in its effect.

For military aircraft in the US Department of Defense (DoD) document, Mil-STD-882 defines the safety requirements during design of an airborne stores. The decision matrix of system safety as per Mil-STD-882 is shown in **Figure 8**. The safety requirement of any failure event is based on the hazard index of the failure which is defined as the product of the probability of the failure and the damage consequent of the failure event. The damage consequences can range from 'catastrophic' (loss life, aircraft and property) to negligible (minor inconvenience). The frequencies of occurrences are grouped under 'frequent' (1 in 10 h of flight) to 'extremely improbable' (1 in 107 flight hours).


**4. Experiment**

In this section, the experimental results are performed on three real hyperspectral datasets to evaluate the performance of the proposed ELP-RGF method.

## *4.1. Datasets Description*

In our experiments, three hyperspectral image datasets including the Indian Pines image, the University of Pavia image and the Kennedy Space Center image are utilized to evaluate the performance of the ELP-RGF.

(1) Indian Pines dataset: This image was acquired by the Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) sensor, which captured an Indian Pines unlabeled agricultural site of northwestern Indiana and contains 220 × 145 × 145 bands. Twenty water absorption bands (Nos. 104–108, 150–163 and 220) were removed before hyperspectral image classification. The spatial resolution of the Indian Pines image is 20 m per pixel, and the spectral coverage ranges from 0.4–2.5 μm. Figure 3 shows a color composite and the corresponding ground-truth data of the Indian Pines image.

(2) University of Pavia dataset: This image capturing the University of Pavia, Italy, was recorded by the Reflective Optics System Imaging Spectrometer (ROSIS). This image contains 115 bands and a size 610 × 340 with a spatial resolution of 1.3 m per pixel and a spectral coverage ranging from 0.43–0.86 μm. Using a standard preprocessing approach before hyperspectral image classification, 12 noisy channels were removed. Nine classes of interest are considered for this image. Figure 4 shows the color composite and the corresponding ground-truth data of the University of Pavia image.

(3) Kennedy Space Center dataset: The Kennedy Space Center (KSC) image was captured by the National Aeronautics and Space Administration (NASA) Airborne Visible/Infrared Imaging Spectrometer instrument at a spatial resolution of 18 m per pixel. The KSC image contains 224 bands with a spatial size of 512 × 614, and the water absorption and low signal-to-noise ratio (SNR) bands

were discarded before the classification. Figure 5 shows the KSC image and the corresponding ground-truth data.

**Figure 4.** University of Pavia image. (**a**) False-color composite; (**b**,**c**) Ground-truth data.

**Figure 5.** (**a**,**b**) Ground truth data of the Kennedy Space Center images.

#### *4.2. Parameter Analysis of the Proposed Method*

In the experiments, the original images were segmented by the multi-scale segmentation method (MSS) [37]. In this section, we fix the shape parameters *v* = 0.1 and the smoothness parameter to *u*1 = 0.5 in MSS. For the proposed method, there are three hyperparameters that have to be adjusted, namely weight parameter *u*, segmentation scale *S* and the width of spatial neighborhood *d*. The three hyperparameters were selected using the cross-validation strategy. Figure 6 shows the classification results obtained by the ELP-RGF method with different weight parameters *μ* and segmentation scales *S*. From Figure 6, we can see that the result of Figure 6a–b is visually more satisfactory than that of Figure 6c–d. If the process of label propagation entirely relied on the spatial graph, that is, u=1 is applied, the result of ELP-RGF is poor, as Figure 6d shows. Therefore, *μ* = 0.001 to *μ* = 0.01

is considered the most optimal weight parameter range. We can see that the classification result of Figure 6f–g is better than Figure 6e,h, especially for the landscape of "Soybeans-min till" and "Hay-windrowed". In addition, Figure 7 shows the OA curves of ELP-RGF in the different u and dto illustrate the spatial weight parameter playing an important role in the process of label propagation. Furthermore, Figure 8 shows that the classification accuracies and computing time of the proposed method are significantly affected by *S*. When the two factors of the classification accuracy and the computing time are taken into full consideration (see Figure 8) and observing the selected parameters obtained by cross-validation, we can know that the optimal parameter range is 4–6.

**Figure 6.** The analysis for the hyperparameters *μ* and *S* for the Indian Pines image. In the first row, *S* is fixed as five. (**a**–**d**) respectively show the classification results obtained by the extended label propagation (ELP)-RGF method with (**a**) u = 0.001, (**b**) u = 0.01, (**c**) u = 0.1 and (**d**) u = 1. In the second row, *μ* is fixed as 0.01; (**e**–**h**) respectively show the classification maps obtained by ELP-RGF method with (**e**) *S* = 1, (**f**) *S* = 3, (**g**) *S* = 5 and (**h**) *S* = 9.

**Figure 7.** Influence of *μ* and *d* on the Kappa coefficient of ELP-RGF for the Indian Pines dataset.

**Figure 8.** The effect of the different segmentation size on OA accuracy and computing times for three datasets.

#### *4.3. Comparison with Other Classification Methods*

In this section, the proposed ELP-RGF method is compared with several hyperspectral image classification methods, i.e., the typical SVM method, more advanced extended random walkers (ERW) [43] and semi-supervised methods (the Laplacian support vector machine (LapSVM) [38] and spectral-spatial label propagation (SSLP-SVM) [24]). In addition, a post-processing-based edge-preserving filtering (EPF) [44] and the rolling guidance filtering method (RGF) [39] are also used as the comparison methods. The parameter settings for the EPF, ERW and SSLP-SVM methods are given in the corresponding papers. The evaluation indexes in Tables 1–4 are given in the form of the mean ± standard deviation.

For the Indian Pines dataset, Table 1 shows the OA, AA and Kappa coefficient of different methods with the 5/10/15 training numbers per class (represented as *s*). From Table 1 we can see that the OA accuracy and Kappa coefficient of the proposed ELP-RGF method are better than other methods when the number of training samples is relatively small. In particular, when the *s* = 5, the OA accuracy of the proposed ELP-RGF method increases 14.29% and 36.63% compared to that of the SSLP-SVM method and the LapSVM method. The Kappa coefficient of ELP-RGF method is 13.52% higher than SSLP-SVM when *s* = 10, which fully shows the superiority of the two-step method proposed in this paper. We can see that the performance of the proposed ELP-RGF is always superior to that of the ERW method. As the number of training samples increases, the accuracy of the increase rate has decreased, however, there is still a large gap compared with other methods. Figure 9 shows the classification maps obtained by different methods. It can be seen that the classification map of the proposed method has less noise, and the boundary region in the classification map is also much clearer.

**Table 1.** Comparison of classification accuracies (in percentage) provided by different methods using different training samples per class (Indian Pines image). EPF, edge-preserving filtering; ERW, extended random walkers; LapSVM, Laplacian SVM; SSLP, spectral-spatial label propagation.

For the University of Pavia image, we randomly selected 5, 10 and 15 samples from each class as the training samples. Table 2 shows the OA, AA and Kappa coefficient of the different methods with different s. According to Table 2, the proposed ELP-RGF, SSLP-SVM, LapSVM and ERW can produce greater classification accuracy than the SVM at the same s. However, the degrees of the improvement of ERW, SSLP-SVM and LapSVM are smaller compared with ELP-RGF. For example, the OA of ERW and SSLP-SVM increased by 1.16% and 10.24%. The experimental result indicated that the proposed ELP-RGF outperforms the compared methods. The results show that the OA of the proposed method is 96.02%, which is 10.25% higher than that of the SSLP-SVM and 0.91% higher than that of the ERW when *s* = 15. The OA accuracy and kappa coefficients of the ELP-RGF method are always the highest, which demonstrates that the ELP-RGF is the most accurate classifier among these methods. We can see that compared with SVM, SSLP-SVM and ERW, the OA accuracy and Kappa coefficients of the ELP method are more competitive. Figure 10 shows the classification maps of different methods when *s* = 15. The figure shows the effectiveness of the proposed method. The proposed method presents more accurate classification results for the class of MetalSheetsand Gravel, and its classification result is better than those of other methods.

**Figure 9.** Classification maps of different methods for the Indian Pines image. (**a**) Reference image, (**b**) SVM method; (**c**) EPF method; (**d**) RGF method; (**e**) ERW method; (**f**) LapSVM method; (**g**) SSLP-SVM method and (**h**) ELP-RGF method.

**Table 2.** Overall accuracy of the various methods for the University of Pavia image (average of 10 runs with thestandard deviation; the bold values indicate the greatest accuracy among the methods in each case).

For the Kennedy Space Center dataset, we evaluated the classification accuracies of different methods using 39 training samples collected from each class. Table 3 shows the OAs, AAs, Kappa and individual classification accuracies obtained for the various methods. From Table 3, it is demonstrated that the OA, AA and Kappa accuracy of the proposed method are the highest in all comparative methods. Most of individual accuracies are significantly higher than other methods. For the class of Willowswamp, the accuracies of the proposed method and SSLP-SVM are 99.75% and 77.38%; thus, the accuracy gain is 22.37%. For the class of Oak/Broadleaf, the proposed method can produce 59.21% and 57.11% OA improvements compared with SSLP-SVM and SVM. Table 4 provides the OA accuracies of the various methods. Observing the values in Table 4, we can see that the classification accuracy is proportional to s. Moreover, the performance of the proposed method is not only higher than the other semi-supervised classification methods, but also can improve more than that of ERW. Figure 11 shows that the proposed ELP-RGF method can achieve better classification performance and produce little noise compared with other methods.

**Figure 10.** Classification maps of different methods for the University of Pavia image (**a**) Reference image; (**b**) SVM method; (**c**) EPF method; (**d**) RGF method; (**e**) ERW method; (**f**) LapSVM method; (**g**) SSLP-SVM method and (**h**) ELP-RGF method.

**Table 3.** Individual class accuracies, OA, AA and Kappa coefficient (in percentage) for the Kennedy Space Center images.

**Table 4.** Overall accuracy of the various methods for the Kennedy Space Center image (average of 10 runs with thestandard deviation; the bold values indicate the greatest accuracy among the method in each case).

**Figure 11.** Classification maps of different methods for the Kennedy Space Center image. (**a**) Reference image; (**b**) SVM method; (**c**) EPF method; (**d**) RGF method; (**e**) ERW method; (**f**) LapSVM method; (**g**) SSLP-SVM method; (**h**) ELP-RGF method.

Table 5 lists the number of samples generated by the two semi-supervised methods (i.e., SSLP-SVM and ELP-RGF) under three different datasets and the correct rate of these new labeled samples. We can see that the total number of labeled samples generated by the ELP-RGF method is almost 7–22-times more than that generated by the SSLP-SVM method for three datasets. Although the correct rate of the SSLP-SVM method is slightly higher, the ELP-RGF method is also competitive. More importantly, the proposed ELP-RGF can produce more labeled samples.

Table 6 illustrates the effect of the superpixel by comparing with the RGF method, the combination of label propagation and RGF and the combination of superpixel propagation and RGF. The table shows that the superpixel propagation plays a major role in the proposed method. For example, for the Indian Pines image, the OA accuracy of the proposed ELP-RGF is 79.13%, while the accuracy obtained by SP-RGF and RGF is 75.62% and 56.14%, respectively. For the Kennedy Space Center image, the OA accuracy of the SP-RGF method is 4.64% higher than that of LP-RGF. As Table 6 shows, the accuracy of the SL-RGF is more than that of the LP-RGF method when RGF is used in those methods. While LP-RGF is higher than SP-RGF, the gap is small. Thus, the process of superpixel propagation is very useful to help improve the classification result.

**Table 5.** Correct rate of samples generated by the two semi-supervised methods of three datasets.

**Figure 12.** The process of modifying the wrongly-labeled samples. (**a**) The five wrongly-labeled pseudo-labeled samples are provided; (**b**) shows that the first and second labels of pseudo-labeled have been modified by superpixel propagation; (**c**) shows the real labels of the provided wrongly-labeled pseudo-labeled samples.

**Table 6.** Overall accuracy of the various combined methods involved in the proposed method for three datasets.


**7.4. Truscan or polarprobe**

This is a portable electronic device designed by Polartechnics, Crystalaid microelectronics in Australia, which allows a non-invasive screening for cervical cancer. Its modality of operation involves emission of low voltage electrical impulses when put in contact with the cervix generate real time images of the cervix which is compared with a data base of thousands of colposcopic and cervical biopsy pictures to give a final diagnosis of the optical image gener‐ ated. This is associated with less pain, removes the challenges encountered in the third world in terms of performance of the conventional Pap smear and is noted to improve compliance to screening in women(Quek et al 1998)

#### **7.5. Ovarian carcinoma screening**

Ovarian cancers are the 4th commonest cause of deaths in women in the USA and the Britain with an overall survival of 35% with about 75% of the women universally presenting in advanced stages. This is largely because of none specificity of presentations, absence of a known precursor, and the absence of specific screening test for ovarian cancers. How fre‐ quently should patients be screened for ovarian cancers is not specific the NIH in 1994 recommended that all women with a family history of hereditary cancer syndrome should have a yearly screening for ovarian cancer using the available screening methods.

#### **7.6. Biochemical markers for ovarian cancer screening**

#### *7.6.1. CA125*

CA 125 is one of the biochemical tumor markers known as cancer antigen or carbohydrate Antigen 125. This high molecular glycoprotein was identified using monoclonal antibody OC 125 was discovered by Bast et al in 1980.This modality of screening is nonspecific, which is elevated in 80% of patients with epithelial cancers and about 50% in the stage 1 cancers. It is expressed by all tissues of mullerian origin but not produced by normal ovarian epithelium This has sufficient sensitivity in post menopausal patients since other conditions that will result in a rise in the value of the CA125 such as missed miscarriages, endometriosis, pelvic inflam‐ matory disease and benign molar pregnancy are absent in postmenopausal women with ovarian cancer.

A value of greater than 35umg/ml is considered significant for further evaluation to be done. The specificity of the screening method is about 99.9% with a poor positive predictive value of 26%. The predictive value can however be increased with serial annual CA125 as against a single value.A clinical programme by Skates et al 2003, confirmed that women with serial annual rising level of CA125 are more at risk of developing ovarian cancers than women with steady level (risk of ovarian cancer Algorithm-ROCA).If the risk is greater than 1% the patient will need evaluation using transvaginal ultrasound to determine whelter further evaluation is necessary.

#### *7.6.2. Other tumor markers useful in the screening of ovarian malignancies*

**Urinary gonadotropin peptides** are peptides with sequence similar to B subunit of human chorionic gonadotropin and present in 70% of women with epithelial tumours. The presence of an identifiable ovarian tumour by ultrasound, an elevated CA125 and normal urinary gonadotropin peptide are most likely a benign tumor. **Carbohydrate Antigen 19-9 i**s increased in patients' mucinous ovarian tumours but not in epithelial tumours

**Carcinoembryonic Antigen (CEA):** This is a high molecular weight glycoprotein, which is a good tumour marker for the detection of mucinous ovarian tumours and noted in 90% of mucinous tumours. **Alpha feto protein-**Albumin like protein, which is increased in patients with germ cell tumours except dysgerminonas and teratomas.

**Human placentae alkaline phosphatase:** This is a glycoprotein molecule which has two sub units and is useful in differentiating germ cell tumours from other ovarian tumors but has the draw back of being expressed by other mullerian structures hence is not specific

**Human chorionic gonadotropin:** This is elevated in non-gestational choriocarcinoma, embryonal and polyembryomas.

In order to improve the sensitivity and specificity of tumour markers use of multimarkers blood test like oviplex which has the incorporation of five tumour markers (CA125,C reactive protein, serum amyloid A(SAA), interleutin 6 and 8 have been shown to improve the sensitivity y 94% and 91% specificity especially when it concerns early ovarian cancer detection(Edgell et al, 2010).

Following diagnosis and treatment tumor markers like macrophage colony stimulating factor (MCSF) can be used in the follow up of patients which has a high predictive value for persistent disease and is raised in 68% of patients with ovarian cancer.

#### **7.7. Genetic screening**

women above the age of 30years can be prolonged to 5years if HPV testing is added to

This is a portable electronic device designed by Polartechnics, Crystalaid microelectronics in Australia, which allows a non-invasive screening for cervical cancer. Its modality of operation involves emission of low voltage electrical impulses when put in contact with the cervix generate real time images of the cervix which is compared with a data base of thousands of colposcopic and cervical biopsy pictures to give a final diagnosis of the optical image gener‐ ated. This is associated with less pain, removes the challenges encountered in the third world in terms of performance of the conventional Pap smear and is noted to improve compliance

Ovarian cancers are the 4th commonest cause of deaths in women in the USA and the Britain with an overall survival of 35% with about 75% of the women universally presenting in advanced stages. This is largely because of none specificity of presentations, absence of a known precursor, and the absence of specific screening test for ovarian cancers. How fre‐ quently should patients be screened for ovarian cancers is not specific the NIH in 1994 recommended that all women with a family history of hereditary cancer syndrome should

CA 125 is one of the biochemical tumor markers known as cancer antigen or carbohydrate Antigen 125. This high molecular glycoprotein was identified using monoclonal antibody OC 125 was discovered by Bast et al in 1980.This modality of screening is nonspecific, which is elevated in 80% of patients with epithelial cancers and about 50% in the stage 1 cancers. It is expressed by all tissues of mullerian origin but not produced by normal ovarian epithelium This has sufficient sensitivity in post menopausal patients since other conditions that will result in a rise in the value of the CA125 such as missed miscarriages, endometriosis, pelvic inflam‐ matory disease and benign molar pregnancy are absent in postmenopausal women with

A value of greater than 35umg/ml is considered significant for further evaluation to be done. The specificity of the screening method is about 99.9% with a poor positive predictive value of 26%. The predictive value can however be increased with serial annual CA125 as against a single value.A clinical programme by Skates et al 2003, confirmed that women with serial annual rising level of CA125 are more at risk of developing ovarian cancers than women with steady level (risk of ovarian cancer Algorithm-ROCA).If the risk is greater than 1% the patient

have a yearly screening for ovarian cancer using the available screening methods.

**7.6. Biochemical markers for ovarian cancer screening**

conventional screening (Grade A recommendation)

**7.4. Truscan or polarprobe**

134 Contemporary Gynecologic Practice

to screening in women(Quek et al 1998)

**7.5. Ovarian carcinoma screening**

*7.6.1. CA125*

ovarian cancer.

This is based on the background information that 5-10% of women have hereditary genetic predisposition and more than 90% of inherited ovarian tumours occur as a result of genetic mutations in the BRCA1 and BRCA2 genes resulting in chromosomal structure dysfunction and increased risk of malignant transformation. The presence of BRCA 1 and 2 confers 16-90% risk of development of ovarian cancers by the age of 70years as against the 1.7% in the general population ((Brose MS et al 2002,Struewing JP et al 1997). In addition Lancaster et al 2007 also estimated that germline mutation of the above mentioned genes confers a 85% lifetime risk of breast cancer and 46% risk of ovarian cancers. Also mutations in the gene mismatch repair gene MLH, MSH or MSH6 associated with lynch /hereditary non polyposis colorectal cancer have a 9-12% of ovarian cancer and advised in favor of genetic risk assessment which will enable the physician to develop strategies prevent these genetically enabled tumour, provide counseling advice, chemoprevention and prophylactic surgeries for the benefit of the patient.


**Author details**

Nataraj Vijapur1 \* and R. Srinivasarao Kunte2

1 KLE Dr. M.S. Sheshgiri College of Engineering and Technology, Belagavi, Karnataka, India

2 Sahyadri College of Engineering and Management, Mangalore, Karnataka, India

\*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.


*3.2. In Vitro Coupling*

We had a series of laboratory experiments to characterise the antibacterial activity of specific fungal species. First assays showed us the ability of TV mycelium grown on GrowLay™ to lyse bacterial cell walls, based on the lytic plate assay, using freeze-dried cells of *M. luteus*. Following this, we carried out the inhibition assay with living *P. larvae* cells. This is different from lytic activity, in that it shows the ability of the samples to inhibit the proliferation of bacterial cells, leading to the death of the bacterium. However, we had yeast contamination in all our plates potentially due to the incubation temperatures (35 ◦C), a much higher temperature than what living mycelium needs to grow. As the third testing method, we homogenised the patches of mycelia of different fungal strains. This process breaks down the cell walls of fungi and frees compounds which might be bioactive and antibacterial, and it potentially kills all living agents in the solution. We first cultured the mycelia of TV, PO, GL, HE, and GF. After we scraped 1 cm × 1 cm mycelium patches from the agar cultures, we diluted them in 1 mL PSB (phosphate solubilising bacteria) water. Then, we used a TissueLyser II to break down the fungal cells, for 15 min and shake with maximum frequency. Since the scraped mycelium culture was mixed with the substrate, the solutions were too thick for the micro filtering process which we needed for the last phase. Therefore, we used a centrifugal shaker to have clearer liquids at the end. After we filtered the solutions, we incubated the *M. luteus* cultured media with these solutions in conditions where the *M. luteus* thrive (35 ◦C). However, this assay did not show us any positive results as opposed to the first assay where we have used a grown and living patch of TV mycelium.

In unsterile conditions, we placed living TV mycelium and propolis in six malt extract agar dishes. The propolis was collected directly from our hives at the HIVEOPOLIS Honeybee Research Field Laboratory at the Botanical Gardens in Graz, Austria and we did not pre-process it to pasteurise or similar. We did not have any control samples, yet from our experience, agar cultures should be prepared in highly sterile environments to avoid contamination. Yet, the mycelium grew fast and healthy in our dishes with propolis. This can be in favour of the mycelium survival in a living fungal honeybee hive. Then, in a closed plastic container, we placed living TV mycelium grown on a solid substrate together with a wax comb piece, collected from one of our hives and not pre-processed to sterilise. We observed a superficial mycelium coverage on beeswax without degradation of the wax, potentially caused by the fattiness of the wax. We have not performed any coupling assays with pollen, honey, or other substances such as bee bread or royal jelly. Among these, we think that the pollen storage area is a good candidate for the fungal mycelia that we introduced to find nutrition and water. Thus, if our mycelial fungi can survive on pollen as a bio-fungicide, the mycotoxin release can be inhibited in favour of the honeybee colony. This also opens mutualism, in terms of an organic coupling towards therapeutic hives indoors. In terms of specific honeybee bacterial pathogens, we concentrated on AFB disease, which can be detrimental to any colony as fast as three weeks. AFB only attacks the honeybee larvae, located in the brood comb area within the nest with stable temperatures between 32–36 ◦C. The conditions that AFB and larvae live in, especially the temperature, are different from the conditions of basidiomycetes fungi habitats. However, in the longer term, as directed evolution techniques become more common, we can improve our materialmaker fungi to survive in higher temperatures and train them to inhibit the growth of pathogenic bacteria. *3.3. First field Experiment with Living Bees: "Beeocompatibility"*  To test if honeybees are tolerant to mycelium materials, we set up a controlled experiment with living bees. We adapted the natural beekeeping friendly and open-source BCN Wárre Hive design and fabrication models provided by the OSBeehives Project [65]. We

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 11 of 21

can improve our material-maker fungi to survive in higher temperatures and train them

### *3.3. First field Experiment with Living Bees: "Beeocompatibility"* selected the mycelium of GL. As growth substrates, we used coconut fibre mats since co-

to inhibit the growth of pathogenic bacteria.

To test if honeybees are tolerant to mycelium materials, we set up a controlled experiment with living bees. We adapted the natural beekeeping friendly and open-source BCN Wárre Hive design and fabrication models provided by the OSBeehives Project [65]. We selected the mycelium of GL. As growth substrates, we used coconut fibre mats since coconut is hostile to bacterial and yeast contamination and has acidity levels suitable for mycelium (5.5–6.5). The cut-out coconut mats were soaked in water for 12 h, hand pressed, and autoclaved at 121 ◦C for 20 min. We laid the pieces from a pregrown GL mycelium on the fibre mats, placed them in shallow plastic boxes without lids, enclosed in large plastic bags and then incubated them for 14 days at 23 ◦C. When we were satisfied with the mycelial colonisation, we dried the pieces in a kitchen oven at 50 ◦C for 2 h each. We used the mycelial side oriented towards the inside of the hive. We stapled the leatherlike edges on the wooden panels and placed a metal mesh to protect the fibres from being eaten by other animals (Figure 7). conut is hostile to bacterial and yeast contamination and has acidity levels suitable for mycelium (5.5–6.5). The cut-out coconut mats were soaked in water for 12 h, hand pressed, and autoclaved at 121 °C for 20 min. We laid the pieces from a pregrown GL mycelium on the fibre mats, placed them in shallow plastic boxes without lids, enclosed in large plastic bags and then incubated them for 14 days at 23 °C. When we were satisfied with the mycelial colonisation, we dried the pieces in a kitchen oven at 50 °C for 2 h each. We used the mycelial side oriented towards the inside of the hive. We stapled the leatherlike edges on the wooden panels and placed a metal mesh to protect the fibres from being eaten by other animals (Figure 7).

**Figure 7.** BCN Wárre Hive with mycelium retrofit. (**a**) Retrofit hive wall with GL mycelium grown on coconut fibre mat. (**b**) Fixing details. (**c**) One of the three mycelium retrofit hives. **Figure 7.** BCN Wárre Hive with mycelium retrofit. (**a**) Retrofit hive wall with GL mycelium grown on coconut fibre mat. (**b**) Fixing details. (**c**) One of the three mycelium retrofit hives.

On 20 July 2019, we introduced six small-sized bee colonies (3000–4000 bees each) into each hive, three of them with mycelial walls and three with regular 18 mm thick wooden walls. We monitored the climate within the hives for two months from July 26th to September 6th, with combined temperature and relative humidity sensors, Beebots, provided by one of the HIVEOPOLIS research groups, Pollenity. The ambient temperature and relative humidity measurements were from near mycelium walls in three of the On 20 July 2019, we introduced six small-sized bee colonies (3000–4000 bees each) into each hive, three of them with mycelial walls and three with regular 18 mm thick wooden walls. We monitored the climate within the hives for two months from 26 July 26 to 6 September, with combined temperature and relative humidity sensors, Beebots, provided by one of the HIVEOPOLIS research groups, Pollenity. The ambient temperature and relative humidity measurements were from near mycelium walls in three of the hives, and from the same locations in the control hives. These measurements were essential for tracking the microclimatic differences that occurred near two materials, as well as for our long-term hive design programme where the mycelium remains alive.

hives, and from the same locations in the control hives. These measurements were essential for tracking the microclimatic differences that occurred near two materials, as well as This experiment gave us several insights into the honeybee and mycelium material coupling and what to consider in experiment set-ups with hives accommodating full colonies.

for our long-term hive design programme where the mycelium remains alive.

This experiment gave us several insights into the honeybee and mycelium material

learned that the contamination can be avoided only with premeditated clean lab protocols, especially in such experimental scenarios in which the materials must be ready to be tested on-site with strict deadlines. The preparation labour and errors cost us a

• Bees chewed out the mycelium from areas that are softer than others. We can only

• Regarding the hive set-up, we realised that the bees should have been blocked from

for the colony given the nutritional and therapeutic benefits of mycelium.

hypothesise about what they did with the mycelium: they might have moved it out from the hive or consumed it. We think that the second possibility could be beneficial

the empty quilt box (a shallow empty volume below the feeder designed to be filled with humidity capturing material). As this area gets warmer and nearer to the feeder,

coupling and what to consider in experiment set-ups with hives accommodating full col-

late start of the whole field experiment.

onies.

Only one of the six hives survived until the following summer season. This survivor hive was one with mycelium attached. We think that the reason is that we populated the hives late in the season, not giving bees enough time to reproduce, collect pollen and nectar, and build wax combs to store enough honey. Additionally, the weather conditions were particularly challenging that year, with heavy rainfall leading to floodings in many adjacent buildings. The colonies and their wax combs were so small that there was too much empty space in the hives before the winter and not enough insulation on the walls.


**9. Future direction and conclusion**

Large irreparable rotator cuff tears in younger and active patients continue to pose a significant clinical challenge to orthopedic surgeons. Arthroplasty treatment option with a reverse shoulder replacement is not ideal in this patient population. Mihata et al. [33] have shown in biomechanical cadaveric studies that a graft reconstruction can restore superior glenohumeral translation when the graft is attached to the glenoid medially and humeral head laterally. However, many technical aspects of this procedure have not been well studied, such as ideal suture and anchor configuration medially or laterally, ideal graft tissue (allograft versus autograft), ideal graft thickness, or ideal tensioning technique.

In the immediate future, we will need larger clinical studies with short, medium, and longterm outcome data demonstrating the effectiveness of superior capsular reconstruction. Radiographic follow-up studies are needed to document graft incorporation or deterioration after this surgery, as well as to monitor the acromiohumeral distance in this patient population. Clinical indications and contraindications, as well as the ideal patient population, for this procedure need be better defined.

Superior capsular reconstruction is a novel technique that may provide a potentially promising solution to a tough problem in the shoulder region. The procedure should be considered for active and physiologically young patients with high functional demand on their upper extremity, and an irreparable rotator cuff tear, and should be performed by surgeons experienced in treating shoulder pathology. More clinical studies are needed before we can advocate widespread use of this procedure in general orthopedic practice.


*3.1.3 Bioconversion processes*

Biodegradation can be featured as a method used by nature to recycle waste and to break down organic materials into compounds by microorganisms such as bacteria, fungi, insects, worms, and others. Through biodegradation processes, it is possible for nature to clean up wastes, to provide nutrients for the growth of new lives, and to produce the energy necessary for various biological processes [46]. Biochemical transformation via fermentation is an attractive way for utilizing recycling textile waste. Cotton is typically composed of 88–96% cellulose, and it is possible to hydrolyze waste cotton by enzymatic or chemical methods to obtain glucose and then ferment it into value-added products. Biogas production from textile waste via anaerobic digestion is an alternative route to utilize solid waste from textile industry. Organic compounds in solid waste can be used as a raw material to produce desired products via bioconversion processes. On the contrary, thermal and chemical processes can convert both organic and inorganic compounds to value-added products [47].

#### *3.1.4 Thermal and thermochemical conversion processes*

Conventional thermal processing refers to the combustion of solid waste and its conversion into energy. Since solid waste from the textile industry contains a high

energy content, it can be used as a raw material to generate heat energy [47]. Solid waste from the textile industry can be used as a raw material to produce briquette. The thermal processes that are performed at high temperature with inadequate oxygen could generate carbon monoxide, which is a greenhouse gas. Therefore, a thermochemical conversion process, such as pyrolysis, is applied. Pyrolysis is referred to the decompositional process with high temperature in the absence of oxygen condition [47]. Products from pyrolysis are various, such as activated carbon fiber, char, bio-oil, and syngas. The variation of product is related to pyrolysis condition.

Denim jeans are characterized by thick lapped seams that create problems during shredding and carding processes. The presence of elastane is another problem. It is more convenient to separate it before shredding and cutting, but this can only be done by chemical recycling. Recycling different colored jeans together results in a multicolored yarn that can create problems in dyeing. Recycled fibers might not meet the quality of virgin ones and could not be spun or woven properly.

*Understanding Denim Recycling: A Quantitative Study with Lifecycle Assessment Methodology*

Life cycle assessment (LCA) is a methodology where the environmental performance of a product or service is assessed starting from the raw material extraction point to the end of life of that product/service, i.e., from "cradle to grave." The

For a pair of denim jeans, the life cycle (**Figure 2**) starts with the production

methodology of LCA is defined under the ISO 14040/44 Standard [50].

of raw materials such as fibers and chemicals. These materials are then transported to fabric manufacturer and processed to become a fabric. During fabric production, energy and water are consumed in addition to raw materials while emission to air and to water and production waste are generated. The following process for the fabric is garment manufacturing in which the fabric is cut, sewn, washed, and accessorized (rivets, buttons, etc.) according to the design. Finally, the finished garment is sent to a warehouse or directly stores to be

sold. After it is bought, the garment is washed and dried (or dry-cleaned depending on its nature) many times throughout its use phase. When it completes its life span, the garment has various "end of life" scenarios such as

recycling, reused, refurbished, and disposed in landfill or incinerated, etc., which

Life cycle assessment (LCA) helps us analyze the environmental performance of denim production in a transparent and systematic way and identify the hot

**4. Life cycle assessment of a denim fabric**

*DOI: http://dx.doi.org/10.5772/intechopen.92793*

were discussed in the previous sections.

spots.

**Figure 2.** *Lifecycle of a jean.*

**69**


**3.3 Cloning and sequencing of Pb-GlcNAcase**

The length of the Pb-GlcNAcase gene was determined to be 1926 bp. It encodes a protein of 642 amino acids. The mRNA sequence was deposited in the GenBank database (LC573548). The amino acid sequence of Pb-GlcNAcase was similar to that of the β-*N*-acetylhexosaminidase from *P. barengoltzii* (99%, WP\_016313754.1), β-*N*-GlcNAcase from *Fontibacillus panacisegetis* (63%, WP\_091231148.1), and β-*N*acetylhexosaminidase from *Gorillbacterium massiliense* (49%, WP\_040950886.1). These β-*N*-GlcNAcase and β-*N*-acetylhexosaminidases belong to the GH family 20, and so Pb-GlcNAcase may belong to this enzyme family. In addition, all catalytically important residues of GlcNAcase from the GH family 20 [22] were conserved in Pb-GlcNAcase (Asp228 and Glu229).

#### **Figure 2.**

*HPLC analysis of products of hydrolysis of chitin oligosaccharides (GlcNAc)3 to (GlcNAc)6 by recombinant Pb-ChiA. The hydrolysis products of (A) (GlcNAc)6, (B) (GlcNAc)5, (C) (GlcNAc)4, and (D) (GlcNAc)3 by the enzyme were detected by HPLC, as described in the materials and methods. Lines: GlcNAc (dark-blue line), (GlcNAc)2 (red line), (GlcNAC)3 (yellowish olive-green line), (GlcNAc)4 (purple line), (GlcNAc)5 (light blue line), and (GlcNAc)6 (mustard-yellow line).*

## **3.4 Expression of Pb-GlcNAcase gene and characterization of recombinant protein**

The molecular mass of the recombinant protein rPb-GlcNAcase was 70 kDa, as determined by SDS-PAGE (data not shown), in good agreement with that predicted from the amino acid sequence. The activity of rPb-GlcNAcase at various temperatures and pH values were determined in enzyme assays using *p*NP-GlcNAc as the substrate. The optimum pH of rPb-GlcNAcase was 6.0 (**Figure 3A**), while the optimal temperature for rPb-Chi activity was 50°C (**Figure 3B**); these values were similar to those of rPb-ChiA.

To recognized the action modes of purified rPb-GlcNAcase, enzyme assays were carried with chitin oligosaccharides of several lengths, such as (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6; buffer aliquots were picked up over time and analyzed by HPLC (**Figure 4**). The main hydrolysis products of (GlcNAc)6 were GlcNAc and (GlcNAc)5 (**Figure 4A**), those of (GlcNAc)5 were GlcNAc and (GlcNAc)4 (**Figure 4B**), and those of (GlcNAc)4 and (GlcNAc)3 were GlcNAc (**Figure 4C, D**). In these experiments, (GlcNAc)2 was degraded to GlcNAc (**Figure 4E**). In addition, the specific activities of (GlcNAc)2 and (GlcNAc)3 were higher than those of (GlcNAc)4–6 (**Table 1**). The present data indicate that this enzyme is an exo-type carbohydrate hydrolase.

#### **3.5 Cloning and sequencing of Pb-LPMO**

The length of the Pb-LPMO gene was determined to be 1350 bp. It encodes a protein of 449 amino acids. The amino acid sequence of Pb-LPMO was the same or very similar to that of the LPMO from *Paenibacillus* sp. (100%, WP\_028538775) and *P. barengoltzii* (99%, WP\_016312786) and the chitin-binding domain from *Paenibacillus* sp. oral taxon 786 (96%, WP\_009222736). As these LPMOs belong to the AA family 10, Pb-LPMO may also belong to this enzyme family. In addition, catalytically important residues of LPMO from the AA family 10 [9] were conserved in Pb-LPMO (His38, His123) as well as the residues of LPMO from *P. barengoltzii* (WP\_016312786), *Paenibacillus* sp. (WP\_028538775), and *S. marcescens* BJL200 (AAU882020.1). Moreover, Pb-LPMO contains a signal peptide, an LPMO domain, two fibronectin type III domains, and a chitin-binding domain, as reported for the LPMOs of *P. barengoltzii* (WP\_016312786) and *Paenibacillus* sp. (WP\_028538775).

#### **Figure 3.**

*Functional properties of purified Pb-GlcNAc. All reactions were conducted with purified enzyme and pNp-GlcNAc as the substrate. (A) Effect of pH on enzyme activity at 37°C in various buffers. The buffer systems comprised x mL of 0.2 M boric acid and 0.05 mM citrate acid, and (200–x) mL of 0.1 M Na3PO4*∙*12H2O (pH 3.0–9.0). (B) Effect of temperature on enzyme activity measured at 20–70°C. The average values of triplicate measurements were used as activity values.*

**81**

**Table 1.**

*Substrate specificity of Pb-GlcNAcase.*

**Figure 4.**

*Effect of LPMO on the Hydrolysis of Crystalline Chitin by Chitinase A…*

*HPLC analysis of products of hydrolysis of chitin oligosaccharides (GlcNAc)3 to (GlcNAc)6) by recombinant Pb-GlcNAcase. The degradation products of (A) (GlcNAc)6, (B) (GlcNAc)5, (C) (GlcNAc)4, (D) (GlcNAc)3, and (E) (GlcNAc)2 by the enzyme were detected by HPLC, as described in the materials and methods. Lines: GlcNAc (dark-blue line), (GlcNAc)2 (red line), (GlcNAC)3 (yellowish olive-green line), (GlcNAc)4 (purple*

**Substrate Activity (units/ mg protein)**

Soluble chitin ND pNP-GlcNAc 111 × 10−3 pNP-GalNAc 73.7 × 10−3 (GlcNAc)2 10.2 (GlcNAc)3 12.7 (GlcNAc)4 5.8 (GlcNAc)5 3.8 (GlcNAc)6 3.7 *The average values of triplicate measurements were used as each activity value.*

*line), (GlcNAc)5 (light-blue line), (GlcNAc)6 (mustard-yellow line).*

*DOI: http://dx.doi.org/10.5772/intechopen.93761*

*Effect of LPMO on the Hydrolysis of Crystalline Chitin by Chitinase A… DOI: http://dx.doi.org/10.5772/intechopen.93761*

#### **Figure 4.**

*Molecular Biotechnology*

similar to those of rPb-ChiA.

**3.5 Cloning and sequencing of Pb-LPMO**

**protein**

hydrolase.

**3.4 Expression of Pb-GlcNAcase gene and characterization of recombinant**

The molecular mass of the recombinant protein rPb-GlcNAcase was 70 kDa, as determined by SDS-PAGE (data not shown), in good agreement with that predicted from the amino acid sequence. The activity of rPb-GlcNAcase at various temperatures and pH values were determined in enzyme assays using *p*NP-GlcNAc as the substrate. The optimum pH of rPb-GlcNAcase was 6.0 (**Figure 3A**), while the optimal temperature for rPb-Chi activity was 50°C (**Figure 3B**); these values were

To recognized the action modes of purified rPb-GlcNAcase, enzyme assays were carried with chitin oligosaccharides of several lengths, such as (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6; buffer aliquots were picked up over time and analyzed by HPLC (**Figure 4**). The main hydrolysis products of (GlcNAc)6 were GlcNAc and (GlcNAc)5 (**Figure 4A**), those of (GlcNAc)5 were GlcNAc and (GlcNAc)4 (**Figure 4B**), and those of (GlcNAc)4 and (GlcNAc)3 were GlcNAc (**Figure 4C, D**). In these experiments, (GlcNAc)2 was degraded to GlcNAc (**Figure 4E**). In addition, the specific activities of (GlcNAc)2 and (GlcNAc)3 were higher than those of (GlcNAc)4–6 (**Table 1**). The present data indicate that this enzyme is an exo-type carbohydrate

The length of the Pb-LPMO gene was determined to be 1350 bp. It encodes a protein of 449 amino acids. The amino acid sequence of Pb-LPMO was the same or very similar to that of the LPMO from *Paenibacillus* sp. (100%, WP\_028538775) and *P. barengoltzii* (99%, WP\_016312786) and the chitin-binding domain from *Paenibacillus* sp. oral taxon 786 (96%, WP\_009222736). As these LPMOs belong to the AA family 10, Pb-LPMO may also belong to this enzyme family. In addition, catalytically important residues of LPMO from the AA family 10 [9] were conserved in Pb-LPMO (His38, His123) as well as the residues of LPMO from *P. barengoltzii* (WP\_016312786), *Paenibacillus* sp. (WP\_028538775), and *S. marcescens* BJL200 (AAU882020.1). Moreover, Pb-LPMO contains a signal peptide, an LPMO domain, two fibronectin type III domains, and a chitin-binding domain, as reported for the LPMOs of *P. barengoltzii* (WP\_016312786) and *Paenibacillus* sp. (WP\_028538775).

*Functional properties of purified Pb-GlcNAc. All reactions were conducted with purified enzyme and pNp-GlcNAc as the substrate. (A) Effect of pH on enzyme activity at 37°C in various buffers. The buffer systems comprised x mL of 0.2 M boric acid and 0.05 mM citrate acid, and (200–x) mL of 0.1 M Na3PO4*∙*12H2O (pH 3.0–9.0). (B) Effect of temperature on enzyme activity measured at 20–70°C. The average values of*

**80**

**Figure 3.**

*triplicate measurements were used as activity values.*

*HPLC analysis of products of hydrolysis of chitin oligosaccharides (GlcNAc)3 to (GlcNAc)6) by recombinant Pb-GlcNAcase. The degradation products of (A) (GlcNAc)6, (B) (GlcNAc)5, (C) (GlcNAc)4, (D) (GlcNAc)3, and (E) (GlcNAc)2 by the enzyme were detected by HPLC, as described in the materials and methods. Lines: GlcNAc (dark-blue line), (GlcNAc)2 (red line), (GlcNAC)3 (yellowish olive-green line), (GlcNAc)4 (purple line), (GlcNAc)5 (light-blue line), (GlcNAc)6 (mustard-yellow line).*

#### **Table 1.**

*Substrate specificity of Pb-GlcNAcase.*


**Achievements and Prospects of Functional Pavement: Materials and Structures**

Editors

**Jian-long Zheng Zhanping You Xueyan Liu**

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

*Editors* Jian-long Zheng School of Traffic and Transportation Engineering Changsha University of Science and Technology Changsha China

Zhanping You Department of Civil, Environmental, and Geospatial Engineering Michigan Technological University Houghton United States

Xueyan Liu Faculty of Civil Engineering and Geosciences Delft University of Technology Delft Netherlands

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Applied Sciences* (ISSN 2076-3417) (available at: www.mdpi.com/journal/applsci/special issues/ Functional Pavement).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

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**ISBN 978-3-0365-5542-3 (Hbk) ISBN 978-3-0365-5541-6 (PDF)**

Cover image courtesy of Zhanping You

© 2022 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.


**7. Acknowledgements**

Work from authors's laboratory was funded by grants from Comunidad de Madrid, Ministerio de Ciencia e Innovación, Fondo de Investigaciones Sanitarias, Fundación MAPFRE Medicina, Fundación Española de Esclerosis Multiple to M.L.Celma and R. Fernandez-Muñoz. We thank Purnell Choppin, Erling Norrby, George Klein, Fabian Wild, Albert Osterhaus, Martin Billeter and Roberto Cattaneo for valuable materials and Concepción Muela and Ricardo Vázquez for skilful art work.

#### **8. References**

178 Non-Flavivirus Encephalitis

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2. Concerning the question of where the virus could persist and replicate during the long latent period, we have observed that at least in the final stages of SSPE, MV is also present in abdominal and thoracic lymph nodes. The comparison of MV genomic RNA from brain and

3. In two SSPE cases, those presenting an average and long disease course, but not in the short disease course case, biased hypermutation U to C was observed in the matrix M gene at a high level (38% U to C) and low level (10%U to C) respectively. The mutation map across the entire genome was the same from distant parts of each brain, supporting the indication of clonal origin of MV brain invasion proposed by V. ter Meulen, M. Billeter and collaborators. After the first decription of biased U to C hypermutation phenomena by M.Billeter and collaborators in one brain from a MIBE case, our results were the first description of biased hypermutation in SSPE brain. Our results indicate that biased hypermutation U to C are found at autopsia in brain of SSPE patients after years of disease, and it is not proportional to the length of the disease. Biased hypermutation U to C is present in MV localized in lymph nodes at similar or higher level than in the respective brain, suggesting that biased hypermutation may take place also in infected lymphoid cells. In the three cases the transcription of M, F and H genes were down-regulated, and M protein ability

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Concepción Muela and Ricardo Vázquez for skilful art work.

**6. Conclusions**

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**9**

*Canada*

**Coronaviruses as Encephalitis**

Pierre J. Talbot, Marc Desforges, Elodie Brison and Hélène Jacomy

Encephalitis usually refers to brain inflammation of various possible causes, including viral infections. Overall, viruses represent the most common cause of encephalitis in humans. The U.S. Center for Disease Control and Prevention (CDC) estimates an annual incidence of usually between 150 and 3000 new cases per year of Arboviral encephalitis in the United States (http://www.cdc.gov/). Although several thousand cases of encephalitis of various viral origins are reported each year, the CDC suspects that many more cases may go unreported. Indeed, encephalitis can follow or accompany common viral illnesses, such as infectious respiratory diseases, and sometimes signs and symptoms of the latter may mask concurrent encephalitis. Most commonly, clinically relevant viral encephalitis affects children, young adults, or elderly patients. The involvement of other determinants, such as the nature of the specific viral agent, the host immune status, and various genetic and

The pathophysiology of viral encephalitis varies according to the virus family involved. Encephalitis occurs in two forms: primary encephalitis involving direct viral infection of the central nervous system (CNS; brain and spinal cord) and secondary encephalitis involving a viral infection which first occurs elsewhere in the body and then travels to the brain. Viruses may enter the CNS through two distinct routes: hematogenous dissemination or neuronal retrograde dissemination. The hematogenous spread, which is the most common path, involves the presence of a given virus in the blood (viremia), where it can either remain free for a period of time or infect leukocytes that will become some sort of viral reservoir. This latter situation, called the Trojan horse, is the route taken by human immunodeficiency virus (HIV) to disseminate to the CNS in humans. Arboviruses also use the hematogenous route to gain access to the CNS, where they can induce a zoonotic encephalitis, with the virus surviving in infection cycles involving bites by arthropods and various vertebrates, especially birds and rodents. After an insect bite, the virus can be transmitted in the blood of

Another form of viral spread towards the CNS is through retrograde neuronal dissemination, where a given virus infects neurons in the periphery and uses the transport

Neuroinvasive viruses can damage the CNS as a result of misdirected host immune responses (virus-induced neuroimmunopathology) and/or viral replication, which will

**1. Introduction**

environmental factors, is also of importance.

a susceptible animal after local replication in the skin.

machinery within those cells in order to gain access to the CNS.

**- Inducing Infectious Agents**

*INRS-Institut Armand-Frappier, Laval (Québec)*

*Laboratory of Neuroimmunovirology,*


**1. Introduction**

Economic dispatch (ED) is one of the most important issues pertaining to the operation and control of power systems [1,2]. It involves finding an optimal scheduling for all committed generators to minimize fuel costs, while satisfying various constraints such as load demand balance and generation capacity constraints. However, owing to increasing global concerns regarding the environmental issues caused by the combustion of fossil fuels, ED has become a significant concern from an economical perspective and for dealing with pollutant emissions from fossil fuel combustion [3]. Some countries have set maximum emission limits and impose fines when these limits are exceeded [4]. Thus, emission constraints should also be considered during operational scheduling. Economic emission dispatch (EED) has been proposed for scheduling to account for the emission of harmful gases such as carbon dioxide (CO2), sulfur oxides (SOx), and nitrogen oxides (NOx), which pollute the air and exacerbate global warming [5]. The objective function of the EED usually adds emission criteria to the fuel cost of the thermal unit. To integrate the emission component with ED, Dhillon et al. [6] and Kulkarni et al. [7] presented a method whereby a penalty factor is multiplied to the emission term in the objective function. This technique allows both components to become commensurately involved in the optimization.

Demand response (DR) is expected to help solve environmental problems, as it is environmentally friendly and offers several benefits to the entire system [8,9]. This is because DR resources are one of

the most inexpensive resources and can react quickly to the commands of the system operator [10]. Furthermore, they do not emit pollutants and help reduce peak loads. In a DR program, consumers sign a contract with the local utility to reduce their demand when requested by the system operator. The utility possesses the advantage of reducing the maximum demand and consequently decreasing operation costs. Moreover, the system operator can use the DR program to satisfy constraints such as maximum pollutant emissions. DR programs are commonly used in small power systems such as microgrids (MGs), because they are difficult to implement in large power systems due to communication problems [11,12].

MGs can be defined as a small-scale form of the centralized power system and typically consist of diesel generation (DG) units, renewable energy resources, and loads that are designed and situated close to customers in small communities [13]. MG operators (MGOs) manage a cluster of loads and power resources, conducting operations to control power locally. MGOs also create contracts with the utility service to meet environmental constraints of the EED, and they take the maximum emission constraints into account in the context of MG operation. To satisfy such constraints, ecofriendly energy resources such as DR should be used appropriately.

Various approaches have been recently adopted to address environmental problems in MG operation. In [14], emissions of harmful gases such as CO2, SOx, and NOx were reduced by installing additional ecofriendly generators that consume less fuel. In [15], a differential evolution technique that combines heat and power was proposed to solve the microgrid economic and emission dispatch problem. In [16], both emission and fuel costs were implemented through different variants of particle swarm optimization. A hybrid algorithm that combines the differential evolution algorithm and particle cluster optimization was used to solve the EED problem [17]. In [18], a price penalty factor, which is the ratio between the maximum fuel cost and the maximum emission of the corresponding generator, was applied for solving the EED problem. However, the studies mentioned above did not take the DR program into account, and the optimal operations were insufficient under environmental considerations. In [19], a multi-objective approach was proposed to optimize microgrid in a short-term with renewable energy sources with a randomized natural behavior. However, pollutant emissions was not considered. Through the implementation of the DR program, in [20], a balanced solution was obtained where a sample hub energy system containing conventional and renewable energy sources was solved with a mixed integer linear program for optimizing operation costs and reducing pollutant emission. In [21], extended game theory was incorporated into the multiobjective dynamic EED optimization problem through a DR model. In [22,23], DR was considered for spinning reserves to solve the EED problem. However, the cost of interruptions and the incentives of the participating subjects were not considered.

Several methods have been considered to solve the EED problem. A flower pollination algorithm was used to solve economic load dispatch and EED problems by considering the power limits of the generator [24]. In [25], economic load and emission dispatch were determined with a bioinspired algorithm in a renewable integrated islanded MG. A converted single objective function comprising fuel cost and emission was optimized using a gravitational search algorithm in [26]. In [27], quantification of the impacts of the CO2 emission has been considered on the planning, economic and scheduling decisions. While most EED problems include pollutant emissions in the objective function, there were no maximum emission constraints on the power system. To operate MGs while accounting for environmental concerns, because maximum emission constraints are practically essential, the optimal operation approach for these problems must be studied. In addition, DR is a considerably ecofriendly energy resource; however, very few studies focus on it to determine volume with respect to environmental constraints. Therefore, it is essential to conduct a study on the optimization of MG operation by utilizing a DR program and considering emission constraints for combined economic emission dispatch (CEED) problems.

In this study, we propose an approach to solve the CEED problem while considering maximum emission constraints. The CEED function consists of the operation costs and pollutant emissions. Here, the penalty factor is used to convert the emission into an equivalent cost value. Various constraints are considered in solving the problem. In particular, the maximum emission constraint is additionally considered. To solve this problem optimally, we propose an environment-based demand response (EBDR) program that determines volume by optimizing economics and reducing the degree of pollutant emissions from the system. A weighting update artificial bee colony algorithm (WU-ABC) is applied to solve the CEED problem under a maximum emission constraint. Here, the penalty factor is updated using the weighting update method until all the constraints are satisfied. By using WU-ABC algorithm, the proposed approach becomes capable of finding the optimal CEED solution of the MG.

The primary contributions of this paper can be summarized as follows:

The proposed approach provides an optimal power scheduling using EBDR for MG systems in CEED problems with maximum emission constraints. In this regard, MGOs can be provided with more reasonable and flexible solutions for optimal operation with environmental constraints.

The remainder of this paper is organized as follows. Section 2 introduces the grid-connected MG system model. Section 3 presents the formulation of the problem for the CEED with a maximum emission constraint. Section 4 outlines the proposed EBDR strategy. Section 5 presents the solution of the WU-ABC algorithm and summarizes the overall process. Section 6 presents the simulation results, and Section 7 presents the conclusions of this study.


**3. Management of human chlamydial infections**

Management of human Chlamydial infections will be discussed as separate sections and will include the most common etiological agents: *C. trochomatis, C. pneumoniae*, and *C. psittaci*, respectively.

#### **3.1 Management of** *C. trochomatis* **infections**

Infections due to *C. trochomatis* are caused by 19 serovars, which affect the ocular, genito-urinary tract, and pulmonary systems. Ocular infections mainly result from serovars A-C, which led to trachoma and to a lesser extent serovars D-K which led to inclusion conjunctivitis and sometimes infant pneumonia [28]. On the other hand, genito-urinary tract infections include chlamydia and lymphogranuloma venereum (LGV), which are caused by serovars D-K and serovars L1-L3, respectively [2, 18, 29].

In most men and women, chlamydia maintains an asymptomatic course and becomes a silent reservoir for infection [5, 30]. Spontaneous clearance of the pathogen may occur gradually over years but if chlamydia is not treated effectively, major medical conditions with both immediate and long-term complications could arise [3, 31, 32]. These health problems include clinical or subclinical pelvic inflammatory disease (PID) leading to chronic pelvic pain, infertility, ectopic pregnancy and Fitzhugh-Curtis syndrome in women; pre-term delivery, inclusion conjunctivitis, and pneumonia in the newborn; and reactive arthritis in both sexes [5, 33, 34]. It was also shown that untreated chlamydia may promote the spread of other STIs, such as human immunodeficiency virus (HIV) [35]. Therefore to prevent chlamydiaassociated diseases and to decrease the degree of transmission to susceptible populations, presumptive or guided therapy should be started in all suspected or confirmed chlamydia infections including asymptomatic diseases.

#### *3.1.1 Treatment of ocular infections due to* C. trachomatis

The most prevalent infectious etiology of blindness all over the world is *C. trachomatis*, which leads to ocular illnesses known as trachoma and adult/neonatal inclusion conjunctivitis*.* Trachoma is transmitted through direct or indirect contact with objects such as hands, fomites, bed sheets, eye-seeking insects, and polluted towels in unsanitary settings [28]. On the other hand, inclusion conjunctivitis is spread by perinatal transmission in neonates or by hand-to-eye inoculation of infected genital secretions in adults.

#### *<IP_ADDRESS> Treatment of trachoma*

Trachoma specifically targets the world's poorest regions. According to data from June 2022 provided by the World Health Organization (WHO), 125 million people reside in areas where trachoma is an endemic disease [36]. Trachoma is initiated in early childhood and recurrent ocular infection leads to conjunctival scarring and eventually blindness. Trachoma incidents decreased as a result of general improvements in living conditions, but recent predictions show that 1.9 million individuals worldwide are blind or have significant vision problems as a result of trachoma. The WHO is implementing the SAFE strategy as part of a global initiative to eradicate blinding trachoma. The SAFE strategy includes early surgical intervention for trichiasis (contact between the eyelids and the eye that results in blindness), widespread use of antibiotics like azithromycin to manage infections, regular facial hygiene, and advancements in living conditions to minimize bacterial spread.

The World Health Organization divides trachoma into five stages, with Stages 1 and 2 characterized by trachomatous inflammation that is follicular (TF) or intense (TI), respectively; Stage 3 by trachomatous scarring (TS); Stage 4 by trachomatous trichiasis (TT); and Stage 5 by corneal opacity (CO), which results in visual impairment and blindness.

Antibacterial treatment is advised in the first two stages of trachoma's inflammatory phase (stages 1 and 2) and before scarring sets in, which is Stage 3 [37, 38]. The antibacterial drug of choice is oral azithromycin because it can be taken as a single dose, has a relatively long half-life, and is more concentrated in tissue than in plasma [39]. Adults and children are given a single oral dose of azithromycin at doses of 1 g and 20 mg/kg, respectively. In case of antibiotic failure, topical 1% tetracycline eye ointment, twice a day, for 6 weeks or erythromycin, 20 mg/kg (maximum 1 gr), orally, twice a day, for 14 days can be given. In stages 3 and 5, no treatment option exists but in Stage 4 surgical treatment is advocated for the protection of the cornea from abrasion.

Because trachoma is a major public health issue, the WHO recommends widespread administration of antibiotics in places with populations of 100,000–250,000 people and when the prevalence of the active trachoma, Stage 1 (TF), is greater than 5% in children aged 1–9 years. According to the most recent estimate of TF prevalence and until the percentage falls below 5%, it is advised that all inhabitants receive antibiotic therapy on an annual basis. It is also recommended that all inhabitants receive antibiotic medication annually up until the most current estimate of the prevalence of TF drops below 5% [40].

#### *<IP_ADDRESS> Treatment of neonatal inclusion conjunctivitis*

Neonatal inclusion conjunctivitis is a preventable yet an undesired complication of active maternal chlamydial infection that affects newborns. Perinatal transmission of

#### *Treatment of Chlamydial Infections DOI: http://dx.doi.org/10.5772/intechopen.109648*

*C. trachomatis* to neonates during vaginal delivery occurs as high as 60% of the cases [41]. Though neonatal infection may retain an asymptomatic course, the infection is usually progressive and mostly affects the conjunctiva, nasopharynx, and lungs. Among infants presenting with conjunctivitis, the likelihood of neonatal-acquired inclusion conjunctivitis varies between 20 and 64% [41, 42]. Conjunctivitis that is left untreated can last for months and cause corneal and conjunctival scarring. Also, the disease may spread to the lungs and cause infantile pneumonia [41]. To prevent the systemic spread of the agent, all infants infected with *C. trachomatis* should receive treatment even if they are asymptomatic.

The neonatal inclusion conjunctivitis incubation period is generally 5–12 days after birth. Conjunctivitis in newborns younger than 30 days should be investigated for chlamydial infection, particularly if the mother has a past history of illness. Infection with ophthalmia neonatorum should also be considered and ocular samples should be examined for *Neisseria gonorrhoeae* [43].

Neonatal inclusion conjunctivitis is treated with oral erythromycin base or ethyl succinate, 50 mg/kg/day in four divided doses for 14 days. The alternative treatment is oral azithromycin 20 mg/kg/day for 3 days [44]. Both antimicrobials have been associated with infantile hypertrophic pyloric stenosis (IHPS) in infants less than 6 weeks of age; consequently, newborns medicated with any of these antimicrobials should be monitored for IHPS [45]. Compared to oral therapy, topical medication has been demonstrated to be ineffective in eliminating nasopharyngeal colonization and linked to persistent infection [46].

There is currently no effective prophylaxis for the prevention of newborn inclusion conjunctivitis, and neonatal gonococcal ophthalmia neonatorum prophylaxis does not offer protection against chlamydial conjunctivitis. Implementing health policies for routine antenatal testing and treatment of pregnant women at risk for chlamydia is advocated as a substitute, however, due to the financial burden of the strategy the screening programs cannot be utilized globally [47].


**3.2 Recommendations**

In line with a globally or regionally accepted management for diabetes in pregnancy—a multidisciplinary team approach, multilevel healthcare system/setting, local policies and standardised clinical practice guidelines should be made available for continuation of care for both mother and child, especially in the postpartum period, to reduce the effect and complications of diabetes in pregnancy.

This is important because most available guidelines are made for the advanced or global north setting, majorly excluding point-of-contact care guidelines (asides from screening and referral) for basic primary or community-based healthcare centres, where most pregnant women first present or have their deliveries in the developing or global south countries. Likewise, more research work needs to be carried out on the social determinants of DM outcomes in mothers and babies, as it pertains to different localities and socio-economic classes across the globe.