**1.   Their temporal and spatial distribution was influenced by environmental, cultural, and political factors with different characteristics. The past, present, and future are in the same chain of time. The study of the spatial and temporal distribution and morphological characteristics of settlements can, therefore, provide a basis for the conservation of their heritage value, as well as be a reference for the reconciliation of human–land relations, thus contributing to global sustainable development.

The spatial distribution of settlements has been a subject of interest for a long time in a number of disciplines, including archaeology, geography, and architecture. Geographic Information Systems (GIS) have been applied to archaeological research since the 1980s. With the further development of GIS technology, there have been many successful examples

**Citation:** Tong, M.; Li, B.; Li, Z. Research on the Spatial–Temporal Distribution and Morphological Characteristics of Ancient Settlements in the Luzhong Region of China. *Land* **2022**, *11*, 1579. https:// doi.org/10.3390/land11091579

Academic Editors: Lucia Della Spina, Paola Pellegrini, Antonia Russo, Maria Rosa Valluzzi and Angela Viglianisi

Received: 11 August 2022 Accepted: 13 September 2022 Published: 15 September 2022

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

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of the application of GIS to settlement archaeological research, and the theory is gradually becoming mature [1]. In recent years, numerous scholars have carried out studies on the Yangtze River basin [2], the Minjiang River basin [3], the Dalian region [4], the Ili River valley [5], and other regions within China. However, relatively few similar studies have been carried out in the lower reaches of the Yellow River Basin. Shandong, located on the lower reaches of the Yellow River, is one of the important birthplaces of Chinese civilisation, and archaeological work has shown that its cultural genealogy is well established. In chronological order, it includes: Yiyuan Man (about 400 ka B.P.), Xintai Wuzhutai Homo Sapiens (about 50–20 ka B.P.), the fine stone tool remains of the Yishu and Wensi river basins (about 10 ka B.P.), the early Neolithic culture (about 8.4–7.7 ka B.P.), the Beixin culture (7.3–6.1 ka B.P.), the Dawenkou culture (6.1–4.6 ka B.P.), the Longshan culture (4.6–4.0 ka B.P.), the Yueshi culture (4.0–3.6 ka B.P.), and all subsequent historical periods. Each of these periods are closely linked, and the cultural traditions have evolved in the same way [6]. Within Shandong province, the Luzhong region (the centre of Shandong Province) is a relatively independent climatic, geographical, and cultural unit. Moreover, the political, economic, and cultural centre of Shandong has been located within this area since Linzi of Qi in the Spring and Autumn period. The Luzhong region is, therefore, a valuable case for studying the spatial and temporal distribution and characteristics of settlements at the core of Chinese civilisation. It can also enrich the case of settlement studies in the lower Yellow River Basin.

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

The spatial extent of this study, the Luzhong region, specifically includes Jinan (except Jiyang District and Shanghe County), Zibo (except Huantai County and Gaoqing County), Tai'an, and Weifang (Figure 1). The climate in the Luzhong region is temperate monsoonal, with a more pronounced continental character. It is cold and dry in winter, and hot and rainy in summer. The average annual temperature is 12 ◦C–14 ◦C, and the annual precipitation is approximately 680–860 m [7]. As can be seen from the map, the region has the Taiyi Mountains in the centre, which are surrounded by hilly undulations that transition into flat plains. In terms of water systems, the Yellow River flows through the territory. In addition, there are dense small- and medium-sized rivers, which have also developed their own water systems. This study covers the period from the Neolithic period to the Qing dynasty and is divided into six time periods according to cultural sequences: Neolithic, Shang and Zhou, Qin to Northern and Southern Dynasties, Sui to Five Dynasties, Song and Yuan, and Ming and Qing.

Based on the temporal and spatial scope of the study, a total of 1972 settlements were selected from the State Administration of Cultural Heritage [8] and the National Cultural Heritage Administration [9] by using "ancient city", "old city", "settlement site", "fortified cities", "fortresses", "castle", and "mountain city" as keywords. These data include all levels of cultural relic protection units and historic cities, towns, and villages. Thus, the data selected balanced representativeness and breadth, and they are able to reflect the spatial and temporal distribution characteristics and evolution patterns of settlements in the central Shandong region. In addition, the Digital Elevation Model (DEM) of the Luzhong region required for the research was derived from the GDEMV3 30 M resolution digital elevation data from the Geospatial Data Cloud website.

This paper analyses the spatial–temporal distribution and morphological characteristics of ancient settlements in the central Shandong region based on data from 1972 settlements and with the aid of a GIS system. Specifically, first, the 1972 data collected were divided into 6 stages according to cultural sequences (Neolithic, Shang and Zhou periods, Qin to North and South Dynasties, Sui to Five Dynasties, Song and Yuan Dynasties, Ming and Qing Dynasties) to establish a database of settlement attributes and perform kernel density calculations. Then, in the QGIS 3.22 software environment, the DEM map of the Luzhong area was overlaid with the settlement points to carry out the calculation of the elevation, slope, aspect, and buffer zone. At the same time, the spatial and temporal

distribution characteristics of the settlements are comprehensively explored in relation to the historical context and dominant cultural forms in which the settlements were located. Based on the above quantitative and qualitative analyses, the distribution and evolutionary characteristics of the settlements are derived.

**Figure 1.** Location and scope of the Luzhong region.


**1. Introduction**

HSG4112 is a new drug candidate which has been developed as a treatment for obesity. Its chemical structure is derived from glabridin. Glabridin is an isoflavane, which is found in *Glycyrrhiza glabra* extract [1–3]. It is known to have whitening activity by suppressing the activity of tyrosinase during the synthesis of melanin and to help alleviate gastroenteric disorders. Recently, it was confirmed that glabridin is effective in metabolic syndromes, including hyperlipidemia, fatty liver, impaired glucose metabolism, diabetes, and obesity and has anti-inflammatory actions, anticancer actions, and the like [4]. However, in spite of useful medicinal efficacy, glabridin is easily broken down by sunlight, moisture, acidity, basicity, oxygen, heat, and the like due to low chemical stability, so it is very difficult to develop a product actually utilizing glabridin [4]. For these reasons, we synthesized a new pyranochromenylphenol derivative, HSG4112, by modifying the structure of glabridin. HSG4112 is stable under various physical conditions, while maintaining or improving its medicinal efficacy [4]. HSG4112 is a racemic compound with a chiral carbon. Thus, glabridin has the structure of R-enantiomer whereas HSG4112 is a mixture of S and R enantiomers (Figure 1).

**Figure 1.** Chemical structures of (**A**) HSG4112 and (**B**) glabridin.

In many cases with chiral drugs, one of the two enantiomers is active and the other is either non-active or even harmful [5–7]. This is because the interaction between a drug molecule and its target is dependent on its three-dimensional environment. The most famous example is thalidomide, which was sold in the 1950s; the drug was introduced as a racemic mixture for use as a sedative but was later withdrawn from the market following the occurrence of birth defects in the children of mothers who took it to treat morning sickness. It was later found that the inactive enantiomer was the cause of the teratogenicity. This disaster was a driving force behind the requirement to strictly test drugs before making them available to the public [8]. In particular, for racemate candidates, both enantiomers should be studied separately as early as possible to assess the relevance of stereoisomerism to effects and fate. Accordingly, a pharmacokinetic evaluation should be provided for each enantiomer [9,10].

In this study, the stereoselective pharmacokinetics of HSG4112 were investigated in rats and beagle dogs. In addition, the metabolic stability was investigated in liver and intestinal microsomes from five different species (rat, mouse, dog, monkey, and human) to evaluate the role of metabolism in the species differences in its stereoselective pharmacokinetic behavior.


**Acknowledgements**

The authors appreciate Prof. Wei-Fang Su, Prof. Yang-Fang Chen and Mr. Meng-Huan Jao at National Taiwan University for useful discussion and suggestions. The authors acknowledge the financial support from the Ministry of Science and Technology, Taiwan (MOST 106-2221-E-182-057-MY3, MOST 106-2119-M-002-030 and MOST 106-2632-E-182-001) and Chang Gung Memorial Hospital, Linkou (CMRPD2F0161 and BMRPC74).


**1. Introduction**

Long-span bridge structures can be affected by various environmental factors during their long-term operations, among which the temperature effect is particularly significant [1–3]. In recent years, numerous studies on bridge temperature effects have been carried out [4–7], mostly considering uniform temperature fields. Some scholars [8,9] have studied the influence of boundary factors such as solar radiation on the bridge temperature field. Some scholars [10–13] studied the time-varying temperature distribution of bridges and established a temperature gradient distribution model. There have also been numerous scholars [14–20] who have studied the long-term measured cross-section temperatures of a large number of bridges with different cross-section forms, observed transverse temperature gradients and proposed two-dimensional temperature field calculation models.

**Citation:** Ma, W.; Wu, B.; Qin, D.; Zhao, B.; Yang, X. Statistical Analyses of the Non-Uniform Longitudinal Temperature Distribution in Steel Box Girder Bridge. *Buildings* **2023**, *13*, 1316. https://doi.org/10.3390/ buildings13051316

Academic Editor: Andrea Benedetti

Received: 12 April 2023 Revised: 8 May 2023 Accepted: 16 May 2023 Published: 18 May 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

1 State Key Laboratory of Mountain Bridge and Tunnel Engineering, Chongqing Jiaotong University, Chongqing 400074, China<EMAIL_ADDRESS>(W.M<EMAIL_ADDRESS>(X.Y.)

It is worth noting that the above studies are based on the assumption that the temperature is uniformly distributed along the longitudinal span direction of the bridge. However, in actual engineering, the temperature field of large-scale structures, such as long-span bridges, is usually non-uniform. It has been shown that the stresses and deformations caused by non-uniform temperature distribution can be comparable to those caused by static and live loads, which may also cause cracks and excessive deformations [21–25].

Actually, the existing standards [26–30] have neglected the influence of the longitudinal non-uniform temperature distribution on bridges, assuming that the temperature field can be represented by one-point measuring data, i.e., a uniform distribution field. Although this assumption simplifies the analysis of bridges affected by temperature in the two-dimensional plane, it may overestimate the temperature effects at some spanwise locations and underestimate them at others. Moreover, the nonlinear effects caused by temperature effects are neglected by the uniform-distribution assumption [31,32]. Therefore, the temperature effects cannot be accurately evaluated when adopting the above assumption. Additionally, the inability to accurately assess the temperature effects of large-span bridges may bring uncertainty to the bridge during the design, construction and operation stages, thus affecting the structural safety of the bridge [33,34].

Therefore, the longitudinal temperature distribution is important for the accurate analysis of bridge temperature effects. Several studies have started to pay attention to this topic. Abid et al. [35] investigated the temperature distribution of bridge segmental models under the influence of solar radiation and air temperature variations through numerical simulations. Based on the health monitoring data of the Aizhai Bridge, Hu et al. [36] found that there were temperature differences at different measuring points along the longitudinal direction of the bridge span. Gu et al. [37] proposed a vertical and lateral temperature gradient model for different longitudinal positions along the bridge span. Liu et al. [38] studied the non-uniform longitudinal distribution temperature of concretefilled steel tubular bridges due to the change in component inclination. Although the above studies considered the variability of temperature distribution, it is limited to a single or several measuring points, and the conclusions may not be well applied to actual bridges. In this context, the longitudinal temperature distribution on the whole bridge and its statistical characteristics was insufficient. Moreover, the role of the non-uniform longitudinal distributions of temperature was not analyzed in depth.

Therefore, this work studies the longitudinal temperature distribution of steel box girders based on the field-measured temperature data. The main objective was to provide a more accurate description of the longitudinal temperature field of long-span bridges.

The paper is organized as follows. In Section 2, the bridge and its health monitoring system are introduced. In Section 3, the probability density characteristic analysis is carried out by selecting typical measuring points in the longitudinal direction of the bridge, and the probability density curves are fitted; next, the temperature of all measurement points is statistically analyzed, and longitudinal distribution curves are fitted; A correlation analysis was then performed, highlighting variability in the longitudinal direction. Finally, the frequency domain features were analyzed, and the space-time contour maps were given to study the mechanisms and processes of the non-uniform distribution of temperature.


**1. Introduction**

124 Cellulose – Medical, Pharmaceutical and Electronic Applications

PP. 645–647, ISSN. 0167-577X.

220, ISSN. 0254-0584.

PP. 2477-2484, ISSN. 0021-8995.

Yang N, Zhu S, Zhang D, & Xu S. (2008). Synthesis and properties of magnetic Fe3O4 activated carbon nanocomposite particles for dye removal. Mater. Lett, Vol. 62, No.4-5,

Yoshida Y, Yanagisawa M, Isogai A, Suguri N, & Sumikawa N. (2005). Preparation of Polymer Brush-Type Cellulose b-Ketoesters using LiCl/1,3-Dimethyl-2-imidazolidinone

Zakaria S, Ong B H, & Van de Ven T G M. (2004a). Lumen loading magnetic paper II: mechanism and kinetics. Colloids. Surf. A, Vol. 251, No. 1-3, PP.31–36, ISSN. 09277757. Zakaria S, Ong B H, & Van de Ven T G M. (2004b). Lumen loading magnetic paper I:

Zakaria S, Ong B H, Ahmad S H, Abdullah M, & Yamauchi T. (2005). Preparation of lumenloaded kenaf pulp with magnetite (Fe3O4). Mater. Chem. Phys, Vol. 89, No.2-3, PP. 216 -

Zhou J, Li R, Liu S, Li Q, & Zhang L. (2009). Structure and Magnetic Properties of Regenerated Cellulose/Fe3O4 Nanocomposite Films. J. Appl. Polym. Sci, Vol. 111, No. 5,

Zhang W, Chen S, Hu W, Zhou B, Yang Z, Yin N, & Wang H. (2011). Facile fabrication of flexible magnetic nanohybrid membrane with amphiphobic surface based on bacterial

cellulose. Carbohydr. Polym, Vol. 86, No.4, PP. 1760-1767, ISSN. 0144-8617.

as a Solvent. Polymer, Vol. 46, No. 8, PP. 2548–2557, ISSN. 0032-3861.

flocculation. Colloids. Surf. A, Vol. 251, No. 1-3, PP. 1–4, ISSN. 09277757.

## **1.1. Cellulose and cellulose biomass**

A cellulose molecule is a linear polymer of D-anhydroglucopyranose units linked by β-1, 4 glucosidic bonds (Figure 1). On its reducing end, a cellulose molecule has an unsubstituted hemiacetal. On its non-reducing end, it has a hydroxyl group.

**Figure 1.** Molecular structure of a cellulose molecule.

Cellulose is the skeleton structure of almost all green plants. It is particularly abundant in non-food plants like trees and grasses, which typically have 40-60% cellulose, 20-40% hemicellulose, and 10-25% lignin (Lynd et al., 2002; Yang et al., 2007). There are four major polymorphs of cellulose: I, II, III, and IV. Cellulose I, often found in native cellulose, contains allomorphs Iα (bacteria and algae) and Iβ (higher plants) (Kontturi et al., 2006; Pérez & Samain, 2010). Cellulose I, when treated with a concentrated alkaline solution, turns into cellulose II, a thermodynamically more stable crystalline form than cellulose I. Cellulose IIII can be obtained when cellulose microcrystal is subjected to supercritical ammonia. The structure of another allomorph of cellulose III, IIIII is still being debated. Cellulose IVI and IVII are formed when cellulose IIIis heated in glycerol at 260°C (Peter, 2001).

Probing the Interaction Between Cellulose and Cellulase with a Nanomechanical Sensor 127

recalcitrance and high cost of cellulase in biomass conversion, the current process for biofuel production is not yet a viable option for the large-scale production (Dwivedi et al., 2009). Much research and developmental efforts have been dedicated to the improvement of the efficiency of cellulase in biomass conversion. One feasible approach is through the incorporation of new features (mutations) into cellulase that accelerate key steps (e.g., rate limiting step) of the enzymatic process. This approach requires a comprehensive

**Figure 3.** The process of biomass conversion through the enzyme-based biochemical method.

Cellulase (Lynd et al., 2002; Mosier et al., 1999; Wilson & Irwin, 1999), like all glycosyl hydrolase enzymes found in bacteria, fungi, plants and some invertebrate animals, breaks down β-1, 4-glycosidic bonds of cellulose through general acid/base catalysis. There are mainly three kinds of cellulases: exo-β-1, 4-D-glucanase, endo-β-1, 4-D-glucanase and β-Dglucosidase. Each enzyme alone cannot hydrolyze the complex crystalline cellulose efficiently but working synergistically with other types of cellulases can increase the rate of

 Exo-β-1, 4-D-glucanase can access individual cellulose chains from the exposed reducing end or non-reducing end and cleave two to four glucose units at a time to

 Endo-β-1, 4-D-glucanase breaks internal glycosidic bonds of individual cellulose chains to disrupt the network structure of cellulose and expose individual polysaccharide

produce tetrasaccharides or disaccharides (i.e., cellobioses) (Figure 4).

β-D-Glucosidase or cellobiase hydrolyzes cellobiose to release D-glucose units.

**Figure 4.** Schematic illustration of cellulose hydrolysis by exo-β-1, 4-D-glucanase and endo-β-1, 4-D-

Most of fungal cellulases have a domain like structure that contains a catalytic domain (CD) and a carbohydrate binding module (CBM) (Martin, 2000). These two domains are

mechanistic understanding of cellulose hydrolysis by cellulase.

**1.3. Cellulase and enzymatic hydrolysis of cellulose**

hydrolysis significantly (Dwivedi et al., 2009; Lynd et al., 2002).

chains (Figure 4).

glucanase.

**Figure 2.** (A) Hydrophilic and hydrophobic sites of cellulose. (B) Schematic drawing of the intrasheet hydrogen-bonding network in cellulose Iα.

Various noncovalent interactions such as hydrogen bonding and van der Waals interactions are present in the ultrastructure of cellulose. While the OH-O hydrogen bonding is mostly responsible for cellulose intrasheet interactions, both the weaker CH-O hydrogen bonding and van der Waals interactions contribute to cellulose intersheet interactions (Li Q. & Renneckar, 2011). Figure 2 shows the arrangement of the intrasheet hydrogen bonding network in cellulose Iα and the resulting hydrophilic and hydrophobic sites of the ring plane (Brown & Saxena, 2007). Overall, because of these noncovalent interactions, cellulose chains aggregate into various forms of ultrastructure, which do not melt or dissolve in any common solvents. Such aggregation prevents the potential cleavage sites (i.e., glycosidic bonds) of a cellulose chain from being accessed by cellulase.

#### **1.2. Degradation of cellulose in biomass conversion**

The biomass conversion is the key step to produce biofuel from cellulosic biomass. Such conversion is often accomplished either through biochemical methods or thermochemical methods, where the polysaccharides in cellulosic biomass are hydrolyzed by biochemical agents such as cellulase enzyme, or by thermal treatment such as gasification to produce simple sugars that are fermentable to produce biofuel products (Dwivedi et al., 2009). For biochemical methods (Gray et al., 2006), cellulases are usually employed to convert the solid cellulosic biomass into glucose or small sugar polymers that can be readily fermented with microorganism to produce ethanol. Compared to thermochemical methods which often require a large amount of acid and energy, biochemical methods are more environmentally friendly and economically feasible because of their better conversion efficiencies and milder operating conditions. By far the enzyme-based biochemical methods are considered as the most promising technologies for biomass conversion. However, because of biomass recalcitrance and high cost of cellulase in biomass conversion, the current process for biofuel production is not yet a viable option for the large-scale production (Dwivedi et al., 2009). Much research and developmental efforts have been dedicated to the improvement of the efficiency of cellulase in biomass conversion. One feasible approach is through the incorporation of new features (mutations) into cellulase that accelerate key steps (e.g., rate limiting step) of the enzymatic process. This approach requires a comprehensive mechanistic understanding of cellulose hydrolysis by cellulase.

**Figure 3.** The process of biomass conversion through the enzyme-based biochemical method.

## **1.3. Cellulase and enzymatic hydrolysis of cellulose**

126 Cellulose – Medical, Pharmaceutical and Electronic Applications

hydrogen-bonding network in cellulose Iα.

bonds) of a cellulose chain from being accessed by cellulase.

**1.2. Degradation of cellulose in biomass conversion**

**Figure 2.** (A) Hydrophilic and hydrophobic sites of cellulose. (B) Schematic drawing of the intrasheet

Various noncovalent interactions such as hydrogen bonding and van der Waals interactions are present in the ultrastructure of cellulose. While the OH-O hydrogen bonding is mostly responsible for cellulose intrasheet interactions, both the weaker CH-O hydrogen bonding and van der Waals interactions contribute to cellulose intersheet interactions (Li Q. & Renneckar, 2011). Figure 2 shows the arrangement of the intrasheet hydrogen bonding network in cellulose Iα and the resulting hydrophilic and hydrophobic sites of the ring plane (Brown & Saxena, 2007). Overall, because of these noncovalent interactions, cellulose chains aggregate into various forms of ultrastructure, which do not melt or dissolve in any common solvents. Such aggregation prevents the potential cleavage sites (i.e., glycosidic

The biomass conversion is the key step to produce biofuel from cellulosic biomass. Such conversion is often accomplished either through biochemical methods or thermochemical methods, where the polysaccharides in cellulosic biomass are hydrolyzed by biochemical agents such as cellulase enzyme, or by thermal treatment such as gasification to produce simple sugars that are fermentable to produce biofuel products (Dwivedi et al., 2009). For biochemical methods (Gray et al., 2006), cellulases are usually employed to convert the solid cellulosic biomass into glucose or small sugar polymers that can be readily fermented with microorganism to produce ethanol. Compared to thermochemical methods which often require a large amount of acid and energy, biochemical methods are more environmentally friendly and economically feasible because of their better conversion efficiencies and milder operating conditions. By far the enzyme-based biochemical methods are considered as the most promising technologies for biomass conversion. However, because of biomass Cellulase (Lynd et al., 2002; Mosier et al., 1999; Wilson & Irwin, 1999), like all glycosyl hydrolase enzymes found in bacteria, fungi, plants and some invertebrate animals, breaks down β-1, 4-glycosidic bonds of cellulose through general acid/base catalysis. There are mainly three kinds of cellulases: exo-β-1, 4-D-glucanase, endo-β-1, 4-D-glucanase and β-Dglucosidase. Each enzyme alone cannot hydrolyze the complex crystalline cellulose efficiently but working synergistically with other types of cellulases can increase the rate of hydrolysis significantly (Dwivedi et al., 2009; Lynd et al., 2002).

**Figure 4.** Schematic illustration of cellulose hydrolysis by exo-β-1, 4-D-glucanase and endo-β-1, 4-Dglucanase.

Most of fungal cellulases have a domain like structure that contains a catalytic domain (CD) and a carbohydrate binding module (CBM) (Martin, 2000). These two domains are

connected by a peptide linker, which is known to maintain the separation between the CD and the CBM (Figure 5). The CD contains the enzyme active site that is responsible for cellulose hydrolysis. The CBM is a contiguous amino acid sequence that anchors the CD onto the surface of cellulose through hydrogen bonding and van der Waals interactions (Boraston et al., 2004; Guillén et al., 2010). Cellulases can be grouped into families according to sequence similarities of their amino acid residues within their CDs and CBMs.

Probing the Interaction Between Cellulose and Cellulase with a Nanomechanical Sensor 129

equilibrium binding models and dynamic binding models have also been proposed to account for the complexities of the binding process including the partially irreversible adsorption of

For cellulose hydrolysis, most recent studies have been focused on elucidation of the mechanism of the hydrolytic cleavage reaction (step 3 in Figure 6) (Divne et al., 1998; Li Y. et al., 2007; Parsiegla et al., 2008). Some of the key amino acid residues involved in cleavage reactions have been identified. The distinctions between endo- and exo-glucanases, and between retention and inversion for the stereo configuration of the products have been made after years of biochemical and biophysical studies. The majority of studies on the hydrolytic cleavage step were done by measuring the concentration of the sugars (P)

Meanwhile, very little success has been achieved in obtaining fundamental knowledge of the enzymatic decrystallization reaction in step 2 (DOE/SC-0095, 2006). In particular, the importance of enzymatic decrystallization was largely unnoticed until very recently (Chundawat et al., 2011; DOE/SC-0095, 2006; Wilson, 2009). Several pieces of biochemical and physical evidence have indicated the presence of such an activity. Back to 1997, Wilson and his coworker showed that cleavage of the β-1, 4-glycosidic linkage in crystalline cellulose is not the rate-limiting step for *T. fusca* endoglucanase E2 (Zhang S. & Wilson, 1997). They speculated that the binding of a cellulose chain from a microfibril into the active site of a cellulase is the rate-limiting step for degradation of crystalline cellulose (Wilson, 2009). Lee and coworkers found indentations and paths on the surface of cotton fibers that had been treated with a cellulase that was incapable of hydrolytic cleavage (Lee et al., 2000). The evidence suggests that such surface modifications on cellulose are likely caused by the

**Figure 7.** Mechanistic models of enzymatic decrystallization. (A) The CBM model: the CBM serves as a wedge to assist the release of a single cellulose chain. (B) The CD model: the protrusion of the CD

The mechanistic model for enzymatic decrystallization proposed by Reilly and coworkers is shown in Figure 7A (Mulakala & Reilly, 2005). In this model, the CBM is inserted under a cellulose chain like a wedge to separate the chain from the cellulose network. Then the released cellulose chain is pulled into the active site of the CD along the top face of the CBM to achieve the decrystallization. In this model, the CBM is essential to enzymatic decrystallization of cellulose. Numerous biochemical studies, however, have shown that in the absence of the CBM, the CD domain alone still retains 20~50% of the hydrolytic activity on crystalline cellulose (Reinikainen et al., 1992; Srisodsuk et al., 1993; Van Tilbeurgh et al.,

domain serves as a wedge to assist the release of a single cellulose chain.

cellulase, multiple types of adsorption site, and so on (Lynd et al., 2002).

released during cleavage of soluble cellodextrins or insoluble cellulose.

decrystallization activity of cellulase.

**Figure 5.** The domain like structure of the cellulase cellobiohydrolase I (CBH I) that is bound to cellulose Iβ microfibril. (Zhong et al., 2008)

Cellulose hydrolysis by cellulase is a multi-step process (Chundawat et al., 2011) that is initiated with the binding of cellulase (E) onto the surface of cellulose (S), shown in Figure 6. After this "initial binding" step, a single cellulose chain will be separated from the cellulose aggregate by cellulase and pulled into the active site of cellulase. This is the "decrystallization" step which forms a pseudo-Michaelis complex (E\*S). E\*S will then undergo "hydrolytic cleavage" to produce cellobiose as the product (P).

**Figure 6.** Mechanism of cellulose hydrolysis by cellulase.

For cellulose hydrolysis, the substrate cellulose is water insoluble and resistant to the attack by biological agents. This makes the formation of E\*S much more difficult compared to those formed with soluble substrates.

#### **1.4. Knowledge gap**

The initial binding (step 1 in Figure 6) has been extensively studied over the years. A Langmuir equation is widely used as a simplified mechanistic model to describe the formation of ES resulting from the initial interaction between the cellulase and the cellulose, which often reaches steady-state within half an hour (Lynd et al., 2002; Zhang Y. H. & Lynd, 2004). Other equilibrium binding models and dynamic binding models have also been proposed to account for the complexities of the binding process including the partially irreversible adsorption of cellulase, multiple types of adsorption site, and so on (Lynd et al., 2002).

128 Cellulose – Medical, Pharmaceutical and Electronic Applications

cellulose Iβ microfibril. (Zhong et al., 2008)

connected by a peptide linker, which is known to maintain the separation between the CD and the CBM (Figure 5). The CD contains the enzyme active site that is responsible for cellulose hydrolysis. The CBM is a contiguous amino acid sequence that anchors the CD onto the surface of cellulose through hydrogen bonding and van der Waals interactions (Boraston et al., 2004; Guillén et al., 2010). Cellulases can be grouped into families according

to sequence similarities of their amino acid residues within their CDs and CBMs.

**Figure 5.** The domain like structure of the cellulase cellobiohydrolase I (CBH I) that is bound to

undergo "hydrolytic cleavage" to produce cellobiose as the product (P).

**Figure 6.** Mechanism of cellulose hydrolysis by cellulase.

those formed with soluble substrates.

**1.4. Knowledge gap**

Cellulose hydrolysis by cellulase is a multi-step process (Chundawat et al., 2011) that is initiated with the binding of cellulase (E) onto the surface of cellulose (S), shown in Figure 6. After this "initial binding" step, a single cellulose chain will be separated from the cellulose aggregate by cellulase and pulled into the active site of cellulase. This is the "decrystallization" step which forms a pseudo-Michaelis complex (E\*S). E\*S will then

For cellulose hydrolysis, the substrate cellulose is water insoluble and resistant to the attack by biological agents. This makes the formation of E\*S much more difficult compared to

The initial binding (step 1 in Figure 6) has been extensively studied over the years. A Langmuir equation is widely used as a simplified mechanistic model to describe the formation of ES resulting from the initial interaction between the cellulase and the cellulose, which often reaches steady-state within half an hour (Lynd et al., 2002; Zhang Y. H. & Lynd, 2004). Other For cellulose hydrolysis, most recent studies have been focused on elucidation of the mechanism of the hydrolytic cleavage reaction (step 3 in Figure 6) (Divne et al., 1998; Li Y. et al., 2007; Parsiegla et al., 2008). Some of the key amino acid residues involved in cleavage reactions have been identified. The distinctions between endo- and exo-glucanases, and between retention and inversion for the stereo configuration of the products have been made after years of biochemical and biophysical studies. The majority of studies on the hydrolytic cleavage step were done by measuring the concentration of the sugars (P) released during cleavage of soluble cellodextrins or insoluble cellulose.

Meanwhile, very little success has been achieved in obtaining fundamental knowledge of the enzymatic decrystallization reaction in step 2 (DOE/SC-0095, 2006). In particular, the importance of enzymatic decrystallization was largely unnoticed until very recently (Chundawat et al., 2011; DOE/SC-0095, 2006; Wilson, 2009). Several pieces of biochemical and physical evidence have indicated the presence of such an activity. Back to 1997, Wilson and his coworker showed that cleavage of the β-1, 4-glycosidic linkage in crystalline cellulose is not the rate-limiting step for *T. fusca* endoglucanase E2 (Zhang S. & Wilson, 1997). They speculated that the binding of a cellulose chain from a microfibril into the active site of a cellulase is the rate-limiting step for degradation of crystalline cellulose (Wilson, 2009). Lee and coworkers found indentations and paths on the surface of cotton fibers that had been treated with a cellulase that was incapable of hydrolytic cleavage (Lee et al., 2000). The evidence suggests that such surface modifications on cellulose are likely caused by the decrystallization activity of cellulase.

**Figure 7.** Mechanistic models of enzymatic decrystallization. (A) The CBM model: the CBM serves as a wedge to assist the release of a single cellulose chain. (B) The CD model: the protrusion of the CD domain serves as a wedge to assist the release of a single cellulose chain.

The mechanistic model for enzymatic decrystallization proposed by Reilly and coworkers is shown in Figure 7A (Mulakala & Reilly, 2005). In this model, the CBM is inserted under a cellulose chain like a wedge to separate the chain from the cellulose network. Then the released cellulose chain is pulled into the active site of the CD along the top face of the CBM to achieve the decrystallization. In this model, the CBM is essential to enzymatic decrystallization of cellulose. Numerous biochemical studies, however, have shown that in the absence of the CBM, the CD domain alone still retains 20~50% of the hydrolytic activity on crystalline cellulose (Reinikainen et al., 1992; Srisodsuk et al., 1993; Van Tilbeurgh et al., 1986). To resolve this discrepancy, we propose an alternative mechanistic model, where a wedge-like structure at the bottom of the CD can be inserted under the cellulose chain to lift it into the active site of the CD (Figure 7B). Since decrystallization by cellulase has been speculated to be the rate limiting step for the degradation of crystalline cellulose (DOE/SC-0095, 2006; Wilson, 2009), understanding the mechanism of this enzymatic activity becomes essential to a comprehensive understanding of cellulose hydrolysis by cellulase.

Probing the Interaction Between Cellulose and Cellulase with a Nanomechanical Sensor 131

The microcantilever is a highly sensitive nanomechanical sensor that originates from a microfabricated AFM probe. Typical dimensions of a microcantilever, which are similar to AFM probes, are 200 μm long, 1 μm thick, and 20 μm wide (Goeders et al., 2008; Lavrik et al., 2004). The microcantilever is capable of detecting minute changes in interaction energy between individual molecules in the thin film of a polymer coating (cellulose, protein, DNA, or polymer brush) in the form of a measurable bending (10-6 to 10-12 m) of the microcantilever (Moulin et al., 1999; Mukhopadhyay et al., 2005; Shu et al., 2005; Yan et al., 2006; Zhao et al., 2010; Zhou et al., 2006). The microcantilever bending can be measured based on the deflection of a laser beam reflecting from the tip of the microcantilever in the AFM (Figure 9). Overall, the

Dynamic mode (Vashist, 2007): When an analyte is adsorbed on an oscillating microcantilever, the microcantilever will oscillate at a lower frequency. The difference in frequency can be used

Static mode (Vashist, 2007): Adsorbates tend to induce a significant change in surface stress of a microcantilever, which results in a deflection of the microcantilever (Lavrik et al., 2004; Shuttleworth, 1950). The surface stress and the deflection of the microcantilever are

> *Et*

Where Δz is the deflection of the microcantilever at the end of the microcantilever, *ν* and *E* are Poisson's ratio and Young's modulus of the microcantilever, *t* and *l* are the thickness and length of the microcantilever, and ∆*σ* is the differential stress on the microcantilever. Using the static mode microcantilever sensor, Ji and coworkers investigated the conformational change of calmodulin (Yan et al., 2006). Sauers and coworkers successfully detected 2 mercaptoethanol using a gold-coated microcantilever (Datskos & Sauers, 1999). Zhou and coworkers successfully demonstrated the use of the microcantilever bending as a means to probe changes in internal structure of polymer brushes in response to changes in pH and

2 2 3(1 )*l*

(1)

to measure properties of adsorbates, such as viscosity and density, etc.

*z*

microcantilever detection has high specificity, high sensitivity, and quick response.

**2.2. Microcantilever and its applications**

**Figure 9.** Working scheme of the microcantilever.

quantitatively related (Yan et al., 2006):

electrolyte concentration (Zhou et al., 2006).

## **2. Innovative approach and microcantilever**

#### **2.1. Existing technologies and their technical limitations**

The conventional approaches to study the cellulose hydrolysis by cellulase are based on the measurement of the concentration of glucose or other simple sugars that are produced in the hydrolytic process. These approaches are not suitable for studying the decrystallization process because no new product is formed and released from this process.  All these techniques focus on the global variations of cellulose. Since cellulose decrystallization only occurs on the outer layer within a relative small region of the surface of cellulose, these techniques are not sensitive enough for such study. Quartz crystal microbalance has been used to study the enzymatic hydrolysis of cellulose (Ahola et al., 2008; Rojas Orlando et al., 2007; Turon et al., 2008), however, its suitability for studying decrystallization has not yet been demonstrated.

**Figure 8.** Various shapes of AFM probes.

Atomic force microscopy (AFM) has been used to examine changes of surface morphology of cellulose. Typical size of AFM probe is 200 μm x 40 μm x 1 μm (length x width x thickness) with various shapes (Figure 8). Lee and coworkers used the tapping mode AFM to exam the effects of three different cellulases on the surface of cotton fibers (Lee et al., 2000). Li and coworkers used the AFM to detect structural changes of cellulose microfibril fragments caused by sonication (Li Q. & Renneckar, 2011). With this powerful surface imaging tool, we can investigate the surface change caused by enzymatic activities. However, AFM imaging is mostly limited to the surface analysis at discrete time points. Additionally a high quality image requires that the surface area of cellulose be prepared prior to imaging. Any extensive handling may potentially alter the surface properties and delay the timely analysis under the assay conditions*.* Therefore, AFM imaging is not an ideal approach for analyzing the dynamic impact of cellulase on cellulose. Both scanning electron microscopy and transmission electron microscopy have similar limitations. This has led to the development of the microcantilever technique for cellulase study.

#### **2.2. Microcantilever and its applications**

130 Cellulose – Medical, Pharmaceutical and Electronic Applications

**2. Innovative approach and microcantilever**

decrystallization has not yet been demonstrated.

**Figure 8.** Various shapes of AFM probes.

**2.1. Existing technologies and their technical limitations**

1986). To resolve this discrepancy, we propose an alternative mechanistic model, where a wedge-like structure at the bottom of the CD can be inserted under the cellulose chain to lift it into the active site of the CD (Figure 7B). Since decrystallization by cellulase has been speculated to be the rate limiting step for the degradation of crystalline cellulose (DOE/SC-0095, 2006; Wilson, 2009), understanding the mechanism of this enzymatic activity becomes

The conventional approaches to study the cellulose hydrolysis by cellulase are based on the measurement of the concentration of glucose or other simple sugars that are produced in the hydrolytic process. These approaches are not suitable for studying the decrystallization process because no new product is formed and released from this process.  All these techniques focus on the global variations of cellulose. Since cellulose decrystallization only occurs on the outer layer within a relative small region of the surface of cellulose, these techniques are not sensitive enough for such study. Quartz crystal microbalance has been used to study the enzymatic hydrolysis of cellulose (Ahola et al., 2008; Rojas Orlando et al., 2007; Turon et al., 2008), however, its suitability for studying

Atomic force microscopy (AFM) has been used to examine changes of surface morphology of cellulose. Typical size of AFM probe is 200 μm x 40 μm x 1 μm (length x width x thickness) with various shapes (Figure 8). Lee and coworkers used the tapping mode AFM to exam the effects of three different cellulases on the surface of cotton fibers (Lee et al., 2000). Li and coworkers used the AFM to detect structural changes of cellulose microfibril fragments caused by sonication (Li Q. & Renneckar, 2011). With this powerful surface imaging tool, we can investigate the surface change caused by enzymatic activities. However, AFM imaging is mostly limited to the surface analysis at discrete time points. Additionally a high quality image requires that the surface area of cellulose be prepared prior to imaging. Any extensive handling may potentially alter the surface properties and delay the timely analysis under the assay conditions*.* Therefore, AFM imaging is not an ideal approach for analyzing the dynamic impact of cellulase on cellulose. Both scanning electron microscopy and transmission electron microscopy have similar limitations. This has

led to the development of the microcantilever technique for cellulase study.

essential to a comprehensive understanding of cellulose hydrolysis by cellulase.

The microcantilever is a highly sensitive nanomechanical sensor that originates from a microfabricated AFM probe. Typical dimensions of a microcantilever, which are similar to AFM probes, are 200 μm long, 1 μm thick, and 20 μm wide (Goeders et al., 2008; Lavrik et al., 2004). The microcantilever is capable of detecting minute changes in interaction energy between individual molecules in the thin film of a polymer coating (cellulose, protein, DNA, or polymer brush) in the form of a measurable bending (10-6 to 10-12 m) of the microcantilever (Moulin et al., 1999; Mukhopadhyay et al., 2005; Shu et al., 2005; Yan et al., 2006; Zhao et al., 2010; Zhou et al., 2006). The microcantilever bending can be measured based on the deflection of a laser beam reflecting from the tip of the microcantilever in the AFM (Figure 9). Overall, the microcantilever detection has high specificity, high sensitivity, and quick response.

**Figure 9.** Working scheme of the microcantilever.

Dynamic mode (Vashist, 2007): When an analyte is adsorbed on an oscillating microcantilever, the microcantilever will oscillate at a lower frequency. The difference in frequency can be used to measure properties of adsorbates, such as viscosity and density, etc.

Static mode (Vashist, 2007): Adsorbates tend to induce a significant change in surface stress of a microcantilever, which results in a deflection of the microcantilever (Lavrik et al., 2004; Shuttleworth, 1950). The surface stress and the deflection of the microcantilever are quantitatively related (Yan et al., 2006):

$$
\Delta z = \frac{\Im(1-\nu)l^2}{Et^2} \Delta \sigma \tag{1}
$$

Where Δz is the deflection of the microcantilever at the end of the microcantilever, *ν* and *E* are Poisson's ratio and Young's modulus of the microcantilever, *t* and *l* are the thickness and length of the microcantilever, and ∆*σ* is the differential stress on the microcantilever. Using the static mode microcantilever sensor, Ji and coworkers investigated the conformational change of calmodulin (Yan et al., 2006). Sauers and coworkers successfully detected 2 mercaptoethanol using a gold-coated microcantilever (Datskos & Sauers, 1999). Zhou and coworkers successfully demonstrated the use of the microcantilever bending as a means to probe changes in internal structure of polymer brushes in response to changes in pH and electrolyte concentration (Zhou et al., 2006).


*<IP_ADDRESS> Tools and methods used*

The private operators companies even though they have limited areas of coverage, these enterprises have been found to have been very effective in their operations. The private operators and the government agencies such as KNUPDA, REMASAB, and Ministry of Environment and of Cape Gate were all found to be involved in public education/awareness campaigns and enlightenment on the need for recovery as well as the importance of recycling recovered waste. They inform and enlighten the general public as well as individuals, families, and or community solid waste stakeholders/groups to improve their environmental health and support the projects of the state government in the area of clean environment and the monthly payment of the agreed fees to the private company. Whereas the benefits of these public education and enlightenment campaign have been recent advances fully highlighted as shown below:-

The benefits of public knowledge are manifold. To effectively reap the benefits, sanitation authorities should develop strategies and procedures for putting them into action. One of these is identifying particular target audiences and planning the methods and media to reach them. Second, should environmental education be incorporated into the curriculum or should it only be used in extracurricular activities like theater and debate? How do we acquire the tools and funds necessary to support the campaigns? Will business stakeholders and charitable people be willing to sponsor the campaigns, or at the very least some radio and television jingles? The benefits of public enlightenment are so many. However, to effectively reap them, sanitation authorities should identify strategies and work out means of achieving them. One of such is the identification of specific target groups and preparing the means and medium of reaching them. Secondly, can we include environmental education in school programs or limit it to extracurricular activities such as drama and debating? How do we find material and financial resources to fund the campaigns? Will corporate stakeholders and philanthropic individuals be willing to sponsor the campaigns or at least some jingles on radio and television? Or limit it to extracurricular activities

such as drama and debating? How do we find material and financial resources to fund the campaigns? Will corporate and philanthropic individuals be willing to sponsor the campaigns or even if it is some jingles on radio and television? [8]. Most especially in the newly introduced on-site recovery and recycling of the recyclables from the waste stream, which has been a challenge for sometimes in the study area until of recent. Therefore, if the solid waste management hierarchy tool concept is fully utilized, the rest is simple; this is because waste will have been reduced by almost 35–40% [9, 10]. A descriptive cross-sectional study was conducted from June to August 2021 in two of the local governments in Nigeria. Kano, Nassarawa, and Municipal local governments are among the 774 local government areas in Nigeria and these two Local Government Areas are within the Kano urban Area in Kano State, Nigeria. Its headquarters are in Kofar Kudu and Giginyu, in the south part of the city of Kano. It has an area of 17 km2 and 18.8 km2 and a population of 465,525 and 464,321, respectively, as at the 2011 population census. The main environmental sanitation scope given attention in Kano Municipal and Nassarawa local government areas is waste collection and disposal. Kano Municipal and Nassarawa, as urban centers, are known for their overpopulation and commercial activities. Kano Municipal and Nassarawa like many other cities in Nigeria are faced with perennial ecological problems favorable to the survival of parasites causing diseases. The major sanitation facilities include: REMASAB bins, private waste management companies, and other SWM stakeholders engaged with evacuating tanks, personal bins, basket, unauthorized dump site plot, and occasionally, fumigation of the markets. The study contained three domains mainly; stakeholders and vital information, mode of refuse disposal, and refuse disposal consistency. Waste that was observed in the study area is mostly from residential areas. Mode of refuse disposal is focused on collecting information on methods of refuse disposal, types of building residents live, and material use for disposal. While refuse are collected and disposed of at a certain fees collected every month by the private companies. Study questionnaires were also used to collect data from the study area. The data sources were measured using a structure questionnaire. Primarily, a structured interview questionnaire, which was made valid and reliable by a team of other solid waste management experts, was used for data collection. The final questionnaire had 16 questions covering information about domains; information and vital information, mode of refuse disposal, and refuse disposal consistency.

### *<IP_ADDRESS> How the practice is being implemented*

The customers who are the citizens are expected to always clean their houses and its surroundings and their establishments and then put the solid wastes in the supplied drums form the private companies. Items to recover from the waste are the usual recyclables of metal and aluminum. Glass, paper, (non-degradable) and the kitchen / food waste which constitute the highest in the waste stream (degradable) [11]. They are to also to ensure that payment for services rendered is regularly made and on time. The waste collection and disposal companies on their part are to regularly convey the solid wastes from their customers' residences and business premises for proper on-site recovery and the recycling process and finally to dumping sites. They are also to enlighten the general public on keeping a clean environment. As for the cost per household, for the service rendered, the waste disposal companies' charges are dependent on the distance from the houses to the disposal sites. At the moment it is a flat rate of N2, 000 per month based on their limited area of coverage for example the Clean Town Company initially started operating using 200 liters drum,


Overarching Goal and Intervention for Healthy Aging in Older People during and after the COVID-19 Pandemic: Impact of Rehabilitation

*Koki Kawamura, Shinichiro Maeshima, Aiko Osawa and Hidenori Arai*


**Author details**

Czerwik-Marcinkowska Joanna\* and Massalski Andrzej

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Botany, Institute of Biology, Jan Kochanowski University, Kielce, Poland


**Preface to "Mesh Methods—Numerical Analysis and Experiments"**

Mathematical models of various natural processes are described by differential equations, systems of partial differential equations and integral equations. In most cases, the exact solution to such problems cannot be determined; therefore, one has to use grid methods to calculate an approximate solution using high-performance computing systems. These methods include the finite element method, the finite difference method, the finite volume method and combined methods. In this Special Issue, we bring to your attention works on theoretical studies of grid methods for approximation, stability and convergence, as well as the results of numerical experiments confirming the effectiveness of the developed methods. Of particular interest are new methods for solving boundary value problems with singularities, the complex geometry of the domain boundary and nonlinear equations. A part of the articles is devoted to the analysis of numerical methods developed for calculating mathematical models in various fields of applied science and engineering applications. As a rule, the ideas of symmetry are present in the design schemes and make the process harmonious and efficient.

> **Viktor A. Rukavishnikov, Pedro M. Lima, Ildar B. Badriev** *Editors*

*Article*