*3.1. Chelators for Copper*

The coordination chemistry of the transition metal copper in an aqueous solution is limited to three accessible oxidation states (I–III). The lowest oxidation state, Cu(I) allows formation of complexes with various ligands, which are however labile and lack sufficient stability for radiopharmaceutical applications. Cu(II) is less labile toward ligand exchange and is optimal for incorporation into radiopharmaceuticals. The Cu(III) oxidation state is relatively rare and difficult to attain and thus not of importance for radiopharmaceuticals [71,72]. To evaluate 67Cu for radioimmunotherapy (RIT), the macrocyclic chelating agent 1,4,7,11-tetraazacyclotetradecane-*N*,*N*0 ,*N*00 ,*N*000-tetraacetic acid (TETA) was designed specifically to bind copper rapidly and selectively for conjugation to MoAbs. The synthesis of this "bifunctional" metal chelator, 6(*p*-bromoacetamidobenzyl)-1,4,8,11 tetraazacyclotetradecane-*N*,*N*0 ,*N*00 ,*N*000-tetraacetic acid, which can be covalently attached to proteins and which binds copper stably in human serum under physiological conditions, was reported by Moi et al., in 1985 [73]. In contrast, other chelators based on ethylenediaminetetraacetic acid or diethylenetriaminepentaacetic acid rapidly lose copper to serum albumin under the same conditions.

Despite their use in several pre-clinical studies, the stability of Cu(II) metal complexes with common macrocyclic ligands, such as DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10 tetraacetic acid) and TETA was evaluated and considered not satisfactory for biomedical applications, demonstrating that nearly 70% of the 64Cu trans-chelated in the liver 20 hours post injection [74–76].

The addition of a bond (or Cross-Bridge, CB) between two opposite nitrogen atoms in the tetraazamacrocyclic skeleton of the macrocycle gives greater stability. The Cu(II) complex with the ligand CB-TE2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo [6.6.2] hexadecane) has been demonstrated to be particularly stable in vivo and to have exceptional inertness kinetics in aqueous solution thanks to the stabilizing effects given by the CBmacrocycle and by the two hanging carboxymethyl arms [77–80]. Unfortunately, the extreme labeling conditions at 95 ◦C do not make it a good candidate for the development of radiopharmaceuticals labile at high temperatures, e.g., antibodies [81]. The replacement

of one or both of the hanging carboxymethyl arms with groups derived from phosphonic acid (–CH2-PO3H2), CB-TE1A1P (4,8,11-tetraazacyclotetradecane-1-(methanophosphonic acid)-8-(methanocarboxylic acid)), and CB-TE2P (1,4,8,11-tetraazacyclotetradecano-1,8 bis(methanephosphonic acid)), has been shown to confer greater thermodynamic and kinetic stability, as well as greater selectivity and faster complexation kinetics [82].

Another class of potential sarcophagus-like cage structure chelators for copper are hexaazamacrobicyclic ligands [83,84]. Labeling conditions of copper complexes with sarcophagine-like ligands are particularly favorable in a wide pH range (4–9) with yields of 100% in a few minutes at room temperature [75]. The complexes obtained possess excellent in vitro and in vivo stability [85–87].

Some N2S2-type acyclic chelators have also been studied, such as PTSM (pyruvaldehydebis(N4-methylthiosemicarbazone)) and ATSM (diacetyl-bis(N4-methylthiosemicarbazone)). This class of chelators, bis (thiosemicarbazones), act as tetradentate ligands and coordinate Cu2+ forming stable and neutral complexes, with a square planar geometry, which show a high permeability of the cell membrane. The preparation of these complexes with radioactive copper isotopes can be carried out under mild conditions in high yield [74,87–89].


**3.1. System configuration-**

The system of the present study is developed by means of Web-GIS, an SNS, and a registration system of walking information, as shown in **Figure 1**. The system enables to visualize the information related to outlines, height, and sightseeing spot on the digital maps of the Web-GIS.-Therefore, it is possible for users to efficiently obtain the necessary information related to walking. Since the system is connected with external SNS (Twitter), it is possible for users to share various information related to walking, and displaying the tweets with a specific hashtag submitted by others. Additionally, it is also possible for users to register their walking information such as the number and time of their steps. Users can freely confirm their registered information, which is maintained in the database of the system.

**Figure 1.** System design.

The usability of the system mentioned in the previous section can be summarized in the following three points, by means of the design described in detail as shown in **Figure 1**.


**Abstract**

Glycans, as potential prognostic biomarkers, deserve attention in clinical glycomics for diseases diagnosis. The variety of glycan chains, attached to proteins and lipids, makes it possible to form unique glycoconjugates with a wide range of cellular functions. Under leukocyte-endothelial interaction, not only the availability of glycoconjugates with sialic acids at the terminal position of glycans are informative, but also the type of glycosidic bond by which sialic acids links to subterminal carbohydrates in structure of glycans. The process of sialylation of leukocyte glycoconjugates undergoes considerable changes in type 1 diabetes mellitus. At early stage of disease without diabetic complications, the pathology is accompanied by the increase of α2,6-linked sialic acids. The quantity of sialic acid-containing glycoconjugates on leukocytes surface increases in condition of disease duration up to five years. However, the quantity of sialic acids linked by α2,6-glycosidic bonds decreases in patients with the disease duration over ten years. Therefore, sialoglycans as marker molecules determine the leukocyte function in patients with type 1 diabetes mellitus, depending on the disease duration. Changes in the glycans structure of membrane glycoconjugates of leukocytes allow understanding the mechanism of diabetic complications development.

**Keywords:** sialic acid, glycans, glycoconjugates, sialyltransferases, sialidases, leukocytes, type 1 diabetes mellitus


*2.1. Study Area and Neighborhoods*

Five megacities (Wuhan, Hefei, Nanjing, Shanghai, and Hangzhou) in the middle and lower reaches of the Yangtze River were selected as the study area (Figure 1); they have the same climate conditions. Due to the extreme high-density construction, these cities experience more serious PM2.5 pollution than other cities in this region [31]. The similar

features of the five cities provide sufficient comparability for this study, including urban form, landform, population, and PM2.5 pollution characteristics.

**Figure 1.** Location of five megacities and distribution of monitoring stations [32].

On average, each city has 10 national PM2.5 monitoring stations that present a relatively uniform distribution throughout the constructed areas. A neighborhood was then defined as a square domain with an area of 1 km2 (1 km × 1 km) centered at a monitoring station because 1 km is a common size for a *neighborhood* division in China and was frequently used in previous studies [32,33]. In addition, the air environment within this size plays an important role in people's quality of daily life [34]. In consideration of the unique locations of 13 neighborhoods and subsequent special land use, such as close to pollution sources (construction sites) and natural land (urban parks or scenic regions), 37 neighborhoods were selected for analysis in this study. Detailed information on the 37 cities is shown in Table S1.