Corporate Governance Ideology, Human Resource Practices and Senior Staff Salaries

*Nicholas Black and Peter Stokes*

### **Abstract**

This chapter examines the link between corporate governance ideology and HR (human resource) practices involved in the important and ongoing issue of senior staff salaries. In the spirit of financialization and hyper-individuals, the mainstream corporate governance ideology promotes beliefs about competitive pay and managerial power. These beliefs shape the design and implementation of HR practices by legitimizing the 'common-sense' assumption that senior staff members should, primarily, be rewarded for meeting corporate goals. However, our discussion critiques the use of this corporate governance ideology for encouraging myopia and silence amongst remuneration committee members in response to growing inequality. This is exemplified by an inductive analysis of remuneration committee minutes taken from British universities (n = 67). Interestingly, this example also highlighted a marginalized belief about sacrificial leadership that countered this growth under alternative ideology in the spirit of altruism. The chapter recommends the radical proposal that remuneration committees should expand their remit beyond only considering senior staff salaries and promote HR practices that will embed altruism and equality.

**Keywords:** HR practices, pay, universities, remuneration committees, corporate governance

### **1. Introduction**

Rising senior staff salaries is a contentious and ongoing human resources management (HRM) issue that received widespread prominence in the United Kingdom (UK) and United States of America (USA) after the 2008 financial crisis [1]. It is also likely to remerge in response to COVID-19 as many on the workforce may be prone to experiencing pay stagnation, zero-hour contracts, redundancies and work intensification in response to falling stock values and the economic recession. For organizations, being able to legitimize such inequality is particularly important as, according to the social contract tradition, socio-economic cooperation is dependent upon everyone believing they receive a fair stake of the economy [2]. However, legitimizing inequality is becoming increasingly harder as important institutions like the UNDP [3] and World Economic Forum [4] are campaigning against it.

Remuneration committees – also known as 'compensation committees' in the USA context – play a key function in determining senior staff salaries [5]. Past

studies focus on their important role in corporate governance, but the conventional understanding of how remuneration committees operate has historically focused primarily on agency theory and quantitative methods approaches [6]. In tandem, HR practices have typically embodied a shareholder-centric ideology rather than wider stewardship considerations [7, 8]. While these predilections may coincide with the spirit of financialization and hyper-individualism, there have recently been calls for HRM to return to its ethical roots – being born out of concern for human welfare – and promote more socially responsible and ethical HR practices [9]. This chapter aspires to respond to this need by exploring what HR practices remuneration committees are using to pay their senior staff and identifying the underpinning ideological norms. Equally, this chapter explores what alternative ideologies of corporate governance exist and theorizes how HR practices could be used to halt rises in senior salaries. Overall, this chapter seeks to answer the research question: How can HR practices be used within remuneration committees to promote equality?


**4. Conclusions**

50 Advances in Hematopoietic Stem Cell Research

Necdin is a member of the melanoma antigen family of molecules, whose physiological roles have not been well characterized (Xiao et al., 2004). Necdin acts as a cell cycle regulator in post-mitotic neurons (Yoshikawa, 2000). Intriguingly, recent genetic analyses show that aberrant genomic imprinting of *NDN* on the human 15q11-q13 chromosomal region is, at least in part, responsible for the pathogenesis of Prader-Willi syndrome (MacDonald & Wevrick, 1997; Nakada et al., 1998; Barker et al., 2002), a disorder associated with a mildly increased risk of myeloid leukemia (Davies et al., 2003). Necdin interacts with multiple cell-cycle related proteins, such as SV-40 large T antigen, adenovirus E1A, E2F1, and p53 (Taniura et al., 1998, 1999, 2005; Hu et al., 2003). As shown in Table 2, necdin is one of 32 genes that show higher expression in HSCs than in differentiated hematopoietic cells (Kubota et al., 2009). Other groups also found that necdin is highly expressed in HSCs (Forsberg et al., 2005; Liu et al., 2009). Necdindeficient mice show accelerated recovery of hematopoietic systems after myelosuppressive stress, such as 5-FU treatment and BM transplantation, whereas no overt abnormality is seen under conditions of steady-state hematopoiesis. Considering necdin as a potential negative cell-cycle regulator, it was reasoned that the enhanced hematologic recovery in necdin-null mice could be the result of an increased number of proliferating HSCs and progenitor cells. As expected, after 5-FU treatment, necdindeficient mice had an increased number of HSCs, but this was only transiently observed during the recovery phase (Kubota et al., 2009). These data suggest that the repression of necdin function in HSCs may present a novel strategy for accelerating hematopoietic recovery, thus providing therapeutic benefits after clinical myelosuppressive treatments

Slug belongs to the highly conserved Slug/Snail family of zinc-finger transcriptional repressors found in diverse species ranging from *C. elegans* to humans. SLUG is a target gene for the E2A-HLF chimeric oncoprotein in pro-B cell acute leukemia (Inukai et al., 1999). Slug-deficient mice show normal peripheral blood counts, but they are very sensitive to γirradiation (Inoue et al., 2002). Slug is induced by p53 and protects primitive hematopoietic cells from apoptosis triggered by DNA damage. Slug exerts this function by repressing Puma, a proapoptotic target of p53 (Wu et al., 2005). Sun et al. recently showed that Slug negatively regulates the repopulating ability of HSCs under conditions of stress. Slug deficiency increases HSC proliferation and reconstitution potential *in vivo* after myelosuppressive treatment, and accelerates HSC expansion during *in vitro* culture (Sun et

Accumulating evidence strongly suggests that tumors are organized into cellular hierarchies initiated and maintained by a small pool of self-renewing cancer stem cells (CSCs) (Dick, 2008; Reya et al., 2001). CSCs are thought to be resistant to various cancer treatments because of their relative quiescence (Komarova & Wodarz., 2007). Cancer relapses may occur because the dormancy of CSCs protects them from elimination by various cancer

(e.g., cytoablative chemotherapy or HSC transplantation).

**2.3.2 Necdin**

**2.3.3 Slug**

al., 2010).

**3. Cancer stem cells**

In this review, we have briefly summarized a number of critical regulators involved in the control of HSC self-renewal, quiescence, survival, and responses to external insults. Recent evidence strongly suggests that the BM niche also plays an integral role by providing critical signals that maintain HSCs in a stat of hibernation, thus preventing them from exhausting themselves. However, HSCs are critical for the maintenance and regeneration of an organism after injury/illness. This process must be tightly regulated and coordinated. Intensive studies have uncovered the molecular signatures and key molecules regulating HSC behavior. Moreover, new systems approaches, such as microRNA expression profiling and protein expression profiling, are expected to provide further useful information about HSC biology in the future. However, the overall picture of the molecular mechanisms that govern HSC fate is still unclear. Further understanding of the systems that regulate HSCs will enable the manipulation of stem cells for use in tissue engineering and cell-based therapies.

### **5. Acknowledgments**

This work was supported by a Grant-in-Aid for Young Scientists to Y.K. (no. 23791083) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

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**3**

Rasmus Freter

*United Kingdom*

**Transcriptional Quiescence of**

*Ludwig Institute for Cancer Research, University of Oxford*

Haematopoietic stem cells (HSC) have the exceptional capacity to undergo continuous selfrenewal and differentiation into multiple lineages, which is essential for haematopoietic homeostasis and response to injury. To achieve this life long function, these cells have to be protected from cytotoxic and genetic damage. On the other hand, rapid activation of haematopoietic stem cell proliferation in response to stimuli must be ensured. While cellular quiescence is thought to be the key mechanism underlying this paradoxical nature of HSC,

Quiescence is commonly defined as a reversible cell cycle exit. Induction and maintenance of stem cell quiescence has been studied at the level of cell cycle regulation (Orford & Scadden, 2008), cellular metabolism (Tothova & Gilliland, 2007) or interaction with the specific niche (Fuchs *et al.*, 2004). Genome-wide association studies have been performed on a variety of quiescent model systems, such as serum starvation of fibroblasts (Coller *et al.*, 2006), primary lymphocytes (Garriga *et al.*, 1998) or yeast in stationary phase (Patturajan *et al.*, 1998, Radonjic *et al.*, 2005). All of these studies revealed a significant decrease of productive mRNA transcription in these model systems. However, if quiescent adult stem

Due to their relative ease of isolation, cells of the haematopoietic lineage have been extensively studied. Importantly, several assays for hematopoietic stem cell function have been developed, such as colony forming ability and rescue of lethally irradiated mice. These functional tests are lacking in most other adult stem cell models, with the exception of spermatogonia and mammary gland stem cells (Brinster & Nagano, 1998, Shackleton *et al.*, 2006). Functional assays for HSC ability have provided us with the notion that most defined populations of long term repopulating HSC still contain progenitor cells, which can only transiently contribute to repopulation of the haematopoietic system. This heterogeneity is not only evident in defined cell populations, but also in the in vivo niche for HSC, the bone marrow. HSC in the bone marrow are interspersed with transient amplifying cells and differentiated cells, complicating stem cell identification by spatial organization of the tissue. Other stem cell systems, such as spermatogonia, keratinocyte or crypt stem cells have a clearly defined niche architecture, enabling stem cell identification by location only (Fuchs *et al.*, 2004). In this case, resting stem cells and activated progenitors can be separated by

the molecular basis of induction and maintenance of quiescence remains unresolved.

cells share this down regulation of mRNA transcription has never been examined.

**1. Introduction**

**Hematopoietic Stem Cells**

(2008) Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. *Cell* 135(6):1118-1129.


**7.1.4. Genetic classification of phosphate rocks**

The genetic classification proposed by EGOROV [50] includes three types of apatite ores according to the mineral assemblages [6],[51],[52],[53],[54],[55]:

**Fig. 8.** The location of upper mantle on the cross-section of the Earth, core and mantle drawn to scale [44] (a). Ternary equilibrium phase diagram of the apatite bearing ijolite-urtite rock (b) approximated by the system NaAlSiO4-CaMg‐ Si2O6-Ca5(PO4)3F [6]: olivine (Ol), melilite (Me), silicophosphate (Sph), apatite (Ap) and immiscibility field (L1+L2).

17 Ijolite, theralite and teschenite belong to phanerites (prelenic rocks characterized by equal or nearly equal amounts of femic components) [55].

18 Urtite is medium-grained and light gray rock composed of about 70% nephelite, 25% aegirite or aegirite-augite [55].

19 In the work [58], the name camaforite, i.e. Russian neologism [59], denotes the calcite-magnetite-forsterite assemblage, used as a synonym with phoscorite.

The endogenous apatite deposits can be classified20 into the following types [60],[61]:

**7.1.4. Genetic classification of phosphate rocks**

**i. Silicate-apatite** (**ijolite**17

urtite.18

femic components) [55].

used as a synonym with phoscorite.

according to the mineral assemblages [6],[51],[52],[53],[54],[55]:

350 Apatites and their Synthetic Analogues - Synthesis, Structure, Properties and Applications

**ii. Silicate-magnetite-apatite** (**phoscorite**): phoscorites19

The genetic classification proposed by EGOROV [50] includes three types of apatite ores

composed of nephelite and aegirite-augite, or other pyroxene, in nearly equal proportions. There are varieties richer in nephelite, approaching the composition of

associated with carbonatites, often forming multiphase phoscorite-carbonatite series [56]. The term "phoscorite" was originally used to describe the magnetite-

temperature carbonate melts that have been evolving from the upper mantle (**Fig. 8**) for at least the past 2 Ga. Many are associated with crustal complexes of alkali-rich

olivine-apatite rock with a carbonate core by RUSSELL et al [57],[58],[59].

**iii. Carbonate-apatite** (**carbonatite**): the crystalline products of low-volume and high-

silicate rocks from which they may have evolved by liquid immiscibility.

**Fig. 8.** The location of upper mantle on the cross-section of the Earth, core and mantle drawn to scale [44] (a). Ternary equilibrium phase diagram of the apatite bearing ijolite-urtite rock (b) approximated by the system NaAlSiO4-CaMg‐ Si2O6-Ca5(PO4)3F [6]: olivine (Ol), melilite (Me), silicophosphate (Sph), apatite (Ap) and immiscibility field (L1+L2).

17 Ijolite, theralite and teschenite belong to phanerites (prelenic rocks characterized by equal or nearly equal amounts of

18 Urtite is medium-grained and light gray rock composed of about 70% nephelite, 25% aegirite or aegirite-augite [55]. 19 In the work [58], the name camaforite, i.e. Russian neologism [59], denotes the calcite-magnetite-forsterite assemblage,

 Nephelite is an equant anhedrons, pyroxene forms euhedral crystals with zonal structure and margin of more sodic pyroxenes than that in the center, which is commonly titaniferous. Some varieties contain titaniferous melanite and iivaarite. Apatite is a prominent constituent and titanate is generally present in small amounts.

): ijolite is a medium- to coarse-grained equigranular rock

are spatially and temporally

	- **Geosynclinal sea**: e.g. in the Late Cretaceous and in Paleogene on the shelf of the Tethys geosyncline with the deposits in Morocco (Khouribga and Youssoufia), Algeria (Djebel Onk, El Kouit), Tunis (Gafsa), of the Permian geosyncline of the Rocky Mountains, with the deposits in the Phosphoria Formation in Idaho, Wyoming, Utah and Montana (USA), of the Caledonian geosyncline Karatau (Russia), large deposits are in the Upper Cretaceous in Kazakhstan, in the neighborhood of Aktyubinsk.
	- **Epicontinental sea**: e.g. in the Cenomanian of the south Russian digression and in the Jurassic of the Moscow region, at the margin of the African Shield in the Eocene complex near Hahotoe (Togo) and Taiba (Senegal), in the upper Cretaceous of Egypt [60].


**4. Conclusions**

In this paper, the thermogenic physical model of the FSW process is established, and the finite element model of FSW is established. The material flow and temperature field in the welding process are studied by a numerical simulation. The finite element model is updated by the results of the axial force and torque tests. Moreover, the overall trend of the material flow in a finite element analysis is verified by the experiments on the weld cross section. The correctness of the finite element model analysis is verified by the microstructure formation and flow trend. Based on the results of this study, the following conclusions were made:

**Author Contributions:** H.L. and T.W. developed the FEM of friction stir welding process and wrote the paper; H.L., T.W. and P.W. conceived and designed the experiments; T.W. and Y.L. analyzed the data and modified the paper. T.W., F.Z. and H.W. performed the experiments.

**Funding:** The authors would like to acknowledge the financial support from the National Natural Science Foundation of China (Grant No. 51505470), the Youth Innovation Promotion Association, CAS and 'JXS' innovation fund, SIA.

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


**5. Conclusions**

There is general agreement that urban water resources need to be managed better. We all depend on clean water for survival and the make-up of our communities reflects the quality of water resources flowing through them. The need for improvement is great and good policy needs both the natural and social sciences to generate good governance of our water resources [98]. But urban water management seems stuck in a state of maladaptation essentially locked into a societal need for large infrastructure and the never ending need to maintain it [99]. Change only occurs in response to some catastrophic or shock event (such as a flood) that in no way bears the needed one-on-one logical change to what triggered the shock event in the first place. This leads to changes in urban infrastructure that may bear no resemblance to real societal needs or watershed management.

 Resources spent on stream restoration and flood mitigation upstream will be well received when urban residents understand the benefits downstream [100]. Local efforts to pick up trash and minimize plastics may have far reaching impact on communities when understood in the context of the world's oceans [101]. Or the necessity to rebuild current infrastructure to deal with ongoing climate driven precipitation change [102]. Good monitoring can expose the need and document the improvement. Urban rivers can be rehabilitated given a dedicated citizenry aided by governmental and scientific support. Our future depends on it.


**Abstract**

This chapter reflects a review of the results obtained in several investigations on the fluidization process of wet biomass particles in a mechanically stirred fluidized bed equipment. For the experimental purposes, an experimental equipment with a drying column of 300 mm in diameter is used. Using the Ergun equation, the fluodynamic behavior of the bed is analyzed to obtain, from the measurements of pressure drops in the bed and imposed velocities, the specific gas-particle contact surface; such interfacial surface varies between 5758 and 7317 m2 m–3 for dry particles and between 2774 and 4444 m2 m–3 for high humidity particles. Subsequently, the phenomena of heat and mass transfer by convection between the fluidizing gas and the biomass particles during the drying process are studied. The gas-particle heat and mass transfer coefficients are determined, considering stirrer rotation speeds between 1 and 2 rev s–1 . The convective coefficients vary between 13 and 25.7 W m–2 K–1 for heat transfer and between 6 x 10–3 and 20 x 10–3 m s–1 for mass transfer; thus, correlations have been obtained between the Nusselt and Reynolds numbers and between the Sherwood and Reynolds numbers, respectively, valid in the Reynolds number range between 102 and 257.

**Keywords:** fluidization, drying, heat and mass transfer, biomass, particle drying, agitated fluidized bed


**About the Editor**

#### **David R. Nalin**

David R. Nalin, MD, Dr.Sci.HC, Professor Emeritus, Center for Immunology and Microbial Diseases, Albany Medical College, Albany NY. After internship and medical residency (Montefiore, NYC) and senior residency (Harvard Service, BCH), Dr. Nalin attained the rank of Senior Surgeon In the U.S. Public Health Service, serving in Dhaka, Bangladesh (then E. Pakistan) as Research Associate, Office of International Research, NIH, and later Assistant Professor, medicine and pathobiology at Johns Hopkins Center for Medical Research and Associate Professor of medicine, international medicine and epidemiology and Director, U. Md. Pakistan Medical Research Center, Lahore. He is currently Professor Emeritus, Center for Immunology and Microbial Diseases, AMC.

Dr. Nalin led the team that first demonstrated that oral glucose-electrolyte solutions(ORS) reduced maintenance intravenous fluid needs by 80% in severe cholera, and published numerous studies demonstrating ORS effectiveness for most acute life-threatening watery diarrhoeal diseases for both rehydration and maintenance therapy. The BMJ estimated that by 1999 ORS saved over 70,000,000 lives.

As Director, Clinical Research, Vaccines and Infectious Diseases and Director, Vaccine Scientific Affairs at Merck (1983–2002) he designed and monitored studies of imipenem-cilastatin, norfloxacin and ivermectin, M-M-R® II, hepatitis A and HPV vaccines.

He has published over 100 peer-reviewed articles, over 200 letters, reviews, book chapters, and abstracts and holds three patents for new vaccination methods. His awards include the Pollin Prize for Pediatric Research and the Prince Mahidol Award 2006, recognizing his development of ORS; the AMC Humanitarian award (2010), the Friends of Liberation War Award Bangladesh (2013) and the Dr. Sci. H.C. degree from the University of Vermont (2017).


**1. Introduction**

In recent years, antibiotics have been extensively used as livestock food additives and to fight infections in animal husbandry [1,2]. The overuse of antibiotics results in their continuous release into the environment in China. Additionally, previous investigations have indicated that antibiotics in animal waste could not be entirely removed using traditional lagoon treatment and in wastewater treatment plants (WWTPs) [3]. As a result, antibiotics were widely detected in wastewater, surface water, and groundwater [4,5].

The wide presence of antibiotics in the environment could cause concern because it not only causes serious toxic effects on organisms, but also promotes the spread of antibiotic-resistant genes

(ARGs) [6], even with low concentrations in the environment [7,8]. ARGs could be spread through horizontal gene transfer (HGT) and vertical gene transfer (VGT), and in many cases, could be maintained in microbial populations, even without selection pressure from antibiotics [9–11]. HGT is a major pathway for the transfer of ARGs, including conjugative transposons, integrons, insertion sequences, and plasmids [11,12]. ARGs have often been detected as part of antibiotic resistance super integrons. Therefore, one antibiotic may coselect resistance to other antibiotics when applying multiple antibiotics [13]. Even if antibiotic-resistant bacteria were damaged or killed, ARGs could still be released to the environment and then transformed into other bacteria [14,15]. Previous studies have revealed the high relative abundances of ARGs in wastewater lagoons and municipal wastewater, even after treatment [16–18]. In recent years, ARGs were regarded as fast-growing potential pollution because of the extensive application of antibiotics in the livestock industry [19–21]. Hence, e ffective treatment processes for antibiotic removal could also prevent the spread of ARGs.

Constructed wetlands (CWs) are designed and constructed exploiting natural processes to treat rural wastewater [22,23]. The advantages of CWs mainly lie in high purification removal, relatively low construction and maintenance costs, reduced energy consumption, convenient operation, and broad application prospects in developing countries or rural areas [24,25]. Recently, CWs were used to remove antibiotics from agricultural and municipal wastewater via natural processes involving plants, soil/sediment, and microorganisms [21,26,27]. CWs have been shown to be more e fficient in the removal of antibiotics and ARGs than conventional wastewater treatment systems [16,28]. Liu et al. (2013) [28] found that the total absolute abundances of tetracycline resistance (*tet*) genes and the 16S rRNA were reduced by 50% from swine wastewater using CWs. Huang et al. (2015) [16] reported that the absolute abundances of the ARGs were greatly reduced, with their log units ranging from 0.26 to 3.3. However, previous studies of ARG reduction have always focused on their elimination, rather than the induction of ARGs along with antibiotics removal by CWs. CWs could also be a significant source of ARGs, and may enhance their spread. Therefore, exploring the induction of ARGs in CWs would greatly assist in evaluating their environmental risks.

Sulfamethoxazole (SMX) is a synthetic antibiotic within the sulfonamide antibiotic family, and is largely consumed in the livestock husbandry [29,30]. *Sul* genes (*sulI* and *sulII*) were chosen as the representatives of ARGs for their frequent use [17,31–33]. In this study, four CWs were established to treat a synthetic wastewater containing SMX for 360 day. The objectives of this study were: (1) to investigate the elimination e fficiencies and products of SMX, (2) to explore the development of *sul* genes in the reactors, (3) to assess the risks of ARGs in e ffluent, and (3) the bacterial community during the treatment process.

#### **2. Results and Discussion**

#### *2.1. SMX Removal E*ffi*ciency*

The concentrations of SMX in the e ffluent, bottom layer, and surface layer of CWs at five sampling points are shown in Figure 1. The concentrations of SMX in the e ffluent ranged 0.051–0.214, 0.047–0.274, 0.0584–0.342, and 0.098–0.574 μg L−1 in CW1, CW2, CW3, and CW4. CW4 fed with 200 μg L−1 SMX exhibited significantly higher concentrations of SMX in the e ffluent than CW1 fed with 20 μg L−1 (*p* < 0.05). On D360, the SMX concentrations in the e ffluent were 0.214, 0.185, 0.342, and 0.574 μg L−1 with influent SMX concentration of 20, 50, 100, and 200 μg L−1, respectively. Temporally speaking, the SMX concentrations in e ffluent gradually increased during the study (*p* < 0.05). In the e ffluent of CW3, SMX concentrations were 0.0584, 0.142, 0.198, 0.247, and 0.342 on D30, D60, D120, D240, and D360, respectively. Notably, excellent removal e fficiencies (>98%) for SMX were obtained using CWs, even when the influent SMX concentration was as high as 200 μg L−1.

**Figure 1.** Concentrations of SMX (mean ± SD, *n* = 3) in the effluent water and layers. (**A**) concentration of SMX in the effluent; (**B**) concentration of SMX in the bottom layer; (**C**) concentration of SMX in the surface layer.

SMX has been reported to be easily biodegradable, especially under anaerobic conditions in the CWs [25]. In the present study, it was noteworthy that the removal efficiencies of antibiotics by different CWs were even better than conventional WWTPs [34]. As demonstrated by previous experiments, biodegradation, absorption, hydrolysis, and photodecomposition played significant roles in the removal of antibiotics in the solid and aqueous phases in CWs [34–36].

Previous reports also showed that the adsorption process only accounted for a minor percentage of the total removal in CWs [12].

The SMX concentrations of the bottom layer ranged 4.256–8.620, 14.213–19.281, 8.546–17.322, and 36.564–47.684 μg Kg−1 in CW1, CW2, CW3, and CW4. The SMX concentrations of the surface layer ranged 0.239–0.852, 0.327–2.652, 2.526–4.365, and 4.531–9.829 μg Kg−1 in CW1, CW2, CW3, and CW4, respectively. The difference of SMX concentration between the bottom and surface layers was affected by the influent SMX concentration. This was probably because the relatively high *Kd* values prompted SMX to be adsorbed in the substrates of CWs [37]. However, the SMX concentrations in the layers did not significantly increase during the treatment (*p* > 0.05). This result was probably due to the fact that other physicochemical and biological processes occurred jointly or separately after SMX adsorption in substrate [16]. Clearly, the concentration of SMX in the bottom layer was higher compared with that in surface layer. This was not surprising, because that the bottom layer was faced with a higher concentration of SMX due to the vertical up-influent.

#### *2.2. SMX Degradation Products*

LC-MS/MS was employed to identify the degradation products in order to clarify the degradation pathway of SMX (C10H11N3O3S, *m*/*z* 252.0450). According to the detected mass/charge ratios under positive mode, several potential metabolites were identified, including 4-amino benzene

sulfinic acid (C6H7NO2S, *m*/*z* 158.0123), 3-amino-5-methylisoxazole (3A5MI, C4H6N2O, *m*/*z* 99.5315), benzenethiol (C6H6S, *m*/*z* 111.0496), and 3-hydroxybutan-1-aminium (C4H11NO, *m*/*z* 90.5263) (Figure 2). A previous report indicated that the removal of antibiotics was mainly caused by adsorption rather than degradation in wastewater treatment plants [38]. However, in this study, it was found that the products of SMX may be transformed by bacterial communities. A previous study also found 4-amino benzene sulfinic acid and 3A5MI during the degradation of SMX [39]. During the degradation process, SMX was initially hydrolyzed into 4-amino benzene sulfinic acid and 3A5MI; then, 4-amino benzene sulfinic acid was further transformed into benzenethiol, which was finally degraded to generate methane or carbon dioxide. During the transformation of 3A5MI to 3-hydroxybutan-1-aminium, the isoxazole ring was opened and its nitrogen atom was removed. Since 3-hydroxybutan-1-aminium has a chain structure, it can be easily mineralized into methane by microbes under anoxic conditions [40].

**Figure 2.** Q-Exactive spectrum degradation products of SMX.

#### *2.3. Sul Genes in the E*ffl*uent and Media*

In order to analyze the changes of corresponding target genes, the relative abundances of *sul* genes were analyzed during the treatment process. The distributions of two *sul* (*sulI* and *sulII*) in the CWs are shown in Figure 3. The relative abundances of *sul* genes showed an obvious increase with the increase of SMX concentrations in the bottom and middle layers. In wastewater treatment installations, the abundances of ARGs were not only determined by their abundances, but were also affected by the concentrations of antibiotics in the influent [6]. Since synthetic wastewater was used in this study, the abundances of *sul* genes in the influent may be ignored. In addition, the *sul* genes were not found in CW0. Therefore, most of the target genes were induced by the antibiotics in the influent. Further, bacteria would also gain the corresponding ARGs via VGT and HGT [10].

**Figure 3.** *Sul* genes (*sulI* and *sulII*) normalized to 16S rRNA genes in the layers (bottom layer, middle layer and surface layer) of CW1, CW2, CW3, and CW4, respectively (mean ± SD, *n* = 3). (**A**) *sulI* genes; (**B**) *sulII* genes.

Notably, the concentration of *sul* genes in the bottom layer was higher than that in the middle layer, with the surface layer containing the least (Figure 3). A similar result was obtained in a previous study, which found that the level of ARGs was high in CWs [28]. This observation was mainly influenced by the level of SMX sources and oxygen transport capacity in the bottom layer [28]. Clearly the concentration of SMX in the bottom layer was the highest, followed by the middle and surface layers (Figure 1). The fate of ARGs in the different layers of CWs was mainly attributable to the accumulation of SMX in the substrate. Interestingly, the relative abundances of *sul* genes in the CWs were in the order of *sulII* > *sulI* (Figure 3). This was not surprising, because the different fates of *sul* genes in CWs were mainly caused by their specific mechanisms [6]. The *sulI* gene was generally found to be associated with other ARGs in class 1 integrons, while *sulII* was usually located on small nonconjugative plasmids, or generally located on large transmissible multi-resistance [41]. Therefore, the persistence of *sulII* genes might be attributed to the successive pressure exerted by antimicrobial agents that were transferred via HGT and VGT between pathogens, nonpathogens, and even distantly related organisms [31,41].

ARGs, as a major source of pollution, may be spread in bioreactors [42]. The relative abundances of corresponding *sul* genes in effluent have been reported (Figure 4). The CWs, indicating the rate of spread for *sul* genes, were probably caused by antibiotics. This was comparable to previous reports in which similar abundances of ARGs were observed in the effluent of CWs [21]. In addition, the relative abundance of most *sul* genes was enhanced with higher concentrations of SMX; *sul* genes were not detected in the effluent of CW0. The relative abundance of *sul* genes exhibited an increase, which tended to be stable among the treatment duration. Meanwhile, the relative abundances of *sul* genes in the effluent were in the order of *sulII* > *sulI*. In a word, vertical up-CWs may be a good choice for application as an effective SMX removal method, but the fate of ARGs remains to be further studied in practical applications.

**Figure 4.** *Sul* genes (*sulI* and *sulII*) normalized to 16S rRNA genes in the effluent of CW1, CW2, CW3, and CW4, respectively (mean ± SD, *n* = 3). (**A**) *sulI* genes; (**B**) *sulII* genes.

#### *2.4. Composition of Bacterial Communities*

The microbial community was evaluated in terms of abundance and bacterial structure in response to the different treatments in CWs [43]. There were 30 dominant genera in the phylum level for the bottom layers, occupying > 94.90% of sequences (Figure 5). Twenty genera were identified while 10 remained unknown by taxonomy assignment. *OD1* (49.32%), *Proteobacteria* (44.59%), *Proteobacteria* (31.36%), and *Proteobacteria* (54.53%) had the maximum amounts of dominant phylum and high relative abundances of detection in CW1, CW2, CW3, and CW4, respectively, followed by*Chloroflexi*, *Bacteroidetes,* and *Acidobacteria*. Previous studies reported that proteobacteria is responsible for the degradation of refractory organic [44]. In addition, *Proteobacteria* (12.79%), *Bacteroidetes*, *Acidobacteria,* and *Planctomycetes* increased after the SMX treatment process. Because of potential coselection on SMX, the accumulation of antibiotics in substrates may also contribute to the enhancement of some dominant bacteria. Our result was similar to that in a previous report, i.e., that *Proteobacteria* occupied the string majority in antibiotic-correlated bacteria, followed by *Bacteroidetes* and *Actinobacteria* [45]. Some specific bacteria in CWs, like *Nitrospirae*, are well-known to be involved in nitrification and ammonia oxidization [46]. In accordance with our result, *Nitrospirae* was a dominant phylum, with a relative abundance of up to 4.45%.

To assess the variation of dominant bacteria after CW treatment, five samples could be divided into three clusters according to the bacterial composition and relative abundance. Cluster I comprised the original sample, cluster II included the CW1 and CW2 samples, and cluster III comprised all of the remaining samples (CW3 and CW4). Previous study has shown that no significant difference was observed in terms of bacterial abundance, richness, or diversity among different treatments of antibiotics [43]. However, in this experiment, the bacterial communities and their relative abundance were influenced by the SMX content of the influent. Therefore, the phenomenon suggested that the original bacterial structure had to adapt to the variation of different SMX concentration conditions, appearing to change over the 360 days of the experiment period.

**Figure 5.** Heat map of bacterial populations in phylum level: Sample names were listed on the *x*-axis, and genus names were listed on the *y*-axis.

## **3. Materials and Methods**


**Salutogenesis and Coping: Ways to Overcome Stress and Conflicts**

Editors

**Orna Braun-Lewensohn Claude-H ´el `ene Mayer**

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

*Editors* Orna Braun-Lewensohn Ben-Gurion University of the Negev Israel

Claude-H´el`ene Mayer University of Johannesburg South Africa

*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 *International Journal of Environmental Research and Public Health* (ISSN 1660-4601) (available at: https: //www.mdpi.com/journal/ijerph/special issues/Salutogenesis Coping).

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

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© 2021 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.

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**4. The past and present of HLA typing**

HLA genes contain 5–8 exons ranging in length from 4 to 17 kb. Most high-resolution fourdigit HLA typing technologies, mainly sequence-based techniques (SBT) or probe-based hybridization techniques, primarily focus on deciphering the sequence of the antigen-binding groove only. This is due to the high cost of complete HLA genotyping and the limited time interval before HSCT. Thus, only exons 2 and 3 (540 bp) for class I molecules and exon 2 (270 bp) of class II molecules are typically analyzed, providing intermediate-resolution typing.

Before these techniques came into light, less informative serology methods were acquired for HLA typing. On the other hand, the future seems very promising thanks to the evolution of sequencing technologies [20].

Next, we will provide a brief review of evolution of the most widely applied techniques for HLA typing, frequently utilized by clinical laboratories.

#### **4.1. Serological methods**

#### *4.1.1. CDC (complement-dependent lympho-cytotoxicity) technique*

Lymphocytes incubated with polyclonal sera in the presence of complement, was the first attempt for determining patient-donor compatibility. CDC utilizes sera from multiparous alloimmunized women, whose HLA specificity (reactivity against a particular HLA type) is determined using a panel of lymphocytes of already known HLA type.

From a population of peripheral blood lymphocytes (PBLs), T lymphocytes are used for determining class I antigens, while B-lymphocytes (professional APCs) are separately isolated for determining class II antigens. The cells are incubated along with the characterized serum and complement. Their reactivity is determined based on the lysis of Ab-covered lymphocytes, from complement components, as shown in **Figure 2**.

**Figure 2.** Example of a serological reaction utilizing the CDC technique. The lysis of the lymphocytes by a serum containing anti-HLA B35 Abs denotes the HLA-B35 positivity of the cells.

The low-resolution two-digit serologic typing this technique offers is further limited by the availability of sera containing various HLA specificities. However, this method has value in confirming the presence or absence of an antigen in case of mutations in promoter regions or genes not otherwise analyzed [5, 18, 21].

A variation of the above technique involves incubation of the donor's cells with serum from the patient in the presence of complement. The results are interpreted the same way as previously described.

Both techniques rely on cell viability in order to be successful and accurate. Also, cell populations need to be lymphocyte-specific so that the results be interpretable. Samples contaminated by other lymphocytes and/or precursor cells lead to inaccuracies [22].

#### *4.1.2. Other serological techniques*

**3.6. Seeking for permissive (relatively well-tolerated) HLA mismatches**

52 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

related mortality, delayed immune reconstitution, but not disease relapse.

sequences, regarding the protein's function.

**4. The past and present of HLA typing**

HLA typing, frequently utilized by clinical laboratories.

from complement components, as shown in **Figure 2**.

*4.1.1. CDC (complement-dependent lympho-cytotoxicity) technique*

determined using a panel of lymphocytes of already known HLA type.

sequencing technologies [20].

**4.1. Serological methods**

It is well understood that even single nucleotide substitutions might impact the course of transplantation significantly either on the aspect of GVHD, engraftment success and transplant

Of course the extent of their impact is driven not only by the kind of genetic alteration (SNP, CNV, InDel, inversion), the effect it exerts on the final protein product (synonymous versus nonsynonymous polymorphisms) and the gene it appears at (HLA class I or II), but also the exact nucleotide position it is located, since this might affect more or less important amino acid

With the advent of novel sequencing techniques allele level typing provides extensive nucleotide sequence data, which in correlation with previously available clinical data, is going

HLA genes contain 5–8 exons ranging in length from 4 to 17 kb. Most high-resolution fourdigit HLA typing technologies, mainly sequence-based techniques (SBT) or probe-based hybridization techniques, primarily focus on deciphering the sequence of the antigen-binding groove only. This is due to the high cost of complete HLA genotyping and the limited time interval before HSCT. Thus, only exons 2 and 3 (540 bp) for class I molecules and exon 2 (270 bp) of class II molecules are typically analyzed, providing intermediate-resolution typing.

Before these techniques came into light, less informative serology methods were acquired for HLA typing. On the other hand, the future seems very promising thanks to the evolution of

Next, we will provide a brief review of evolution of the most widely applied techniques for

Lymphocytes incubated with polyclonal sera in the presence of complement, was the first attempt for determining patient-donor compatibility. CDC utilizes sera from multiparous alloimmunized women, whose HLA specificity (reactivity against a particular HLA type) is

From a population of peripheral blood lymphocytes (PBLs), T lymphocytes are used for determining class I antigens, while B-lymphocytes (professional APCs) are separately isolated for determining class II antigens. The cells are incubated along with the characterized serum and complement. Their reactivity is determined based on the lysis of Ab-covered lymphocytes,

to provide significant information in the context of retrospective studies [8, 12, 19].

FACS overcomes two additional obstacles of the CDC method. The first refers to the positive reactions mediated through cytotoxic Abs directed against non-HLA molecules (lower specificity). The second regards positive reactions that are driven only by the complementactivating Abs, thus failing to detect complement-independent acting donor-specific Abs (lower sensitivity).

However cell viability dependence is still an obstacle. Combined with its high cost, this technique is prohibitive for HLA-typing in a routine use by a clinical laboratory.

Solid phase assays, such as ELISA (enzyme-linked immunosorbent assays) and the Luminex technology, the last utilizing fluorescent dye impregnated beads bound to HLA molecules, have also been developed for HLA typing. These have mainly, but not exclusively, been studied in solid organ transplantation studies. In HSCT studies, these techniques mainly focus on HLA-Ab screening due to the renewed interest on donor-specific Abs (DSA) and their importance in graft failure [22–27].

Since molecular methods have proven to be more reliable, we will focus on them for a much more comprehensive analysis [28].

#### **4.2. Molecular methods**

The development and extensive usage of molecular methods soon substituted serologic techniques, for determination of individuals' HLA type.

Molecular methods provided higher resolution typing, without the need for preserving characterized sera of all HLA types, even those not very common ones, and without the prerequisite of cell viability for test success.

Molecular methods of typing are still the main approaches to HLA typing resolution of low (two-digit) or intermediate (partial four-digit) level. The four-digit resolution these techniques offer for analysis of limited but HSCT important loci, contributed to the identification of serologically indistinguishable variants of HLA class I and II molecules with few, but detrimental, amino acid changes.

High-resolution typing at the four-digit level for all HLA loci is an unrealistic goal with these techniques. Molecular methods mainly focus on identifying polymorphisms in exons 2 and 3 of the class I locus and exon 2 of the class II locus, which are crucial for HSCT, as mentioned before [5, 18, 29].

#### *4.2.1. PCR/SSP (sequence-specific priming PCR)*

Genomic DNA (gDNA) is isolated from the under investigation sample and HLA regions of interest are amplified using PCR technology.

PCR utilizes in vitro prepared small oligonucleotide sequences (primers or oligos). These oligos bind to an exact location of a DNvA molecule, according to complementarity rules, acting as starting points for the production of multiple complementary copies of the intermediate region between a pair of them (amplification). More primers per reaction or more pairs of primers may be included in a single PCR, depending on the purpose of each protocol [29–32].

Sequence specific PCR (PCR/SSP), whether characterized as allele-specific amplification PCR (PCR/AS), amplification refractory mutation system PCR (PCR/ARMS) or multiplex PCR, exploits PCR for the amplification of specific HLA regions with minor alterations in primer design each time.

The amplification primers are polymorphic-specific, meaning that they only extend and form a product if the targeted polymorphism exists. The primers are designed in such a way, so that their 3' end nucleotide is complementary to the investigated genomic alteration. Thus products of specific length are produced depending on the polymorphism and the primer design. Afterward, these are visualized using gel electrophoresis.

This technique might result in no amplification at all if none of the polymorphisms analyzed exists, thus another set of primers is coamplified. This corresponds to a monomorphic target sequence that produces an extra fragment of distinguishable length. The last determines the quality of DNA and the validity of the technique (successful reaction) [24].

PCR/SSP may become a time and labor intense technique, when not used in a multiplexed format for the analysis of many polymorphic sites. If multiplex format is preferred, the conditions for a successful PCR need to be very stringent. Also, this technique is prone to falsepositive bands and false-negative results, especially for degraded samples.

However, this method is especially useful if applied in conjunction with PCR/SSOP (sequence specific oligonucleotide probes) hybridization typing, providing higher resolution, since it allows the separate amplification of the two alleles in a heterozygote [28, 33–35].

#### *4.2.2. PCR/SSOP (sequence-specific oligonucleotide probes)*

#### *<IP_ADDRESS>. Reverse hybridization (reverse dot blot)*

Since molecular methods have proven to be more reliable, we will focus on them for a much

The development and extensive usage of molecular methods soon substituted serologic

Molecular methods provided higher resolution typing, without the need for preserving characterized sera of all HLA types, even those not very common ones, and without the

Molecular methods of typing are still the main approaches to HLA typing resolution of low (two-digit) or intermediate (partial four-digit) level. The four-digit resolution these techniques offer for analysis of limited but HSCT important loci, contributed to the identification of serologically indistinguishable variants of HLA class I and II molecules with few, but detri-

High-resolution typing at the four-digit level for all HLA loci is an unrealistic goal with these techniques. Molecular methods mainly focus on identifying polymorphisms in exons 2 and 3 of the class I locus and exon 2 of the class II locus, which are crucial for HSCT, as mentioned

Genomic DNA (gDNA) is isolated from the under investigation sample and HLA regions of

PCR utilizes in vitro prepared small oligonucleotide sequences (primers or oligos). These oligos bind to an exact location of a DNvA molecule, according to complementarity rules, acting as starting points for the production of multiple complementary copies of the intermediate region between a pair of them (amplification). More primers per reaction or more pairs of primers may be included in a single PCR, depending on the purpose of each protocol

Sequence specific PCR (PCR/SSP), whether characterized as allele-specific amplification PCR (PCR/AS), amplification refractory mutation system PCR (PCR/ARMS) or multiplex PCR, exploits PCR for the amplification of specific HLA regions with minor alterations in primer

The amplification primers are polymorphic-specific, meaning that they only extend and form a product if the targeted polymorphism exists. The primers are designed in such a way, so that their 3' end nucleotide is complementary to the investigated genomic alteration. Thus products of specific length are produced depending on the polymorphism and the primer design.

This technique might result in no amplification at all if none of the polymorphisms analyzed exists, thus another set of primers is coamplified. This corresponds to a monomorphic target

more comprehensive analysis [28].

techniques, for determination of individuals' HLA type.

54 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

prerequisite of cell viability for test success.

*4.2.1. PCR/SSP (sequence-specific priming PCR)*

interest are amplified using PCR technology.

Afterward, these are visualized using gel electrophoresis.

**4.2. Molecular methods**

mental, amino acid changes.

before [5, 18, 29].

[29–32].

design each time.

The PCR step of SSOP utilizes chemically modified (biotin labeled) primers nonpolymorphicspecific. Under normal circumstances, meaning the DNA is intact and no random polymorphism exists in the 3' end of a single primer, the biotinylated amplified products (amplicons) are produced by each primer pair. This way, the primer pair is designed in such a way in order to produce an amplicon that includes polymorphisms inside its sequence.

The PCR product is afterward incubated with a panel of already known polymorphic HLA sequence molecules (SSO probes), which are enzymatically poly-thymidine (poly-T) tailed. This enzymatic tailing enables their prestabilization on a solid surface, usually a nylon membrane.

The modified amplicon will cross react with only one of these probes (complete complementarity) during their incubation along with horseradish peroxidase (HRP)-conjugated streptavidin and a chromogenic or chemiluminescent substrate. HRP-conjugated streptavidin binds on the biotinylated product. In the presence of biotin, HRP enzyme is activated and metabolizes the substrate in order to emit light signal. This light is device detected and computer analyzed later on, to determine the polymorphism of the unknown sample, based on position analysis of the signal, since the stabilization position of each probe on the membrane is previously known.

As one can conclude the specific sequence of gDNA where the primers will bind, need to be known in advance. The polymorphisms under investigation also need to be already known, in order to prepare and stabilize the suitable set of probes.

Interpretation difficulties may arise from less intense, absent or dubious hybridization patterns, due to poor-quality and low-quantity DNA amplification or background signals due to poor membrane washing or temperature variation during hybridization.

Another limitation of this technique is the fact that the extremely polymorphic HLA alleles, especially those of class I, are impossible to analyze due to the very large number of probes such a design would require. However, as a general rule, wider number of probes for every HLA loci and larger number of unknown HLA regions investigated provides higher resolution level [29–31, 36].

#### *<IP_ADDRESS>. Direct hybridization (conventional dot-blot)*

An alternative to reverse dot-blot PCR-SSOP, is the conventional dot-blot technique, where the PCR amplified regions are the ones immobilized on the solid surface, and biotin-labeled SSO probes are incubated along with HRP-conjugated streptavidin and a substrate. The ones that bind the unknown amplicons emit light of specific wavelength identified in the same manner as previously described.

In addition to the previously described limitations this technique is also more cumbersome, since the number of SSOPs required for typing vastly increases, due to the high polymorphic state of HLA loci [30, 31].

#### *4.2.3. PCR/RFLP (restriction fragment length polymorphism)*

RFLP analysis involves digestion of gDNA with endonucleases, one at a time, which cleaved a specific nucleotide sequence motif, in order to produce fragments of various lengths. This variation can be detected by Southern blot analysis of the digested fragments. Suitable probes for detection are either cloned cDNA, or genomic DNA sequences, complementary to mainly HLA class II regions which were better studied. In fact, this technique was the first that revealed the incredible variation of HLA class II region.

However, it is cumbersome, it requires a large amount of high molecular weight genomic DNA, and it can only be applied to regions that bear the respective restriction sites, which is not always the case and that is why it did not replace serological typing, but was rather used complementary.

PCR-combined RFLP (PCR/RFLP) analysis is an improvement to the previous method. The amplified HLA region is incubated with a restriction endonuclease that recognizes specific nucleotide sequence. The digestion reaction is performed whether a polymorphism exists or not, producing amplicon fragments of various lengths. The products are analyzed by gel electrophoresis.

However, like RFLP analysis, PCR/RFLP may lead to inconsistencies during HLA typing due to complex manipulation steps and possible incomplete digestion reactions. Also, this technique is unable to detect multiple recognition sites simultaneously [29, 33, 36, 37].

#### *4.2.4. PCR/SBT (sequencing-based typing)*

Another approach to high-resolution HLA typing is the PCR amplification and subsequent direct sequencing of previously described class I and II exons. Dideoxy-based Sanger sequencing, using capillary electrophoresis, provides increased reliability especially when applied after SSO or SSP.

The two alleles of heterozygous samples, which represent a substantial source of ambiguities, are usually sequenced separately following SSO or SSP typing, thus increasing the resolution of possible genotypes [29, 37].

This technique, although more automated, easier to implement and less prone to technical and interpretational errors, is less sensitive than others, like SSO, which when optimized provide more accurate results and less ambiguous results [24].

## *4.2.5. PCR/RSCA (reference strand-mediated conformational analysis)*

HLA loci and larger number of unknown HLA regions investigated provides higher resolution

An alternative to reverse dot-blot PCR-SSOP, is the conventional dot-blot technique, where the PCR amplified regions are the ones immobilized on the solid surface, and biotin-labeled SSO probes are incubated along with HRP-conjugated streptavidin and a substrate. The ones that bind the unknown amplicons emit light of specific wavelength identified in the same manner

In addition to the previously described limitations this technique is also more cumbersome, since the number of SSOPs required for typing vastly increases, due to the high polymorphic

RFLP analysis involves digestion of gDNA with endonucleases, one at a time, which cleaved a specific nucleotide sequence motif, in order to produce fragments of various lengths. This variation can be detected by Southern blot analysis of the digested fragments. Suitable probes for detection are either cloned cDNA, or genomic DNA sequences, complementary to mainly HLA class II regions which were better studied. In fact, this technique was the first that revealed

However, it is cumbersome, it requires a large amount of high molecular weight genomic DNA, and it can only be applied to regions that bear the respective restriction sites, which is not always the case and that is why it did not replace serological typing, but was rather used

PCR-combined RFLP (PCR/RFLP) analysis is an improvement to the previous method. The amplified HLA region is incubated with a restriction endonuclease that recognizes specific nucleotide sequence. The digestion reaction is performed whether a polymorphism exists or not, producing amplicon fragments of various lengths. The products are analyzed by gel

However, like RFLP analysis, PCR/RFLP may lead to inconsistencies during HLA typing due to complex manipulation steps and possible incomplete digestion reactions. Also, this

Another approach to high-resolution HLA typing is the PCR amplification and subsequent direct sequencing of previously described class I and II exons. Dideoxy-based Sanger sequencing, using capillary electrophoresis, provides increased reliability especially when applied

technique is unable to detect multiple recognition sites simultaneously [29, 33, 36, 37].

level [29–31, 36].

as previously described.

state of HLA loci [30, 31].

complementary.

electrophoresis.

after SSO or SSP.

*<IP_ADDRESS>. Direct hybridization (conventional dot-blot)*

56 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

*4.2.3. PCR/RFLP (restriction fragment length polymorphism)*

the incredible variation of HLA class II region.

*4.2.4. PCR/SBT (sequencing-based typing)*

Although PCR/SBT is able to detect unknown HLA polymorphisms, the problem of not being entirely able to resolve novel arrangements of known polymorphisms, also known as ambiguity, can be overcome by the PCR/RSCA technique [19].

PCR/RSCA can achieve high-resolution results without the ambiguities seen in the previously described methods. This technique is based on the principles that DNA fragments that differ in nucleotide composition exhibit different motilities after separation by nondenaturing polyacrylamide gel electrophoresis (PAGE). The amplified alleles under investigation are hybridized with a fluorescent labeled reference strand, forming a double stranded DNA with unique conformation (double strand conformation analysis; DSCA).

PCR/RSCA is capable of resolving even single base alterations and of course, identification of new mutations thanks to the different spatial structure of the newly formed DNA-probe duplexes [38].


*4.2. SC-M\* Description*

SC-M\* is a general solver to the MAPP problem in Equation (11) by adjusting M\* to the soft-collision constraints on common resources. The pseudocode for SC-M\* is presented in Algorithm 2, where critical commands relative to the soft-collision constraint are underscored. In this algorithm, Lines 1–7 initialize each vertex *v* in the vertex set *V* with infinite cost from the source *vs* (the cost of *vs* itself is zero), set dissatisfying experience to zero and make collision set *Ck* empty. The initial open list contains *vs* only (Line 8). In each iteration, SC-M\* expands the first-ranked vertex *vk* in the open list ordered by the total cost *vk*.*cost* + *heuristic*[*vk*] (Lines 10 and 11). The algorithm terminates and returns the result if the expanded *vk* is the destination *vd* (Lines 12–14) or jumps to the next iteration if immediate collision occurs at *vk*, i.e., *ψ* (*vk*) = ∅ (Lines 15–17). Line 18 constructs the *limit neighbors Vnbh k* of *vk* using Equation (4). For each vertex *vl* in *Vnbh k* (Line 19), it adds *vl* to the descendant set *Vk* of *vk* (Line 20), updates the dissatisfying experience of *vl* using Algorithm 1 (Line 21), and merges the immediate collision at *vl* to its soft-collision set *Cl* (Line 22). On top of the new collision set of *vl*, SC-M\* backpropagates to update all the affected ancestor vertexes from *vl* (see Equation (3)) and adds them back to the open list for re-expanding (Line 23). After this collision set updating operation, if *vl* is free from collision and has improved cost, the algorithm accepts the new cost by save the trace-back information and adding *vl* to the open list for expansion (Lines 24–28). This process repeats until the open list is empty (Line 9) when no solution exists or the optimal solution is found (Lines 12–14).