**1. Introduction**

The development of infectious diseases in animals is often accompanied by the proliferation of complex concomitant microorganisms in addition to the development of the main pathogen. In particular, mycoses of insects may develop as mixed infections when opportunistic bacteria are actively involved in the pathogenesis. This occurs due to tissue damage [1] and deregulation of host immune reactions in response to the pathogenic fungi [2,3]. Direct and indirect interactions between fungi and bacteria may lead to both antagonistic and synergistic e ffects on survival [2,4–6]. In addition, interrelations between fungi and bacteria in insect hosts may be mediated by complex environmental factors, such as temperature, chemicals, or parasitoids, that have an influence on the outcome of the disease [7,8]. However, these immune-ecological studies are just beginning to develop.

Temperature is one of the crucial factors that influences the development of mycoses and bacterioses in insects. Temperature acts on both microorganism growth and on insect immune and

behavioral reactions [9–14]. Entomopathogenic ascomycetes usually have optimal growth between 20–30 ◦C [15]. In contrast, many bacteria that are associated with terrestrial insects exhibit more active growth between 28–37 ◦C [16,17]. In many cases, host cellular and humoral antifungal reactions and resistance to fungi are increased with a short or prolonged elevation of temperature [18–24]; however, cold stresses may also activate antifungal systems [18]. Insect antibacterial responses are also dependent on environmental temperatures [25–27] and elevated temperature often promotes bacterial infection [16,17]. There are examples of increased antibacterial responses in insects under short-term or prolonged exposure to low temperatures [16,26,28,29]. Importantly, in a state of cold diapause, cellular immunity continues to work [30] and changes in microbiome composition, immune response and susceptibility to fungal and bacterial infections may also occur [27,31]. It is likely that the outcome of complex infections may be shifted toward mycoses under low temperatures and toward bacterioses under high temperatures. However, the changes in immune response and microbiota composition during complex infections under different temperature conditions are insufficiently understood.

Various antimicrobial peptides (AMPs) of insects have key roles in both antibacterial and antifungal responses [32]. Some AMPs, such as gallerimycin and galiomycin, which are regulated by the Toll immune signaling pathway, exhibit activities against filamentous fungi, but not against bacteria [33,34]. Many AMPs (e.g., cecropins, gloverins, lysozymes) synthesized via the IMD and Toll pathways have a broad spectrum of activities predominantly against gram-positive and gram-negative bacteria as well as against fungi [35,36]. It is likely that AMPs control the proliferation of bacteria during the development of mycoses. In fact, the level of AMP gene expression clearly responds to changes in the microbial community during fungal infections [3]. Moreover, AMP gene expression is dependent on temperature [23,29].

The expression of apoptosis, reactive oxygen species (ROS) and stress-related genes may be crucial in the development of infections caused by entomopathogenic fungi and concomitant bacteria. In particular, a key regulator of programmed cell death, inhibitor of apoptosis (IAP), has paramount physiological importance, including in the antifungal response as was recently shown by Zhang and coworkers [37]. RNAi-mediated knockdown of the IAP homologue in locusts led to a decrease in the total hemocyte count, a degeneration of the gut, a shift in the microbiota, and increased susceptibility to fungal infection. In addition, IAP is involved in immunity to bacterial infections, as shown for *Drosophila* [38]. The generation of ROS has a large impact in reactions against different pathogens [39], as well as in maintaining microbial homeostasis, especially in the insect gut [40–42]. The main source of ROS in insect hemolymph is the prophenoloxidase cascade. As a result of its activation, ROS (primarily semi-quinone radicals and H2O2) are formed [43–45]. In the gut, fat body and in other tissues, the formation of ROS occurs with the participation of members of the NADPH oxidase (NOX) family, such as dual oxidases (DUOX). The enzyme generates superoxide and H2O2, which are powerful oxidants that exhibit microbicidal activity [40]. Both fungal and bacterial infections led to changes in DUOX activity in the gut and hemocoel tissues [2,3,42]. RNAi knockdown of the DUOX system caused a decrease in ROS and uncontrolled proliferation of bacteria [42]. Heat shock proteins (HSPs) have functions in protein folding and unfolding, and participate in immune signaling pathways and other processes [46]. HSPs are important stress markers, which sense different thermal actions, diapause formation [47] and infections [12].

It is important to note that entomopathogenic fungi produce various secondary metabolites and enzymes (oosporeins, destruxins, different proteinases, AMPs) for inhibiting both host immune responses and competitive microorganisms [36,48,49]. The set of enzymes and secondary metabolites present is significantly different between fungal species and depends on host and habitat specificity. As a rule, generalist species have a broader spectrum of metabolites compared to species with restricted host ranges [50–52].

The ascomycete *Cordyceps militaris* is characterized by a restricted host and habitat range and has a highly reduced number of genes involved in secondary metabolism and the synthesis of proteases compared to generalists such as *Metarhizium robertsii* and *Beauveria bassiana* [50,53]. This fungus mainly infects forest lepidopterans (Lepidoptera, Macroheterocera) in the larval and pupal stages which are located in the soil, forest flour and fallen wood [54–56]. Previously, natural infections of insects with *C. militaris* were studied insufficiently. It is known that larvae and pupae could be infected by topical application with ascospores or conidia in a laboratory [22,57]. However this method is difficult to reproduce, and the outcomes strongly depend on the physiological state of the host [22]. In contrast, injection of lepidopteran larvae and pupae with blastospores or conidia has led to more stable development of the mycosis [55,58,59]. Importantly, spontaneous bacterial infections have been constantly documented after infection of *C. militaris* with insects in laboratory conditions [22,58,59]. Therefore, *C. militaris* is a convenient model to study fungal–bacteria interactions in insects. The optimal temperature for mycelial growth of *C. militaris* palearctic isolates is approximately 20 ◦C [22,60]. In a previous study [22], we showed in a model insect, the wax moth *Galleria mellonella,* that larvae in a state of facultative diapause induced by a low temperature (15 ◦C) are most susceptible to the fungus. Mycosis successfully developed after injection with *C. militaris* blastospores at 15 ◦C. By contrast, at 25 ◦C (active state), larvae were able to overcome the infection and complete metamorphosis, although, the infection may persist in pupae and adults and could still be activated by a low temperature. Activation of the antifungal response (encapsulation and phenoloxidase activity) in response to *C. militaris* infection was observed in wax moth larvae at 25 ◦C, while inhibition of these parameters occurred at 15 ◦C. We suggested that *C. militaris* uses fewer universal tools for evasion and inhibition of host immunity compared to generalists fungi, such as *Metarhizium* and *Beauveria,* that induce prolonged mycosis development, persist in hosts and have a specialization in killing dormant insects with reduced immune activity [22,59]. Moreover, because we registered spontaneous bacterioses in the wax moths post injection of *C. militaris* blastospores and conidia, we hypothesized that *C. militaris* has poorly developed mechanisms for manipulating the host microbiota and requires a low temperature for its normal development.

In the present study we investigated the microbial communities of the wax moth larvae hemolymph and midgut, as well as the expression of AMP, apoptosis, ROS and stress-related genes in the wax moth midgut and fat body after injection with *C. militaris* and incubation under two temperatures, 15 ◦C (state of facultative diapause) and 25 ◦C (active state). We found significant changes in these parameters in response to infection at different temperatures, which support the hypothesis mentioned above.

#### **2. Material and Methods**

#### *2.1. Fungi and Insects*

*C. militaris* isolate CNAp (GenBank No MF073255.1), from the microorganism collection of the Institute of Systematics and Ecology of Animals SB RAS, was used in this work. Conidia had been stored at −80 ◦C since 2015. For infections, conidia were cultivated on Sabouraud dextrose agar with yeast extract (2.5 g/L) (SDAY) for 22 days at 23 ◦C and a photoperiod of 8:16 (light:dark). Conidia were suspended in saline (0.9% NaCl) without any detergents and filtered through a sterile cloth to remove mycelial clumps. Concentrations of conidia were determined using a Neubauer hemocytometer. A Siberian line of wax moth larvae was maintained on artificial media as described previously [61]. Larvae of the sixth instar were used in experiments.

#### *2.2. Procedures for Infection and Bioassays*

Larvae were injected with 4 μL of a suspension containing 1250, 2500 or 5000 conidia. Control larvae were injected with saline. Punctures were made between the sixth and seventh abdominal segments using a microinjector with an insulin syringe. The needle was sterilized with 96% ethanol before each injection. Infected and control larvae were placed at two constant temperatures (25 ◦C and 15 ◦C) immediately after injection. Larvae were maintained in 90 mm glass Petri dishes (12 larvae per dish) with artificial media (3 g per one Petri dish) in the dark. Temperature in the Petri dishes at such an insect density corresponded with environmental temperatures. Ventilation of the Petri dishes and

registration of mortality was conducted every day over 10 days. To determine the causes of death, cadavers were placed on moist filter paper in the Petri dishes and maintained at the temperatures indicated above. Three replicates (one replicate = 12 larvae) were used to assay mortality after injection with each dose, and the whole experiment was repeated twice.

For detection of hyphal bodies and bacteria in the hemolymph of infected larvae, we used gradient centrifugation of the hemolymph in Percol followed by electron transmission microscopy as described previously [59]. To determine the yield of conidia on the larvae, the cadavers were incubated for 30 days in moist chambers at 25 ◦C and 15 ◦C. Then, each cadaver was placed in a tube with 20 mL of a 0.1% water-Tween 20 solution and vortexed for 3 min until the mycelia and conidia were completely washed off. Conidia were counted using a hemocytometer and the concentrations were calculated for each cadaver.

#### *2.3. Bacterial Colony Forming Unit (CFU) Counts*

At 96 h after injection with a dose of 2500 conidia per larvae, control and infected insects were surface sterilized by 3% H2O2 and 70% ethanol. Forty five μL of hemolymph from three larvae were placed in 100 μL of 150 mM cool NaCl and immediately homogenized using an ultrasonic homogenizer (Sonopuls, Bandelin electronic GmbH & Co. KG, Berlin, Germany). Midguts were pooled in the same NaCl (one sample = three larvae) and homogenized by the same technique. Samples were diluted with the same NaCl by 10, 100, and 1000-fold and a 100 μL aliquot was plated on media (Bile esculin azide agar for enterococci and Endo agar for enterobacteria) in 90 mm Petri dishes. The cultures were incubated for 2 days at 35 ◦C and then CFUs were counted. CFU counts were calculated for each midgut or 10 μL of hemolymph. A total of 5–6 samples from each treatment were used for analysis.

#### *2.4. Analysis of Bacterial Communities*

At 96 h post-treatment, infected (2500 conidia) and control larvae were surface-sterilized by 3% H2O2 and 70% ethanol and dissected. Midguts with content were isolated and frozen in liquid nitrogen (one sample = 5 midguts). In addition, decomposed cadavers (6–7 d post infection) were analyzed. Whole cadavers were frozen in liquid nitrogen (one sample = 3 whole bodies). Three biological replicates from each treatment were used.

DNA was isolated using a DNeasy PowerSoil DNA Isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The 16S rRNA region was amplified with the primer pair V3–V4 combined with Illumina adapter sequences [62]. PCR amplification was performed as described previously [63]. A total of 200 ng of PCR product from each sample was pooled together and purified using a MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). The obtained libraries were sequenced with 2 × 300 bp paired-end reagents on a MiSeq (Illumina Inc., San-Diego, California, USA) in the SB RAS Genomics Core Facility (ICBFM SB RAS, Novosibirsk, Russia). The sequencing data reported in this study were submitted to GenBank under the study accession PRJNA650299.

Raw sequences were analyzed with the UPARSE pipeline [64] using Usearch v11.0. The UPARSE pipeline included a merging of paired reads, read quality filtering, length trimming, merging of identical reads (dereplication), discarding singleton reads, removing chimeras, and operational taxonomic unit (OTU) clustering using the UPARSE-OTU algorithm. The OTU sequences were assigned a taxonomy using the SINTAX [65] and 16S RDP training set v.16 [66]. The final dataset included 384,875 reads (mean ± SE = 20,532 ± 630 per midgut sample and 46,162 ± 1453 per cadaver sample, see Dataset). All rarefaction curves showed a trend of approaching the saturation plateau (Figure S1), which indicated a reasonable volume of the sequenced reads.


Food Health with Increased Probiotic Survival During Storage

*Fatemeh Shoaei*

#### **Abstract**

In recent years, due to the increasing concern of consumers about their food health. Pay attention to foods not only as a source of nutrients but also as promoters of health and wellness-hence the increase in demand for foods that have active or functional ingredients (especially natural ingredients). They increase nutritional value and nutritional health. Changes in food consumption, disorder the intestinal microbial system. Maintaining the health benefits of consuming beneficial bacteria that are present in the intestinal system. Probiotics are essential for improving intestinal microbial homeostasis. Probiotics are living microorganisms that, if recommended in sufficient quantities, can have positive effects on human health. Lowers cholesterol, improves lactose intolerance, increases nutritional value and prevents cancer. Probiotics are unstable during storage and the gastrointestinal tract (pH and bile salts). For this reason, the survival of probiotic cells and the absence of changes in the sensory properties of the product during storage are of have fundamental importance. Encapsulation and co-encapsulation with prebiotics are often a good way to increase the resistance of probiotic bacteria to difficult conditions and their survival. This leads to improved production of probiotic products and increased food health in the world.

**Keywords:** probiotic, encapsulation, survival, functional foods, intestine

#### **1. Introduction**

Probiotics are living microbes that must be in the number of 106 log Cfu/gr at the time of entering the intestinal environment to have their beneficial effects on human health [1]. Probiotics are usually one or mixture of several microorganisms, when consumed by humans or animals, they have many beneficial effects on the body. Therefore, researchers are trying to add it to food the survival of probiotics. Since dairy products are Suitable for the transmission and survival of probiotic bacteria, most probiotic bacteria enter dairy products such as yogurt, Dough and various dairy desserts [2]. Due to the presence of animal cholesterol, lactose intolerance and sensitivity of some people to dairy products, it is necessary to study probiotic products with new flavors and non-dairy products, especially herbal [2]. Researchers are always looking for ways to improve the survival of probiotic bacteria to increase the survival of probiotics in Unfavorable environmental conditions, including during the production and storage of food products, as well as acidic and biliary conditions in the gastrointestinal tract [3, 4]. it is recommended that probiotic foods contain at least 108 log Cfu / gr at the time of consumption [1]. One of

the newest methods that has had significant effects in this regard is the microencapsulation of bacteria in different ways by different coatings. From a microbiological point of view, microencapsulation is the monopoly of bacterial cells with hydrocolloid coatings that are used to separate and protect it from the external environment. The main purpose of microencapsulation is to increase the survival of bacterial cells during storage, passage and release in the gastrointestinal tract [5]. Symbiotic, on the other hand, are a mixture of probiotics and prebiotics that affect the health of the host, selectively stimulating probiotic growth. And activate, metabolize beneficial intestinal bacteria, thus improving beneficial effects on the host [5]. By adhering to intestinal epithelial cells, probiotics can improve micro biota and digestion. Provides protection against pathogens and carcinogenic properties [1]. In this chapter, a brief description of probiotics and their increase in survival in different ways are depicted.

### **2. Definition and role of probiotics**

The word probiotic, meaning to live, is derived from the Greece language. For more than 4,000 years, lactic acid bacteria have been used to increase the shelf life of various foods through fermentation processes. In 1857, Pasteur discovered that microorganisms were responsible for fermenting milk and play role in the production of lactic acid, which was eliminated by boiling milk. The Clinton Processing Company (USA), for first in 1881, produced lactic acid by fermentation Process. The idea of using one-way primer cultures in the decades 1940 and 1950 consisting of lactic acid bacteria was evolving and becoming commercially available [3]. Researchers have observed widespread in the decades 1980, use of lactic acid bacteria in biomedicine, food preservation, food processing, and fermentation and animal husbandry [3]. In 2002, the World Health Organization provided a comprehensive definition of probiotics: Probiotics are living microorganisms that, if taken in sufficient amounts, have beneficial effects on host health [6, 7].

The gastrointestinal tract contains millions of bacteria, the balance of which is very important for the gastrointestinal tract and the functioning of the immune system. During the day, the intestinal microflora is exposed to various stresses (use of antibiotics, anxiety and food poisoning). Which can create an imbalance between the so-called (good) and (bad) bacteria. However, eating foods that contain extra probiotics can increase the level of healthy or "good" bacteria in the gut which can change the microbial balance in this way [8]. Probiotics are classified as "safe" bacteria because their metabolism is saccharolytic. Probiotics are classified as "safe" bacteria because their metabolism is saccharolytic, (That is, they break down carbohydrates in the large intestine to produce short-chain fatty acids). This process is also known as fermentation and is beneficial to the host [8].

#### **3. Selection criteria and requirements for probiotic strains**

In the process of selecting probiotic strains for consumption, it must be approved by the WHO, FAO and the European Food Safety Authority (EFSA) for their safe status (GRAS) and (QPS) [4]. Recommended properties for a probiotic bacterium that have shown good and prominent effects on human health include [9]:


**5. Antimicrobial peptides in** *Escherichia coli* **clinical trials**

Naturally occurring cationic antimicrobial peptides (AMPs) and their mimics form a diverse class of antibacterial agents currently validated in preclinical and clinical settings for the treatment of infections caused by antimicrobial-resistant bacteria [56]. Clinical trials have been cautious of toxicity at these doses, as other peptide-based antibiotics (such as colistin) are toxic in high concentrations.

AMPs can be classified into three distinct approaches based on their clinical development: (i) direct antimicrobial effect on the cell membrane, (ii) indirect antimicrobial activity through immune regulation, and (iii) blocking the intracellular functions. Among forty-four peptides that have been undergoing clinical and preclinical trials, 35 target the bacterial cell membrane directly, eight affect the immune system to regulate the response of the body to infection, and three act on intracellular targets. Sixteen of these that show broad-spectrum activity, have been considered for treatment of Gram-negative infections [50].

Until 2020, FDA approves seven AMPs for clinical usages. Vancomycin and dalbavancin (vancomycin derivative) block bacterial wall synthesis, while oritavancin and telavancin (other derivatives) have both membranolytic and cell wall synthesis inhibition actions. Gramicidin D is a linear peptide that forms in the membrane. Daptomycin, colistin (polymyxin E), and cyclic lipopeptide lysis the membrane [7]. Among the AMPs that FDA approves, Buforin II that binds to nucleic acid, Colicin E1 and Bac8c that disrupt the bacterial membrane, specifically are used for *E. coli* treatment [41]. LPS in the outer membrane of Gram-negative bacteria, act as a protective shield and prevent from transporting the large glycopeptide antibiotics, such as vancomycin to intracellular targets. Recent studies have shown that vancomycin, when given together with other AMPs acts against vancomycin-resistant Grampositive bacteria [57]. Corbett et al. reported SPR741 (polymyxin B derivative) that potentiates the efficacy of conventional antibiotics on Gram-negative bacteria whose spectrum of activity is limited because of bacterial outer membrane permeability obstacles [58]. Studies show the MICs in eight out of 35 antibiotics while combined with SPR741 were reduced 32 to 8000-fold against *E. coli* and *Klebsiella pneumoniae*. Interestingly, based on research *E. coli* becomes susceptible to vancomycin under cold stress. Moreover, the mechanism of vancomycin action to eradicate *E. coli* is similar to the Gram-positive bacteria, which is through inhibition of peptidoglycan biosynthesis [59]. It was also shown that silver ions can increase membrane permeability of Gram-negative bacteria and can potentiate the Gram-positive-specific antibiotic vancomycin against Gram-negative bacteria [60].

The permeability barrier of the outer membrane of Gram-negative bacteria limits the efficacy of vancomycin. So, a synergistic mechanism of action for P-113 derivatives (e.g., Bip-P-113, Dip-P-113, and Nal-P-113) and vancomycin was proposed. Study results showed, however, P-113 derivatives could perturb the outer membrane of Gram-negative bacteria and increase vancomycin entry into the resistant species. In addition, P-113 derivatives bind to the extra hydrophobic motif of lipid A and neutralize LPS protective actions [61].

Employing long-chain amino acid sequences increases the output cost of peptides and thereby the cost of research; hence, synthetic short-chain cationic peptides with potential antimicrobial activity have been attempted [62].

In particular, Indolicidin, a tridecapeptide isolated from the cytoplasmic granules of bovine neutrophils, was reported to exhibit membrane permeabilization effects and antimicrobial activity against Gram-negative and Gram-positive bacteria, fungi, HIV-1 virus, and protozoa [63, 64].

Enteroaggregative *Escherichia coli* (EAEC), an emerging foodborne pathogen, is implicated in endemic and epidemic diarrheal episodes. Multidrug resistance toward the antibiotics of first-line empirical therapy (fluoroquinolones and b-lactams) has been evident globally among the EAEC isolates [65, 66]. Indolicidin as an antimicrobial peptide exhibited a complete elimination of multidrug-resistant EAEC isolates in the time-kill kinetic assay by 2 h pi, while meropenem represented a similar effect after 60 min. These results indicate a unique advantage of AMPs over conventional antibiotics for better treatment of resistant antibacterial species. Studies about the antimicrobial effect of Indolicidin against MDR-EAEC strains in the *G. mellonella* larval model reported that Indolicidin is stable at high temperatures, in the presence of proteinase K and at physiological concentration of cationic salts. In addition, results demonstrated that while Indolicidin could eliminate MDR-EAEC completely, to be safe for commensal gut flora and eukaryotic cells [67].

Peptide 35,409 contains 20 amino acid residues and has been exhibited antibacterial activity against *Escherichia coli* ML35 at 22 ìM minimum inhibitory concentration (MIC). In spite, this peptide did not have cytotoxic activity against human cell lines such as HeLa and HepG2, showing hemolytic effects on human red blood cells at 1.5 μM minimum concentration. According to the low selectivity of peptide 35,409 at the therapeutic index for *E. coli* ML35 (calculated equal 0.045), its therapeutic use is restricted [7]. However, considering the essential need for developing new compounds with activity against microorganisms, 17-residue-long peptide 35,409-1 was obtained from peptide 35,409. This shorter peptide synthesized chemically with less charge but had greater hydrophobicity and amphipathicity properties than the original sequence. Peptide 35,409-1 sequence could inhibit *E. coli* multiresistant isolates and seemed to be highly selective for Gram-negative *E. coli* bacteria because it does not act against Gram-positive bacteria or human red blood cells. Peptide 35,409-1 permeabilizes into the bacterial membrane and leads to *E. coli* cytoplasmatic content leakage [7]. The interactions of AMPs with membranes have been very considered due to serious implications regarding AMPs therapeutic advantages [68, 69]. Five of the seven AMPs that are approved by the FDA are active on the membrane [70], so this mechanism must be surveyed for 35,409-1 profoundly. In comparison to conventional antibiotics, peptide 35,409-1 exhibited a lower potential for inducing resistance significantly. Therefore, it seems that peptide 35,409-1 could be a potential candidate for clinical therapy usages or developing highly selective new AMPs against Gram-negative *E. coli*. The stability in the presence of sera, efficacy against MRD- *E. coli*, and low inducing resistance of peptide 35,409-1 propose its significant clinical advantages for overcoming recent antibacterial *E. coli* resistance [71].

Some substitutes of histidine-rich antimicrobial peptide P-113 were developed recently [72]. Among them, Bip-P-113 showed serum proteolytic stability, enhanced salt resistance, peptide-induced permeabilization, zeta potential measurements, LPS condensed, and *in vitro* and *in vivo* neutralizing activities against LPS [70].

Polymyxin B and its derivatives are able to interact with anionic LPS in the outer membrane (OM) of Gram-negative bacteria. The derivatives of polymyxin B act as "permeabilizers" or "potentiators" and sensitize bacteria to antibiotics, Moreover, reinforce the action of other antibiotics [58]. Studies showed synergistic effects between colistin and bacteriocins that led to inhibit Gram-negative bacteria and reduction of antibiotic toxicity [73]. Ionic silver (Ag+) in silver nitrate salt (AgNO3) *Antibiotic Resistance among* Escherichia coli *Isolates, Antimicrobial Peptides and Cell Membrane… DOI: http://dx.doi.org/10.5772/intechopen.101936*

was found to increase the permeability of the bacterial outer membrane and sensitize Gram-negative bacteria to vancomycin [60]. Synergistic effects also have been proved between highly membrane-active AMPs and intracellular targeting antibiotics [61].

Stationary phase bacteria are much more resistant than exponentially growing cells to killing by conventional antibiotics, such as ampicillin, tetracycline, ciprofloxacin, and streptomycin [74]. The susceptibility of *E. coli* to human α-defensin 5 (HD5ox) was shown to be lower in the stationary phase compared to mid-log phase cells [75]. The authors suspected a correlation between bacterial susceptibility and altered cellular morphology [39]. Treated β-lactam resistant *E. coli* with ampicillin displayed changes in cell elasticity, membrane permeability, nanoscale morphology, and hydrophilic/hydrophobic interactions. Moreover, different ampicillin-resistant *E. coli* strains exhibited different traits phenotypically [76]. Therefore, exploring the interactions of conjugated molecules with wild-type and ampicillin-resistant bacterial strains is crucial since the cell drug interaction is highly dependent on the type of strains and the drug molecules applied [29].


*Article* **Flotation of Copper Ores with High Cu/Zn Ratio: Effects of Pyrite on Cu/Zn Separation and an Efficient Method to Enhance Sphalerite Depression**

**Kosei Aikawa 1,\* , Mayumi Ito 2 , Nodoka Orii 1 , Sanghee Jeon 2 , Ilhwan Park 2 , Kazutoshi Haga 3 , Taro Kamiya 4 , Tatsuru Takahashi 5 , Kazuya Sunada 6 , Taisuke Sakakibara 7 , Tatsuhiro Ono 4 , Refilwe S. Magwaneng 4 and Naoki Hiroyoshi 2**

**Abstract:** Porphyry copper deposits are important sources of copper and typically processed by flotation to produce copper concentrates. As mining areas become deeper, the amounts of impurities, such as sphalerite, can be increased in copper ores, so the appropriate depression of sphalerite floatability should be achieved to obtain saleable copper concentrates. In this study, the flotation behaviors of chalcopyrite and sphalerite in model samples mimicking copper ores with high Cu/Zn ratios (i.e., the ratio of chalcopyrite/sphalerite = 13:1) were investigated with zinc sulfate as a depressant for sphalerite. In addition, the effect of pyrite—a major gangue mineral in copper ores on the depression of sphalerite floatability with zinc sulfate was examined. When sphalerite and chalcopyrite coexisted, the floatability of the former was effectively depressed by zinc sulfate (Zn recovery: <12%), whereas the presence of pyrite promoted the release of Cu2+ due to the galvanic interaction with chalcopyrite, which resulted in the elimination of the effectiveness of zinc sulfate in depressing sphalerite floatability (Zn recovery: >90%). Despite the presence of much higher amounts of chalcopyrite and pyrite than sphalerite, the application of nitrogen (N2 ) gas limiting the galvanic interaction between pyrite and chalcopyrite by reducing the dissolved oxygen (DO) concentration in the system effectively depressed the floatability of sphalerite (Zn recovery: <30%).

**Keywords:** flotation; porphyry copper deposits; chalcopyrite; sphalerite; pyrite; galvanic interaction


**5. Pathological and physiological relevance of redox signal-regulated TRPM2 activation**

that human TRPA1 and monkey TRPA1 are not activated by cold stimulus whereas rat TRPA1 and mouse TRPA1 are. Mutation of Gly878 in S5 of rat TRPA1 to Val that is present in human TRPA1 abolished cold sensitivity. A proline hydroxylation-deficient mutant of human TRPA1 (Pro394Ala) was activated by cold stimulation in the presence of low concentrations of H2

(0.1 μM), but wild-type TRPA1 showed no activation [50]. PHD inhibitors could result in activation of wild-type TRPA1 by cold temperature, which is attenuated by mitochondrial ROS

wild type, and a PHD inhibitor increased the response of wild-type TRPA1. In contrast, mouse wild-type TRPA1 was activated by cold temperature and effects of the PHD inhibitor and ROS scavengers were also observed in a mouse TRPA1 clone, confirming that the inhibitory effect of proline hydroxylation was conserved between human and mouse. Several chemotherapeutic agents can cause cold allodynia [51–53]. For instance, the drug oxaliplatin potentiates H2

TRPA1 is also reported to play a physiological role in artery vasodilation, although its activity varies among different types of vascular beds. In cerebral arteries, TRPA1 expressed in the endothelium mediates "endothelium-dependent" vasodilation. ROS such as superoxide anions

cerebral microcirculation [54]. TRPA1 mRNA expression was observed in the endothelium of cerebral arteries, but not in peripheral vascular beds in renal, coronary and mesenteric arteries [55]. TRPA1 protein in the endothelium of cerebral arteries preferentially colocalizes with NOX2, a ROS-generating enzyme, in fenestration of internal elastic lamina where plasma membranes of endothelium and smooth muscle cell are in close contact. ROS generated by NADPH-induced NOX activity led to cerebral artery vasodilation following TRPA1 activation. This vasodilation could be abolished in a variety of ways, including by a NOX inhibitor,

of the intermediate conductance Ca2+-sensitive K channel (IK). Moreover, vasodilation could be mimicked by application of 4-hydroxy-nonenal (4-HNE), a product of lipid peroxidation. These results suggest that ROS-derived lipid peroxidation products activate TRPA1, leading to cytosolic Ca2+ elevation, which in turn activates intermediate conductance Ca2+-sensitive K channels (IK) and membrane hyperpolarization in the endothelium. This change in membrane potential is propagated through gap junctions to smooth muscle cells to promote additional vasodilation. On the other hand, in peripheral arteries, TRPA1 expressed in primary sensory neurons is reportedly involved in "neurogenic" vasodilation. TRPA1 expressed in sensory neurons likely mediates vasodilation in peripheral arteries because TRPA1 is not expressed in the endothelium of peripheral arteries [56]. Topical application of cinnamaldehyde, a TRPA1 agonist, onto mouse ears caused vasodilation in wild-type mice, but not in TRPA1KO mice. TRPA1 agonist-induced vasodilation could be attenuated by a CGRP antagonist, a nonselective NOS or a neuronal NOS (nNOS) inhibitor, suggesting the possible involvement of CGRP and NO release from sensory neurons. In addition, TRPA1 agonist-induced vasodilation is

Taken together, TRPA1 expressed in the endothelium of central arteries is involved in endothelial-dependent vasodilation, whereas TRPA1 expressed in vagal and primary sensory neu-

induced responses in mouse DRG neurons possibly by inhibiting PHD [50].

O2

mediated by formation of superoxide and peroxynitrite.

rons functions in neurogenic vasodilation in peripheral arteries.

O2

) are known to cause vasodilation, leading to increases in

, deferoxamine, which inhibits the Fenton reaction that

), the TRPA1 inhibitor HC-030031 and TRAM34, a blocker

scavenging. H2

(O2 − O2

210 Redox - Principles and Advanced Applications

) and hydrogen peroxide (H2

catalase-mediated degradation of H2

generates hydroxyl radical (OH<sup>−</sup>

O2

O2 - TRPM2 activation induced by H2 O2 promotes cell death due to sustained elevation of intracellular Ca2+ [17] and also increases inflammation and tissue injury [18]. Therefore, numerous reports have shown the implications of TRPM2 activity in oxidative stress-induced cell death of many kinds of tissues and cells [57]. Endogenous and exogenous agents that promote ROS production such as TNFα, β-amyloid, and neurotoxin MPTP also cause neurotoxicity in a TRPM2-dependent manner [17, 58–61]. In addition, ischemia/reperfusion (I/R) injury is a major pathological situation involving unregulated ROS production in stroke, brain trauma, cardiac arrest and other disorders and diseases. Ischemia is the restriction of blood flow that diminishes oxygen (hypoxia/anoxia) and glucose supplies to tissues. When blood supply is restored to the affected tissues, secondary effects associated with I/R injury can occur wherein inflammatory agents and ROS that can cause tissue damage are produced. ROS-evoked TRPM2 activity was reported to aggravate tissue damage in the presence of I/R injury [62–64], and this situation can be explained in part by increased neutrophil migration toward affected tissues [63, 65].

On the other hand, more recent reports suggest that TRPM2 could exert protective roles in I/R injury. One group reported that TRPM2 channels are expressed in the sarcolemma and transverse tubules of adult cardiac myocytes [66, 67]. They also showed that TRPM2KO heart is vulnerable to I/R injury and I/R-induced prolongation of action potential duration was enhanced in TRPM2KO myocytes compared with wild type [67]. Proteomic analysis of I/Raffected ventricles from wild-type and TRPM2KO mice revealed that, relative to wild-type heart, mitochondrial respiratory complex dysfunction in TRPM2KO heart is more severe and is associated with altered expression levels of proteins localized in the mitochondrial inner membrane [68]. Under I/R conditions, TRPM2KO myocyte mitochondria showed lower mitochondrial membrane potential, Ca2+ uniporter activity, ATP levels and oxygen consumption as well as higher ROS levels compared to those seen for wild type. These data suggest that Ca2+ supplied by TRPM2 activity can have a protective role by ameliorating mitochondrial dysfunction and diminishing mitochondrial ROS levels in I/R situations. Similar results were recapitulated in intact and TRPM2-depleted SH-SY5Y neuroblastoma cells treated with the ROS-generating chemotherapeutic agent doxorubicin [69]. Interestingly, these diminished parameters of mitochondrial function in TRPM2KO myocytes were also seen under normoxic conditions [68]. Considering these results, TRPM2 may contribute to the maintenance of basal mitochondrial bioenergetics by supplying Ca2+, which has numerous functions in mitochondrial metabolism [70].

TRPM2 also has roles in inflammation and infection as evidenced by its expression in immunocytes (**Table 1**). In inflammatory situations, ROS are produced and ROS-evoked TRPM2 activity can aggravate inflammation. At sites of inflammation, phagocytes such as neutrophils and macrophages digest deleterious agents and increase oxygen consumption that enhances NOX-mediated production of ROS, phagocytized agents can then be cleared by ROS. TRPM2 is widely expressed in leukocytes, including lymphocytes, neutrophils, monocytes/macrophages, dendritic cells, microglia and mast cells [19, 25, 71–75]. TRPM2 activation by ROS is

**Table 1.** Reports showing the implication of TRPM2 in immune reactivities using *in vivo* and *in vitro* models.

reported to worsen inflammation by elevating cytokine release. Indeed, TRPM2 activation in mouse monocytes elevates H2 O2 -induced release of the neutrophil-attracting chemokine macrophage inflammatory protein-2 (CXCL2) through activation of the Ca2+-dependent tyrosine kinase (Pyk2) and nuclear factor κB (NFκB), and this elevation was attenuated in TRPM2KO monocytes [73]. Supporting these *in vitro* experiments, wild-type mice showed elevated CXCL2 levels and neutrophil infiltration in inflamed colon tissues with ulcerative colitis in a dextran sulfate sodium (DSS)-induced experimental colitis model. These conditions are suppressed in TRPM2KO mice, suggesting that TRPM2 activation in monocytes increased chemokine release and neutrophil migration followed by aggravation of inflammation. In our experiments, TRPM2 activity elevated release of the cytokines granulocyte colony stimulating factor (G-CSF), interleukin-1α (IL-1α), and CXCL2 from macrophages [19]. TRPM2-mediated exacerbation of inflammation also explains pathological conditions associated with chronic pain. TRPM2KO mice showed impaired pain responses in inflammatory pain induced by intraplantar injection of formalin or carrageenan in mice, and neuropathic pain models induced by partial sciatic nerve ligation or spinal nerve transaction [76]. TRPM2 activity in macrophages and microglia is suggested to aggravate chronic pain through CXCL2 release and neutrophil infiltration toward inflamed tissues.

Nod-like receptor family pyrin domain containing-3 (NLRP3) inflammasomes are activated by several conditions of cellular stress, including microbial products, particulate substances, elevated plasma glucose levels that accompany metabolic disorders, intracellular [K+ ] reduction and [Ca2+] i increase [77, 78]. NLRP3 inflammasome is a complex of NLRP3, apoptosisassociated speck-like protein (ASC) and caspase-1 to activate caspase-1 that in turn promotes release of pro-inflammatory cytokines, IL-1β and IL-18. TRPM2 is reported to be involved in the inflammasome activation in macrophages/monocytes. Charged liposomes evoke ROS production, caspase-1 activation and IL-1β release from bone-marrow-derived macrophages (BMDM) [79]. Caspase-1 activation and IL-1β release evoked by liposomes are attenuated by TRPM2 deficiency, although mitochondrial ROS production is comparable to that of wild type. Thioredoxin-interacting protein (TXNIP) is known to bind to NLRP3 and participate in ROS-dependent inflammasome activation [80]. TXNIP expression is up-regulated by high glucose and is involved in ROS production induced by high glucose [81]. Treatment of U937 monocytes with high levels of glucose induced TRPM2 up-regulation, ROS production, caspase-1 activation and IL-1β release, which can be attenuated by TRPM2-siRNA or TRPM2 inhibitors [82]. TXNIP up-regulation by high glucose is also inhibited by TRPM2-siRNA. Upon activation of NOX, p47phox, a cytosolic subunit of NOX, translocates to the plasma membrane. Under high glucose conditions, p47phox translocation is increased as is its colocalization with TRPM2. In addition, co-localization of TXNIP and NLRP3 is enhanced by high glucose conditions, which could also be suppressed by TRPM2-siRNA. These data suggest that Ca2+ influx through TRPM2 can contribute to high glucose-induced ROS production as well as inflammasome activation and amplification of TRPM2 activation to further exacerbate oxidative stress in diseases such as type 2 diabetes.

An important role of macrophages, phagocytosis, is regulated by TRPM2 activity. As mentioned above, macrophages phagocytose deleterious agents such as exogenous pathogens and digest them in phagosomes by producing ROS levels that are toxic to the pathogens. This phagocytosis function is known to be enhanced by temperatures that are associated with fever [83]. Phagocytosis induced by toll-like receptor 2 agonist was enhanced by temperature elevation to febrile range in wild-type macrophages which can detect slight temperature change through TRPM2 activity in the presence of low concentration of ROS [19]. On the other hand, TRPM2KO macrophages showed no such temperature-dependent elevation of phagocytosis, suggesting that ROS generated in phagosomes increases TRPM2 sensitivity to body temperature and that TRPM2 activation by temperature increases phagocytosis. Because TRPM2 is highly localized in phagosomal membranes [84], functional cooperation between TRPM2 and NOX is suggested to increase phagocytosis. As many reports have shown roles for TRPM2 in innate immunity, the physiological roles of TRPM2 have been studied in microbial infection models. In a *Listeria monocytogenes* (Lm) infection model, TRPM2KO mice show lower serum IL-12 and IFNγ levels and a higher mortality rate than wild-type mice; these conditions can be reversed by the application of IFNγ [85]. In contrast to the DSS-induced experimental colitis model [73], Lm-infected TRPM2KO mice have CXCL2 expression and neutrophil infiltration in the spleen that is comparable to wild-type mice. Given that the ratio of activated monocyte (iNOS+ ) is decreased and IL-12 release is unchanged in an *in vitro* cytokine release assay using BMDMs from Lm-infected TRPM2KO, TRPM2 activation is suggested to have important roles in reciprocal activation among macrophages, natural killer cells, and CD8+ T cells mediated by IL-12 and IFNγ during the early phase of Lm infection [86]. TRPM2 deficiency is also

reported to worsen inflammation by elevating cytokine release. Indeed, TRPM2 activation

**Table 1.** Reports showing the implication of TRPM2 in immune reactivities using *in vivo* and *in vitro* models.

macrophage inflammatory protein-2 (CXCL2) through activation of the Ca2+-dependent tyrosine kinase (Pyk2) and nuclear factor κB (NFκB), and this elevation was attenuated in TRPM2KO monocytes [73]. Supporting these *in vitro* experiments, wild-type mice showed elevated CXCL2 levels and neutrophil infiltration in inflamed colon tissues with ulcerative colitis in a dextran sulfate sodium (DSS)-induced experimental colitis model. These conditions are suppressed in TRPM2KO mice, suggesting that TRPM2 activation in monocytes increased chemokine release and neutrophil migration followed by aggravation of inflammation. In our experiments, TRPM2 activity elevated release of the cytokines granulocyte colony stimulating factor (G-CSF), interleukin-1α (IL-1α), and CXCL2 from macrophages [19]. TRPM2-mediated exacerbation of inflammation also explains pathological conditions associated with chronic pain. TRPM2KO mice showed impaired pain responses in inflammatory pain induced by intraplantar injection of formalin or carrageenan in mice, and neuropathic pain models induced by partial sciatic nerve ligation or spinal nerve transaction [76]. TRPM2 activity in macrophages and microglia is suggested to aggravate chronic pain through CXCL2

Nod-like receptor family pyrin domain containing-3 (NLRP3) inflammasomes are activated by several conditions of cellular stress, including microbial products, particulate substances,

monocyte↓

,

O2

*In vivo* **model The effects of TRPM2 down regulation** DSS-induced colitis [73] CXCL2↓, neutrophil infiltration↓

*Listeria monocytogenes* [85] Survival rate↓, CXCL2→,

CLP-induced sepsis [87] Survival rate↓, inflammation↑,

PA i.t., CLP [84] Survival rate↓, bacterial clearance↓

inflammation↓

LPS i.p. [89] Survival rate↓, inflammation↑, CXCL2↑, TNFα↑, IL-6↑, neutrophil

*In vitro* **model Stimulus The effect of TRPM2 down regulation**

Peritoneal macrophage [19] Zymosan CXCL2↓, GCSF↓, IL-1α↓, IL-1β↓#

BMDM [76] Liposome Inflammasome activation↓, IL-1β↓,

U937 monocyte [82] High glucose Inflammasome activation↓,

BMDM [84] PA, SA Phagosomal acidification↓,

neutrophil infiltration→, iNOS+

infiltration↑, ROS production↑

O2 CXCL8 (human homolog of CXCL2)↓

TNFα→, Temp-phagocytosis↓

TNFα→, ROS production→

NOX activation↓, IL-1β↓

Bacterial clearance↓

IL-6↑, HMGB↑, bacterial clearance↓

release and neutrophil infiltration toward inflamed tissues.

in mouse monocytes elevates H2

U937 monocyte [73] H2

212 Redox - Principles and Advanced Applications

reported to increase the mortality rate in a cecal ligation and puncture (CLP)-induced polymicrobial sepsis model [87]. Moreover, TRPM2KO mice show increased bacterial burden and enhanced inflammation and injury in tissues. TRPM2-deficient BMDM show lower bacterial killing than that of wild type without noticeable effects on phagocytosis. Lipopolysaccharide (LPS) treatment of BMDM from wild-type and TRPM2KO mice revealed that heme oxygenase-1 (HO-1) induction observed in wild-type cells did not occur in TRPM2KO cells and the induction in wild-type cells is inhibited by chelating Ca2+, suggesting that [Ca2+]i increases mediated through TRPM2 lead to HO-1 expression. Bacterial killing in phagosomes is also reported to be regulated by TRPM2 activity [84]. TRPM2 deficiency increases the bacterial burden of *Pseudomonas aeruginosa* (PA) and *Staphylococcus aureus* (SA) in BMDM without affecting phagocytosis. TRPM2 functionally expressed in phagosomal membrane promotes acidification of SA-internalized phagosomes that is a hallmark of phagosomal maturation and is regarded to be crucial for degradation of phagocytosed particles and up-regulation of bacterial killing [88].

Contrary to reports showing that TRPM2 up-regulates inflammation/innate immunity, protective anti-inflammatory roles of TRPM2 were also reported in a LPS-induced lung inflammatory model [89]. After intraperitoneal injection with LPS, TRPM2KO mice had higher mortality with pronounced lung edema than did wild-type mice. Histopathology of tissues from TRPM2KO mice showed increased levels of tissue cytokines including CXCL2, TNFα, IL-6 and myeloperoxidase (MPO) activity, suggesting enhanced neutrophil infiltration in mice lacking TRPM2. ROS release and oxidative DNA damage in TRPM2KO cells are significantly larger than those in wild-type cells, and a TRPM2 inhibitor increased ROS release from wild-type cells that was equivalent to that of TRPM2KO, thus indicating the inhibitory effects of TRPM2 activity. As activity of the electrogenic NOX enzyme is known to be voltage-sensitive [90], membrane depolarization through TRPM2 is suggested to negatively regulate NOX activity, and the absence of this regulatory mechanism in TRPM2KO mice could enhance ROS production and aggravate inflammation. Nonetheless, the role of TRPM2 in immunity remains controversial, and its activation could depend on the stimulus type, particularly since immunogens can activate multiple cascades (**Table 1**). In addition, immune reactivity is strongly related to body temperature [83, 91]. Thus, the temperature dependence of TRPM2 must be considered [19]. Outcomes could also be affected depending on the extent of TRPM2 activity, e.g., Ca2+ influx through TRPM2 could be beneficial for many Ca2+-dependent events, whereas large Ca2+ influx could cause Ca2+ overload.

TRPM2 is also known to contribute to the regulation of blood glucose levels (**Figure 4**) likely because of its functional expression in pancreatic β-cells [92], which secrete insulin in response to elevated blood glucose. Although the primary pathway of glucose-stimulated insulin secretion is through ATP-sensitive K+ (KATP) channel closure and L-type voltage-gated Ca2+ channel activation to increase [Ca2+] i , many other ion channels, including TRPM2, are reported to be involved in [Ca2+] i -increases to evoke insulin secretion [93]. ROS are produced in response to extracellular signals such as insulin, cytokines, hormones and blood glucose elevation in pancreatic β-cells [94, 95]. In addition, expression of the antioxidant enzyme catalase and glutathione reductase in the pancreas is very low [96], indicating that the intracellular redox state of β-cells could be affected at a physiological level by the systemic metabolic state. Intracellular Ca2+-oscillations evoked by high glucose concentrations are attenuated in TRPM2KO pancreatic β-cells [97]. In

reported to increase the mortality rate in a cecal ligation and puncture (CLP)-induced polymicrobial sepsis model [87]. Moreover, TRPM2KO mice show increased bacterial burden and enhanced inflammation and injury in tissues. TRPM2-deficient BMDM show lower bacterial killing than that of wild type without noticeable effects on phagocytosis. Lipopolysaccharide (LPS) treatment of BMDM from wild-type and TRPM2KO mice revealed that heme oxygenase-1 (HO-1) induction observed in wild-type cells did not occur in TRPM2KO cells and the

mediated through TRPM2 lead to HO-1 expression. Bacterial killing in phagosomes is also reported to be regulated by TRPM2 activity [84]. TRPM2 deficiency increases the bacterial burden of *Pseudomonas aeruginosa* (PA) and *Staphylococcus aureus* (SA) in BMDM without affecting phagocytosis. TRPM2 functionally expressed in phagosomal membrane promotes acidification of SA-internalized phagosomes that is a hallmark of phagosomal maturation and is regarded to be crucial for degradation of phagocytosed particles and up-regulation of

Contrary to reports showing that TRPM2 up-regulates inflammation/innate immunity, protective anti-inflammatory roles of TRPM2 were also reported in a LPS-induced lung inflammatory model [89]. After intraperitoneal injection with LPS, TRPM2KO mice had higher mortality with pronounced lung edema than did wild-type mice. Histopathology of tissues from TRPM2KO mice showed increased levels of tissue cytokines including CXCL2, TNFα, IL-6 and myeloperoxidase (MPO) activity, suggesting enhanced neutrophil infiltration in mice lacking TRPM2. ROS release and oxidative DNA damage in TRPM2KO cells are significantly larger than those in wild-type cells, and a TRPM2 inhibitor increased ROS release from wild-type cells that was equivalent to that of TRPM2KO, thus indicating the inhibitory effects of TRPM2 activity. As activity of the electrogenic NOX enzyme is known to be voltage-sensitive [90], membrane depolarization through TRPM2 is suggested to negatively regulate NOX activity, and the absence of this regulatory mechanism in TRPM2KO mice could enhance ROS production and aggravate inflammation. Nonetheless, the role of TRPM2 in immunity remains controversial, and its activation could depend on the stimulus type, particularly since immunogens can activate multiple cascades (**Table 1**). In addition, immune reactivity is strongly related to body temperature [83, 91]. Thus, the temperature dependence of TRPM2 must be considered [19]. Outcomes could also be affected depending on the extent of TRPM2 activity, e.g., Ca2+ influx through TRPM2 could be beneficial for many Ca2+-dependent events,

TRPM2 is also known to contribute to the regulation of blood glucose levels (**Figure 4**) likely because of its functional expression in pancreatic β-cells [92], which secrete insulin in response to elevated blood glucose. Although the primary pathway of glucose-stimulated insulin secretion

signals such as insulin, cytokines, hormones and blood glucose elevation in pancreatic β-cells [94, 95]. In addition, expression of the antioxidant enzyme catalase and glutathione reductase in the pancreas is very low [96], indicating that the intracellular redox state of β-cells could be affected at a physiological level by the systemic metabolic state. Intracellular Ca2+-oscillations evoked by high glucose concentrations are attenuated in TRPM2KO pancreatic β-cells [97]. In

(KATP) channel closure and L-type voltage-gated Ca2+ channel activa-

, many other ion channels, including TRPM2, are reported to be involved

increases

induction in wild-type cells is inhibited by chelating Ca2+, suggesting that [Ca2+]i

bacterial killing [88].

214 Redox - Principles and Advanced Applications

whereas large Ca2+ influx could cause Ca2+ overload.

is through ATP-sensitive K+

i

tion to increase [Ca2+]

in [Ca2+] i

**Figure 4.** Functional involvement of TRPM2 in regulating insulin secretion from pancreatic β-cells. GLUT2, glucose transporter 2; KATP, ATP-sensitive K+ channel; VGCC, voltage-gated Ca2+ channel; GHSR, growth hormone secretagogue receptor; α2R, α2 adrenergic receptor; IncretinR, incretin receptor; EPAC, exchange protein directly activated by cAMP; Mito, mitochondria.

addition, heat-evoked responses in Wt b-cells were enhanced by H2 O2 in a dose-dependent manner, however, TRPM2KO cells completely lacked the response [20]. Glucose-stimulated insulin secretion from pancreatic islets was attenuated by TRPM2 deficiency [97], and the N-acetyl cysteine-sensitive fraction of glucose-stimulated insulin secretion was increased by temperature elevation from 33 °C to 40 °C in wild type, but not TRPM2KO cells, suggesting insulin secretion could be up-regulated by ROS and temperature-dependent TRPM2 activity [20]. TRPM2KO mice show blunted insulin secretion and higher blood glucose levels in oral and intraperitoneal glucose tolerance tests relative to wild-type mice, which highlights TRPM2 function in blood glucose regulation *in vivo* [97].

TRPM2 could also be involved in hormone-regulated insulin secretion. Glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) are incretin hormones released from the intestine to enhance glucose-stimulated insulin secretion from pancreatic islets [98]. The up-regulation of insulin release by incretin was shown to be TRPM2-dependent [97, 99]. This increase in insulin secretion is mediated by generation of cAMP and activation of exchange protein directly activated by cAMP (EPAC). By inhibiting the same pathway, insulinostatic effects of ghrelin and adrenaline are reportedly achieved [100, 101]. Although the involvement of ROS-evoked TRPM2 activity in such hormone-regulated insulin secretion has not been defined, TRPM2 participation in glucose metabolism seems likely. In contrast, TRPM2 mediated aggravation of inflammation was reported to cause insulin resistance in peripheral tissues [102]. High-fat diet (HFD)-induced obesity, chronic inflammation and insulin resistance with elevated plasma and tissue cytokines are also reported to be related [103]. TRPM2KO mice show resistance to HFD-induced obesity, as well as lower cytokine levels and macrophage infiltration in adipose tissue compared with wild-type mice [102]. Energy expenditure and glucose uptake to muscle and heart are higher in TRPM2KO mice than in wildtype animals, and this increase is accompanied by elevated expression levels of metabolic and mitochondrial genes. These data suggest that, in addition to regulating insulin secretion, TRPM2 could control glucose metabolism in glucose-consuming tissues.


**Meet the editor**

Alemayhu Lemma, DVM, PhD born in Ethiopia in January, 1967, graduated with DVM from Addis Ababa University (AAU) in 1990. He subsequently worked as a field veterinarian and later joined AAU in 1995. He completed his PhD studies in Animal Reproduction from Humboldt University of Berlin in 2004. Since then, he is working in the same university with continued

researches in livestock reproduction mainly focusing on ovarian activities, AI, embryo transfer, and semen cryopreservation. He has authored/co-authored several scientific publications and contributed a chapter in a book on Artificial Insemination in farm Animals. He has over 20 years of expereince in veterinay science. He is currently head of the veterinary teaching hospital, and an associate professor in animal reproduction at AAU.

Contents

**Preface VII**

**Assessment 1**

**In cattle 39**

Chapter 1 **The Importance of Semen Quality in AI Programs and Advances in Laboratory Analyses for Semen Characteristics**

Arruda and Eneiva Carla Carvalho Celeghini

**Semen Cryopreservation in Swine 17**

Tantasuparuk and Nutthee Am-In

Chapter 2 **Improvement of Semen Quality by Feed Supplement and**

Chapter 3 **The Use Of Sex-Sorted Sperm For Reproductive Programs**

Chapter 4 **Fertility Results After Artificial Insemination with Bull Semen Frozen with Low Density Lipoprotein Extender 63** L. Briand-Amirat, D. Bencharif, S. Pineau and D. Tainturier

Rita Payan-Carreira, Paulo Borges, Fernando Mir and Alain

Campos Filho and Pietro Sampaio Baruselli

Chapter 5 **Nonsteroid Anti-Inflammatory Drugs to Improve**

**Fertility in Cows 73**

Fontbonne

Zahid Paksoy and Hüseyin Daş

Chapter 6 **Molecular Markers in Sperm Analysis 93**

Mongkol Techakumphu, Kakanang Buranaamnuay, Wichai

Gustavo Guerino Macedo, Manoel Francisco de Sá Filho, Rodrigo Vasconcelos Sala, Márcio Ferreira Mendanha, Evanil Pires de

Leticia Zoccolaro Oliveira, Fabio Morato Monteiro, Rubens Paes de


*4.3.3 Light color (wavelength)*

*Smart Nanosystems for Biomedicine, Optoelectronics and Catalysis*

*4.2.5 Boiling*

**4.3 Various conditions**

been discussed.

*4.3.1 Dark condition*

*4.3.2 Sun light conditions*

mixture of metallic salt and plant extract is heated to a burner for particular temperature (10–100 °C) for certain period of time until the solution boils. Care must be taken not to overheat the reaction mixture as it comes out of the bottle. After boiling, the mixture of the solution may cool down at room temperature. To confirm that the synthesis of nanoparticles has been completed, the solution is observed to change the color from light green to dark brown. It may be possible to notice a change of color within minutes or it may take hours a day for a change of color depending on the reducing and capping agent involved in the process [18].

The boiling method could be an approach in which the heat is given up to the boiling point in order to transform the liquid sample into a vapor state. Phytochemicals, secondary metabolites as well as metallic salt reactants are activated by boiling. The free electron of the secondary metabolite or phytochemicals reacts with metallic salt and helps to maintain stability. The reaction mixture of metallic salt AgNO3 and plant extract is boiled on a burner or gas in a beaker for 5–10 min, the boiled solution that come out of a beaker, so care must be taken and the beaker is put out of a gas or a burner so that it may not falls out of a beaker. Once the solution boils, allow the mixture solution to cool down to reach room temperature and the color change from light green to dark brown is observed for verification of the synthesized nanoparticles [18].

The general condition that can be used in a laboratory is discussed in the following paragraph. In general, light of different wavelengths activates the enzyme and activated caused the reduction of silver or metal salts to the formation of nanoparticles and by-products. In this section, light-induced nanoparticle systems have

Until going to light condition, it is very important to understand the effect of the dark condition on nanoparticle synthesis. Thus, the reaction mixture (extract + AgNO3) is kept in a dark state. The nanoparticle synthesis reaction could be performed in dark where the conical flask is covered with aluminum foil to confer the darkness. Originally, the drops of AgNO3 (1 mM) 5 ml are continuously applied to the known concentration of leaf extracts of a specific plant (*Passiflora. foetida*) 95 ml in conical flask. Such mixtures are kept at continuous stirring using a magnetic stirrer. The reaction must be conducted under dark conditions as light oxidized metal, resulting in photo-leaching due to its photo-reactivity properties. Some studies have indicated that synergistic antimicrobial activity is not linked to photo-activity. Nevertheless, the dark state of treated nanoparticles was found to be more stable than UV-light-synthesized particles [14, 18]. The [20] analysis is of synergistic bactericidal activity of silver titanium oxide (AgTiO2) nanoparticles in both light and dark conditions. Therefore, the synthesis of nanoparticles using dark

Corresponding to the dark environment, when the reaction mixture (extract +

silver salt) is maintained under sunlight conditions, it may activate certain

could be a convenient method in certain areas of application.

**102**

The light of different colors could produce different sizes and shapes of nanoparticles. Red, green, and blue are the primary colors of light that are used in different conditions in different ratios to create certain light colors. It is noted that silver nanoparticles under different light colors, such as green, blue, red, yellow and orange 15 W, result in silver nanoparticle synthesis where the blue color light of 15 W shows good UV absorption at 400 nm compared to other color lights. Therefore, the size and shape as well as the stability of nanoparticles could be altered by different wavelengths of light. Like the various wavelength of light, we would like to explore the idea of the influence of sound on nanoparticles [18]. **Figure 4** shows the silver nanoparticle synthesis using various methods and conformation by visual change in colors.

#### **Figure 4.**

*Synthesis of silver nanoparticles using various methods/conditions: sun, microwave oven, sonication, autoclave, yellow bulb, white bulb, UV light and dark (from left to right) [18].*


**2.1. Over voltages and under voltages**

On one side, voltage quality focuses on variations of voltage from its ideal waveform (i.e., characterized by a sine wave of constant magnitude and frequency), while current quality is concerned with the deviation of the current from the ideal sinusoidal waveform. On the other side, discrimination of power quality as a voltage quality or current quality is an ambiguous way of thinking as a deviation in voltage can result in a deviation in current and vice versa. Thus, in order to keep generality, and as the power is mathematically the voltage times current, power quality should be the combination of both voltage and current qualities [3] and is signified by a set of electrical limitations (reference boundaries/margins) that enable an equipment to operate in its planned manner without major operating losses [4, 5] to long live

2 Power System Harmonics - Analysis, Effects and Mitigation Solutions for Power Quality Improvement

All electrical equipment may fail or malfunction when come across power quality disturbances, depending on the severity of the disturbance. It is essential for engineers, technicians, manufacturers, and power system operators to well understand and face the several power

Power quality issues include voltage variations (dips, interruptions, flicker, etc.), transients (surges, lightning, and switching events), and grounding issues. **Figure 1** summarizes the

as possible.

**2. Disturbances**

quality disturbances.

common power quality problems.

**Figure 1.** The common power quality problems.

Over voltages are defined as any voltage greater than the equipment nominal operating voltage when the equipment is specified to operate at for a time period that exceeds 1 min. While, the under voltage can be defined as any voltage below the nominal operating voltage of the equipment for a time period that exceeds 1 min.

Over-voltage phenomenon has many causes in power system networks such as sudden changes in the system operating settings, abrupt load rejection, series/parallel harmonic resonance cases, sudden line-to-ground faults, improper earthing schemes, poor voltage regulation throughout the system, and overcompensation of the reactive power support provided by capacitor banks. Under voltages can result from improper power cables sizing, long feeder routes with high loading capacities, and large motor starting conditions.

Over voltage has a serious impact on electrical equipment and power systems as it stresses the equipment's insulation and may damage it, in addition to protective devices tripping because of dielectric failure. Also, over voltage may lead to flashover between line and ground at the weakest point in the system and can cause breakdown of the equipment insulation. On the other hand, under voltage causes an increase in the system losses and results in voltage stability problems. Also, different operational problems may arise due to under voltages such as motor starting problems and protection relay tripping [7].


**7. Conclusions**

*Gene Expression and Control*

**Figure 2.**

summarized in **Figure 2**.

*Saccharomyces cerevisiae*.

regulation of RPGs and rRNAs [56].

**6. The TCT-motif module in metazoan RPG**

essential for accurate translation activity. The coordinated regulation and expression of the RPG with other ribosomal components is still poorly understood. However, in the fission yeast *Schizosaccharomyces pombe*, it is known that rRNAs, RPGs, and box C/D snoRNAs contain in their promoters a HomolD-box [39, 53], which might be able to control the expression of those genes. Moreover, the Rrn7 transcription factor, which is the HomolD-box-binding protein in RPG, was found to be responsible for the control of the gene expression of box C/D snoRNAs and RPGs *in vivo* in *Schizosaccharomyces pombe* cells [53]. Interestingly, the yeast orthologs of the human RECQL/DDB1 complex may recognize the HomolD-box and down-regulate RPG expression [53]. Taking all these results together, we propose a model, in which the HomolD-box is bound by Rrn7 and co-regulates the transcription of RPG, box C/D snoRNA and rRNA genes in the fission yeast. This model is

*promoters contain a HomolD-box, which is critical for in vivo transcription.*

Role of HomolD-box-containing promoters in ribosome biogenesis**.** *It is believed that the coordinated expression of genes that encode ribosome components (rRNA, RPG, box C/D snoRNA) is due to a common DNA element (HomolD-box) that is able to bind a protein factor (Rrn7). The presence of the HomolD-box in the promoters of several genes encoding ribosome components indicates a common regulation. Until now, experiments have demonstrated that Rrn7 binds to rRNA and RPG promoters in Schizosaccharomyces pombe. However, binding of Rrn7 to box C/D snoRNA promoters has not been demonstrated yet, although those*

Unlike *Schizosaccharomyces pombe* in the case of *Saccharomyces cerevisiae*, there is accumulated evidence that show a putative coordinated model to regulate biogenesis of ribosome components. In this model, CK2 protein kinase is part of protein complexes that regulate RPG expression and rRNA synthesis [54] and interact with the protein Fhl1p that is associated to Ifh1p, which binds to the IFH1 element near to the Rap1 sequence. CK2 and Ifh1p are part of the complex CURI associated to rRNA processing and RPG transcription [55]. Also, in *S. cerevisiae,* another protein has been identified and named protein HmoI, which is associated with the transcription

In addition to the role of CK2 to modulate Rrn7 function in *Schizosaccharomyces pombe* during HomolD-box directed transcription, there might be another points of regulation related to protein complexes, such as those described in the yeast

The analyses of insect and mammalian RPG promoters have shown the presence of a common core promoter element that is part of the poly-pyrimidine initiator (TCT)-motif family, which is a novel core promoter element necessary to initiate transcription in those genes [35, 57]. In these promoters, the transcription start site (TSS) involves the TCT motif and is positioned around −2 to +6 relative to TSS, competing with exactly the same position as the Inr. However, the features of

**158**

The fission yeast *Schizosaccharomyces pombe* provides an excellent biological model to study the coordinated expression of ribosome components. The finding that rDNAs, RPGs, and box C/D snoRNAs genes contain a HomolD-box, which is most likely bound by Rrn7, provides the starting point to investigate this issue. The most important questions to answer are: (i) to determine whether or not box C/D snoRNA genes are transcribed by the same transcription apparatus that transcribes RPG; (ii) to identify the signal that activates transcription of HomolD-box containing genes, and (iii) to identify the HomolE-binding protein. The resolution of all these issues would contribute to understand the regulation of RPG transcription in the fission yeast and most likely could be extrapolated to metazoan organisms.

## **Acknowledgements**

This work has been funded by the Corporación Nacional de Ciencia y Tecnología de Chile (CONICYT), Fondo de Desarrollo de Ciencia y Tecnología (FONDECYT), and FONDEF (ID16I10145). We are very grateful to Mrs. Teresa Donoso for her continued support to F. U. and E. M.


**Acknowledgements**

This research was supported by Operation Subsidy of Inner Mongolia Dengkou Desert Ecosystem National Observation Research Station<PHONE_NUMBER>), Science and Technology Planning Project of Inner Mongolia Autonomous Region (2020GG0125).


**Abstract**

Human body temperature continues to be of interest to researchers with the newest discovery that it had been steadily decreasing since the mid-1800s, thus affecting our definition of normothermia and the cutoff for fever. Further, body temperature constitutes an explicit manifestation of our circadian rhythm, with temperature trough occurring in early morning and peak in the evening in healthy individuals. On the other hand, human illness, as seen among patients ranging from stable hospitalized ones to the critically ill, was shown to correlate with disturbance or even complete disappearance of the body temperature circadian rhythm. Also, night shift work affects the rhythm and had been associated with increased risk for malignancies, gastroenterological and metabolic disorders. Importantly, quantification of the observed rhythm deviations appears to have diagnostic and prognostic value in medicine. This chapter reviews the determinants of body temperature and the body temperature circadian rhythm, discusses the most prominent published research on associations between the rhythm and human disease, and concludes by outlining possible new research avenues and promising clinical applications in the field of chronotherapy.

**Keywords:** circadian rhythm, body temperature, chronotherapy, fever, hypothermia, personalized medicine


**1. Introduction**

The usage of language, both in its written and oral expressions (texts and speech), follows very strong statistical regularities. One of the goals of quantitative linguistics is to unveil, analyze, explain, and exploit those linguistic statistical laws. Perhaps the clearest example of a statistical law in language usage is Zipf's law, which quantifies the frequency of occurrence of words in such written and oral forms [1–6], establishing that there is no unarbitrary way to distinguish between rare and common words (due to the absence of a characteristic scale in "rarity"). Surprisingly, Zipf's law is not only a linguistic law, but seems to be a rather common phenomenon in complex systems where discrete units self-organize into groups, or types (persons into cities, money into persons, etc. [7]).

Zipf's law can be considered as the "tip of the iceberg" of text statistics. Another well-known pattern of this sort is Herdan's law, also called Heaps' law [2,8,9], which states that the growth of vocabulary with text length is sublinear (however, the precise mathematical dependence has been debated [10]). Herdan's law has been related to Zipf's law, sometimes with too simple arguments, although rigorous connections have been established as well [8,10]. The authors of [11] provide another example of relations between linguistic laws, but, in general, no general framework encompassing all laws exists.

Two other laws—the law of word length and the so-called Zipf's law of abbreviation or brevity law— are of particular interest in this work. As far as we know, and in contrast to the Zipf's law of word frequency, these two laws do not have non-linguistic counterparts. The law of word length finds that the length of words (measured in number of letter tokens, for instance) is lognormally distributed [12,13], whereas the brevity law determines that more frequent words tend to be shorter, and rarer words tend to be longer. This is usually quantified between a negative correlation between word frequency and word length [14].

Very recently, Torre et al. [13] parameterized the dependence between mean frequency and length, obtaining (using a speech corpus) that the frequency averaged for fixed length decays exponentially with length. This is in contrast with a result suggested by Herdan (to the best of our knowledge not directly supported by empirical analysis), who proposed a power-law decay, with exponent between 2 and 3 [12]. This result probably arose from an analogy with the word-frequency distribution derived by Simon [15], with an exponential tail that was neglected.

The purpose of our paper is to put these three important linguistic laws (Zipf's law of word frequency, the word-length law, and the brevity law) into a broader context. By means of considering word frequency and word length as two random variables associated to word types, we will see how the bivariate distribution of those two variables is the appropriate framework to describe the brevity-frequency phenomenon. This leads us to several findings: (i) a gamma law for the word-length distribution, in contrast to the previously proposed lognormal shape; (ii) a well-defined functional form for the word-frequency distributions conditioned to fixed length, where a power-law decay with exponent *α* for the bulk frequencies becomes dominant; (iii) a scaling law for those distributions, apparent as a collapse of data under rescaling; (iv) an approximate power-law decay of the characteristic scale of frequency as a function of length, with exponent *δ*; and (v) a possible explanation for Zipf's law of word frequency as arising from the mixture of conditional distributions of frequency at different lengths, where Zipf's exponent is determined by the exponents *α* and *δ*.


**1. Introduction**

Accountability refers to 'being answerable for one's behaviour or actions' [1, 2]. Financial accountability describes the process accounting by officials of the manner in which public funds, in this case, at local government or municipal level, have been used to implement municipal policies as approved by its council [2, 3]. In a 2019 media release by the Auditor-General South Africa (AGSA), the lack of accountability was flagged as the major cause of poor local government audit results in South Africa [4].

The AGSA reports on municipal performance have consistently over the years highlighted governance issues affecting municipalities; however, analysis of the audit result trends over the period 2015/16−2021/22 demonstrated limited action on recommendations by the AGSA to address the identified shortcomings. According to AGSA, widespread monitoring and control breakdowns were found at a significant number of the 257 municipalities within South African local government environment [5], resulting in fiscal resources either misused or misappropriated or not appropriately accounted for as required by South African public finance management laws. 'Evidence of these is largely incomplete projects, unsupervised projects, lack of maintenance of significant service delivery infrastructure and haphazard road maintenance projects and infrastructure' [4].

#### **2. Corruption contextualised**

The Prevention and Combating of Corrupt Activities Act 12, 2004 (PCCAA) criminalises corruption in South Africa [6]. The PCCAA, as cited by Bernstein and Shaw, defines corruption as 'where an individual either directly or indirectly receiving or giving an intention to receive a form of gratitude from another individual or offers or approve to provide a gratitude to some other individual for his or her benefit or for another, and the offering or acceptance is conducted in order to influence the other party to perform in an inappropriate way, in the functioning of that individual's duties [7]'.

As a member state to the New Partnership for Africa's Development (NEPAD), South Africa prescribes to its strategic priorities, which amongst others require adherence to good governance values including but not limited to political, economic and corporate governance [8]. Poor levels of corporate governance, resulting in corruption without apparent and decisive consequence management, are currently some of the issues dominating the news and public discourse in South Africa and beyond its borders.

The Constitution of the Republic of South Africa, 1996, with specific reference to Section 195, prescribes public administration governance in line with democratic values and principles, being '(i) the promotion of a high standard of professional ethics; (ii) the promotion of efficient, economic, and effective use of resources; (iii) transparency must be fostered by providing the public with timely, accessible and accurate information; and (iv) public administration must be accountable [9]'. These values and principles embody the aspirations of sound corporate governance.

Corporate governance embodies processes and systems by which corporate enterprises are directed, controlled and held to account. The Institute of Directors of South Africa [10] defines corporate governance as 'the exercise of ethical and effective leadership by the governing body towards the achievement of ethical culture, good performance, effective control and legitimacy'.

## **3. Methodology**

This chapter is based on an exploratory desktop literature review of numerous publications, such as academic journals, official South African public sector reports and publications, legislation and policies, newspaper articles, as well as documentation obtained from various official websites in the field of public governance and accountability. Comprehensive insight was gained from the analysis of the documents into the current governance frameworks versus the situation pertaining to

*Addressing the Endemic Consequences of Corruption in the South African Local Government… DOI: http://dx.doi.org/10.5772/intechopen.108154*

the prevention and/or addressing of corruption within the South African Local Government Public Sector.

Analysis of the literature reveals numerous intricate and well-designed frameworks, mechanisms and public institutions in place to firstly prevent acts of corruption and mismanagement of resources and secondarily to deal with such acts, should they occur. As revealed by the various AGSA municipal audit reports, a lack of consequence for mismanagement of resources evidenced by the failure to investigate findings and/or failure to act on findings resulted in deterioration of the financial health of municipalities and in the delivery and maintenance of municipal infrastructure.

A qualitative research approach using unobtrusive methods was used at it was viewed as being the most suitable approach towards understanding and unpacking public sector corruption and consequence management practices in South Africa. As stated by Auriacombe [11], 'a theoretical framework is essential to gain a clear understanding of the relationships manifested between the various elements and issues of an identified phenomenon', which in the case of this study is the public sector and public financial management and accountability.

All the documentary sources used in this study are available in the public domain and easily accessible, which allowed for a detailed contextual and content analysis. The focus was on official publications which would ensure validity and trustworthiness, such as the Auditor General's reports and peer-reviewed journals pertaining to the subject matter at hand. A process of analysis of the phenomenon (public corruption and consequence management), reflection and synthesis to make meaningful deductions was followed towards creating the information and positioned in the article. After analysis of the trustworthy sources [12], the authors were able to gain an understanding of the phenomenon under investigation, to make relevant recommendations towards improved consequence management aimed at addressing corruption within the South African Local Government Public Sector and to develop various recommendations to improve public sector accountability and consequence management.

#### **4. Discussion**

#### **4.1 Legislation for good governance**

According to the White Paper on Local Government [13], 'a developmental municipality should play a strategic policy-making and visionary role and seek to mobilise a range of resources to meet basic needs and achieve developmental goals'. The developmental duties of local government are set out in Section 153 of the Constitution, namely that a municipality must structure and manage its administration, budgeting and planning processes to prioritise the basic needs of the community and to promote its social and economic development [9]. In addition, Sections 152, 153 and 154 of the Constitution indicate that local government must promote social and economic development, a safe and healthy environment, and encourage the involvement of communities and community organisations in the matters of local government [9]. Section 152 of the Constitution determines that local government is responsible for:

Section 195 stipulates that public administration must be governed by the democratic values and principles enshrined in the Constitution, including that a high standard of professional ethics must be promoted and maintained. According to the section, all three levels of the state, including public enterprises and state-owned entities, are subjected to the principles and values of a democracy [9]:

The following legislative frameworks and guidelines as per the National Treasury Guideline for Accounting Officers underpin corporate governance in the public sector [15]:


2.2.6. Iron (II) Chelating Activity

Iron (II) chelating activity was conducted by the procedure described by Dinis et al. [38] with minor modifications. An amount of 400 μL of the extract was mixed with 50 μL of 2 mM ferrous chloride. The reaction was started by adding 200 μL of 5 mM ferrozine to the reaction liquid and incubating it at room temperature for 10 min. The mixture's absorbance was measured at 562 nm in comparison to a blank sample. The decrease in absorbance was due to increased iron chelating activity. The following Equation (2) was used to calculate the iron (II) chelating activity:

$$\text{Chelating (\%)} = \left(1 - \text{A562 Sample/A562 Control} \right) \times 100\tag{2}$$

## *2.3. Experimental Design and Statistical Analysis*

The data were analyzed using the Statistical Analysis System (SAS) ver. 9.4. All of the experiments were performed in triplicates in a two or three-factorial Completely Randomized Design (CRD) and the results are expressed as mean ± SE. Duncan's Multiple Range Test (DMRT) was used to compare means at a *p* = 0.05 level. Pearson's correlation analysis was used to determine the correlation between variables with indicator r < 0.25 indicating a weak correlation, r < 0.75 indicating an intermediate correlation, r < 1 indicating a strong correlation,r=1 indicating perfect correlation.

#### **3. Results**

#### *3.1. In Vitro Callus Induction of Bougainvillea glabra via Nodal Segment*

3.1.1. The Main Effect of 2,4-D, BAP and Light Regimes on Callus Induction of *B. glabra*

Results from the present study showed that from the different concentrations of 2,4-D, the minimum days for callus initiation was obtained in the treatment of 5 μM 2,4-D but it was not significantly different from the treatment of 2.5 μM 2,4-D. Meanwhile, the maximum days for callus initiation were recorded from the treatment of 7.5 μM 2,4-D. However, for the treatments of BAP, the minimum days for callus initiation was exhibited from the 1.5 μM BAP but it was not significantly different from 0.5 and 1 μM BAP, respectively. The type of explants to induce callus is also an important factor that needs to be considered in callus induction experiments due to various factors affecting the development of cell culture systems, such as genotype, explant type, plant growth regulators (PGR), culture medium, and culture condition. Based on the results in (Table 1), with WPM medium supplemented with various concentrations of 2,4-D, an increment of the callus frequency was observed as 2,4-D concentration increased until 5 μM. The highest callus frequency was recorded using 5 μM 2,4-D, which is significantly different from 7.5 μM 2,4-D, but it was not significantly different from 2.5 μM 2,4-D. On the other hand, there was no callus formation on the control media without plant growth regulators, indicating that growth regulators had a significant effect on callus induction on the nodal explant. Following that, there was a decrease in percent callus induction growth, followed by an increase of the concentrations of 2, 4-D, and BAP.

**Table 1.** Main effects of 2,4-D and BAP on callus induction in the nodal segment of *Bougainvillea glabra* under different light conditions.

Values are means ± SE. the same letter within the same column for each factor indicates no significant difference (*p* < 0.05). The means separation is done by using Duncan's multiple range test (DMRT). F value represented \* = <0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001 and ns = no significant differences. 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl amino purine (BAP), FW (Fresh Weight), and DW (Dry Weight).

In terms of biomass accumulation, maximum callus biomass was recorded when the nodal explants were cultured on WPM medium supplemented with 5 μM 2,4-D. However, for the treatments of BAP, 1.5 μM BAP exhibited significantly higher callus biomass, respectively. Based on the results, it was seen that there was an increase in the fresh and dry weight of callus as the concentration of BAP was increased up to 1.5 μM, but for the

treatments of 2,4-D, the callus biomass increased as the 2,4-D increased until 5 μM and significantly decreased with 7.5 μM. Primary metabolism, particularly carbon and nitrogen metabolism, is closely linked to biomass accumulation. Carbon metabolism meets the energy requirements resulting from carbohydrate synthesis, and hence, contributes to cell growth and structural components [39]. The products of nitrogen metabolism, on the other hand, are primarily amino acids and proteins, which are then used to regulate cellular processes [40]. Over all, both cellular development and division result in increased fresh and dry weight.

The callus induction of *B. glabra* was also evaluated under two growth conditions, dark and light. The results showed that the dark condition was more favorable for earlier callus initiation and higher callus frequency than the light condition. However, the calluses induced under light incubation conditions produced significantly higher callus fresh and dry weight than the calluses induced under complete dark incubation conditions. Based on the observation in this experiment, the nodal segments incubated under complete dark conditions grew faster than in a light-containing photoperiod. In the dark condition, the minimum days for callus initiation was (8.45), which is significantly different from the light condition with (14.73) days. In addition, the higher callus frequency was also obtained from the cultured incubated under the dark condition, which is significantly different from the cultured incubated under the light condition, respectively. However, callus grown under a set photoperiod presented the higher callus fresh and dry weight than callus incubated under the complete dark condition.

Based on the observation in this experiment, the interaction effects of various concentrations of 2,4-D and BAP were significant (*p* < 0.05) for all parameters, but the interaction effect between 2,4-D \*Condition and BAP \*Condition were only significant for the parameters such as days to callus initiation and callus frequency; also, the statistical analysis indicated that the triple interaction of 2,4-D, BAP, and culture condition was significantly different for days to callus initiation and callus induction rate, but was not significantly different for the biomass production (Table 1). A similar trend also was observed in the WPM medium supplemented with different concentrations of BAP. The maximum callus frequency was obtained from 0.5 μM and 1 μM BAP, respectively. However, by increasing the BAP concentration, the callus frequency decreased.

#### 3.1.2. Synergistic Effect of Cytokinin, Auxins, and Light Regime on Callus Induction

The results in Table 2 exhibit that the minimum days of callus initiation were recorded from the treatments of 2.5 μM 2,4-D + 1.5 μM BAP and 5 μM 2,4-D + 1 μM BAP under the dark incubation condition, respectively. However, the maximum days for callus initiation were recorded from the treatment of 7.5 μM 2,4-D + 0.5 μM BAP under light incubation conditions. As shown, the period of callus induction and growth of callus varied. They depend on the type and concentration of growth regulators and lighting conditions. Meanwhile, the highest callus induction rate was produced from the combination of 2.5 μM 2,4-D + 1.5 μM BAP under a dark incubation condition and 5 μM 2,4-D + 0.5 μM BAP under both light and dark incubation conditions, respectively. However, the lowest callus induction rate was obtained from the treatments of 7.5 μM 2,4-D + 1.5 μM BAP under both light and dark conditions.

In this experiment, for the WPM medium fortified with various concentrations of 2,4-D and BAP, a different trend of callus fresh and dry weight was observed. The callus fresh and dry weight was drastically increased by three-fold in the treatment of 7.5 μM 2,4-D + 1.5 μM BAP under dark incubation condition but it was not significantly different from the same treatment under the light condition. As the concentration decreased to 2.5 μM 2,4-D and 0.5 μM BAP, the callus fresh weight was decreased under dark and light incubation conditions, respectively. Overall, no significant biomass differences were observed for light regimes.

**Table 2.** Interaction effects of 2,4-D and BAP on callus induction in the nodal segment of *Bougainvillea glabra* under different light conditions.

Values are means ± SE. the same letter within the same column for each factor indicates no significant difference (*p* < 0.05). The means separation is done by using Duncan's multiple range test (DMRT). 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). W white, Y yellow, R red, B brown, F friable, and C compact.

Based on the results, the morphogenesis responses based on the intensity of callus formation, texture, types, and concentration of PGRs, cultural condition, and in vitro morphogenesis were different according to the treatment used and are presented in (Table 2) and (Figures 1 and 2A–E). The cultural condition was capable of inducing a callus that could be classified into two types. The first type, which was induced under light incubation conditions, was compact and yellow to brown and red (Figure 1A–E). The second type, which was induced under dark incubation conditions, was friable and yellow to white and brown (Figure 2A–E).


**Conflict of interest**

The authors declare no conflict of interest.

*Data Mining for Source Apportionment of Trace Elements in Water and Solid Matrix DOI: http://dx.doi.org/10.5772/intechopen.88818*