**4.2 Experiences of the course**

How do the students and mentors experience the programme integrating courses? Some results from the mandatory survey of PIC2 at the end of the academic year 2018/2019 are shown in **Table 5**. In each of these five questions, the students should answer on a Likert scale from 1 to 7 whether they agree or not to a statement. In the scale, 1 means *totally disagree* and 7 means *fully agree*. The same questions have been asked for a sequence of years, and the results are almost stable.

We can see that already from year 1, the students understand the aims of the seminars. They also, throughout the 3 years, appreciate listening to elder or younger students. The interviewed students confirmed this and even expressed that sharing an experience that could evoke change was the main benefit of the course.

The students value the programme integrating course more and more during the course of the course. At the end of the course, a majority of the students rank the fruitfulness of the course to 6 or 7 on the Likert scale. Increase of the knowledge of the education through PIC is also something that students rank higher in the third year than in the first year.

The student interviews showed that discussing the courses of the programme and how they link to each other was considered to be an important part of PIC, where the mentors were seen as gateways to change things. Some interviewed students considered some seminar topics to be nontechnical and far from what they chose to study and therefore not that valuable. The interviewed mentors confirmed that a few students' attitudes towards some topics were disappointing. Some mentors expressed that discussing these topics could be out of their own comfort zone. The proportion of students answering below 4 (i.e. were negative) to the fruitfulness of the 8 first seminars of the course varied between 8% (master programmes topic) and 28% (learning objectives, criteria and assessment topic).

Some of the interviewed students expressed that timing of the reflection assignments always was the worst possible—when all parallel courses had assignments due. One should note that the students get the assignment about 10 days before the deadline and that the assignment will take about 3 hours to complete.

Experienced mentors expressed that they were fortunate to be able to follow the development of their students throughout the 2 or 3 years of the course, to be able to learn their names, which is often not possible in the ordinary courses where the number of students is often over 200.

#### **Table 5.**

*Results from the survey 2019. The students were asked how well they agree with a set of statements on a Likert scale from 1 (I totally disagree) to 7 (I fully agree). The mean values of the answers are shown in the table.*

**211**

**Table 6.**

*Programme Integrating Courses Making Engineering Students Reflect*

Many more experiences of PIC are reported in [28, 29].

least one new study skill to try for the next months.

3.The students read each other's texts within the group.

The students often described a good relationship with the mentor, a relationship of trust. However, not all mentors seem to be engaged to the same extent in the course.

Hedin and Kann [30] have studied the effects of the programme integrating course PIC2 with respect to study skills. The course starts with a learning-to-learn

1.The students are instructed to look at least four of nine short videos, where Björn Liljeqvist, a young specialist in study skills, explains and motivates the use of a number of study skills. They are also instructed to read a short book on

2.The students write a reflective text about their own study habits and choose at

4.The students in the group meet and discuss the topic and their reflections in a

The evaluation shows, among other things, which effects the students believed the study skills had after trying them (see **Table 6**). No significant change was found in how satisfied the students were with their overall study technique immediately after the initial module, but in the long-term, 77% of the students believed the course had promoted their ability to analyse and adapt their study habits [30]. The proportion of students who believe that PIC has promoted this is largely the same in

5.About 6 weeks later, the students write a new text, reflecting on how the attempt to try a new study skill fell out, and discuss this at a new seminar.

We wanted the students to improve their ability to reflect more deeply. Therefore, we in 2012 developed and introduced a four-level model for reflections

Preparing before lectures? 23 69 8 Taking smart notes at lectures? 23 57 21 Going through the previous day's and week's teaching? 23 63 15 Planning my studies the upcoming week? 49 40 11 Maintaining a study diary? 23 45 32 Reading the course literature in three steps? 44 37 19 Trying to stop procrastinating? 59 31 10 Doing some other change? 43 57 0 In total (mean values) 35 51 14

**Obvious effect (%)** **Most likely effect (%)**

**No noticed effect (%)**

*DOI: http://dx.doi.org/10.5772/intechopen.88253*

module, consisting of the following parts:

**4.3 Improving study skills**

how to study.

one-hour seminar.

different years and in different surveys.

**What is your perception of the effects on your**

*Results from the postquestionnaire on the effects on the students' learning.*

**4.4 Progression of reflection**

**learning of**

The students often described a good relationship with the mentor, a relationship of trust. However, not all mentors seem to be engaged to the same extent in the course.

Many more experiences of PIC are reported in [28, 29].


**Author details**

Mizuo Mifune1 and Yoshihiko Kanno2 \*

1 Shinozaki HD Clinic, Tokyo, Japan

2 Department of Nephrology, Tokyo Medical University, Tokyo, Japan

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**Table 4.**

*Sediment texture and organic matter (OM) in %.*

Porvenir" site with 24.20 μg/g. The results for Fe in the rainy season were higher at the "El Porvenir" site with 224.75 μg/g.

To analyze a contaminated site, it is important to establish correlations with the physical and chemical characteristics of the sediments, because different contaminants are related to organic matter and fine fractions. In this study, the texture of the sediments, organic matter, and their relationships with heavy metals (**Table 4**). The organic matter content is high and is related to the extensive area of mangroves, various species of macrophytes, and human settlements, which contributes to the variability of organic matter.


2.2 Reversible addition-fragmentation chain transfer (RAFT) polymerization

RAFT polymerization is an alternative method to synthesize well-defined and narrow distributed polymers with complex topological architectures by choosing a proper chain transfer agent (CTA). RAFT polymerization can be conducted without using any metal catalyst, and thus it is convenient and easy to purify the resultant polymers, which is the biggest advantage over ATRP. To date, only limited attention has been paid to cellulose graft copolymers through homogeneous RAFT polymerization in the literature [78–81]. The combination of cellulose and RAFT polymerization can provide new opportunities for graft copolymers, especially those that could not be synthesized directly via ATRP strategy. Recently, our group designed a novel cellulose-based macromolecular chain transfer agent by introducing a trithiocarbonate derivative with dodecyl as stabilizing group on cellulose backbone from Cell-BiB for the synthesis of Cell-g-P(BA-co-AM) copolymers as strong materials from thermoplastics to elastomers via RAFT polymerization [82]. As shown in Figure 5, the bromine groups on Cell-BiB can be substituted by reacting with 1-dodecanethiol, carbon disulfide (CS2), and triethylamine (TEA) in DMSO. This Cell-CTA is versatile and suitable for a lot of monomers, and the DS of Cell-CTA can be manipulated by changing the molar ratios of above chemical reagents. AM and BA were chosen as the rigid and soft segments in the grafted side chains. PAM can provide reversible physical network structure in the cellulose graft copolymers. The N▬H and C=O groups in AM units can form strong selfcomplementary hydrogen bonds, and they are distributed homogeneously in the polymer matrix, leading to the strong and tough elastomer materials. Inspired by this work, we propose that high-performance cellulose graft copolymer can be

Cellulose-Based Thermoplastics and Elastomers via Controlled Radical Polymerization DOI: http://dx.doi.org/10.5772/intechopen.89436

Figure 5.

(N-isopropylacrylamide) (PNIPAM) [45–48], poly(4-vinylpyridine) (P4VP) [51], poly(N,N-dimethylamino-2-ethyl methacrylate) (PMDAEMA) [34], and poly(2-

(a) Schematic illustration of the synthesis of Cell-g-PI by ATRP and (b) fabrication of Cell-g-PI core-shell

nanoparticles with a PI core and cellulose shell (adapted with permission from Ref. [36]).

2.2 Reversible addition-fragmentation chain transfer (RAFT) polymerization

copolymers. The N▬H and C=O groups in AM units can form strong selfcomplementary hydrogen bonds, and they are distributed homogeneously in the polymer matrix, leading to the strong and tough elastomer materials. Inspired by this work, we propose that high-performance cellulose graft copolymer can be

RAFT polymerization is an alternative method to synthesize well-defined and narrow distributed polymers with complex topological architectures by choosing a proper chain transfer agent (CTA). RAFT polymerization can be conducted without using any metal catalyst, and thus it is convenient and easy to purify the resultant polymers, which is the biggest advantage over ATRP. To date, only limited attention has been paid to cellulose graft copolymers through homogeneous RAFT polymerization in the literature [78–81]. The combination of cellulose and RAFT polymerization can provide new opportunities for graft copolymers, especially those that could not be synthesized directly via ATRP strategy. Recently, our group designed a novel cellulose-based macromolecular chain transfer agent by introducing a trithiocarbonate derivative with dodecyl as stabilizing group on cellulose backbone from Cell-BiB for the synthesis of Cell-g-P(BA-co-AM) copolymers as strong materials from thermoplastics to elastomers via RAFT polymerization [82]. As shown in Figure 5, the bromine groups on Cell-BiB can be substituted by reacting with 1-dodecanethiol, carbon disulfide (CS2), and triethylamine (TEA) in DMSO. This Cell-CTA is versatile and suitable for a lot of monomers, and the DS of Cell-CTA can be manipulated by changing the molar ratios of above chemical reagents. AM and BA were chosen as the rigid and soft segments in the grafted side chains. PAM can provide reversible physical network structure in the cellulose graft

(diethylamino)ethyl methacrylate) (PDEAEMA) [56].

Figure 4.

Thermosoftening Plastics

40

Illustrations for the synthesis of Cell-CTA, Cell-g-P(BA-co-AM) copolymer, and the self-complementary hydrogen bonding in the cellulose graft copolymer (adapted from Ref. [82]).

accessed by introducing other supramolecular interactions into the matrix as reversible physical networks, such as metal-ligand coordination, π-π stacking, and host-guest complexation.

## 2.3 Nitroxide-mediated polymerization (NMP)

NMP is the first and easiest CRP technology controlled by a reversible termination mechanism between the nitroxide moieties and growing propagating macroradicals. A wide range of monomers, including acrylates, styrene derivatives, vinylpyridines, acrylonitrile, acryl acid, acrylamide derivatives, cyclic ketene acetals, and miscellaneous, can be polymerized via NMP to develop well-defined polymers [31]. The primary advantage of NMP is the absence of post-treatment since no catalyst or bimolecular exchange is needed during the polymerization. However, the higher polymerization temperatures and lower polymerization rates limit the wide applications of NMP. To the best of our knowledge, only one study reported the synthesis of cellulose graft copolymers through homogeneous NMP strategy started from microcrystalline cellulose [83]. In this work, pretreated cellulose was dispersed in anhydrous tetrahydrofuran and reacted with 2-bromoisobutyryl bromide and pyridine to prepare Cell-BiB. As shown in Figure 6, Cell-BiB could react with 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) in DMF in the presence of sodium hydride (NaH) to obtain functionalized cellulose (Cell-TEMPOL). At first, PS was grafted from cellulose backbone with Cell-TEMPOL as macroinitiator in the absence of any catalyst to produce Cell-g-PS. Cell-g-(PMMAb-PS) copolymer was synthesized by chain extension of above Cell-g-PS via the second NMP. Such kind of cellulose graft block copolymers may be applied as reinforcing agents, packing materials, and membrane materials. More efforts can be

backbone, the grafting density (the average number of grafts per anhydroglucose unit), the degree of polymerization, and distribution of grafts. Various block and random copolymers have been grafted from cellulose via CRPs as described above. However, as exhibited in Figure 7a, heterograft and graft-on-graft architectures with cellulose as backbone remain unreported, and these interesting graft copolymers can be synthesized by the combination of RAFT polymerization and ATRP, ring-opening polymerization (ROP) and ATRP or ROP and RAFT polymerization. Due to the increased attention paid on biomass-derived thermoplastics and elastomers, it becomes necessary and desirable to explore new monomers, which can be synthesized from bioresources, including lignins, terpenes, plant oils, rosin acids, and coumarins. For instance, bioresources, 7-hydroxyl-4-methylcoumarin, vanillin, guaiacol, and oleyl alcohol, can be used to synthesize sustainable monomers by reacting with acryloyl chloride, methacryloyl chloride or methacrylate anhydride as shown in Figure 7b. Among these monomers, 7-acryloyloxy-4 methylcoumarin (AMC), 7-methacryloyloxy-4-methylcoumarin (MMC), acrylated

Cellulose-Based Thermoplastics and Elastomers via Controlled Radical Polymerization

DOI: http://dx.doi.org/10.5772/intechopen.89436

vanillin (AV), methacrylated vanillin (MV), acrylated guaiacol (AG), and

Figure 7.

43

derived from different bioresources.

methacylated guaiacol (MG) can be utilized as rigid segments, while oleyl acrylate (OA) and oleyl methacrylate (OM) can be utilized as soft segments in the graft copolymers. Though there has been considerable progress, it is still challenging to

(a) Heterograft and graft-on-graft architectures of cellulose graft copolymers. (b) Sustainable monomers

#### Figure 6.

Schematic illustration for the synthesis of Cell-g-PS and Cell-g-(PMMA-b-PS) copolymers through NMP technique.

made in the future by grafting functional block polymers from cellulose via NMP to design novel stimuli-responsive hybrid materials with excellent macroscopic mechanical properties.

## 3. Perspective

CRPs are versatile and robust techniques to develop well-defined cellulose graft copolymers as novel thermoplastic and elastomers with desired properties for particular application demands. As the most adopted technique for cellulose graft copolymers, conventional ATRP suffers from the limitation of removing metal catalysts from the resultant products, which may cause undesired toxicity and coloration. Recently, new advances in ARTP have been reported, and metal-free ATRP has been developed to synthesize narrowly distributed polymers with welldefined structures by using photoredox organic catalysts under light irradiation instead of metal catalysts [84–86]. This new strategy can greatly promote the design and preparation of cellulose graft copolymers for different application fields. Cellulose graft copolymers show improved properties compared to the linear counterparts due to their unique molecular architectures, and the macroscopic performance of cellulose graft copolymers is affected by the degree of polymerization of cellulose

### Cellulose-Based Thermoplastics and Elastomers via Controlled Radical Polymerization DOI: http://dx.doi.org/10.5772/intechopen.89436

backbone, the grafting density (the average number of grafts per anhydroglucose unit), the degree of polymerization, and distribution of grafts. Various block and random copolymers have been grafted from cellulose via CRPs as described above. However, as exhibited in Figure 7a, heterograft and graft-on-graft architectures with cellulose as backbone remain unreported, and these interesting graft copolymers can be synthesized by the combination of RAFT polymerization and ATRP, ring-opening polymerization (ROP) and ATRP or ROP and RAFT polymerization.

Due to the increased attention paid on biomass-derived thermoplastics and elastomers, it becomes necessary and desirable to explore new monomers, which can be synthesized from bioresources, including lignins, terpenes, plant oils, rosin acids, and coumarins. For instance, bioresources, 7-hydroxyl-4-methylcoumarin, vanillin, guaiacol, and oleyl alcohol, can be used to synthesize sustainable monomers by reacting with acryloyl chloride, methacryloyl chloride or methacrylate anhydride as shown in Figure 7b. Among these monomers, 7-acryloyloxy-4 methylcoumarin (AMC), 7-methacryloyloxy-4-methylcoumarin (MMC), acrylated vanillin (AV), methacrylated vanillin (MV), acrylated guaiacol (AG), and methacylated guaiacol (MG) can be utilized as rigid segments, while oleyl acrylate (OA) and oleyl methacrylate (OM) can be utilized as soft segments in the graft copolymers. Though there has been considerable progress, it is still challenging to

Figure 7.

(a) Heterograft and graft-on-graft architectures of cellulose graft copolymers. (b) Sustainable monomers derived from different bioresources.

made in the future by grafting functional block polymers from cellulose via NMP to design novel stimuli-responsive hybrid materials with excellent macroscopic

Schematic illustration for the synthesis of Cell-g-PS and Cell-g-(PMMA-b-PS) copolymers through NMP

CRPs are versatile and robust techniques to develop well-defined cellulose graft copolymers as novel thermoplastic and elastomers with desired properties for particular application demands. As the most adopted technique for cellulose graft copolymers, conventional ATRP suffers from the limitation of removing metal catalysts from the resultant products, which may cause undesired toxicity and coloration. Recently, new advances in ARTP have been reported, and metal-free ATRP has been developed to synthesize narrowly distributed polymers with welldefined structures by using photoredox organic catalysts under light irradiation instead of metal catalysts [84–86]. This new strategy can greatly promote the design and preparation of cellulose graft copolymers for different application fields. Cellulose graft copolymers show improved properties compared to the linear counterparts due to their unique molecular architectures, and the macroscopic performance of cellulose graft copolymers is affected by the degree of polymerization of cellulose

mechanical properties.

Thermosoftening Plastics

3. Perspective

42

Figure 6.

technique.

achieve breakthroughs for the development of cellulose graft copolymers with ultra-strong mechanical properties comparable to commercial petroleum-based products. The marriage of cellulose and novel bio-based monomers via CRPs can provide a variety of opportunities for sustainable materials ranging from thermoplastics to elastomers, and these fascinating materials can find a pyramid of applications in our daily life in the near future.

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**2. Cognitive deficit in schizophrenia**

One of the main recently advances of the concept of schizophrenia has been the confirmation that this disorder is primarily associated to cognitive deficits, do not being a consequence to symptoms or drugs [21]. Nevertheless, the idea that cognitive domains played a fundamental role in this disorder was not so recent. Since the first descriptions of schizophrenia, which was known under the Dementia praecox label, the aspects related to what it is now understood as cognitive deficit were considered as central symptoms of the picture. Besides the essential idea of Bleuler was that the core of schizophrenia, its fundamental symptoms, was the fragmentation of the thought process and delusions and hallucinations were accessory symptoms, a consequence of the main process [22, 23]. It has also been shown that this cognitive deficit has not only been described in long-standing schizophrenic patients [24], but is also present in patients with a first psychotic episode [25–27], in remission [28], in patients without antipsychotic medication [21, 29] and even in studies in high-risk subjects [30] and in close relatives and healthy patients with schizophrenia [12].

It is estimated that among 61–78% of patients with schizophrenia manifest a significant level of cognitive deficit [1] reaching between 1 and 2 standard deviations below the control groups of the same age [6, 31]. These cognitive deficits seem independent of positive symptoms [32] and are maintained throughout the course of the disease.

Although some early investigations [33] showed that about 27% of schizophrenic patients, after neuropsychological evaluation, could not be considered deficit, in fact, it has been proven that they would continue to present neuropsychological deficit compared to healthy subjects, even matching IQ measurements [34]. This group of patients, without supposed deterioration, would show high levels of premorbid functioning, but once the disease was diagnosed, this would be considerably lower [35]. In addition, discordant monozygotic twin comparisons for schizophrenia suggest that almost all affected twins perform worse on cognitive tests than their unaffected twin [36]. In this sense, it can be safely talked about cognitive deficits in schizophrenic patients although, in any case, it is very likely that their cognitive performance is below than what was expected in the absence of disease. It is also interesting to mention that several authors have found a worse performance in tasks of work memory (especially in visual) and in learning verbal tests and free memories in unaffected monozygotic twin brothers and, even a lesser degree, in not affected dizygotic twin brothers against controls [37–40].

It is interesting to point out that many patients have a lack of awareness of cognitive dysfunction. Those who are better aware of their deficits are not associated with a lower use of treatment, nor with a lesser deterioration of executive function. But they do have better results in the rehabilitation of some cognitive domains, in adherence to treatment and in their functional capacity [41, 42].

The neurocognitive dysfunction affects the ability to perform activities of daily living, impairs the ability to solve social problems [15, 16] and has proved to be the better predictor of reincorporation to activities in social and community settings [43, 44] and, especially, in terms of work rehabilitation and maintenance works [45–48]. These data review the importance of cognitive domains as a reliable scale of clinical improvement [15, 49–52] and, as is to be expected, the degree of cognitive impairment implies a worse adjustment in the quality of life of these patients [52–54]. Some authors also consider cognitive functions as an integral part of the concept of resistance to treatment [55]. On the other hand, several studies emphasize the importance and efficacy of cognitive rehabilitation treatments in early stages of psychosis [56, 57].

As expected with these data, the cognitive alteration in schizophrenia is the current focus of attention for the research of therapeutic strategies, both pharmacological and psychological. Regarding the interventions on cognitive domains, the pharmacological treatment, although it has offered certain results, has not been very encouraging [58], however studies using cognitive stimulation (training in executive skills, memory and other cognitive processes) have achieved more hopeful data [59–62].


*2.3. Evaluation of ER Stress*

The effects of C-PE on the PERK/p-eIF2α (Ser52)/ATF4 and PERK/p-eIF2α (Ser52)/ ATF6α signaling pathways is portrayed in Figure 8. HgCl2-induced AKI was manifested as an overexpression of PERK (A), p-eIF2α (Ser 52) (B), ATF4 (C), GADD153 (D), GADD34 (E), and ATF6α (F). The C-PE treatment did not prevent the alteration in the expression of these proteins in both pathways. A representative Western blot of the marker for the PERK/eIF2α/ATF4 and PERK/eIF2α/ATF6α signaling pathways is shown in Figure 9.

Figure 10 shows the effect of C-PE on the IRE1α pathway and the proteins associated with cellular damage. HgCl2 exposure generated an overexpression of IRE1α (panel A), XBP1 (panel B), caspase 12 (panel C), Bax (panel D), p-p53 (Thr 155) (panel G), and p53 (panel H). It also increased the Bax/Bcl2 and p-p53 (Thr 155)/p53 ratios (panels F and I, respectively) and reduced the expression of Bcl2 (panel E). With C-PE treatment, there was no alteration in the level of any of the proteins evaluated, which is observed in the corresponding Western blot depicted in Figure 11.

**Figure 8.** Effect of C-phycoerythrin (C-PE) on HgCl2-induced endoplasmic reticulum stress through the PERK/p-eIF2α (Ser 52)/ATF4-GADD153 and PERK/p-eIF2α (Ser 52)/ATF6α/GADD153 pathways in the kidney. An evaluation was made of the expression of PERK (**A**), p-eIF2α (Ser 52) (**B**), ATF4 (**C**), GADD153 (**D**), GADD34 (**E**), and ATF6α (**F**). Data are expressed as the mean ± SEM (*n* = 3 mice/group). OD, optical density. \* *p* < 0.05 vs. the control group.

**Figure 9.** Representative Western blot of the effect of C-phycoerythrin (C-PE) on HgCl2-induced endoplasmic reticulum stress through the PERK/p-eIF2α (Ser 52)/ATF4/GADD153 and PERK/p-eIF2α (Ser 52)/ATF6α/GADD153 pathways.

**Figure 10.** Effect of C-phycoerythrin (C-PE) on HgCl2-induced endoplasmic reticulum stress and cell death. Protein expression was evaluated for IRE1α (**A**), XBP1 (**B**), caspase 12 (**C**), Bax (**D**), Bcl2 (**E**), the Bax/Bcl2 ratio (**F**), p53 (**G**), p-p53 (Thr 155) (**H**), and the p53/p-p53 (Thr 155) ratio (**I**). Data are expressed as the mean ± SEM (*n* = 3 mice/group). OD, optical density. \* *p* < 0.05 vs. the control group. \*\* *p* < 0.05 vs. the HgCl2 group.

**Figure 11.** Representative Western blot of the effect of C-phycoerythrin (C-PE) on HgCl2-induced endoplasmic reticulum stress through the IRE1α pathway and the attenuation of cell death.

#### **3. Discussion**

C-PE is reported to have nutraceutical activity against the damage resulting from cell insult [12,13]. Our group has demonstrated that treatment with a protein extract rich in C-PE prevented oxidative stress and cellular damage in an animal model of HgCl2-induced AKI [16]. This model was chosen because mercury produces ER stress, which leads to renal damage. However, the aforementioned study only associated the nutraceutical properties of C-PE with scavenging and antioxidant activity. Thus, the aim of the current contribution was to explore the molecular mechanism of action of C-PE (purified from *P. persicinum*) by examining its nephroprotective activity against HgCl2-induced ER stress, oxidative stress, and alterations in the redox environment in the same animal model.

HgCl2 produces oxidative stress and alterations in the redox environment by three mechanisms: Fenton and Haber-Weiss reactions that generate free radicals and ROS [17], the activation of ER stress [3], and the binding of Hg2+ with intracellular sulfhydrylcontaining proteins and low-molecular-weight compounds (e.g., GSH) capable of affecting the redox environment and protein function [18]. As a consequence of these reactions, nephrin and podocin are downregulated, and the slit diaphragm is injured, which is observed as HgCl2-induced AKI. The resulting inflammatory process participates in the progression of AKI [19].

In recent years, the use of nutraceuticals from cyanobacteria and their metabolites has proven effective against renal damage (e.g., AKI) stemming from toxicants or chronic kidney disease [16,20–22]. Purified C-PE presently demonstrated nephroprotective activity when tested against HgCl2-induced AKI, as evidenced by the reduction found in oxidative stress and ER stress.

C-PE, a protein with a molecular weight of ~240 KDa, has nutraceutical properties in vitro as an ROS scavenger [23]. Moreover, it prevents oxidative stress and cellular damage in vivo [12,13]. All reports on C-PE suggest that it is a potent antioxidant. By scavenging ROS, it avoids alterations in the redox environment and therefore impedes cellular damage [12,13,24]. However, animal studies have not yet completely defined the nutraceutical protection mechanism.

C-PE may act as a prodrug that leads to the release of the phycoerythrobilin moiety into the gastrointestinal tract, as previously demonstrated by our group for C-PC and phycocyanobilin [22]. C-PC is known to break down into chromo-peptides that contain phycocyanobilin, followed by the apparent absorption of linear tetrapyrrole compounds facilitated by the action of intestinal peptidases [24,25]. Once in serum, phycoerythrobilin could bind to albumin due to its low water solubility, which would extend its therapeutic activity into the entire organism [26].

The protective effect of C-PE against HgCl2-induced AKI is associated with antioxidant, anti-inflammatory, and chelation mechanisms. C-PE acts as an antioxidant because it contains PEB. In addition, the chemical structure of phycoerythrobilin acts as a nucleophilic compound, neutralizing free radicals and ROS [24]. According to an in vitro model, the chelation of Hg2+ by PEB suppresses the degranulation of RBL-2H3 mast cells and decreases the intracellular concentration of Ca2+ [27], giving rise to anti-inflammatory and nephroprotective effects. Hg2+ binds to PEB thioether bridges in C-PE, which assume a cyclic helical form capable of chelation [28]. The antioxidant and chelating activity of C-PE can avoid Fenton and Haber-Weiss reactions and consequently ameliorate the production of free radicals, the generation of oxidative stress, and the alteration of the redox environment in kidney cells. All the aforementioned mechanisms of C-PE are related to the maintenance of the redox environment and therefore prevent the dysfunction of organelles such as the ER.

In the current evaluation of proteostasis, HgCl2-induced ER stress was found to activate the IRE1α pathway and promote cell death. At the same time, mercury activated the PERK pathway, which restored proteostasis through PERK/eIF2α/ATF-4/GADD153. When the cell was incapable of compensating for imbalances in proteostasis, the activation of ATF4 and GADD153 in the same pathway led to the expression of proapoptotic proteins and the triggering of cell death. As can be appreciated, PERK and IRE1α have a synergic effect in prompting kidney cell death by increasing the Bax/Bcl-2 ratio and the level of caspases 3, 8, 9, and 12 [3,10]. Hence, HgCl2 was capable of generating AKI in the present study by fomenting oxidative stress, an alteration in the redox environment, and ER stress. The resulting histological damage was considerable (grade 4), affecting over 75% of tubular and glomerular cells.

C-PE treatment enhanced the canonical ER response through the PERK/p-eIF2α (ser 52)/ATF-4/GADD153 pathway, involving ER-associated degradation (ERAD), known to process misfolded and unfolded proteins. The phosphorylation of eIF2α (ser 52) is able to suppress the overall translation of mRNA, thus reducing protein stress in the ER. Furthermore, the moderate increment in ATF6α upregulates several genes that participate in the adaptative phase of the unfolded protein response [29]. C-PE treatment is herein proposed to have activated the PERK and ATF6 signaling pathways, maintaining proteostasis by avoiding oxidative stress and alterations in the redox environment and by activating the unfolded protein response [30,31].

The response elicited by C-PE is distinct from that of other phycobiliproteins. For instance, C-PC averts the overexpression of GADD34 by activating GADD153, which is related to the inhibition of apoptosis [11,32]. On the other hand, both C-PC and C-PE maintain proteostasis. The differences between these two responses should be explored in depth in future research.

C-PE and C-PC have a similar effect on the IREα pathway, decreasing cell death mediated by caspases 3, 9, and 12 as well as reducing the disruption in p53 activation and the alteration of the Bax/Bcl2 ratio [10,11]. This idea is supported by neurotoxicological models, where C-PE prevents ER stress linked to calcium deregulation and mitochondrial dysfunction [33].

In the control group, interestingly, C-PE per se increased the phosphorylation of p53 (Thr 155), which is a genome gatekeeper because it is a master transcriptional factor that induces cellular senescence and suppresses cell growth and tumor formation. Exposure to various cellular stressors, however, causes p53 to be overexpressed and phosphorylated in several regions, leading to cell cycle arrest or apoptosis. Accordingly, p53 is phosphorylated by the C-Jun activation domain-binding protein-1 (Jab1) in Thr 155, promoting its translocation into the cytoplasm to favor interaction with the COP9 signalosome complex. These nuclear export mechanisms of p53 provide a practical future approach to a possible C-PE-induced activation of anti-cancer therapy by p53 [34], as evidenced by the lack of histological irregularities in the C-PE control group as well as the capacity of C-PE treatment of AKI mice to prevent oxidative stress, ER stress, and alterations in the redox environment and cell death markers.


*Review* **Enzymatic Bioreactors: An Electrochemical Perspective**

#### **Simin Arshi 1 , Mehran Nozari-Asbemarz 2 and Edmond Magner 1, \***

Received: 30 September 2020; Accepted: 18 October 2020; Published: 24 October 2020

**Abstract:** Biocatalysts provide a number of advantages such as high selectivity, the ability to operate under mild reaction conditions and availability from renewable resources that are of interest in the development of bioreactors for applications in the pharmaceutical and other sectors. The use of oxidoreductases in biocatalytic reactors is primarily focused on the use of NAD(P)-dependent enzymes, with the recycling of the cofactor occurring via an additional enzymatic system. The use of electrochemically based systems has been limited. This review focuses on the development of electrochemically based biocatalytic reactors. The mechanisms of mediated and direct electron transfer together with methods of immobilising enzymes are briefly reviewed. The use of electrochemically based batch and flow reactors is reviewed in detail with a focus on recent developments in the use of high surface area electrodes, enzyme engineering and enzyme cascades. A future perspective on electrochemically based bioreactors is presented.

**Keywords:** bioelectrocatalysts; oxidoreductases; biocatalytic reactors; electrochemical reactors