**3. The determinants of immunoparalysis**

Only recently it became clear that the CARS does not represent only a physiologic counterbalance to the inflammatory response to PAMP and DAMP but that it can determine a critical condition in and by itself [13, 15].

Actually, different experimental and clinical studies indicate that the advanced stage of sepsis and SS is characterized by a reduction of both the innate and adaptive immune responses (**Table 4**). Extensive evidence supports this model, even if large inter-patient differences exist. First, monocytes present a reduced expression of membrane HLA-DR in association to either a decreased secretion of inflammatory mediators when stimulated or a diminished antigen presentation. Second, different membrane-bound receptors able to potentiate the immune response, including IL-2α, IL-7R α, CD86, etc., are reduced. Third, the production of immunosuppressant substances, such and programmed death 1 (PD1) and its ligand (PD-L1), is increased in antigenpresenting cells, thus inhibiting the activation of T lymphocytes. Fourth, there is an increased appearance of immunosuppressive T-cell subpopulations, such as myeloid-derived suppressor cell and CD4+ and CD25+ T-regulatory cells (Treg), which suppress adaptive immunity. These appear to be particularly relevant, as Treg (a) actively produce anti-inflammatory cytokines including TGF-β and IL-10, (b) downregulate the secretion of pro-inflammatory mediators, (c)

neutralize cytotoxic T cells, and (d) deactivate the monocytes. Fourth, immune cells present an increased apoptosis, and their loss is not replaced enough by the production of new ones. Finally, the phagocytosis of apoptotic cells by fixed and circulating macrophages leads to a switch of the latter to the M2 phenotype, whose feature is an increased production of the antiinflammatory substances IL-10 and IL-1ra. Put briefly, all these mechanisms exert their action via relatively few common pathways, which include the increased apoptosis determining the reduction of immune cells, the loss of antigen presentation, the blunted response to PAMP, and the reduction of energy production caused by the impairment of the glucose metabolism (**Table 5**) [16, 17]. All these reactions are driven by epigenetic changes causing in different time frames the activation or deactivation of genes involved in the immune response, and the resulting phenotype is an intense inflammatory response or, conversely, an immunoparalysis.

Endotoxin tolerance ↑ Anti-inflammatory mediators, ↓ pro-inflammatory mediators

Apoptosis

↓ Antigen presentation

Immune cell number anergy

Immunoparalysis in Septic Shock Patients http://dx.doi.org/10.5772/intechopen.88866 13

The recognition of sepsis-induced immunoparalysis is not straightforward because the clinical manifestations associated with the switch from the hyperinflammatory state to CARS and the full-blown depression of the immune capabilities are not so protean as the symptoms of SS [18]. Moreover, the SSC guidelines focus almost exclusively on the former and pay much less attention, if any, to the latter. From a practical and clinical point view, some issues appear

The transition from the hyperinflammatory phase to immunoparalysis can be challenging to identify and to monitor at the bedside and represents a kind of no man's land in the clinical

The onset is highly variable. Actually, although the secretion of immunomodulatory substances can occur relatively early, their clinical consequences present wide variations. Some authors [19] observed a substantial difference of mHLA-DR starting from 3 to 7 days in a small group of surgical septic patients, and other authors demonstrated that significant decrease of

**4. The diagnosis of immunoparalysis**

**Mechanisms Effect**

**Table 5.** Mechanisms of immunoparalysis.

Apoptosis ↓ Immune cell number

Energy failure Immune cell anergy

Epigenetic regulation ↓ Pro-inflammatory mediators

course of patients which survived from the initial phase of SS.

particularly relevant.

**4.1. Timing of onset**

mHLA-DR, human leukocyte antigen on the monocyte surface; PD-(L1), programmed death ligand; CTLA-4, cytotoxic lymphocyte antigen 4; BTLA, B and T lymphocyte attenuator; LAG-3, lymphocyte activation gene 3; TIM-3, T lymphocyte immunoglobulin protein 3; sFAS, soluble FAS ligand; TGF-β, transforming growth factor-β.

**Table 4.** Factors of immunosuppression.

**Table 5.** Mechanisms of immunoparalysis.

toward the baseline immune function; and in the third one, the CARS prevails and causes the

Only recently it became clear that the CARS does not represent only a physiologic counterbalance to the inflammatory response to PAMP and DAMP but that it can determine a critical

Actually, different experimental and clinical studies indicate that the advanced stage of sepsis and SS is characterized by a reduction of both the innate and adaptive immune responses (**Table 4**). Extensive evidence supports this model, even if large inter-patient differences exist. First, monocytes present a reduced expression of membrane HLA-DR in association to either a decreased secretion of inflammatory mediators when stimulated or a diminished antigen presentation. Second, different membrane-bound receptors able to potentiate the immune response, including IL-2α, IL-7R α, CD86, etc., are reduced. Third, the production of immunosuppressant substances, such and programmed death 1 (PD1) and its ligand (PD-L1), is increased in antigenpresenting cells, thus inhibiting the activation of T lymphocytes. Fourth, there is an increased appearance of immunosuppressive T-cell subpopulations, such as myeloid-derived suppressor

appear to be particularly relevant, as Treg (a) actively produce anti-inflammatory cytokines including TGF-β and IL-10, (b) downregulate the secretion of pro-inflammatory mediators, (c)

T-regulatory cells (Treg), which suppress adaptive immunity. These

↓ TNF-α production

↓ lymphocytes

↑ IL-10/TNF-α

Anti-inflammatory cytokines ↑ IL-10, IL-13, IL-4, IL1 receptor antagonists, TGF-β

immunoglobulin protein 3; sFAS, soluble FAS ligand; TGF-β, transforming growth factor-β.

mHLA-DR, human leukocyte antigen on the monocyte surface; PD-(L1), programmed death ligand; CTLA-4, cytotoxic lymphocyte antigen 4; BTLA, B and T lymphocyte attenuator; LAG-3, lymphocyte activation gene 3; TIM-3, T lymphocyte

↑ CTL-4, BTLA expression ↑ LAG-3 and TIM-3 expression

↑ myeloid-derived suppressor cells

loss of the immune capabilities.

12 Infectious Process and Sepsis

condition in and by itself [13, 15].

and CD25+

**Factors involved Marker**

Tissue macrophage dysfunction Presently none Negative regulatory mediators ↑ PD-(L)1 expression

Receptors downregulation ↓ IL-7 receptor

Suppression of immune cells ↑ CD-4, CD-25

Apoptosis ↑ FAS

**Table 4.** Factors of immunosuppression.

Monocyte deactivation ↓ mHLA-DR expression

cell and CD4+

**3. The determinants of immunoparalysis**

neutralize cytotoxic T cells, and (d) deactivate the monocytes. Fourth, immune cells present an increased apoptosis, and their loss is not replaced enough by the production of new ones. Finally, the phagocytosis of apoptotic cells by fixed and circulating macrophages leads to a switch of the latter to the M2 phenotype, whose feature is an increased production of the antiinflammatory substances IL-10 and IL-1ra. Put briefly, all these mechanisms exert their action via relatively few common pathways, which include the increased apoptosis determining the reduction of immune cells, the loss of antigen presentation, the blunted response to PAMP, and the reduction of energy production caused by the impairment of the glucose metabolism (**Table 5**) [16, 17]. All these reactions are driven by epigenetic changes causing in different time frames the activation or deactivation of genes involved in the immune response, and the resulting phenotype is an intense inflammatory response or, conversely, an immunoparalysis.

### **4. The diagnosis of immunoparalysis**

The recognition of sepsis-induced immunoparalysis is not straightforward because the clinical manifestations associated with the switch from the hyperinflammatory state to CARS and the full-blown depression of the immune capabilities are not so protean as the symptoms of SS [18]. Moreover, the SSC guidelines focus almost exclusively on the former and pay much less attention, if any, to the latter. From a practical and clinical point view, some issues appear particularly relevant.

### **4.1. Timing of onset**

The transition from the hyperinflammatory phase to immunoparalysis can be challenging to identify and to monitor at the bedside and represents a kind of no man's land in the clinical course of patients which survived from the initial phase of SS.

The onset is highly variable. Actually, although the secretion of immunomodulatory substances can occur relatively early, their clinical consequences present wide variations. Some authors [19] observed a substantial difference of mHLA-DR starting from 3 to 7 days in a small group of surgical septic patients, and other authors demonstrated that significant decrease of the CD14/HLA-DR and of heat-shock proteins (HSP) 70 and 90 was present already within 24 hours from the onset of sepsis [5]; in both studies, these alterations were more marked in patients who developed SS later on. More recently, Morris et al. [20] in association with raised percentage of regulatory T cells (Treg) were predictive for infections occurring between 3 and 9 days after ICU admission, and a similar timing has been demonstrated also in another study in which the mortally rate of secondary infection was ~14% [17]. On the basis of these findings, it is reasonable to hypothesize that (a) a combination of cellular and soluble factors able to blunt the immune response is present since the very initial phase of sepsis; (b) their effects on the clinical course, namely, the appearance of secondary infections and/or viral reactivation, can occur within the initial 10 days from the admission; and (c) these are associated with a substantial mortality of patients surviving the initial insult.

challenges, and the measurement of the blood concentrations of soluble mediators involved in the different clinical frames [14, 15, 21, 22]. It could be useful to describe separately those currently available and those which will be used likely in the next future. Most of the former (**Table 6**) can be obtained cheaply and on a daily basis; among all, the neutrophil-to-lymphocyte ratio has been indicated as the less costly and more rapidly available monitoring tool [23, 24]. Other advanced, expensive, and not yet widely available monitoring tools take advantage of more sophisticated lab techniques (**Table 7**) requiring lab expertise and financial resources putting them at risk of not being used outside the research center. Another dynamic approach, which shares the very same limitations of the previously described advanced techniques, consists in challenging the immune cells with substances able to trigger their activation, including LPS, other PAMP, and phytohemoagglutinin; actually, a number of investigators demonstrated that a blunted response to the stimulation is associated with an increased rate of severe infec-

Independently from the systems used, it should be clear that the monitoring of the immune response in septic as well in other clinical conditions (a) is based on the time variations of a panel of indicators and not on a single one and (b) due to their direct and indirect costs,

**Function Cell Marker Outcome Lab technique Runaround (h)**

↑ PD-L1 Secondary

All lymphocytes ↑ CTLA 4, BTLA Not clear FC, IHC 1.5

Treg ↑ Treg Death FC 1.5

FC, flow cytometry; IHC, immunohistochemistry; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent

CD 127 Death,

Both Transcriptomic CD 74, CX3CR1 Not clear PCR, microarray 72

secondary infections

infections

secondary infections

secondary infections

secondary infections MODS

IL10/TNF ratio Death ELISA 5

↑ PD Death FC, IHC 1.5

Not clear Cell culture,

FC, PCR, IHC, ELISA

ELISA, FC, IHC

FC, IHC 1.5

FC 1.5

FC, IHC 1.5

Cell culture + FC 72

5

Immunoparalysis in Septic Shock Patients http://dx.doi.org/10.5772/intechopen.88866 15

72

tious complications in different patient populations [25–27].

Monocytes ↓ sCD127 Death,

Dendritic cells ↓ Count Death,

T cells Proliferation Death,

**Table 7.** Some promising, yet not currently available, markers of immunoparalysis.

Endotoxin tolerance

Innate immunity

Adaptive immunity

assay.

### **4.2. Monitoring of the immune function**

In ICU patients, every organ system is monitored to allow a change in the treatment tailored on the variation observed. An ideal monitoring system should be accurate, cheap, and not labor-intensive, and the information gathered should be readily if not continuously available. Since it has become clear that the immune system in sepsis undergoes modifications not reflected by the commonly measured biological variables such as the arterial pressure, the heart rate, the urinary output, etc., different investigations aimed to identify one or more markers of changes of its functions whose follow-up could be valuable to modify the therapy according to its changes: as an example, the occurrence of immunoparalysis contraindicates the administration of steroids whose use is recommended by the SSC guidelines.

Several monitoring systems exploring both legs of the immune response have been developed so far, based on the repeated assessments of the cells involved, their response to different

**Table 6.** Some currently available indicators of immune function.

challenges, and the measurement of the blood concentrations of soluble mediators involved in the different clinical frames [14, 15, 21, 22]. It could be useful to describe separately those currently available and those which will be used likely in the next future. Most of the former (**Table 6**) can be obtained cheaply and on a daily basis; among all, the neutrophil-to-lymphocyte ratio has been indicated as the less costly and more rapidly available monitoring tool [23, 24]. Other advanced, expensive, and not yet widely available monitoring tools take advantage of more sophisticated lab techniques (**Table 7**) requiring lab expertise and financial resources putting them at risk of not being used outside the research center. Another dynamic approach, which shares the very same limitations of the previously described advanced techniques, consists in challenging the immune cells with substances able to trigger their activation, including LPS, other PAMP, and phytohemoagglutinin; actually, a number of investigators demonstrated that a blunted response to the stimulation is associated with an increased rate of severe infectious complications in different patient populations [25–27].

the CD14/HLA-DR and of heat-shock proteins (HSP) 70 and 90 was present already within 24 hours from the onset of sepsis [5]; in both studies, these alterations were more marked in patients who developed SS later on. More recently, Morris et al. [20] in association with raised percentage of regulatory T cells (Treg) were predictive for infections occurring between 3 and 9 days after ICU admission, and a similar timing has been demonstrated also in another study in which the mortally rate of secondary infection was ~14% [17]. On the basis of these findings, it is reasonable to hypothesize that (a) a combination of cellular and soluble factors able to blunt the immune response is present since the very initial phase of sepsis; (b) their effects on the clinical course, namely, the appearance of secondary infections and/or viral reactivation, can occur within the initial 10 days from the admission; and (c) these are associated with

In ICU patients, every organ system is monitored to allow a change in the treatment tailored on the variation observed. An ideal monitoring system should be accurate, cheap, and not labor-intensive, and the information gathered should be readily if not continuously available. Since it has become clear that the immune system in sepsis undergoes modifications not reflected by the commonly measured biological variables such as the arterial pressure, the heart rate, the urinary output, etc., different investigations aimed to identify one or more markers of changes of its functions whose follow-up could be valuable to modify the therapy according to its changes: as an example, the occurrence of immunoparalysis contraindicates the administration of steroids whose use is recommended by the SSC guidelines. Several monitoring systems exploring both legs of the immune response have been developed so far, based on the repeated assessments of the cells involved, their response to different

**Function Cell Marker Outcome Lab technique Runaround (h)**

Secondary infections

Secondary infections

Secondary infections

Secondary infections

FC. Hematology analyzer

FC. Hematology analyzer

FC.  Some currently available indicators of immune function.

Both Lymphocytes Viral reactivation Death PCR 12

FC, flow cytometry; IHC, immunohistochemistry; PCR, polymerase chain reaction; NTL, neutrophil/lymphocyte ratio.

a substantial mortality of patients surviving the initial insult.

**4.2. Monitoring of the immune function**

14 Infectious Process and Sepsis

Innate immunity

Adaptive immunity Independently from the systems used, it should be clear that the monitoring of the immune response in septic as well in other clinical conditions (a) is based on the time variations of a panel of indicators and not on a single one and (b) due to their direct and indirect costs,

FC, flow cytometry; IHC, immunohistochemistry; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.

**Table 7.** Some promising, yet not currently available, markers of immunoparalysis.

demonstrating the tumor cells are able to suppress in many different ways the host's immune response against themselves. Independently from the substance use and the molecular target, these innovative treatments have been demonstrated to be effective but somehow difficult to handle, as they are associated with a number of side effects ranging from mild to life-threatening [31]. As several similarities exist between tumor- and sepsis-induced blunting of the immune response [32], it is likely that in the next future the immune-boosting treatments will be developed to treat the latter, aiming to develop a precision medicine also in ICU patients

Presently, according to the SSC guidelines [2], the immune-targeted approaches are limited to the administration of steroids in not fluid and catecholamine-responding SS, whereas the use of intravenous immunoglobulins (IvIg) is discouraged. Actually, this latter position is questionable as a number of trials performed in several thousands of patients demonstrated that (a) the administration of IvIg is associated with the reduction of mortality in different subsets of SS patients; (b) among the different preparations available, the only ones containing supranormal concentrations of IgM and IgA appears more effective, and (c) the improvement of survival is time-dependent, as a ~6% increase of mortality has been observed for every day of

Besides steroids and IvIg, other treatments aimed to modulate the immune response include

GM-CSF

FTL3L TNF

Anti-PD1 ab Anti-PDL 1 ab

GM-CSF TLR agonists FT3L TNF

Toll-like receptor antagonists

Immunoparalysis in Septic Shock Patients http://dx.doi.org/10.5772/intechopen.88866 17

Anti CTLA4, TIM3, LAG3 ab

blood purification (BPT) techniques and a number of substances able to boost it.

**Cells/factors involved Alterations Possible therapies**

↑ Tolerant dendritic cells

Altered metabolism ↓ Proliferation

↑ PGE 2 ↑ TGFβ

**Table 8.**

[33] (**Table 8**).

delay in the administration [34].

Myeloid cells ↑ Immature neutrophils

Lymphocytes ↓ Cytokine production

↑ Systemic cytokine release ↑ IL-10

**Figure 2.** The multiple hits phenomenon ultimately leading to the exhaustion of the immune response.

it should be limited to the subjects at risk; as an example, it is worthwhile to monitor the immune function in patients undergoing multiple abdominal surgical procedures for suture dehiscence but not in another one safely recovering after peritonitis.

### **4.3. The identification of patients at risk of immunoparalysis**

Even with the exclusion of clinical conditions and/or treatments known to cause an immunoparalysis (i.e., solid and hematologic cancers, autoimmune disorders), etc., this circumstance can occur in virtually all ICU patients; however, different studies identified some predisposing factors that should be considered particularly relevant, including septic shock, advanced age, health care-associated infections, elevated Charlson's score indicating a substantial underlying fragility, comorbidities, prolonged hospital and ICU length of stay, and multiple surgical procedures [17, 28, 29]. The latter, which are associated with the repeated activation of the inflammatory and anti-inflammatory responses, according to the multiple hits model, ultimately lead to the exhaustion of the immune response [30] (**Figure 2**).


**1. Introduction**

Plasma concentration (>5 to >100 μg/dl) of lead can cause neurological, cardiovascular, hematological, reproductive, renal, immunological, and respiratory problems [1]. The reported lead concentrations of surface sample of soil was 23–35 mg/kg soil, whereas the concentration of the lead elute from soil was 0.6 mg/L, respectively [2]. Lead has the ability to penetrate brain having molecular weight of 207.28 g [3] and elimination half-life of 18 months. Highly contaminated soil could have the concentration of lead 3.03 times higher than the maximum limit for agricultural soil and 1.97 times higher than the value limit for fodder [4], suggesting that lead is very stable in soil and toxic to human [5]. However, soil concentration of lead (11.42 mg/L) could decrease during rainy season relative to dry season [6]. Lead exerts opposite effects on antibody response and phagocytosis [7]. Some plants such as *Cyamopsis tetragonoloba* and *Sesamum indicum* could tolerate lead concentration of up to 1000 mg/kg; hence, they could be used for bioremediation of soil heavily contaminated by lead [8]. The factors responsible for penetration of central nervous system acting agents are lipid solubility, pH, and molecular weight [9]. Hence, toxicological study of chemicals is necessary for the identification of potential toxicants [10]. Severe lead poisoning in young children and neonatal rats may cross microvessel endothelium of the brain. There is evidence that lead can cause brain damage, and lead uptake in the endothelium is reduced by calcium adenosine triphosphatase (ATPase) pump [11]. In view of this, transport of lead through brain capillary was mathematically assessed with a view to identifying pathogenesis of brain injury caused by lead in humans.


**Contents**

Reprinted from: *Membranes* **2020**, *10*, 200, doi:10.3390/membranes10090200 ............ **157**

#### **Violetta Gil, Mikhail Porozhnyy, Olesya Rybalkina, Dmitrii Butylskii and Natalia Pismenskaya**

The Development of Electroconvection at the Surface of a Heterogeneous Cation-Exchange Membrane Modified with Perfluorosulfonic Acid Polymer Film Containing Titanium Oxide Reprinted from: *Membranes* **2020**, *10*, 125, doi:10.3390/membranes10060125 ............ **175**

#### **Myriam Bdiri, Asma Bensghaier, Lobna Chaabane, Anton Kozmai, Lassaad Baklouti and Christian Larchet**

Preliminary Study on Enzymatic-Based Cleaning of Cation-Exchange Membranes Used in Electrodialysis System in Red Wine Production

```
Reprinted from: Membranes 2019, 9, 114, doi:10.3390/membranes9090114 .............. 195
```
## **About the Editors**

**Lasˆaad Dammak** is a professor specialized in Materials Science and in Industrial Process Engineering, at the Institut de Chimie et des Matériaux Paris-Est, which is a joint research unit between the Centre National de le Recherche Scientifique (CNRS, France) and the Université Paris-Est Créteil (UPEC, France). His research activities are mainly focused on ion-exchange materials and their applications in membrane processes in various fields, such as water treatment and purification, food industry, and energy production. These activities concern the synthesis, physicochemical, mechanical, and structural characterization of membranes, the modeling of their microstructures, and the study of their fouling throughout their life cycle, whereby antifouling solutions are provided to extend the life of these materials. He has participated in many national and international projects in this field.

**Natalia Pismenskaya** is a professor at the Department of Physical Chemistry and Head of the Laboratory of Electromembrane Phenomena at the Institute of Membranes, Kuban State University (Russia). Her research interests are mainly focused on the fundamental aspects of mass transfer in systems with ion-exchange membranes. These activities concern the development of experimental methods for investigating the phenomena of concentration polarization and studying the transfer of substances capable of participating in protonation–deprotonation reactions toward revealing the mechanisms of fouling during electrodialysis purification and concentration of various solutions, including the processing of liquid media in the food industry. She leads and participates in many national and international scientific projects in addition to teaching postgraduate and undergraduate students in this field.

## **Preface to "In-Depth on the Fouling and Antifouling of Ion-Exchange Membranes"**

The use of ion-exchange membranes (IEMs) has accelerated over the past two decades in a wide variety of industrial processes (electrodialysis, electro-electrodialysis, electrolysis, dialysis, etc.) for applications related to the chemical, pharmaceutical, and food industries, energy production, and water treatments, among others. Organic and mineral fouling (or scaling) phenomena are two major factors limiting the efficiencies of IEM processes and performances (which reduce the selectivity and stability of IEMs, increase their electrical resistance, decrease the energy efficiency of the process, etc.) leading to significant economic losses. While the current washing, cleaning, and sterilization processes (antifouling treatments) make it possible to restore some of the loss in IEM performance, they frequently result in the degradation of membrane material.

Another essential point in fouling studies is to choose the best analysis and diagnostic technique to evaluate any degree of magnitude or to observe any type of object on the surface or within the membrane mass.

This book is a collection of the scientific contributions to the Special Issue "Fouling and Antifouling of Ion-Exchange Membranes" of the journal *Membranes*. It focuses on recent advancements in techniques for diagnosing and characterizing the fouling effects on membranes, the mechanisms governing this complex phenomenon, and the various innovative and economically viable solutions for reducing fouling.

> **Las ˆaad Dammak, Natalia Pismenskaya** *Editors*

#### *Editorial* **In-Depth on the Fouling and Antifouling of Ion-Exchange Membranes**

**Lasâad Dammak 1,\* and Natalia Pismenskaya 2**

This work is a synthesis of several in-depth studies on fouling and antifouling phenomena of ion-exchange membranes (IEMs). It is motivated by the increasing interest of the scientific and industrial communities in the diagnosis, quantification, and understanding of the different fouling aspects, and also in applications for its reduction. The main aim of such a synthesis is to help users of processes associated with ion-exchange membranes to find the best solution in order to extend the duration of membrane life and to reduce the operating costs of industrial processes.

Separation processes associated with ion-exchange membranes (IEM-processes), and with their two main families of dialysis (absence of electric current) and electrodialysis (application of an electric current) membranes, were first established for water treatment and desalination, and are still highly recommended in this field for their high water recovery, long life, and acceptable electricity consumption. Today, thanks to technological progress, these IEM-processes, especially electrodialysis, and the emerging new IEMs, have been extended to many other applications (chemical, food, pharmaceutical, and energy industries, etc.). This expansion of uses has also generated several problems, such as limitation of IEMs' lifetimes due to different ageing phenomena (because of organic and/or mineral compounds). If these aspects are not sufficiently controlled and mastered, the use and the efficiency of IEM-processes will be limited since they will no longer be competitive or profitable compared to other separation methods. The current commercial IEMs have excellent performances in IEM-processes; however, organic foulants such as proteins, surfactants, polyphenols, or other natural organic matter, can adhere to their surface (especially when using anion-exchange membranes (AEMs)) forming a colloid layer or they can infiltrate the membrane matrix, which leads to an increase in ion-transport resistance, resulting in higher energy consumption, lower water recovery, loss of membrane permselectivity and current efficiency as well as lifetime limitation. It has therefore become necessary and very useful to develop research areas to understand these fouling phenomena in order to act appropriately to limit their negative effects. Significant developments have been achieved in recent years and, currently, research is very active on this topic.

In this Special Issue, we have collected contributions on recent advancements in techniques for diagnosis and characterization of the fouling effects on ion-exchange membranes, the mechanisms governing this complex phenomenon, and the various innovative and economically viable solutions for reducing fouling. The Special Issue contains nine articles. Two reviews on ion-exchange membrane fouling during the electrodialysis process in the food industry are included; three studies are focused on the formation of fouling both on the surface and inside the IEMs, one of which involves a mathematical modeling of this phenomenon; three other studies are devoted to the antifouling processes, and the ninth study is dedicated to scaling (mineral fouling).

Dammak et al. [1] and Pismenskaya et al. [2] reviewed a significant number (about 380) of recent and relevant high-quality scientific papers, research articles and reviews studying the phenomena of IEM fouling during the ED process in the food industry with a special

**Citation:** Dammak, L.; Pismenskaya, N. In-Depth on the Fouling and Antifouling of Ion-Exchange Membranes. *Membranes* **2021**, *11*, 962. https:// doi.org/10.3390/membranes11120962

Received: 1 December 2021 Accepted: 3 December 2021 Published: 7 December 2021

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

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

focus on the last decade. They first classified the different types of fouling according to the most commonly used classifications. Then, the fouling effects and the characterization methods and techniques as well as the different fouling mechanisms and interactions where presented, analyzed, discussed, and illustrated in a very in-depth manner. The relationships between the nature of the foulant and the structural, physicochemical, and transport properties and behaviors of ion-exchange membranes in an electric field were analyzed using experimental data and modern mathematical models. The implications of traditional chemical cleaning are taken into account in this analysis and modern "mild" membrane cleaning methods are discussed. Therefore, conclusions and outlook are provided, highlighting the main technical challenges, the current status of the fouling and antifouling phenomena of ion-exchange membranes in the food industry, and the key points for the future R&D. Finally, many challenges for the near future are identified and realistic suggestions are made.

Kozmai et al. [3] show the broad possibilities of electrochemical impedance spectroscopy for assessing the capacitance of interphase boundaries and the resistance and thickness of the foulant layer through the example of an AMX-Sb homogeneous membrane separating a red-wine solution from a 0.02 M sodium chloride solution. This enabled them to determine to what extent foulants affect the electrical resistance of ion-exchange membranes, the ohmic resistance and the thickness of diffusion layers, the intensity of water splitting, and the electroconvection in under- and over-limiting current modes. The authors established that short-term (10 h) contact of the AMX-Sb membrane with wine reduces the water splitting due to the screening of fixed groups on the membrane surface by wine components. On the contrary, biofouling, which develops upon a longer membrane operation, enhances water splitting, due to the formation of a bipolar structure on the AMX-Sb surface. This bipolar structure is composed of a positively charged surface of the anion-exchange membrane and negatively charged outer membranes of microorganisms. The island-like distribution of foulants on the membrane surface contributes to the development of electroconvection. Using optical microscopy and microbiological analysis, Kozmai et al. found that more intense biofouling was observed on the AMX-Sb surface that had not been in contact with wine.

Todeschini et al. [4] were interested in the herring milt hydrolysate (HMH) which, like many fish products, has the drawback of being associated with off-flavors. As odor is an important criterion, an effective deodorization method targeting the volatile compounds responsible for off-flavors was developed. First, the authors identified 15 volatile compounds known for their main contribution to the odor of HMH. Then, they evaluated the performances of the electrodialysis (ED) process to remove these 15 volatile compounds as well as trimethylamine, dimethylamine, and trimethylamine oxide, by testing the impact of both hydrolysate pH (4 and 7) and pulsed electric field (PEF) current modes (duration of a DC pulse and pause). The ED performance was compared with that of a deaerator by assessing three hydrolysate pH values (4, 7, and 10). The initial pH of HMH had a huge impact on the targeted compounds, while ED had no effect. Finally, the authors showed that the fouling formation, resulting from electrostatic and hydrophobic interactions between HMH constituents and ion-exchange membranes (IEM), the occurrence of water splitting at IEM interfaces, due to the reaching of the limiting current density, and the presence of water dissociation catalyzers were considered as the major limiting ED-process conditions.

Skolotneva et al. [5] focused on the electrochemical methods utilizing reactive electrochemical membranes (REMs) as a promising technology for efficient degradation and mineralization of organic compounds in natural, industrial, and municipal wastewaters. They proposed a two-dimensional (2D) model considering the transport and reaction of organic species with hydroxyl radicals generated at a TiOx REM operated in flow-through mode, which takes into account convection, diffusion, and chemical reaction constants. This model allows the determination of unknown parameters of the system by treatment of experimental data and predicts the behavior of the electrolysis setup. The authors obtained a good agreement in the calculated and experimental degradation rate of a model pollutant

(foulants molecules) at different permeate fluxes and current densities. The model also provides an understanding of the current density distribution over an electrically heterogeneous surface and its effect on the distribution profile of hydroxyl radicals and diluted species. Skolotneva et al. proved that the removal percentage of paracetamol increases with decreasing the pore radius by foulant molecules and/or increasing the porosity of the membrane. This effect becomes more pronounced as the current density increases. The model highlights how convection, diffusion, and reaction limitations have to be taken into consideration for understanding the effectiveness of the process.

Andreeva et al. [6] investigated scaling on the surface of a heterogeneous MK-40 cationexchange membrane and on the same membrane, the surface layer of which contained polyaniline. The studies were carried out using a solution of a mixture of Na2CO3, KCl, CaCl2, and MgCl2 salts. Voltammetry and chronopotentiometry with the simultaneous control of the desalted solution pH provided information on the effect of current density on the concentration polarization of the membrane systems under study. A mineral scale on the surfaces of the studied membranes was found by scanning electron microscopy. Two limiting currents, which were observed on the current–voltage curve of the modified membrane, indicated the formation of a bipolar interface between the polyaniline layer with positively charged amino groups and the pristine membrane with negatively charged sulfone fixed groups. Enhanced water splitting due to the formation of this bipolar interface caused more significant scaling on the modified membrane compared to the commercial MK-40.

Nichka et al. [7] compared the effect of five different solution flow rates, corresponding to Reynolds numbers of 162, 242, 323, 404, and 485, combined with the use of pulsed electric field current mode to avoid protein fouling of the bipolar membrane (BPM) during electrodialysis of skim milk. They proved that application of PEF almost completely prevented fouling formation by proteins on the cationic interface of the BPM, regardless of the flow rate or Reynolds number. Indeed, under the PEF mode of current, the weight of protein fouling was negligible in comparison with the continuous current (CC) mode (0.07 ± 0.08 mg/cm2 vs. 5.56 ± 2.40 mg/cm2). When CC mode was applied, a Reynolds number equal to or higher than 323 corresponded to a minimal value of protein fouling of the BPM. The authors explain that the positive effect of both increasing the flow rate and using a PEF is due to the fact that during pauses, the solution flow flushes the accumulated protein from the membrane while at the same time there is a decrease in concentration polarization (CP) and consequently a decrease in H+ flux at the feed solution/cationic interface of the BPM, minimizing fouling formation and accumulation.

Gil et al. [8] focused on modifying the surface of the MK-40 cation-exchange membrane to reduce fouling in wastewater ED by enhancing electroconvection. The surface of this membrane was covered with the perfluorosulfonic acid (PFSA) polymer film doped with TiO2 nanoparticles. It was found that changes in surface characteristics conditioned by such modification lead to an increase in the limiting current density due to the stimulation of electroconvection, which develops according to the mechanism of electroosmosis of the first kind. The greatest increase in the current compared to the pristine membrane was obtained by modification with the film being 20 μm thick and containing 3 wt% of TiO2. The sample containing 6 wt% of TiO2 provided higher mass transfer in over-limiting current modes due to the development of nonequilibrium electroconvection. A 1.5-fold increase in the thickness of the modifying film reduced the positive effect of introducing TiO2 nanoparticles due to (1) partial shielding of the nanoparticles on the surface of the modified membrane and (2) a decrease in the tangential component of the electric force, which affects the development of electroconvection.

Bdiri et al. [9] used enzymatic agents as biological solutions for cleaning ion-exchange membranes fouled by organic compounds during ED treatments in the food industry. They tested the cleaning efficiency of three enzyme classes (β-glucanase, protease, and polyphenol oxidase) chosen for their specific actions on polysaccharides, proteins, and phenolic compounds, respectively, fouled on a homogeneous cation-exchange membrane

(referred to as CMX-Sb) and used for tartaric stabilization of red wine by ED in industry. First, enzymatic cleaning tests were performed using each enzyme solution separately with two different concentrations (0.1 and 1.0 g/L) at different incubation temperatures (30, 35, 40, 45, and 50 ◦C). The evolution of membrane parameters, such as electrical conductivity, ion-exchange capacity, and contact angle was determined to estimate the efficiency of the membranes principal action as well as its side activities. Based on these tests, these authors determined the optimal operating conditions for optimal recovery of the studied characteristics. Then, they tested two strategies combining these enzymes. The first one consisted of cleaning the fouled membranes with three successive enzyme solutions. The second one combined the use of two enzymes simultaneously in an enzyme mixture. In each case they took into account the optimal conditions of the enzymatic activity (concentration, temperatures, and pH). This study led to significant results, indicating effective external and internal cleaning by the studied enzymes. For example, they obtained a recovery rate of at least 25% of the electrical conductivity, 14% of the ion-exchange capacity, and 12% of the contact angle, and demonstrated the presence of possible enzyme combinations for the enhancement of the global cleaning efficiency or reducing cleaning durations. These important results prove, for the first time, the applicability of enzymatic cleanings to membranes and the inertia of their action towards the polymer matrix to the extent that the choice of enzymes is specific to the fouling substrates.

**Funding:** This research received no external funding.

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


**1. Introduction**

During the service period of buffer materials for nuclear waste repository [1–3], thermal prefabricated vertical drains [4,5], geothermal energy piles [6], and buried cables and pipelines [7], the temperature of the surrounding soil may change significantly. However, when the soil temperature changes, thermal pore water pressure will be generated in the soil due to the difference of thermal expansion characteristics between the pore fluid and soil particles. In case of geological formations with low hydraulic conductivity, the thermal pore water can cause the loss of effective stress. Furthermore, with the dissipation of thermal pore water pressure, the soil will produce additional thermal volume change. It is found that the magnitude of thermal pore water pressure mainly depends on the stress history. Knowledge of the volume change of saturated soil under different stress states, which is a key factor that needs to be considered in design of thermo-active structures, must accurately estimate the thermal pore water pressure with different degrees of overconsolidation.

A series of studies have investigated the thermal response of saturated clay soils. To explore the evolution law of thermal pore water pressure under undrained conditions, Campanella and Mitchell [8], Ghaaowd et al. [9], Abuel-Naga et al. [10], and Ghabezloo et al. [11] carried out related experimental investigations. These experimental studies found that the magnitude of thermal pore water pressure depends on the compressibility of the soil, the physicochemical coefficient of structural volume change, initial void ratio, initial effective stress, the change in temperature, and thermal expansion coefficients of pore water and soil particles. Furthermore, Abuel-Naga et al. [10], Ghabezloo et al. [11],

**Citation:** Tian, G.; Zhang, Z. A Calculation Method of Thermal Pore Water Pressure Considering Overconsolidation Effect for Saturated Clay. *Appl. Sci.* **2022**, *12*, 6325. https://doi.org/10.3390/ app12136325

Academic Editor: Bing Bai

Received: 20 May 2022 Accepted: 19 June 2022 Published: 21 June 2022

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

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

Burghignoli et al. [12], and McCartney [13] explored the thermally induced volume change of soil by conducting drainage heating tests. Among them, Abuel-Naga et al. [10] used the theoretical microstructure mechanism to explain the thermally induced volume change behavior under different stresses. Based on the principle of particle rearrangement in porous granular materials undergoing thermodynamic process, Bai et al. [14,15] established a thermo-hydro-mechanical constitutive model. This model can accurately describe the irreversible consolidation of normally consolidated saturated soils induced under thermal loading and the aging effect caused by cyclic thermal loading. Subsequently, under the framework of granular thermodynamics, Bai et al. [16] derived a generalized effective stress principle, which can automatically consider the influence of the stress path, temperature path, and soil structure.

It should be noted that both the magnitude of thermal pore water pressure and the sign of the thermally induced volume change are affected by the degree of overconsolidation [10,12,14,17]. With the dissipation of the thermal pore water pressure, normally consolidated clays will exhibit irreversible volume shrinkage, highly overconsolidated clays will undergo elastic thermal expansion, and slightly overconsolidated clays will show a volume change trend that expands first and then shrinks [13,18–20]. Meanwhile, for overconsolidated clays, there is a transition temperature. When the soil temperature exceeds the transition temperature, soil deformation will change from expansion to contraction [18,21–23]. The reason for the different deformation laws of soils with different overconsolidation degrees may be due to the difference in thermal pore water pressure [21]. To explore the effect of overconsolidation ratio on the changes of thermal pore water pressure and thermal volume, constitutive models that can consider the influence of stress history are established based on the Cambridge model or modified Cambridge model [3,10,24,25]. However, because the yield caused by temperature and stress is considered, many parameters are involved in these constitutive models, which makes the solution process extremely cumbersome. Furthermore, these constitutive models cannot obtain an explicit expression of thermal pore water pressure [26], which makes engineering applications very inconvenient. To conveniently and quickly predict the thermal pore water pressure, Campanella and Mitchell [8] proposed a thermo-porous-mechanical model by using concepts of thermoelasticity and linear elasticity. However, for highly overconsolidated soils, the thermo-porous-mechanical model may no longer be applicable. Based on the unified hardening model established by Yao and Zhou [25], Wang et al. [26] established a calculation method of thermal pore water pressure including *OCR*, but when the soil is in highly overconsolidated state, there is a large gap between the prediction results and the experimental results. In addition, the acquisition of thermal parameters in this calculation method requires additional thermal tests.

In summary, various methods for calculating thermal pore water pressure have been developed. However, the overconsolidation effect has not been well explored. To conveniently and quickly predict the thermal pore water pressure in overconsolidated clay and quantify overconsolidation effect on the thermal pore water pressure, by introducing the parameter *Λ* affected by *OCR* and the nonlinear relationship between *OCR* and the thermal pore water pressure, a calculation method of thermal pore water pressure considering overconsolidation effect for saturated clay is proposed. In addition, the calculation method is applied to predict the thermal pore water pressure, and the predicted results are compared with experimental data.


*2.2. CYGNSS Data*

CYGNSS is a constellation of eight satellites, each of which has four channels; i.e., thirty-two observations per second can be received. Due to the increase in CYGNSS sampling frequency after July 2019 (i.e., with 500 ms non-coherent accumulation), the resolution of CYGNSS is 3.5 × 0.5 km at this time. The CYGNSS data used in this study are the L1-level data of version 3.0 from July 2019 to December 2021, which can be downloaded from the official CYGNSS website (https://cygnss.engin.umich.edu/data-products/ (accessed on 6 November 2021)). To improve the quality of CYGNSS data, standard quality control and empirical quality control were performed [19]. The higher launch power of the GPS Block IIF satellite introduces uncertainty at peak power [25]. Removing these data will

reduce the overall data volume by 30%. Therefore, this part of the data was chosen to be retained in this study. The power received by CYGNSS consists of both incoherent and coherent scattering components [26]. Most studies have been based on the assumption that the incoherent component is ignored and only the coherent scattering component is retained [11,12,14,20]. Therefore, based on the land scattering model of GNSS-R, the surface reflectivity Γ(θ) of CYGNSS can be readily derived, following the assumption that the coherent component dominates:

$$\Gamma(\theta) = \frac{P\_r (R\_t + R\_r)^2 (4\pi)^2}{P\_t G\_t G\_r \lambda^2} \tag{1}$$

where *Pr* and *Pt* are, respectively, the reflected power received by the receiver and the transmitted power. λ is the carrier signal wavelength. *Rr* and *Rt* are, respectively, the distances from the receiver and transmitter to the specular reflection point. *Gt* and *Gr* are the transmitting antenna gain and receiving antenna gain, respectively.


**2. The network infrastructure and QoS system**

Although the main purpose of this work is to present measurements, it is important to describe the testbed setup and some related installation problems, as well as the QoS system and its main principles.


**Genome-Wide Analysis of the** *Rhododendron* **AP2/ERF Gene Family: Identification and Expression Profiles in Response to Cold, Salt and Drought Stress**

**Zhenhao Guo 1,2, Lisi He 1,2, Xiaobo Sun 1,2, Chang Li 1,2, Jiale Su 1,2, Huimin Zhou 1,2 and Xiaoqing Liu 1,2,\***

1 Institute of Leisure Agriculture, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

2 Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014, China

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** The AP2/ERF gene family is one of the most conserved and important transcription factor families mainly occurring in plants with various functions in regulating plant biological and physiological processes. However, little comprehensive research has been conducted on the AP2/ERF gene family in *Rhododendron* (specifically, *Rhododendron simsii*), an important ornamental plant. The existing whole-genome sequence of *Rhododendron* provided data to investigate the AP2/ERF genes in *Rhododendron* on a genome-wide scale. A total of 120 *Rhododendron* AP2/ERF genes were identified. The phylogenetic analysis showed that RsAP2 genes were classified into five main subfamilies, AP2, ERF, DREB, RAV and soloist. Cis-acting elements involving plant growth regulators, response to abiotic stress and MYB binding sites were detected in the upstream sequences of RsAP2 genes. A heatmap of RsAP2 gene expression levels showed that these genes had different expression patterns in the five developmental stages of *Rhododendron* flowers. Twenty RsAP2 genes were selected for quantitative RT-PCR experiments to clarify the expression level changes under cold, salt and drought stress treatments, and the results showed that most of the RsAP2 genes responded to these abiotic stresses. This study generated comprehensive information on the RsAP2 gene family and provides a theoretical basis for future genetic improvement.

**Keywords:** *Rhododendron*; transcription factor; genome-wide; abiotic stress; gene duplication; gene expression


**3. System design of a large diameter MCM UUV unit**

Current status of unmanned system technology and participation of unmanned vehicle fleets to naval operations can transform the concepts of future navy MCM tactics, weapons, and man machine interoperability in the field. In the near future, MCM vessels with C4I capability and MIW data base that act a main control center for a variety of unmanned vehicles in the enemy territory and doing a MCM operations without the presence of men in the mine field [9].

## **3.1. Large diameter UUV [16]**

The conventional smaller diameter unmanned vehicle system has major operational difficul‐ ties in sensor system and vehicle endurance that give limited search, launch and recovery operations. If we can design larger unmanned vehicle, we will have more payload and energy storage for longer endurance. They can be a force multiplier for increasing the operational capability of submarines and surface ships [7].

The options for hull shape and various subsystem configuration of vehicle define the set of vehicle design options which are evaluated by the design requirements. Some important design options are the fineness ratio and block coefficient, which dictate the basic packaging spaces and sizing of all other vehicle subsystems [12].

#### **3.2. Main control center configurations**

The autonomy of the system and the spectrum of operations are fundamental characteristics on MCM operations. The main platform has mission management blocks that automatically perform various MCM operational procedures such as contact of mines, obstacles from multiple sensor data sources and management of neutralization process, environmental data and bottom mapping [17]. For the mission accomplishment, organic MCM platforms, and operation with various sensors must undergo guidance of system management command, which is from rigorous analysis, experimentation, modeling and simulation on board [18]. The missions of MCM fleet operation is divided into five segments; launch, transit, execution of mission, return transit and recovery, each defined by key mission, and environmental parameters such as range, speed, ocean current and salinity and various MCM mission-related execution orders [12].

Advanced technologies applied for MCM main mission management system include situa‐ tional reactive mission control suits, smart sensor system of systems, dynamic intelligent mine classification processors with MCM data bases. Vehicle management system controls precise intelligent undersea navigation, intelligent sensor systems and obstacle avoidance measures. Application of up to date AI technologies to the vehicle's functionality and mission effective‐ ness of MCM UUV system are implemented with expert system blocks, AI pattern classifica‐ tion and efficient power management systems in our works [12]. The MCM UUV unit provides autonomy of vehicle systems through incorporation with platform-independent autonomous controller that supports high degree of autonomy, highly precise low-power navigation and machine vision in support of automatic classification [26]. Mission management function block diagram of MCM UUV is given in **Figure 5**.

**Figure 4.** Concept of MCM UUV system operations.

138 Autonomous Vehicle

**3.1. Large diameter UUV [16]**

capability of submarines and surface ships [7].

**3.2. Main control center configurations**

spaces and sizing of all other vehicle subsystems [12].

**3. System design of a large diameter MCM UUV unit**

Current status of unmanned system technology and participation of unmanned vehicle fleets to naval operations can transform the concepts of future navy MCM tactics, weapons, and man machine interoperability in the field. In the near future, MCM vessels with C4I capability and MIW data base that act a main control center for a variety of unmanned vehicles in the enemy territory and doing a MCM operations without the presence of men in the mine field [9].

The conventional smaller diameter unmanned vehicle system has major operational difficul‐ ties in sensor system and vehicle endurance that give limited search, launch and recovery operations. If we can design larger unmanned vehicle, we will have more payload and energy storage for longer endurance. They can be a force multiplier for increasing the operational

The options for hull shape and various subsystem configuration of vehicle define the set of vehicle design options which are evaluated by the design requirements. Some important design options are the fineness ratio and block coefficient, which dictate the basic packaging

The autonomy of the system and the spectrum of operations are fundamental characteristics on MCM operations. The main platform has mission management blocks that automatically

**Figure 5.** Function diagram of AI mission management system.

A capability of directing all aspects of the multifaceted MIW campaign plan is needed to bring the various MCM capabilities together, providing unity of effort in defeating the mine threat.

## **3.3. Neutralization weapon**

There are several state-of-the-art weapon systems to dispose or detonate mines effectively, and economically such as the use of a laser gun, acquire gun and small charge delivery devices. Furthermore, the confidence for job completion requires the capability of accurate battle damage assessment (BDA). Underwater motion projectile is multipurpose in formed cavity water, due to its density, has a profound impact upon the terminal velocity of the implant at the target. A suitable weapon technology applied to MCM UUV is a lightweight composite 30 mm launcher that would implant a round filled with either high explosives (HE) for an explosive hard-kill or reactive material for a soft kill burn [3].

Similar technology was developed to counter roadside improvised explosive devices using 50 caliber weapons. A 30 mm implant would be usefully larger and could integrate a compliant fusing device, utilizing a detonator enables digital fusing, and affords either timed or control‐ led detonation, including detonation by an acoustically transmitted command. A 30 mm launcher provides sufficient terminal velocity to penetrate 5/10 inch cold rolled steel from a range of 30 feet [13, 26].

The currently achieved standoff range of 30 feet which the UUV should shot detonate the mine is not sufficient to ensure safety of MCM UUVs. Shooting from longer ranges requires significant basic research, and development, both in material strengths, and in achieving precise sonar fire-control accuracies before truly safe standoff ranges are achievable.

## **3.4. Energy and power managing section**

Considering the operational combat field endurance limit of more than 50 days of MCM UUV and the current status of the battery systems technology, the combination of diesel internal combustion engine (ICE) and effective battery systems could become reality. The high specific power generation of the internal combustion system gives effective operation of the vehicle and can provide a stable recharge power source of the battery system. Integration of a small diesel engine connected to the battery systems, and modification of the UUV hull structure for the snorkeling operation could give better alternatives for both recharging and propulsion of the MCM UUV in the meantime [25].

A diesel submarine is a very good example of hybrid power supplying and sharing systems. The two or more sets of diesel engines in most diesel submarines can run propellers or can run generators that recharge a huge battery bank, or work in combination mode; one engine driving a propeller and others driving a generator. The submarines should run the diesel engines, they must surface or cruise just below the surface of water using snorkeling, and once the batteries are fully charged, the submarine can dive to underwater operations [8]. These diesel battery hybrid power systems are controlled by vehicle management computers and a main AI expert mission management system. Combined power generation, and the control system structure are given in **Figure 6**.

**Figure 6.** Power management system of MCM UUV.


**1. Introduction**

Kelp forests are highly productive three-dimensional coastal marine habitats [1,2] that provide a number of environmental services and contribute substantial economic value to coastal communities globally [3]. In the northeast Pacific, the two dominant surfacecanopy forming kelp species, *Nereocystis luetkeana* and *Macrocystis pyrifera* [4], stabilize shorelines via wave dampening [5,6], support economically important fisheries [7,8], and are commercially harvested for various purposes [9,10]. However, both kelp species are subject to high spatial and temporal variability, correlated with biotic and abiotic drivers of change [11,12]. As such, resource managers are incentivized to monitor the status of these kelp forests, and the corollary effects of the ecosystem services they provide [10,12,13], a task that has been facilitated by remote sensing since the mid-20th century [12,14].

Generally, the remote sensing of surface-canopy forming kelp forests aims to detect the portion of the kelp that forms a canopy, floating at the water's surface; using sensors

**Citation:** Timmer, B.; Reshitnyk, L.Y.; Hessing-Lewis, M.; Juanes, F.; Costa, M. Comparing the Use of Red-Edge and Near-Infrared Wavelength Ranges for Detecting Submerged Kelp Canopy. *Remote Sens.* **2022**, *14*, 2241. https://doi.org/10.3390/ rs14092241

Academic Editors: Simona Niculescu, Junshi Xia and Dar Roberts

Received: 11 March 2022 Accepted: 5 May 2022 Published: 7 May 2022

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

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

aboard Earth Observation Satellites (EOS) [15,16], piloted aircraft [10,12], and Uncrewed Aerial Vehicles (UAVs) [17]. In order to use the data provided by remote sensing platforms effectively, it is crucial to understand factors that influence the spectral signature of kelp canopy in water [16]. Floating kelp canopy has high reflectance in the near-infrared wavelength range (NIR) (700–1000 nm), which contrasts with the high NIR absorption by the surrounding water, allowing for binary classification of floating kelp canopy and water within an image [14]. However, there are numerous considerations (e.g., sun glint, bathymetry, turbidity; see [13,16]) that can reduce the separability between the spectral values of kelp canopy and water. One crucial factor that can affect the ability to detect kelp canopy is the submersion of the canopy by tides and associated tidal currents, which can dampen the NIR reflectance of kelp and lead to potential errors when estimating kelp area or biomass [17,18].

In an attempt to minimize classification errors associated with kelp submergence, remote sensing imagery is often acquired at low tides during the peak growing season (mid-late summer) when the majority of the kelp canopy is floating at the water's surface [10,18,19]. However, there are multiple reasons why a remote sensor may also want to detect the submerged portion of the kelp canopy. For example, the northeast Pacific coastline often experiences non-ideal weather conditions for remote sensing data acquisition, leading to imagery being opportunistically collected at higher than ideal tidal heights when more kelp canopy is more likely to be submerged compared to ideal low tide conditions [13,16]. Further, the fixed rate of EOS orbits may result in some regions only having imagery available during high tides even if acquisition conditions are otherwise ideal [13]. Even if remote sensing imagery is captured during ideal tide and weather conditions, portions of kelp canopy may also be continuously submerged depending on the species being targeted. Specifically, if a remote sensor is targeting detection of *Nereocystis luetkeana* (hereafter, *Nereocystis*) surface canopy, one has to consider the two distinct structures with varying buoyancy, the bulb and blades. The bulb is a roughly cylindrical gas-filled structure that floats on the surface of the water and is anchored to the sea floor by a stipe and holdfast [9]. The blades are long thin structures that trail from the end of the bulb, often with many individuals around four meters long per bulb [9]. The blades are not buoyant and are likely to remain submerged below the water's surface regardless of tidal height [16,20]. In addition, floating portions of kelp canopy may be periodically submerged in areas with especially strong currents [20]. Therefore, it is important to understand how submersion of kelp canopy affects the reflectance in the NIR range, as well as whether certain spectral features may allow for higher detectability of kelp when collecting remote sensing imagery from different platforms.

In the past, the red-edge (RE) spectral region (670–750 nm), which includes a range of the shortest NIR wavelengths, has traditionally been used to determine health characteristics of terrestrial plants [21]. However, these wavelength ranges also penetrate deeper into the water column than longer NIR wavelength ranges [22], resulting in the potential for higher above water reflectance in the RE than the longer NIR for submerged vegetation [23–26]. Therefore, given the spectral similarities between kelps and other types of vegetation, it is reasonable to assume that the RE wavelength range may also be beneficial for detecting submerged kelp canopy. Hereafter, the term NIR will refer to only the longer wavelength range above 751 nm, to avoid confusion with the NIR wavelength range that overlaps the RE wavelength range.

To date, there have been no direct comparisons of the ability to detect submerged kelp when using RE or NIR wavelength ranges. Additionally, while the submersion kelp canopy due to tides and currents is well documented using various sensors with different spatial and spectral resolutions [11,17–20,27], there has been no characterization of the changes to the above water spectra of kelp as the canopy is submerged, nor any investigation of the band combinations used in vegetation indices in relationship to accurate detection of the submerged kelp canopy. With this is mind, our goal was to characterize changes to abovewater reflectance of different *Nereocystis* canopy structures as they were submerged and to

relate those changes to depth detection limits. To accomplish this goal, we performed (1) an experiment that documents the effects of kelp submersion on the above-water hyperspectral reflectance of both *Nereocystis* bulb and blade structures. We also compared (2) the detection limits of submerged kelp using RE and NIR vegetation indices, which were calculated from the simulated multispectral bands of high spatial-resolution air- and space-borne sensors.

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


**3. Results**

The error changes (G) in the operation of the water meter for status Q2=0 were tested for two cases: without UFR and with UFR in operation at water meter no. 3.

**Figure 4.** Error changes (G) in the operation of water meter no. 3 during time in the function of Q3 flow without UFR in operation (circles) and with the UFR in operation (crosses).

With the criterion described by equation (2), steady state flow stabilisation time, tst was de‐ fined:

Water volume flown through the water meter was defined by:

ume in the vessel, the water flow rate Q was calculated.

measuring at water meter propeller in stillstand [12].

) and 0.1 kg (for Qn).

cases: without UFR and with UFR in operation at water meter no. 3.

Qmin), 0.01 kg (for Qt

138 Water Supply System Analysis - Selected Topics

**3. Results**

in operation (circles) and with the UFR in operation (crosses).

**G (%)**

**•** the difference in reading on the water meter prior and after measuring, and

**•** measuring water quantity in the vessel (of 15 to 200 litres in volume) and water density. By measuring time (with stop-watch) between two readings, through the defined water vol‐

During accuracy measurements, water meter readings were as follows: 2.5 centilitres, 1 deci‐ litre and 1 litre. Measurement accuracy of water quantity in the vessel was 0.005 kg (for

Error changes in the operation of the water meter described by equation (1), were tested by applying two method by stopping the water meter: according to the valid Protocol of the Republic of Serbia, the status on the water meter and the scale was read prior and after

The error changes (G) in the operation of the water meter for status Q2=0 were tested for two

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375

Q3=1,3 l/h Q3=1,3 l/h Q3=3,0 l/h Q3=2,8 l/h Q3=5,2 l/h Q3=5,0 l/h Q3=7,1 l/h Q3=6,7 l/h Q3=12,7 l/h Q3=12,4 l/h Q3=19,6 l/h Q3=18,7 l/h Q3=25,5-25,6 l/h Q3=24,4-24,6 l/h

**t (min)**

**Figure 4.** Error changes (G) in the operation of water meter no. 3 during time in the function of Q3 flow without UFR

**Figure 5.** Stabilisation time of water flow in the installation tst in the function of flow Q3.

**Table 2.** Stabilisation time of water flow in the installation tst in the function of flow Q3.

UFR (right) in operation.

balancing decrease.

99.5-28.93=70.57%.

flow rates Q2=0.03-0.0385 m3

**G (%)**

0 100 200 300 400 500 600 700<PHONE_NUMBER> 1100 1200

Error in Water Meter Measuring Due to Shorter Flow and Consumption Shorter Than the Time...

http://dx.doi.org/10.5772/51046

141

Q3=1,1-1,3 l/h Q3=1,1-1,2 l/h UFR Q3=2,8-3,0 l/h Q3=2,8-2,9 l/h UFR Q3=4,7-5,1 l/h Q3=4,6-5,1 l/h UFR Q3=6,1-6,3 l/h Q3=6,1 l/h UFR Q3=11,2-12,3 l/h Q3=11,1-12,0 l/h UFR Q3=19,7-21,0 l/h Q3=19,2-20,6 l/h UFR Q3=23,6-25,4 l/h Q3=23,0-24,6 l/h UFR Q3=5,1-5,3 l/h Q3=4,9-5,3 l/h UFR

**Q2 (l/h)**

**Figure 7.** Error (Gb) in water balancing on the rig in the function of flow rates Q2 and Q3, without UFR (left) and with

The highest balancing error values for the tested statuses are +0.9 and -16.6%. By increasing flow rate Q2 for both statuses (without UFR and with UFR at water meter no. 3) errors in

With UFR in operation, the most significant contribution in measuring water volume by wa‐ ter meter (15.7-0=15.7%, mean value of 30 measurments) was determined at operations at

At steady flow slightly lesser than Qa, UFR efficiency increases from T10 to T30: water meter equipped with UFR type T10 measures for 99.53-84.14=15.39% more water than in status without UFR, while the increase with type T20 is 99.33-68.55=30.78% and with type T30 it is

This conclusion was verified for a single household water supply network by the results of five measurements for each status: water meter equipped with type T10 UFR measured (at flow rates Q2=51.3-51.4 l/h and Q3=5.2-5.6 l/h) maximum 9.89-6.84=3.05% more water con‐ sumption of than without UFR, with type T20 (at flow rate of Q2=56.9-60 l/h and Q3=5.3-5.5 l/h) the most significant improvement was 9.06-4.19=4.87%, while with type T30 (at flow

/h.

/h and Q3=0.0049-0.0053 m3

rates of Q2=55.9-58.5 l/h and Q3=4.9-5.1 l/h) the result is 8.4-0.97=7.43%.

Error in the operation of water meter at steady state flow defined by equation (1) is:

**Figure 6.** Error (G) in the operation of water meter no. 3 at steady state flow in function of flow rate Q3, without UFR and with UFR.

The error in the operation of water meter no. 3 without UFR is bigger than with UFR.

The measurements proved that Qa<0.01 m3 /h is between 0.0052 and 0.0067 m3 /h.

By increasing flow rate Q3 towards flow rate Qa, the influence of UFR operation on meas‐ uring water volume increases: for Q3=0.0013 m3 /h the contribution is 99.59-78.49=21.1%, and for Q3=0.005-0.0052 m3 /h it is 99.5-28.93=70.57%. For flow rates Q3>Qa up to Q3=0.026 m3 /h the contribution of UFR to measuring water volume by water meter is decreasing: for flow rate Q3=0.0067-0.0071 m3 /h the contribution is 37.67-4.07=33.6%, and for Q3=0.0244-0.0255 m3 /h it is 0.47+0.36=0.83%.

Without the UFR in operation, the water meter always shows lower water volume than the real value. With the UFR in operation, at flow rate Q3=0.01 m3 /h, the operation of the wa‐ ter meter is changing: for flow rate Q3<0.01 m3 /h the water meter shows lower volume than the real value, while for Q3>0.01 m3 /h it shows higher value (not exceeding 2.7%) than the real one.

For status Qmin≤Q2≤Qn balancing errors described by equation (3) are:

Error in the operation of water meter at steady state flow defined by equation (1) is:

0 5 10 15 20 25 30

**Q3 (l/h)**

/h is between 0.0052 and 0.0067 m3

/h it is 99.5-28.93=70.57%. For flow rates Q3>Qa up to Q3=0.026

/h the contribution is 37.67-4.07=33.6%, and for

/h it shows higher value (not exceeding 2.7%) than the

**Figure 6.** Error (G) in the operation of water meter no. 3 at steady state flow in function of flow rate Q3, without UFR

By increasing flow rate Q3 towards flow rate Qa, the influence of UFR operation on meas‐

/h the contribution of UFR to measuring water volume by water meter is decreasing:

Without the UFR in operation, the water meter always shows lower water volume than the

The error in the operation of water meter no. 3 without UFR is bigger than with UFR.

without UFR with UFR

/h.

/h, the operation of the wa‐

/h the contribution is 99.59-78.49=21.1%,

/h the water meter shows lower volume than

and with UFR.

m3

real one.

The measurements proved that Qa<0.01 m3

and for Q3=0.005-0.0052 m3

Q3=0.0244-0.0255 m3

for flow rate Q3=0.0067-0.0071 m3

the real value, while for Q3>0.01 m3

uring water volume increases: for Q3=0.0013 m3

ter meter is changing: for flow rate Q3<0.01 m3

/h it is 0.47+0.36=0.83%.

real value. With the UFR in operation, at flow rate Q3=0.01 m3

For status Qmin≤Q2≤Qn balancing errors described by equation (3) are:

**G (%)**

140 Water Supply System Analysis - Selected Topics

**Figure 7.** Error (Gb) in water balancing on the rig in the function of flow rates Q2 and Q3, without UFR (left) and with UFR (right) in operation.

The highest balancing error values for the tested statuses are +0.9 and -16.6%. By increasing flow rate Q2 for both statuses (without UFR and with UFR at water meter no. 3) errors in balancing decrease.

With UFR in operation, the most significant contribution in measuring water volume by wa‐ ter meter (15.7-0=15.7%, mean value of 30 measurments) was determined at operations at flow rates Q2=0.03-0.0385 m3 /h and Q3=0.0049-0.0053 m3 /h.

At steady flow slightly lesser than Qa, UFR efficiency increases from T10 to T30: water meter equipped with UFR type T10 measures for 99.53-84.14=15.39% more water than in status without UFR, while the increase with type T20 is 99.33-68.55=30.78% and with type T30 it is 99.5-28.93=70.57%.

This conclusion was verified for a single household water supply network by the results of five measurements for each status: water meter equipped with type T10 UFR measured (at flow rates Q2=51.3-51.4 l/h and Q3=5.2-5.6 l/h) maximum 9.89-6.84=3.05% more water con‐ sumption of than without UFR, with type T20 (at flow rate of Q2=56.9-60 l/h and Q3=5.3-5.5 l/h) the most significant improvement was 9.06-4.19=4.87%, while with type T30 (at flow rates of Q2=55.9-58.5 l/h and Q3=4.9-5.1 l/h) the result is 8.4-0.97=7.43%.

**Figure 8.** Error (G) in the operation of water meter no. 3 at steady state flow in function of flow rate Q3=Qa without UFR (left) and with UFR (right) type T10, T20 and T30.

**Figure 10.** Error in water meter measuring at Qmin flow during water flow shorter than the time for which the meter

Error in Water Meter Measuring Due to Shorter Flow and Consumption Shorter Than the Time...

http://dx.doi.org/10.5772/51046

143

**Figure 11.** Error in water meter measuring at Qt flow during water flow shorter than the time for which the meter was

was calibrated (10 min.) in the function of water meter reading accuracy.

calibrated (12.5 min.) in the function of water meter reading accuracy.

**Figure 9.** Water balance error (Gb) of the tested water supply network without UFR type T10, T20 and T30 (quadrat) and equipped with one of the UFR type (cross).

Error in water meter measuring due to consumption shorter than the time the meter was calibrated for: each measurement was repeated 5-30 times.

Error in Water Meter Measuring Due to Shorter Flow and Consumption Shorter Than the Time... http://dx.doi.org/10.5772/51046 143

calibrated for: each measurement was repeated 5-30 times.

**Gb (%)**

UFR (left) and with UFR (right) type T10, T20 and T30.

142 Water Supply System Analysis - Selected Topics

and equipped with one of the UFR type (cross).

**G (%)**

10 20 30

Q3=5,8-5,9 l/h Q3=5,6 l/h Q3=5,2-5,3 l/h Q3=4,8-4,9 l/h Q3=5,2 l/h Q3=5 l/h

**T10, T20 and T30**

10 20 30

**T10, T20 and T30**

**Figure 9.** Water balance error (Gb) of the tested water supply network without UFR type T10, T20 and T30 (quadrat)

Error in water meter measuring due to consumption shorter than the time the meter was

Q2=51,2-56,9 l/h, Q3=5,4-5,6 l/h Q2=51,4-53,2 l/h, Q3=5,2-5,4 l/h Q2=55,3-58,8 l/h, Q3=4,9-5,6 l/h Q2=58,9-60,3 l/h, Q3=5,3 l/h" Q2=54-60,7 l/h, Q3=4,9-5 l/h Q2=54,3-58,5 l/h, Q3=4,9-5,1 l/h

**Figure 8.** Error (G) in the operation of water meter no. 3 at steady state flow in function of flow rate Q3=Qa without

**Figure 10.** Error in water meter measuring at Qmin flow during water flow shorter than the time for which the meter was calibrated (10 min.) in the function of water meter reading accuracy.

**Figure 11.** Error in water meter measuring at Qt flow during water flow shorter than the time for which the meter was calibrated (12.5 min.) in the function of water meter reading accuracy.

**4. Discussion**

bution is decreasing from flow Qa towards flow Qmin.

out UFR and with UFR enabled specifying the results.

Measurement results confirm and specify the conclusions of tests made in Udine in relation to the contribution of UFR to water meter operation: a) that the most significant contribution is at flow, when water meter propeller is steady (specifically for Qa), and b) that this contri‐

Error in Water Meter Measuring Due to Shorter Flow and Consumption Shorter Than the Time...

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145

The same conclusion is valid for test results on the UFR contribution at parts of the water supply network. Providing similar water consumption during testings on the test rig with‐

In the case, when consumption time was shorter than the time the meter was calibrated for,

**Accuracy charge During experiment During calibration**

Qt from 1.9 to 12.1 ±2 Qn from 1.6 to 3.2 ±2

Qt from 4.2 to 7.5 Qn from 1.2 to 2.8 1 decilitar Qmin from -25.9 to 50.9 ±5

> Qt from -8.3 to 86.9 Qn from -4.8 to 4.8

**Table 4.** Error ranges of water meter measuring for calibrated discharges in the function of water meter reading accuracy (class B, rated diameter of 20 mm and discharge of Qn=1.5 m3/h), for a duration of 0.5 minutes flow.

It means that during consumption shorter than the time the water meter was calibrated for, measuring by the meter is unreliable in 95% of the consumption shorter than 1 minute. Dur‐

In order to improve water consumption measuring in households, it is necessary to provide conditions for measuring consumption at flow lower than Qmin and duration shorter than the time the meter has been calibrated for. Such conditions may be created in supply pipe‐ lines with water storage tanks in households. Only such systems are appropriate in which

The UFR should be installed on the outlet pipe from the tank to the household. Signalising the start of the UFR's operation may initiate works on eliminating water losses due to leak‐ age on the tap, bathroom battery and the flushing cistern. This way, the use of UFR would

the range of the meter measuring error exceeded the range of permitted errors.

**Reading Dis- Error range (%)**

2.5 centilitar Qmin from -7.4 to 32.1

1 litar Qmin from -100 to 277.4

ing a discharge of 0.5 minutes, the error may be even 277.4%.

serve to protect the interest of the households.

all the water needed in a household flows through this storage tank [19].

**Figure 12.** Error in water meter measuring at Qn flow during water flow shorter than the time for which the meter was calibrated (4 min.) in the function of water meter reading accuracy.

The minimum time at which measuring errors undoubtedly are within the permitted limit is defined for the calibrated flows of the water meter as follows:

**Table 3.** Minimum calibrated flow duration time in the function of reading accuracy.


*2.3. Bioactivity*

Litoralimycins A (**1**) and B (**2**) were evaluated for their minimum inhibitory concentration (MIC). Both compounds showed only a very weak activity against *Staphylococcus aureus* Newman and *Bacillus subtilis* DSM10T with MIC values of 66.7μg/mL, respectively. No further effects were detected against any Gram-negative bacteria or fungi. (Table S1). However, cytotoxic effects against different cell lines were detected for **1** (Table 2). Litoralimycin B (**2**) with its truncated side chain showed much weaker cytotoxic activity.

**Table 2.** Cytotoxic activities of litoralimycins A (**1**) and B (**2**) against different cell lines. Values indicate IC50 in μg/mL.


*3.2. Morphological Observation*

The morphological changes of the algal cells were observed using a Leica DM750 light microscope (Leica Microsystems, Wetzlar, Germany) and photos were taken with a Leica ICC50 W camera (Leica Microsystems, Wetzlar, Germany).

#### *3.3. Determination of Cell Number*

The samples were fixed with Lugol's iodine solution first. Then, cell numbers were counted using a Neubauer improved cell counting chamber under Leica DM750 light microscope and measured as cells mL−1.

The daily percentage growth rate of nonmotile cells (% day−1) was calculated as follows:

$$\text{Daily percentage growth rate of nonmotile cells } \left( \% \text{ day}^{-1} \right) = \frac{\frac{\mathcal{C}\_{\text{Ml}}}{\mathcal{C}\_{\text{Tl}}} \times 100\%}{t} \tag{1}$$

where *CNt* and *CTt* were the nonmotile cells number and total cells number on day t, respectively.


**6. Conclusion and future research**

The use of linkage analysis, candidate gene studies, and GWAS has led to the identification of several causal loci and genes for fALS and sALS. The overview above clearly shows the extent of heterogeneity in genes that underlie fALS, let alone sALS, illustrating the complex molecular basis of this disease. There is not one dominant biological process that is represented by these genes, although RNA-processing, axonal transport and synaptic dysfunction appear to emerge as being relevant in ALS etiology. Interestingly, several of the genes implicated in these processes are already known to be causal or have been implicated in other neurodegenerative diseases which suggests that there is, at least in part, a common underlying mechanism.

Genetics of Amyotrophic Lateral Sclerosis 499

This work has received funding from the European Community's Health Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 259867, VSB fonds, EURO-MOTOR FP7, The Netherlands ALS Foundation (J.H.V, and L.H.v.d.B.), Neuroscience and Cognition Utrecht (NCU), the Prinses Beatrix Fonds (Kersten Foundation) and the Adessium Foundation (R.J.P., J.H.V. and L.H.v.d.B.). J.H.V. is supported by the Brain Foundation of The Netherlands and J.H.V, R.J.P., and M.K are supported by the

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**7. Acknowledgements**

Thierry Latran Foundation.

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**8. References**

Since these findings explain only about a third of the genetic variability in fALS and a small percentage of the genetic contribution to sALS, there is a clear need for the identification of additional causal loci. This would require the collection of large family pedigrees with many affected individuals, which is difficult in ALS considering the adult onset with rapid disease progression. However, the development of next generation sequencing techniques provides a possible solution to this problem. Using exome and whole-genome sequencing, causal genes can be identified with a small number of affected and unaffected individuals as has been shown in several, mostly autosomal recessive disorders (Choi et al., 2009; Ng et al., 2009). Recently, exome sequencing in two affected individuals from the same family identified *VCP* as a causal gene for fALS, illustrating that this technique is a promising tool for gene identification in ALS as well (Johnson et al., 2010). In addition, the repeat expansion in C9ORF72 was also discovered with the use of whole-genome sequencing (Renton et al., 2011).

The identification of causal genes for ALS has broadened our understanding of this motor neuron degenerative disease. Studying the function of associated genes in neurons and animal models has revealed several possible processes underlying ALS such as RNA processing, axonal transport, glutamate regulation, oxidative stress and synaptic dysfunction. However, the contribution of most genes to ALS pathogenesis has not been resolved. SOD1 and TDP-43 transgenic animal models have provided valuable insights into ALS pathogenesis. Further research using existing animal models of ALS associated genes and the generation of new animal models are needed to further determine their role in the disease. Generation of animal models harbouring repeat expansions in C9ORF72 and ATXN2 could help to reveal the pathogenic mechanisms behind these repeats. The effect of overexpression or knockdown of ALS associated genes and the expression of repeat expansions in motor neurons or motor neuron-like cell lines on protein aggregation and cell survival could also help to unravel the contribution of these genes to ALS. In addition, some associated genes (e.g. *DCTN1*, *PON1/2/3*, *TAF15*, and *VCP*) remain to be sequenced in larger cohorts from different populations in order to determine the actual contribution of these genes to ALS.

Additional new strategies in sALS include a more network oriented approach to gene identification. It is possible to detect networks of genes, proteins and metabolites that are misregulated in ALS, or that determine disease progression. By searching for subtle genetic variation that drives these network perturbations, new genes might be identified that are hard to detect with GWAS. Also, the focus in ALS genetics thus far has been on common variation in exonic DNA. The regulatory part of the genome is challenging to study, but might be relevant as well. This also requires the combined analysis of gene-expression and protein data with data on genetic variation. In addition, recent studies show that tandem repeats in DNA might be also relevant, as exemplified by the ATXN2 and C9ORF72 findings. Typically, this type of variation is hard to detect by current high-throughput methods. Lastly, the type of copy number variation that has not yet been covered very well to date, including variation in microRNAs or inversions, deserves more attention.

In summary, impressive progress in the understanding of the genetics of ALS has been made over the past several years with the identification of several causal genes. However, most of the genetic variability underlying ALS remains to be identified. The use of deep sequencing techniques and functional research will be needed to further broaden our understanding of ALS pathogenesis.


**Author details**

Emanuela Balestrieri1 \*, Chiara Cipriani1 , Enrico Garaci2,3, Claudia Matteucci1 and Paola Sinibaldi-Vallebona1,4

1 Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy

2 University San Raffaele, Rome, Italy

3 IRCCS San Raffaele Pisana, Rome, Italy

4 Institute of Translational Pharmacology, National Research Council, Rome, Italy

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 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.


Preface

Paraffin oil and mineral oil are synonymous terms. Fluid paraffin oil is a mineral oil that is a byproduct of unrefined petroleum refining. It is a straightforward, lackluster, unscented, and dull oil, chiefly made from high-bubbling alkane subsidiaries. However, it has the potential as a low-cost energy storage material.

Different forms of energy can be stored, including mechanical, electrical, and thermal energy. Energy storage not only reduces the mismatch between supply and demand but also improves the performance and reliability of energy systems and plays an important role in conserving energy. Thermal energy can be stored during the off-peak period, usually at night, and re-used during the peak period.

This book focuses on the application of paraffin for thermal energy storage. It examines the preparation of paraffin via encapsulation to develop a nonconventional energy storage material.

> **ElSayed G. Zaki** Associate Professor, Petroleum Applications Department, Egyptian Petroleum Research Institute, Cairo, Egypt

#### **Abdelghaffar S. Dhmees**

Associate Professor, Analysis and Evaluation Department, Egyptian Petroleum Research Institute, Cairo, Egypt


*Edited by Abhay Nanda Srivastva*

Analytical insight of materials provides a lucid pathway for further opportunities in the development of high-potential modified materials. The analytical assessment also enhances the probability of finding suitable materials for various applications. This book presents the latest advancements and applications of analytical chemistry in a systematic manner. It is an anthology of scientific findings and views of researchers from various research centers across the globe on emerging topics of instrumentation, energy, environment, biotechnology, and synthetic enhancement analysis techniques related to analytical chemistry. The volume contains twelve chapters containing discussion, analogies, and graphics for a better understanding of the presented concepts.

Published in London, UK © 2021 IntechOpen © onsuda / iStock

Analytical Chemistry - Advancement, Perspectives and Applications

Analytical Chemistry

Advancement, Perspectives and Applications

*Edited by Abhay Nanda Srivastva*


**4.1 OriP/EBNA-1 episomal vectors**

The best characterized system for episomal maintenance is based on sequences derived from the EBV genome. EBV is a member of the herpesvirus family with a 172-kb genome that is latently maintained as an independently replicating episome in a small percentage of infected lymphocytes (Masucci & Ernberg, 1994). During the latent phase of its cycle, DNA replication occurs from the origin of replication *oriP* and only about 10 proteins are produced of which the only protein that is required for replication at *oriP* is the Epstein Barr Nuclear Antigen-1 (EBNA-1) (Yates et al., 1985). The interaction of *oriP* with EBNA-1 also enables the segregation of the viral genome between the daughter cells through the association of EBNA-1 with host metaphase chromosomes (Harris et al., 1985).

These features of the EBV have been exploited to develop a system for episomal maintenance of foreign DNA delivered into cells. It has been shown that plasmids carrying *oriP* and expressing EBNA-1 can replicate autonomously once per cell cycle when delivered into human cells and can segregate by attaching to the host chromosomes (Haase & Calos, 1991). The *oriP*/EBNA-1 system has also been shown to support long-term episomal maintenance without selection and expression of very large human genes, such as the *CFTR* (Huertas et al., 2000), the human hypoxanthine phosphoribosyltransferase (*HPRT*) (Wade-Martins et al., 2000) and the β*-globin* gene (Black & Vos, 2002). Following these promising results, a convenient system for adding the *oriP*/EBNA-1 sequences onto any BAC already containing a therapeutic gene has been developed (Magin-Lachmann et al., 2003) and will be analyzed later on.

The *oriP*/EBNA-1 retention system is easy to use and can provide extra-chromosomal maintenance to foreign DNA of hundred kilobases delivered into cells but has some major disadvantages. It provides random rather than equal segregation of the episomal vector to daughter cells which results in loss of the episomes at a rate of 2-8% per cell division (Sclimenti & Calos, 1998). This, along with the fact that it involves viral sequences particularly from the EBV which has been associated to several types of human malignancies (Cohen, 2000) limits the use of EBV vectors for safe gene therapy.


*Article* **S100A6, Calumenin and Cytohesin 2 as Biomarkers for Cutaneous Involvement in Systemic Sclerosis Patients: A Case Control Study**

**Paul Balanescu 1,2,3, \*, Eugenia Balanescu 2 , Cristian Baicus 1,2,3 and Anca Balanescu 4**

**Abstract:** Background: Systemic sclerosis (Ssc) is an autoimmune disease with incomplete known physiopathology. There is a high number of candidate proteomic biomarkers for Ssc that have not yet been confirmed on independent Ssc cohorts. The aim of the study was to confirm circulating S100A6, calumenin, and cytohesin 2 as biomarkers for Ssc. Methods: 53 Ssc patients and 26 age- and gender-matched controls were included. Serum S100A6, calumenin, and cytohesin 2 were evaluated with commercial ELISA kits. Associations between serum expression and clinical Ssc characteristics were evaluated. Results: Serum calumenin, S100A6, and cytohesin 2 were higher in Ssc patients compared to controls. Calumenin associated with extensive cutaneous fibrosis, frequency of Raynaud phenomenon, and low complement level, and had a tendency to be higher in Ssc patients with pulmonary fibrosis. S100A6 correlated with the number of active digital ulcers. Serum cytohesin 2 levels were higher in patients with teleangiectasia and associated with pulmonary artery pressure. Conclusions: Serum calumenin, S100A6, and cytohesin 2 were confirmed as biomarkers on an independent group of Ssc patients. Calumenin had the best predictive capacity for cutaneous Ssc manifestations. Future studies are needed to evaluate the prognostic value of these biomarkers and evaluate them as possible therapeutic targets.

**Keywords:** systemic sclerosis; biomarker; calumenin; S100A6; cytohesin 2