*3.1. Separation*

A III-V device structure can be separated from its growth substrate by a technique called epitaxial lift-off. It involves the selective lateral wet etching of a thin sacrificial layer. Once the layer is completely etched away, the III-V structure becomes separated from the underlying layers and growth substrate [21,22]. We tailored this technique to detach our HEMT structures from GaAs substrate. The rate of the heterostructure release was studied with dependence on the composition of an etchant based on hydrofluoric acid (HF). The thickness of the AlAs interlayer was the parameter (three values), and the etching was carried out at room temperature. The results are presented in Figure 1.

−

**Figure 1.** Dependence of the release etching rate of HEMT heterostructure nanomembranes on the composition of an HF-based solution with the thickness of the AlAs sacrificial layer as the parameter.

Thin AlAs layers (<100 nm) were not conducive to the HEMT heterostructure release because the sacrificial etching self-terminated at short distances from the edges of lithographically defined nanomembrane areas (a similar outcome was published previously) [23]. The self-termination of the etching was attributed to a very low wettability of the channel formed as the result of the AlAs etching, as the separation between the underlying GaAs buffer layer and the HEMT heterostructure was too narrow. The situation where the etching of the sacrificial layer (thickness of AlAs is 20 nm) was stopped at a short distance is documented in Figure 2. The wettability problem was alleviated by adding a small amount of a hydrophilic agent to the etching solution.

**Figure 2.** SEM image of a sample where the sacrificial etching of a 20 nm thick AlAs separation layer was self-terminated.

Nanomembranes of the HEMT heterostructure that were processed and studied in this experiment were 1.5 × 1.5 cm2 in size. Their release was achieved by the sacrificial etching

of a 300 nm thick AlAs layer. To assure a successful transfer of the nanomembranes, it was necessary to have all surfaces of the nanomembranes as smooth and clean as possible. This would guarantee that the heterostructure adhered well to the host substrate by means of van der Waals forces, especially the surface that was exposed by the sacrificial etching at the original interface between the GsAs layer; the heterostructure had to be clean from any remnants of the AlAs sacrificial etching. To achieve this, the AlAs layer had to have very sharp interfaces with the overlying HEMT heterostructure and underlying GaAs buffer layer.

To look at the bottom surface of the nanomembranes, some were flipped over and attached upside down to sapphire host substrate, as is shown in the SEM micrographs of Figure 3. The white arrows in the main picture and the inset point to the edge of an HEMT nanomembrane where the heterostructure was delineated. The main SEM image shows the back side of a nanomembrane surface, which is that of the GaAs layer, after the sacrificial AlAs layer was completely etched away. This surface was originally the interface between the GaAs layer and the AlAl sacrificial layer, and it should ideally be completely flat and smooth. However, the SEM image revealed that it was not fully smooth but contained a shallow wavy structure with prolonged shallow depressions. The relatively large shallow depressions may have originated due to etching at crystal defects. The shallow wavy structure may have stemmed from the intermixing of Al and Ga atoms as the epitaxial growth was switched from AlAs to GaAs. As a result, a very narrow inhomogeneous AlGaAs layer was formed. As the HF-based etchant etched the AlAs layer away during the release process, it reached this inhomogeneous intermixed layer, etched it off (although more slowly compared with the AlAs layer), and left the shallow surface imprint behind. We believe that it did not significantly hamper the bonding of the nanomembranes to the sapphire substrate.

**Figure 3.** SEM images of an HEMT nanomembrane that was flipped over to expose the surface of its GaAs back side. It contained a shallow wavy structure with prolonged shallow depressions. The arrows mark an edge of the nanomembrane with the HEMT heterostructure delineated.

Figure 4a shows an SEM image of a GaAs nanomembrane attached to the host sapphire substrate. The nanomembrane evidently adhered well to the substrate. Figure 4b shows a detail of the transferred heterostructure nanomembrane with sharp interfaces between the layers of the heterostructure.

Once the nanomembranes became separated from the growth substrate, the etching solution was carefully diluted with water. The membranes were then taken out of the solution and transferred to sapphire substrate. They were dried and thoroughly cleaned in preparation for HEMT processing. Van der Waals forces between the HEMT nanomembranes and sapphire were strong enough for the subsequent processing steps.

**Figure 4.** (**a**) SEM image of a GaAs nanomembrane attached to the host sapphire substrate by van der Waals forces. (**b**) SEM close-up of the transferred heterostructure nanomembrane with sharp interfaces between the layers.


**5. Conclusions**

Our updated systematic review and meta-analysis suggested that moxibustion has a positive effect on correcting breech presentation. However, more randomized, controlled clinical trials are needed to evaluate whether our estimate of the magnitude of the effect of moxibustion remains constant.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/1 0.3390/healthcare9060619/s1, Table S1: Literature search strategy, Table S2: Summary of risk of bias assessment, Figure S1: Forest plot of comparison: Moxibustion versus Control; Outcome: Preterm delivery & Premature rupture of membranes.

**Author Contributions:** J.-A.L., S.-C.S. and Y.-C.C. designed this study; C.-T.C., P.Y.-C.C., K.-L.O., T.-H.H., C.-H.Y., T.-J.L. analyzed and interpreted study results. J.-A.L. and S.-C.S. were major contributors in writing the manuscript. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank all researchers involved in the individual trials.

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


*Communication* **Flooding Tolerance in Sweet Potato (***Ipomoea batatas* **(L.) Lam) Is Mediated by Reactive Oxygen Species and Nitric Oxide**

**Sul-U Park 1,2,†, Chan-Ju Lee 1,†, Sung-Chul Park 3, Ki Jung Nam 4, Kang-Lok Lee 4, Sang-Soo Kwak 1,2, Ho Soo Kim 1,\* and Yun-Hee Kim 4,\***

**Abstract:** Flooding is harmful to almost all higher plants, including crop species. Most cultivars of the root crop sweet potato are able to tolerate environmental stresses such as drought, high temperature, and high salinity. They are, however, relatively sensitive to flooding stress, which greatly reduces yield and commercial value. Previous transcriptomic analysis of flood-sensitive and flood-resistant sweet potato cultivars identified genes that were likely to contribute to protection against flooding stress, including genes related to ethylene (ET), reactive oxygen species (ROS), and nitric oxide (NO) metabolism. Although each sweet potato cultivar can be classified as either tolerant or sensitive to flooding stress, the molecular mechanisms of flooding resistance in ET, ROS, and NO regulationmediated responses have not yet been reported. Therefore, this study characterized the regulation of ET, ROS, and NO metabolism in two sweet potato cultivars—one flood-tolerant cultivar and one flood-sensitive cultivar—under early flooding treatment conditions. The expression of *ERFVII* genes, which are involved in low oxygen signaling, was upregulated in leaves during flooding stress treatments. In addition, levels of respiratory burst oxidase homologs and metallothionein-mediated ROS scavenging were greatly increased in the early stage of flooding in the flood-tolerant sweet potato cultivar compared with the flood-sensitive cultivar. The expression of genes involved in NO biosynthesis and scavenging was also upregulated in the tolerant cultivar. Finally, NO scavengingrelated *MDHAR* expressions and enzymatic activity were higher in the flood-tolerant cultivar than in the flood-sensitive cultivar. These results indicate that, in sweet potato, genes involved in ET, ROS, and NO regulation play an important part in response mechanisms against flooding stress.

**Keywords:** ethylene response factor; flooding stress; metallothionein; monodehydroascorbic acid reductase; resistant cultivar; respiratory burst oxidase; sensitive cultivar


**Citation:** Galber, C.; Carissimi, S.; Baracca, A.; Giorgio, V. The ATP Synthase Deficiency in Human Diseases. *Life* **2021**, *11*, 325. https://doi.org/10.3390/ life11040325

Academic Editor: Gopal J. Babu; Giorgio Lenaz and Salvatore Nesci

Received: 8 March 2021 Accepted: 3 April 2021 Published: 8 April 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/).


**1. Introduction**

Polarized environment, from the beginning of life on our planet, imposes on living organisms a necessity to read directions. Light and gravity are the two most important oriented signals that come from well-defined sources. The response to these primary polar signals allowed for development of the secondary, more sophisticated reactions to the network of other polar signals like gradients of chemical molecules or mechanical stresses. The signals of chemical and physical nature provide the extrinsic but also an intrinsic information for biological systems.

One of the basic features of the organism developing in polar environment is its axiality. This brings in consequence following possibilities: 1) multiplication of repetitive units of the body and their special alignment along the axis (segmentation, metamerism) and 2) deviations of structures from the axis to the left or to the right (development of L/R symmetry).

The main axis in the motionless plants, fastened to the ground, is mostly vertical, extending between the apical and basal poles. The animal main axis, regardless its position in the gravity field, connects the anterior and posterior poles. The axis

formation starts at the very early stages of development. The identity of segments formed iteratively along the axis in both plants and animals is genetically controlled and their evolutionary multiplication creates a great potential for morphological and functional diversity through many useful modifications. This process is in a sense similar to the effects of gene duplication on the molecular level.

Subsequent emergence of L/R symmetry may be observed on all, hierarchically different levels of body organization. Some general principles, like minimum energy rule, are universal in nature leading to the identical solutions on all these levels. Good example represent spherical geodesic shapes. The structure of carbon allotrope - C60 closed fullerene, is also present in coated endocytic vesicles reinforced by the clathrin cage [1], in regularly sculptured surface of pollen grains [2] and in a cellular pattern on the surface of the plant paraboloid apical meristem [3].

Other universal basic forms are chiral helices and spirals commonly observed on molecular, cellular and organismal levels. They are of particular interest here because of distinct mirror symmetry they have, which is the main focus of this chapter. Chirality of many macromolecules: nucleic acids, proteins or cellulose fibers [4], coiled coils of collagen, or such structures as tubulin cytoskeleton, thickenings of plant cell wall, plant tendrils, spiral snail shells or narwhal tusks are but a few examples. Not only structures but also some developmental processes may be chiral. The apical cell divisions in moss gametophores [5], cell cleavage in the embryos of snails [6, 7] or lateral organ initiation on plant shoot apical meristem (SAM) proceed clockwise (CW) or counterclockwise (CCW). Two interesting problems may be addressed while considering chiral structures in biological systems – mechanism of their formation and proportion between the two chiral configurations.

The aim of this chapter is to provide the readers with the overview of some examples of bio-chirality discovered over the years both in animals and plants. The stronger accent will be placed on the latter because they are less known and because they have always been in a focus of the author's research. The mechanisms of many cases of mirror-symmetry presence in plants are yet to be elucidated.

#### **2. Mirror symmetry on cellular level**

Cell chirality or handedness is a newly discovered phenomenon, which nowadays is intensely studied, mostly in animal cells [7–10]. It is manifested in the presence of chiral structures within the cells but also in the cell behavior that may lead to directional movements or assuming L or R orientation of cell alignment. In animals it affects organs laterality [10], in plants results in development of spiral, helical or wavy patterns [3, 11–15].

During primary axis formation on the cellular level the polarity of the cell is manifested in an uneven distribution of receptors, ion channels and hormone carriers on plasma membrane, and internally in the ion currents and cytoskeletal fibers parallel to the developing axis but also in the polar distribution of ultrastructural components like cell organelles or nutrients. All these sophisticated processes have been investigated mainly in plant egg cells or fucoid zygotes [16–18] and animal oocytes [19, 20]. However, even in the integrated system of multicellular organism, singular cell polarity is often a case. In animal body the epithelial cells of intestines constituting a planar 2D barrier, have their polarity unified. It is manifested in nonrandom distribution of glucose transporters which facilitates the oriented transepithelial sugar transport [21]. In plants the polar distribution of the auxin influx (AUX) and efflux (PIN) carriers in plasma membrane results in polar transport of the hormone between the cells [22]. In L1 layer of SAM auxin is transported

**165**

**Figure 1.**

*watched from the inside of the cell.*

*Mirror Symmetry of Life*

of the roots [24].

undiscovered.

*DOI: http://dx.doi.org/10.5772/intechopen.96507*

acropetally, whereas inside of the plant body, in the provascular tissues and later in cambium, the hormone transport is basipetal. Change in the distribution of carriers and thus of the cell polarity redirects the transport, often affecting the directions of plant organ growth [23]. This, for instance, has been noted in gravitropic response

Many elements of the cell ultrastructure are spectacularly chiral. In some green algae exemplified by multicellular filamentous *Spirogyra* or unicellular *Spirotaenia* and *Chlamydomonas spirogyroides* the chloroplasts are of considerable length, flat and ribbon-like. They assume helical course in the cortical cytoplasm of the cell. Not much is known about the chiral configurations of their coiling. Images available in various data bases suggest that in *Spirogyra* both configurations may be present in different filaments [25], in different cells of the same filament or even within the same cell [26]. However, the error resulting from improper focusing during microscopic observations cannot be excluded. The sample taken for analysis from the aquarium of the Botanical Garden of Wrocław showed under light microscope hundreds of cells of the same S configuration of chloroplasts coiling from the right to the left (**Figure 1**). Mechanisms by which the configuration is regulated remain

Another clearly chiral component of the cell is basal body. In eukaryotic, plant and animal cells some identical structures bear different names although they look the same. Two centrioles of the centrosome, basal bodies or kinetosomes in the motile or ciliary epithelial cells have the same architecture. Composed of 9 triplets of microtubules (MT), overlapping on all available images either CW or CCW, they resemble a pinwheel toy. It is unclear, however, whether both configurations, being a mirror-image of one another, are indeed present in all different types of the cells. Transmission electron micrographs (TEM) of tangentially sectioned cell surface show that in a particular cell all basal bodies underlying cilia are of the same chirality [27]. However, unless it is clearly stated like in [28], it is not known whether

*The same filament of* Spirogyra *photographed under fluorescent microscope at different optical levels: at its upper surface (left) and well below, close to its opposite side (right). S helix of the chloroplast, visualized here by the red autofuorescence of chlorophyll, may be falsely interpreted as Z, when due to the changing focus is*

#### *Mirror Symmetry of Life DOI: http://dx.doi.org/10.5772/intechopen.96507*

*Current Topics in Chirality - From Chemistry to Biology*

formation starts at the very early stages of development. The identity of segments formed iteratively along the axis in both plants and animals is genetically controlled and their evolutionary multiplication creates a great potential for morphological and functional diversity through many useful modifications. This process is in a

Subsequent emergence of L/R symmetry may be observed on all, hierarchically different levels of body organization. Some general principles, like minimum energy rule, are universal in nature leading to the identical solutions on all these levels. Good example represent spherical geodesic shapes. The structure of carbon allotrope - C60 closed fullerene, is also present in coated endocytic vesicles reinforced by the clathrin cage [1], in regularly sculptured surface of pollen grains [2] and in a cellular pattern on the surface of the plant paraboloid apical meristem [3]. Other universal basic forms are chiral helices and spirals commonly observed on molecular, cellular and organismal levels. They are of particular interest here because of distinct mirror symmetry they have, which is the main focus of this chapter. Chirality of many macromolecules: nucleic acids, proteins or cellulose fibers [4], coiled coils of collagen, or such structures as tubulin cytoskeleton, thickenings of plant cell wall, plant tendrils, spiral snail shells or narwhal tusks are but a few examples. Not only structures but also some developmental processes may be chiral. The apical cell divisions in moss gametophores [5], cell cleavage in the embryos of snails [6, 7] or lateral organ initiation on plant shoot apical meristem (SAM) proceed clockwise (CW) or counterclockwise (CCW). Two interesting problems may be addressed while considering chiral structures in biological systems – mechanism of their formation and proportion between the two chiral

The aim of this chapter is to provide the readers with the overview of some examples of bio-chirality discovered over the years both in animals and plants. The stronger accent will be placed on the latter because they are less known and because they have always been in a focus of the author's research. The mechanisms of many

Cell chirality or handedness is a newly discovered phenomenon, which nowadays is intensely studied, mostly in animal cells [7–10]. It is manifested in the presence of chiral structures within the cells but also in the cell behavior that may lead to directional movements or assuming L or R orientation of cell alignment. In animals it affects organs laterality [10], in plants results in development of spiral,

During primary axis formation on the cellular level the polarity of the cell is manifested in an uneven distribution of receptors, ion channels and hormone carriers on plasma membrane, and internally in the ion currents and cytoskeletal fibers parallel to the developing axis but also in the polar distribution of ultrastructural components like cell organelles or nutrients. All these sophisticated processes have been investigated mainly in plant egg cells or fucoid zygotes [16–18] and animal oocytes [19, 20]. However, even in the integrated system of multicellular organism, singular cell polarity is often a case. In animal body the epithelial cells of intestines constituting a planar 2D barrier, have their polarity unified. It is manifested in nonrandom distribution of glucose transporters which facilitates the oriented transepithelial sugar transport [21]. In plants the polar distribution of the auxin influx (AUX) and efflux (PIN) carriers in plasma membrane results in polar transport of the hormone between the cells [22]. In L1 layer of SAM auxin is transported

cases of mirror-symmetry presence in plants are yet to be elucidated.

**2. Mirror symmetry on cellular level**

helical or wavy patterns [3, 11–15].

sense similar to the effects of gene duplication on the molecular level.

**164**

configurations.

acropetally, whereas inside of the plant body, in the provascular tissues and later in cambium, the hormone transport is basipetal. Change in the distribution of carriers and thus of the cell polarity redirects the transport, often affecting the directions of plant organ growth [23]. This, for instance, has been noted in gravitropic response of the roots [24].

Many elements of the cell ultrastructure are spectacularly chiral. In some green algae exemplified by multicellular filamentous *Spirogyra* or unicellular *Spirotaenia* and *Chlamydomonas spirogyroides* the chloroplasts are of considerable length, flat and ribbon-like. They assume helical course in the cortical cytoplasm of the cell. Not much is known about the chiral configurations of their coiling. Images available in various data bases suggest that in *Spirogyra* both configurations may be present in different filaments [25], in different cells of the same filament or even within the same cell [26]. However, the error resulting from improper focusing during microscopic observations cannot be excluded. The sample taken for analysis from the aquarium of the Botanical Garden of Wrocław showed under light microscope hundreds of cells of the same S configuration of chloroplasts coiling from the right to the left (**Figure 1**). Mechanisms by which the configuration is regulated remain undiscovered.

Another clearly chiral component of the cell is basal body. In eukaryotic, plant and animal cells some identical structures bear different names although they look the same. Two centrioles of the centrosome, basal bodies or kinetosomes in the motile or ciliary epithelial cells have the same architecture. Composed of 9 triplets of microtubules (MT), overlapping on all available images either CW or CCW, they resemble a pinwheel toy. It is unclear, however, whether both configurations, being a mirror-image of one another, are indeed present in all different types of the cells. Transmission electron micrographs (TEM) of tangentially sectioned cell surface show that in a particular cell all basal bodies underlying cilia are of the same chirality [27]. However, unless it is clearly stated like in [28], it is not known whether

#### **Figure 1.**

*The same filament of* Spirogyra *photographed under fluorescent microscope at different optical levels: at its upper surface (left) and well below, close to its opposite side (right). S helix of the chloroplast, visualized here by the red autofuorescence of chlorophyll, may be falsely interpreted as Z, when due to the changing focus is watched from the inside of the cell.*

basal body is seen on TEM from the surface of the cell or from its inside. This is the reason for chiral configurations of basal bodies being uncertain. The image of *Paramaecium micronucleatum* by Dennis Kunkel [27] shows CCW overlapping triplets, whereas in *Paramecium tetraurelia* [29] the triplets overlap CW.

In flagellar apparatus of *Chlamydomonas* or *Acrosiphonia* gamete both chiral configurations of basal bodies are present on the same electron micrograph [30, 31] but in these cases it is certainly an effect of opposite orientation of flagella and their two basal bodies facing each other horizontally. The bodies are in fact of the same chirality. This case shows again how careful one must be analyzing and interpreting the examples of mirror-symmetry of the chiral ultrastructural components of the cell on TEM images, that can be easily flipped and show the same structure either from above or from the bottom side. There is one additional aspect of mirror symmetry presence in the locomotion apparatus of green algae. The flagellar roots of the two basal bodies are slightly rotated one with respect to the other – CW in representatives of Chlorophyceae and CCW in Ulvophyceae [30, 32, 33]. This finding, among the others, was the foundation of the profound revision of green algae taxonomy [30, 33].

The process of cilia beating is chiral. Both ciliates, motile spermatozoids and stationary epithelial cells readily change the direction of cilia movement either to navigate or alter the current of surrounding fluids [34]. Interestingly, the latter has been employed by unicellular *Stentor rosei*, to avoid, in quite deliberate and calculated manner, the irritating particles experimentally added to the medium [35]. The cell, however, is not always in full control over the cilia beating. The doublets of some ciliates, having notably the same chirality of basal bodies, show that in the form being a mirror image of the typical one, food particles are expelled from the oral apparatus instead of being directed towards it [28]. Typical chiral form of *Paramaecium* swims forward employing leftward rotation. Stressful conditions like low temperature or heavy metals force the ciliate to spin in opposite direction [36].

Among fibrillar elements constituting cytoskeleton, a tensegral structure of eukaryotic cytoplasm it is actin microfilament (MF) that is chiral. Mammalian cells exhibit specific, actin dependent L/R asymmetry which is different in normal and cancerous cells and changes when inhibitors of actin function are applied [37]. In bacteria changeable chirality of actin homologs MreB has impact on their growth and cell shape [38]. Actin is also responsible for directional, chiral movement of cytoplasm in the cells of charophytes [39]. Another important component of cytoskeleton, MTs. *per se* are not chiral. However, their arrangement in the cortical cytoplasm, beneath plasma membrane, is often oriented in plant cells (**Figure 2**). The elongating cells in the axial organs of the model plant *Arabidopsis thaliana* apparently have the chirality of their cortical cytoskeleton genetically controlled. In *spiral 1* and *spiral 2* mutants the cortical MTs, watched from the cell surface, form an ascending S helix, whereas in *lefty* mutant the helix is Z. The mutations lead to abnormal growth of the plant axial organs which, strangely enough, become twisted oppositely to the configuration of MT helix in their cells – Z in the *spiral*, S in the *lefty* mutants [40–42].

Configuration of cortical cytoskeleton, below plasma membrane, in some differentiating plant cells may change with time causing the development of interlocked pattern of the cellulose microfibrils deposited in the secondary wall [43]. Both chiral systems: intracellular and extracellular, parallel each other in consecutive stages of the secondary cell wall formation. The cycle of changes in microfibrils orientation always starts from the ascending S helix in S1 layer of the secondary wall (watched from the outside of the cell), it alters to Z in the S2 layer and returns to S helix in the S3 layer. No exception from this rule has been found

**167**

*Mirror Symmetry of Life*

**Figure 2.**

from breaking (**Figure 2**).

recently shown in chicken embryos [48].

function [45].

*DOI: http://dx.doi.org/10.5772/intechopen.96507*

*surface of the thickenings seen from their outside.*

even though, at least theoretically, the opposite sequence of changes in microfibrils orientation is possible. Moreover, the cycle of changes in the chirality of microfibrillar helix appears to be independent of the overall orientation of the differentiating cell within figured wood, which also exhibits L/R symmetry [44]. This astounding regularity points to existence of yet undiscovered mechanism that must precisely regulate the phases sequence in the full cycle of changes in the cortical MTs orientation. Must be also independent of the other, hypothetical one, which controls L/R symmetry of cambial cellular events such as oriented anticlinal divisions and intrusive growth. This assumption is supported even further by the S helix of the secondary wall thickenings in differentiating protoxylem. The first, S1 deposit of this wall, not yet completely covering the primary wall surface prevents the cells, exposed to mechanical stress caused by longitudinal expansion of growing shoot,

*Immunofluorescent visualization of Z helical cortical cytoskeleton in the elongated cambial cell of*  Cinnamomum camphora *(left) and two S helices of the secondary cell wall thickenings isolated from the protoxylem cells of* Scindapsus *sp. photographed at two different optical levels. The middle photo shows the*

The discovery of cell intrinsic chirality resulted from the fundamental question how the laterality of organs within the animal body is accomplished. Over the years much attention has been paid to this phenomenon and its connection with the development of L/R symmetry of the whole multicellular organism. It was found that blood neutrophiles polarity, defined by position of centrosomes with regard to the cell nucleus, makes them capable of directional movements in absence of polar external signals. This property disappears after application of drugs affecting MT

The model invertebrate organism *Drosophila melanogaster* provided evidence that myosin encoding gene mutation switches cell chirality and results in the development of *situs inversus* phenotype of the hindguts or genitalia [7, 8, 10, 46]. In vertebrates the development of typical L/R asymmetry from anteceding state of embryo bilateral symmetry is generated by various mechanisms. One of them is based on function of axonemal dynein. This motor-protein is responsible for appropriate beating of cilia in nodal epithelial cells, causing the directed ion current. Defects in the gene structure encoding for the dynein results in random selection of heart position in mouse embryo [47]. The involvement of C kinase signaling pathway in the reversal of cells chirality leading to mirrored position of heart was

#### **Figure 2.**

*Current Topics in Chirality - From Chemistry to Biology*

Z in the *spiral*, S in the *lefty* mutants [40–42].

taxonomy [30, 33].

basal body is seen on TEM from the surface of the cell or from its inside. This is the reason for chiral configurations of basal bodies being uncertain. The image of *Paramaecium micronucleatum* by Dennis Kunkel [27] shows CCW overlapping

In flagellar apparatus of *Chlamydomonas* or *Acrosiphonia* gamete both chiral configurations of basal bodies are present on the same electron micrograph [30, 31] but in these cases it is certainly an effect of opposite orientation of flagella and their two basal bodies facing each other horizontally. The bodies are in fact of the same chirality. This case shows again how careful one must be analyzing and interpreting the examples of mirror-symmetry of the chiral ultrastructural components of the cell on TEM images, that can be easily flipped and show the same structure either from above or from the bottom side. There is one additional aspect of mirror symmetry presence in the locomotion apparatus of green algae. The flagellar roots of the two basal bodies are slightly rotated one with respect to the other – CW in representatives of Chlorophyceae and CCW in Ulvophyceae [30, 32, 33]. This finding, among the others, was the foundation of the profound revision of green algae

The process of cilia beating is chiral. Both ciliates, motile spermatozoids and stationary epithelial cells readily change the direction of cilia movement either to navigate or alter the current of surrounding fluids [34]. Interestingly, the latter has been employed by unicellular *Stentor rosei*, to avoid, in quite deliberate and calculated manner, the irritating particles experimentally added to the medium [35]. The cell, however, is not always in full control over the cilia beating. The doublets of some ciliates, having notably the same chirality of basal bodies, show that in the form being a mirror image of the typical one, food particles are expelled from the oral apparatus instead of being directed towards it [28]. Typical chiral form of *Paramaecium* swims forward employing leftward rotation. Stressful conditions like low temperature or heavy metals force the ciliate to spin in opposite direction [36]. Among fibrillar elements constituting cytoskeleton, a tensegral structure of eukaryotic cytoplasm it is actin microfilament (MF) that is chiral. Mammalian cells exhibit specific, actin dependent L/R asymmetry which is different in normal and cancerous cells and changes when inhibitors of actin function are applied [37]. In bacteria changeable chirality of actin homologs MreB has impact on their growth and cell shape [38]. Actin is also responsible for directional, chiral movement of cytoplasm in the cells of charophytes [39]. Another important component of cytoskeleton, MTs. *per se* are not chiral. However, their arrangement in the cortical cytoplasm, beneath plasma membrane, is often oriented in plant cells (**Figure 2**). The elongating cells in the axial organs of the model plant *Arabidopsis thaliana* apparently have the chirality of their cortical cytoskeleton genetically controlled. In *spiral 1* and *spiral 2* mutants the cortical MTs, watched from the cell surface, form an ascending S helix, whereas in *lefty* mutant the helix is Z. The mutations lead to abnormal growth of the plant axial organs which, strangely enough, become twisted oppositely to the configuration of MT helix in their cells –

Configuration of cortical cytoskeleton, below plasma membrane, in some differentiating plant cells may change with time causing the development of interlocked pattern of the cellulose microfibrils deposited in the secondary wall [43]. Both chiral systems: intracellular and extracellular, parallel each other in consecutive stages of the secondary cell wall formation. The cycle of changes in microfibrils orientation always starts from the ascending S helix in S1 layer of the secondary wall (watched from the outside of the cell), it alters to Z in the S2 layer and returns to S helix in the S3 layer. No exception from this rule has been found

triplets, whereas in *Paramecium tetraurelia* [29] the triplets overlap CW.

**166**

*Immunofluorescent visualization of Z helical cortical cytoskeleton in the elongated cambial cell of*  Cinnamomum camphora *(left) and two S helices of the secondary cell wall thickenings isolated from the protoxylem cells of* Scindapsus *sp. photographed at two different optical levels. The middle photo shows the surface of the thickenings seen from their outside.*

even though, at least theoretically, the opposite sequence of changes in microfibrils orientation is possible. Moreover, the cycle of changes in the chirality of microfibrillar helix appears to be independent of the overall orientation of the differentiating cell within figured wood, which also exhibits L/R symmetry [44]. This astounding regularity points to existence of yet undiscovered mechanism that must precisely regulate the phases sequence in the full cycle of changes in the cortical MTs orientation. Must be also independent of the other, hypothetical one, which controls L/R symmetry of cambial cellular events such as oriented anticlinal divisions and intrusive growth. This assumption is supported even further by the S helix of the secondary wall thickenings in differentiating protoxylem. The first, S1 deposit of this wall, not yet completely covering the primary wall surface prevents the cells, exposed to mechanical stress caused by longitudinal expansion of growing shoot, from breaking (**Figure 2**).

The discovery of cell intrinsic chirality resulted from the fundamental question how the laterality of organs within the animal body is accomplished. Over the years much attention has been paid to this phenomenon and its connection with the development of L/R symmetry of the whole multicellular organism. It was found that blood neutrophiles polarity, defined by position of centrosomes with regard to the cell nucleus, makes them capable of directional movements in absence of polar external signals. This property disappears after application of drugs affecting MT function [45].

The model invertebrate organism *Drosophila melanogaster* provided evidence that myosin encoding gene mutation switches cell chirality and results in the development of *situs inversus* phenotype of the hindguts or genitalia [7, 8, 10, 46]. In vertebrates the development of typical L/R asymmetry from anteceding state of embryo bilateral symmetry is generated by various mechanisms. One of them is based on function of axonemal dynein. This motor-protein is responsible for appropriate beating of cilia in nodal epithelial cells, causing the directed ion current. Defects in the gene structure encoding for the dynein results in random selection of heart position in mouse embryo [47]. The involvement of C kinase signaling pathway in the reversal of cells chirality leading to mirrored position of heart was recently shown in chicken embryos [48].

All the above studies show how intrinsic cell polarity may be translated onto the higher level of multicellular organism organization in animals. Very little though is known about L/R symmetry regulation in multicellular plant organisms.


*Article* **Elucidation of Melanogenesis-Associated Signaling Pathways Regulated by Argan Press Cake in B16 Melanoma Cells**

**Thouria Bourhim 1,‡, Myra O. Villareal 2,3,‡, Chemseddoha Gadhi 1,2,\* and Hiroko Isoda 2,3,\***

**Abstract:** The beneficial effect on health of argan oil is recognized worldwide. We have previously reported that the cake that remains after argan oil extraction (argan press-cake or APC) inhibits melanogenesis in B16 melanoma cells in a time-dependent manner without cytotoxicity. In this study, the global gene expression profile of B16 melanoma cells treated with APC extract was determined in order to gain an understanding of the possible mechanisms of action of APC. The results suggest that APC extract inhibits melanin biosynthesis by down-regulating microphthalmiaassociated transcription factor (*Mitf*) and its downstream signaling pathway through JNK signaling activation, and the inhibition of Wnt/β-catenin and cAMP/PKA signaling pathways. APC extract also prevented the transport of melanosomes by down-regulating *Rab27a* expression. These results suggest that APC may be an important natural skin whitening product and pharmacological agent used for clinical treatment of pigmentary disorders.

**Keywords:** argan press-cake; MITF; JNK; cAMP/PKA; Wnt/β-catenin; microarray analysis

#### **1. Introduction**

*Argania spinosa* L. (family Sapotaceae) is a Moroccan endemic tree. It covers more than 800,000 hectares in the south-western region of the country [1]. In 1998, it was recognized as a biosphere reserve by the UNESCO and is known worldwide for its edible oil, which is now one of the world's priciest oils [2]. Argan oil is extremely rich in unsaturated fatty acids and bioactive phytochemicals including tocopherols, phenolic compounds, and carotenoids [3] contributing to its pharmacological and cosmetic properties [4]. Traditionally, this oil is used as an anti-aging skin care [5]. Consumption of argan oil and/or topical application decrease trans-epidermal water loss and improve skin elasticity [6,7].

At agronomic level, this species has led to great financial returns, significantly reducing the poverty of the local population. In addition to its considerable socio-economic importance, argan trees also contribute to preventing soil erosion and desertification in the Southern part of Morocco [1]. However, destructive logging, climate change, and overexploitation have negatively affected the argan tree ecosystem and about 44% of the argan forest was lost between 1970 and 2007 [8]. The increase in demand for argan oil now requires a more efficient use of this valuable resource.

One of the by-products of argan oil production is argan press-cake (APC). While it was used previously as an animal feed, the discovery that it is abundant in functional secondary metabolites that give argan oil its functional properties made it a possible source of health benefits that argan oil users are looking for. APC contains a high phenolic compounds content that includes procyanidin B1 and B2, catechin, epicatechin, epigallocatechin gallate,

**Citation:** Bourhim, T.; Villareal, M.O.; Gadhi, C.; Isoda, H. Elucidation of Melanogenesis-Associated Signaling Pathways Regulated by Argan Press Cake in B16 Melanoma Cells. *Nutrients* **2021**, *13*, 2697. https:// doi.org/10.3390/nu13082697

Academic Editor: Yoshinori Katakura

Received: 19 June 2021 Accepted: 2 August 2021 Published: 4 August 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/).

phloridzin, myricetin, and quercitrin [9]. It also has a large amount of saponins, steroids, and triterpenoids [10], suggesting that APC could have the same biological effects as argan oil.

Skin exposure to ultraviolet (UV) radiation generates reactive oxygen species (ROS) that may cause skin aging and increased pigmentation [11]. Skin pigmentation is a result of melanin synthesis in the melanocytes, and its subsequent distribution to keratinocytes [12]. Melanin protects the skin against harmful UV radiation and stress resulting from exposure to various environmental pollutants [13]. However, increased production of melanin could cause several unwanted localized skin hyperpigmentation issues such as freckles and age spots, and these could be due to melasma and other post-inflammatory-associated hyperpigmentation [14]. The unwanted side effects of existing treatments for hyperpigmentation increase the demand for safe melanogenesis regulators of plant origin to treat skin hyperpigmentation diseases.

Melanin biosynthesis is regulated by a variety of signal transduction pathways that include cyclic adenosine monophosphate (cAMP) and mitogen activated protein kinase (MAPK), as well as the Wnt signaling pathway [15,16]. cAMP, via PKA, induces cAMPresponse element-binding protein (CREB) family phosphorylation and activation, which then leads to the expression of microphthalmia transcription factor (MITF) [17], with CREB being one of the transcription factors that regulate MITF expression [18]. The transcription factor MITF is responsible for the expression of tyrosinase (TYR), tyrosinase related protein (TRP1), and dopachrome tautomerase (DCT), the major enzymes that catalyze relevant reaction in the melanogenesis process [17]. The Wnt pathway regulates MITF expression through the ß-catenin, the pivotal component of the Wnt pathway. When Wnt proteins bind to their receptors, it stabilizes the cytoplasmic ß-catenin, which then leads to its localization in the nucleus, where it regulates the expression of MITF [19]. The effect of the Wnt signaling pathway on MITF makes it an important pathway for regulating melanocyte differentiation [20].

Extracellular signal-regulated protein kinase (ERK), p38, and c-jun N-terminal kinase (JNK) have essential roles in melanogenesis regulation [21–23]. Interfering with p38, MAPK expression has been reported to promote melanogenesis and tyrosinase expression [24], while the active form of ERK, on the other hand, phosphorylates MITF at serine-73 during the posttranslational process, leading to its ubiquitination and, subsequently, its degradation [25]. JNK can interfere with CREB-regulated transcription co-activator 3 (CRTC3)-dependent MITF expression leading to melanogenesis inhibition [26].

Another key factor in melanogenesis regulation is the melanosome pH, which controls the maturation of melanosome in melanocytes and the rate of melanogenesis, as well as the ratio of eumelanin to phaeomelanin [27].

We have demonstrated the melanogenesis inhibitory effect of APC extract via *Mitf* expression down-regulation in B16 murine melanoma cells [28]. However, the mechanism by which APC regulates melanogenesis is not yet understood. In this study, the global gene expression analysis was done to identify the signaling involved in the melanogenesis inhibitory effect of APC extract.

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

#### *2.1. Chemicals and Reagents*

The Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), and L-glutamine were from Sigma-Aldrich (Burlington, MA, USA). Penicillin/streptomycin solution was from Lonza, Walkersville Inc., (Walkersville, MD, USA). All the other chemicals were from Wako (Saitama, Japan).

#### *2.2. Extraction of APC*

Mature *Argania spinosa* fruits of the spherical type were harvested in July 2012 from the Sidi Ifni region (southwest of Morocco). Voucher samples (MARK10888-1) kept at the regional herbarium of Marrakech (Marrakesh, Morocco) were verified by Prof. Ahmed Ouhammou. The samples were dried at 25 ◦C, after which the argan fruits were manually peeled, and the nut's shells cracked. The press-cake was obtained by extracting the oil from argan kernels using the mechanical press Komet DD 85 G press (IBG Monforts Oekotec GmbH & Co. KG, Mönchengladbach, Germany).

APC (10 g) was extracted with 100 mL ethanol 70% for 2 weeks at room temperature, after which it was centrifuged (1000× *g*, 15 min). The supernatant was then filter-sterilized using a 0.45 μm pore size filter (Millipore, Billerica, MA, USA) and kept at −80 ◦C in a freezer until use.

#### *2.3. Cells and Cell Culture*

The B16 murine melanoma cells used in this study were purchased from Riken Cell Bank (Tsukuba, Japan). Cells were cultured in DMEM supplemented with 10% FBS, 4 mmol/L L-glutamine, 50 units ⁄ml penicillin, and 50 μg/mL streptomycin, and they were maintained in a humidified incubator at 37 ◦C with 5% CO2.

#### *2.4. Total RNA Extraction*

B16 cells were seeded at a density of 3 × 106 cells per 100-mm Petri-dish and were allowed to attach before treatment. The growth medium was replaced with a fresh one containing 0, 50 μg/mL of APC extract or 100 μmol/L of arbutin. The extraction of total RNA was done as described previously [28]. After the specified treatment time, the cells were washed twice with cold PBS before RNA extraction using the ISOGEN kit (Nippon Gene, Tokyo, Japan). The quality and quantity of the RNA was examined using a Nanodrop 2000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). The RNAs used were reverse transcribed using the SuperScript III Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA).

#### *2.5. Quantitative Real-Time PCR Analysis*

The effect of APC on gene expression in B16 cells was determined by real-time PCR (Applied Biosystems, Carlsbad, CA, USA) using TaqMan master mix, and performed using the TaqMan 7500 Fast Real-time PCR System (Applied Biosystems, Carlsbad, CA, USA). Cycling conditions were as follows: 2 min at 50 ◦C and 10 min at 95 ◦C, followed by 40 cycles of 95 ◦C for 15 s and 60 ◦C for 1 min. The assay IDs of the TaqMan primers used were: *Rab27a*—Mm00469997\_m1, *Ctnnb1*—Mm00483039\_m1, *Map3k12*—Mm00437378\_m1, and *Gapdh*–Mm99999915\_g1. *Gapdh* was used as the internal control. All the reactions were run in triplicates.

#### *2.6. DNA Microarrays*

The microarray analysis was performed to determine the global transcriptional response of B16 melanoma cells to argan press-cake or arbutin (positive control) treatment. Total RNA (100 ng) was reverse transcribed to synthesize the first-strand cDNA, which was then converted into double-stranded cDNA (ds-cDNA). The ds-cDNA template and biotin-labeled aRNA was generated using the 30 IVT Express Labeling Kit (Affymetrix, Santa Clara, CA, USA). The ds-cDNA was used to synthesize biotin-modified aRNA, 10 μg of which was fragmented using the GeneAtlas 3 IVT Express Kit, before hybridization to the Affymetrix Mouse 430 PM Array strips (Affymetrix) for 16 h at 45 ◦C. Following hybridization, the microarray DNA array was washed and stained in the GeneAtlas Fluidics Station 400 (Affymetrix), and then scanned using the GeneAtlas Imaging Station (Affymetrix). Data analysis was carried out using Affymetrix Expression Console Software and Affymetrix Transcriptome Analysis Console (TAC) 2.0 Software (Affymetrix). Hierarchical clustering was performed using Euclidean distance by TIGR's MultiExperiment Viewer v4.9.0 software. The rows represent genes while columns represent the experimental samples. The heat map represents the gene expression ratios with the green and red color of cells indicating gene down- and up-regulation. In addition, the DNA microarray data have been deposited in the ArrayExpress database at EMBI-EBI (Available online: www.ebi.ac.uk/arrayexpress (accessed on 27 May 2020)), under the reference number E-MTAB-9089.

#### *2.7. Statistical Analysis*

The results were expressed as mean ± standard deviation (SD) of three independent experiments. The differences between means were analyzed for significance using one-way analysis of variance (ANOVA) with a Fisher's Least Significant Difference (LSD) post hoc test. A value of *p* ≤ 0.05 was considered significant.

#### **3. Results**

#### *3.1. Gene Expression Profile of APC Extract-Treated Cells*

The expression level of genes in APC extract- or arbutin-treated cells was determined. Furthermore, genes expressed in APC-treated cells that were 1.5-fold different from the untreated controls were subjected to GO Enrichment Analysis. GO Enrichment Analysis identifies the genes that are relevant to biological processes and that were affected by APC treatment. The genes that were significantly changed in expression are presented in Table 1. The processes down-regulated by APC extract included those that are associated with pigmentation, melanosome transport, keratinocyte differentiation, melanocyte differentiation, cell differentiation, and apoptosis. Moreover, nervous system development, Wnt receptor signaling pathway through beta-catenin, the glutathione metabolic process, and MAP kinase kinase kinase activity were differentially regulated by APC extract.

**Table 1.** List of genes that were differentially expressed (1.5-fold change in expression) in B16 melanoma cells treated with argan press-cake (APC) or arbutin (ARB) as determined by DNA microarray (*p* < 0.05) 1.

#### **Table 1.** *Cont.*

**Table 1.** *Cont.*

1 Based on gene ontology annotations in Mouse Genome Informatics (MGI).

DNA microarray analysis also revealed that several Wnt signaling pathway associated genes were significantly changed in response to APC extract treatment (Table 1). Among those genes, several genes were down-regulated (*Ctnnb1* and *Cxxc4*), while some were up-regulated (*Tcf7L2*). Interestingly, a number of intermediate genes of the MAPK pathway were up-regulated (*Akap9*, *Rasa1*, *Spry4*), while *Map3k12* was down-regulated by APC extract treatment. Moreover, the microarray dataset revealed an up-regulation by the APC extract of the *Prkar1b* gene, a regulatory subunit of PKA inactivating its catalytic domains. Additionally, several genes under the transcriptional regulation of MITF, such as *Tyr*, *Trpm1*, *Vat1*, *Atp6v0b*, *Rbm39*, and *Usp9x*, were significantly down-regulated, including *Rab27a*, which is a melanosome transport protein. The obtained data also showed that many of the solute carrier genes (SLCs) (*Slc24a4*, *Slc35a5*, *Slc12a6*, *Slc6a6*, *Slc4a4*, *and Slc7a11*) were differentially expressed following APC extract treatment (Table 1).

Genes that were significantly modulated by APC extract or arbutin were subjected to hierarchical clustering and the results grouped the genes into two main groups: APC-downregulated and APC-up-regulated genes (Figure 1). The down-regulated genes formed five subgroups, while up-regulated genes formed three subgroups. The first cluster is composed of genes relevant to transporter activity represented by two solute carrier genes *Slc24a4* and *Slc6a17* (*p* = 0.52). The second cluster are genes significant for DNA repair (*Brca1*, *p* = 1.04). The next cluster constituted of genes that are relevant in melanin synthesis, melanosome transport, ATP binding, ion transport, and metal binding (*Tyr*, *Rab27a*, *Trpm1*, *Vat1*, *Atp6v0b*, *Cxxc4*, *Adam10*, *Epb41l2* (*p* = 0.30)). In addition, a regulation of *Ctnnb1*, *Map3k12* genes that play a role in the Wnt signaling pathway and MAP kinase activity was observed in cluster four (*p* = 0.34). The fifth cluster are genes in the Wnt signaling pathway and c-AMP signaling pathway, or those that function in metal binding, protein binding (*p* = 0.33). The last three clusters represent the up-regulated genes by APC extract treatment. Those genes play roles in the c-AMP signaling pathway, Wnt signaling pathway, transporter activity, protein binding, and kinase activity (Figure 1, Table 2).

**Table 2.** Gene clusters obtained by hierarchical clustering of significantly expressed genes in APC-treated B16 cells a,b.

a Euclidean distance by TIGR's MultiExperiment Viewer v4.9.0 software. b Based on gene ontology annotations in Mouse Genome Informatics (MGI).

**Figure 1.** Heat map and hierarchical clustering of 34 genes in response to treatment with argan presscake (APC) or by arbutin (ARB) in B16 melanoma cells. Clustering was calculated using Euclidian distance in the TIGR's MultiExperiment Viewer v4.9.0 software. Rows and columns represent genes and experimental samples, respectively. Gene expression ratios are presented in the heat map with green and red color indicating down and up-regulation, respectively.

#### *3.2. Validation of Global Gene Expression Results*

The DNA microarray results were validated using rt-PCR. *Rab27a* was significantly down-regulated by 51.7%. *Ctnnb1* (catenin cadherin associated protein beta1) and *Map3k12* (mitogen-activated protein kinase kinase kinase 12), the genes that code for proteins that regulate *Mitf* expression, were decreased significantly (Figure 2). The changes in genes expression were consistent with the microarray data.

**Figure 2.** Effect of argan press-cake (APC) on the expression of (**a**) *Rab27a* (member RAS oncogene family), (**b**) mitogenactivated protein kinase kinase kinase 12 (*Map3k12*), and (**c**) catenin (cadherin associated protein) beta1 (*Ctnnb1*) genes in B16 cells. Cells were seeded onto a 100-mm dish at a density of 3 × 106. B16 cells were treated with or without arbutin (100 μM) or argan press-cake extract (50 μg/mL) for 24 h. Rt-PCR was used to determine the select genes expression level. Data are expressed as mean ± SD (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01.

#### **4. Discussion**

Previously, we demonstrated that APC extract has a melanogenesis inhibitory effect in B16 cells. APC inhibits the melanogenic enzymes expression through *Mitf* downregulation [28]. In this study, the global gene expression in the B16 cells in response to APC treatment was analyzed in order to fully understand the effect of APC on B16 cells and to identify APC targets that may have directly or indirectly contributed to its inhibitory effect, specifically on MITF and on melanogenesis in general.

As presented in Table 1, APC up-regulated the *Prkar1b* gene, which codes for the protein that is a regulatory subunit of cyclic AMP-dependent protein kinase A (PKA) [29]. As previously reported, APC treatment inhibits the *Mitf* expression [28], which could be due to the inhibition of signals that are upstream of *Mitf*. In short, an inactivation of PKA and the down-regulation of cAMP due to APC treatment could explain the decreased *Mitf* expression. Down-regulation of *Mitf* also led to inhibition of melanosome transport by its direct regulatory effect on *Rab27a* expression [30]. In this study, *Rab27a* expression was down-regulated by 51.7%. In addition, it has also been observed that *β-catenin* was down-regulated. β-catenin, as a transcriptional regulator, can be redirected by MITF away from genes that are under the regulation of the Wnt signaling toward *Mitf*-specific targets [31]. Additionally, β-catenin is also known to regulate *Mitf* expression [32]. βcatenin may be degraded via a ubiquitin-dependent, PKA-attenuated GSK3β (glycogen synthase kinase-3β) action [24]. MITF-regulated gene *Rab27a*, in addition to its function in melanosome transport, also has a significant role in various cell activities that include

cell growth, invasion, and metastasis [33,34]. Several studies have associated increased expression of *Rab27a* with carcinogenesis, and it has been reported to promote the stemness of colon cancer cells [35], leading to poor survival in pancreatic cancer [36]. The inhibition of *Rab27a* by APC therefore suggests an anti-cancer effect of APC by targeting *Rab27a.*

Cheli et al. (2009) showed that cAMP controls the melanosome pH through PKAindependent mechanism. It has also been suggested that cAMP modulates vacuolar ATPases and ion transporters expression [37]. The melanosome pH of light-colored human skin melanocytes is more acidic, which explains the observed low tyrosinase activity observed in Caucasian skin. In contrast, melanosomes in dark human skin are less acidic and have higher tyrosinase activity [38]. The results of this study revealed that many of the solute carrier genes (*Slc24a4*, *Slc35a5*, *Slc12a6*, *Slc6a6*, *Slc4a4*, *and Slc7a11*), several vacuolar ATPases, and ion transporters were differentially expressed following APC extract treatment. In addition to its action at the mRNA level, the results of this study further suggest that APC extract may decrease the activity of TYR by reducing the pH of melanosome. This may also explain why despite the fact that there was no change in the TYR expression, a decrease in B16 cells melanin content at 48 h was observed [28]. Moreover, a growing number of solute carrier genes have been reported to play pivotal roles in melanogenesis regulation and in ethnic skin color determination [39,40], and this includes *Slc24a4*, which was down-regulated by 2-fold in APC extract-treated cells.

The effect of APC extract on the JNK signaling most likely contributed to the decreased melanogenesis. JNK is regulated by several molecules, but in this study, a down-regulation of the expression of *Map3k12* and up-regulation of *Plcb1* could induce the activation of the JNK pathway (Figure 3). The activation of JNK causes phosphorylation of MITF at serine 73, which could then result to subsequent ubiquitin-dependent proteasomal degradation of MITF [21]. Although MITF was observed to be down-regulated at the transcriptional level, it is also possible that post-transcriptional modifications occurred in response to APC extract treatment. That effect could be through the several pathways mentioned earlier, which include the MAPK (JNK) pathway.

**Figure 3.** Argan press-cake (APC) down-regulated genes relevant to melanogenesis. This figure is adapted from Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway for melanogenesis (Available online: https://www.genome.jp/pathway/map04916 (accessed on 15 February 2021)).

DNA microarray results also showed that the Wnt/β-catenin and the cAMP/PKA signaling pathways were inactivated (Figure 3). The cAMP's effect on melanogenesis is mainly of its effect on the process' rate limiting enzyme tyrosinase, and cAMP stimulating tyrosinase activity [41]. However, what has been well reported is its effect on PKA activation that will then lead to CREB activation. CREB activates MITF expression. Compounds that inhibit the cAMP pathway are expected to inhibit melanogenesis [42].

This complex effect of APC extract on B16 cells could be attributed to the presence of different bioactive compounds, polyphenols, saponins, steroids, and triterpenoids.

In this study, the APC-treated cells were not just compared to untreated cells, but also to arbutin, the positive control. Arbutin is a D-glucopyranoside derivative of hydroquinone, which is a widely used skin lightning agent in cosmetic and healthcare industry [43,44]. Our microarray data revealed that *Nfkbia*, which is involved in nuclear factor kappa B (NF-κB) binding, and cytoplasmic sequestering were down-regulated by arbutin. NF-κB signaling has been reported to play a key role in melanogenesis regulation [45–47]. In addition, Ahn et al. (2003) reported that the treatment of human keratinocytes with arbutin effectively down-regulate NF-κB activation, which is consistent with our results [48]. It is well-known that the MAP3K-related kinase is associated with NF-κB stimulation by TNF, CD95, and IL-1 [49,50]. Therefore, *Map3k12*, which was significantly decreased by arbutin, appears to be a plausible target in regulating melanogenesis through NF-κB inhibition.

#### **5. Conclusions**

Argan press-cake extract significantly decreases melanin synthesis in B16 cells [28]. In this study, microarray analysis sheds light on the exact mechanism by which the antimelanogenesis effect occurs. The inhibitory effect on melanogenesis was a result of the regulation of several signals at once, including (i) the inactivation of the cAMP/PKA signaling pathway through an up-regulation of *Prkar1b* gene that lead to PKA downregulation and subsequently inhibition of *Mitf* expression; (ii) the down-regulation of the Wnt/β-catenin signaling pathway; and (iii) the activation of the JNK MAP kinase pathway. JNK activation causes a subsequent ubiquitin-dependent proteasomal degradation of MITF. In addition, APC may inhibit not only the melanin production but also melanosome transport by *Rab27a* down-regulation.

By inhibiting the overproduction and accumulation of melanin in the skin that cause numerous related pigment disorders like melasma, freckles or lentigines, dermatitis, and geriatric skin pigmentation [14], APC may become an alternative natural and noncytotoxic therapeutic agent against hyperpigmentation disorders. It also has a potential use in cosmetics like argan oil [51] and other argan oil extraction by-products, argan fruit shells [52], argan leaves [53], etc.

**Author Contributions:** Conceptualization: H.I. and C.G.; methodology: H.I. and M.O.V.; funding acquisition: H.I. and C.G; investigation: T.B. and M.O.V.; supervision: C.G. and H.I.; writing original draft: T.B.; writing—review and editing: T.B., M.O.V. and C.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Japan International Cooperation Agency (JICA)-Japan Science and Technology Agency (JST)'s Science and Technology Research Partnership for Sustainable Development (SATREPS) project entitled, "Valorization of Bioresources Based on Scientific Evidence in Semi- and Arid Land for Creation of New Industry"; and by Ministry of Higher Education, Scientific Research and Executive Training (MHESRET) of Kingdom of Morocco.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The DNA microarray data have been deposited in the ArrayExpress database at EMBI-EBI (Available online: www.ebi.ac.uk/arrayexpress (accessed on 27 May 2020)), under the reference number E-MTAB-9089.

**Acknowledgments:** Authors are grateful to the following organizations for their support by providing grants to the first author (TB): Japan Student Services Organization (JASSO); and Centre National pour la Recherche Scientifique et Technique (CNRST) Morocco.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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**3. Apolipoproteins effects on secondary drug response**

#### **3.1. Tamoxifen**

256 Lipoproteins – Role in Health and Diseases

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**2. Chemotherapy,** *ApoE* **and memory**

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In rodent models, the lack of *ApoE* or ApoE4 protein expression leads to destabilization of cell membranes, increased apoptosis, and heightened sensitivity to neuronal trauma; whereas, ApoE3 and ApoE2 protein expression allow for healthy cell functioning and neuroprotection [4]. ApoE4 has detrimental effects in transgenic mice, including behavioral abnormalities, such as deficits in spatial learning and memory using Morris water maze (MWM) [6], as well as significant alterations in the hippocampus and cortex [4,7]. The studies in mice are consistent with clinical studies indicating reduced spatial learning and memory in those who carry the *ApoE4* allele [8]. Experiments have also demonstrated that the three isoforms of human *ApoE* gene have different effects on the development of neurodegenerative diseases. Those individuals that are *ApoE4* carriers have an increased risk of age-related mild cognitive impairments (MCI) and the development of Alzheimer's

In ovarian cancer, ApoE protein levels act as a potential tumor-associated marker as found in serous carcinomas, but not in serious borderline or normal ovarian surface epithelium cells [10].  Blockage of ApoE expression in the serous carcinoma cell lines leads to cell cycle arrest and apoptosis. Women infused with ApoE protein at the time of diagnosis showed significantly higher survival rates [10]. This data suggest that upregulation of ApoE expression may be a defense

Over the last 20 years it has become apparent that chemotherapy drugs not only attack cancer cells, but also cross the blood brain barrier (BBB) leading to negative effects on cognitive processing which is known as Chemo-brain or Chem-fog [11,12]. Methotrexate, 5- Flourouracil (5-FU) the most common chemotherapy drugs used to treat breast, colorectal, head and neck cancers been shown in both rodent models and clinical studies to lead to neurocognitive deficits in a variety of domains including visual memory and visuospatial functioning [11,13,14]. In clinical studies, there is considerable variability between studies as to the extent and frequency of such impairments heretofore mentioned. However, rodent studies are clearly show that the drugs in the CMF (cyclophosphamide, methotrexate, 5-FU) regiment lead to decreased hippocampus cell proliferation and induce MWM memory impairments [15,16]. In addition, cytarabine (cytosine arabinoside) and ifosfamide among other chemotherapy have been shown to lead to memory impairments, hemiparesis, aphasia and progressive dementia [17]. However, there is significant lack of pre-clinical testing of

most chemotherapeutic agents and their long-term effects on memory.

Other confounding factors that can affect cognitive status after chemotherapy include other medications patients are taking to control comorbid conditions or to treat the tumor itself. Tamoxifen is used as an estrogen receptor modulator (SERM) in estrogen receptor (ER) positive breast cancer carcinomas. Tamoxifen is a pure estrogen receptor blocker. As with other drugs, tamoxifen and its metabolites can cross the BBB affecting ER in various brain regions including the cerebral cortex, hippocampus and amygdala [23]. In combination with chemotherapy, tamoxifen appears to intensify the cognitive impairments, particularly in visual memory, verbal working memory and visuospatial ability [24]. The Anatrozole, Tamoxifen Combined (ATAC) trial, also found verbal memory and processing speed impairments post-chemotherapy treated only with tamoxifen compared to women only on a combined ATAC treatment (Table 1) [25]. This suggests that tamoxifen has confounding effects when given as part of the chemotherapy regiment. Tamoxifen also appears to have both agonist and antagonist properties in the brain with reported up-regulation of proinflammatory cytokines shown to be related to cognitive dysfunction [26]. Positron emission tomography (PET) imaging of survivors does show higher hypometabolism with dual chemotherapy and tamoxifen, not seen in women treated only with tamoxifen [27]. Animal models using repeated tamoxifen or combinations of methotrexate and 5-FU injections both produced deficits in acquisition and retention in an operant learning paradigm (Table 1) [28]. These studies were conducted when women were still in treatment, leaving the question of potential long-term effects. There is a study that examined women who used tamoxifen for <4 years compared to >6 years of exposure. The study found that the current exposure led to greater memory deficits compared to non-users (Table 1) [29]. This suggests that while on therapy, patients may have acute memory impairments and that alternative drugs should be seriously considered.

In breast cancer survivors undergoing tamoxifen treatment, their total cholesterol, VLDL, high density lipoproteins (HDL) and Apolipoprotein B (ApoB) protein levels have been shown to decrease in both *ApoE4* carriers and non-*ApoE4* carriers (Table 1) [30,31]. Breast cancer patients who are *ApoE4* carriers have higher plasma triglyceride levels and altered ApoA-1/ApoB ratio. Both are risk factors for cardiovascular events, after tamoxifen treatment (Table 1) [30,31]. In non-*ApoE4* carriers, there were lower levels of lipoprotein (a) after treatment, but no effect on triglycerides or the ApoA-1/ApoB ratio (Table 1). This suggests that non-*ApoE4* carriers have a more positive response to tamoxifen with respect to lipid profiles and risk for cardiovascular complications [31]. Considering tamoxifen and *ApoE4* both have a deleterious effect on cognition and tamoxifen has an *ApoE* genotype dependent effect on lipid profiles, suggest further investigations are warranted to understand the mechanistic relationship.

#### **3.2. Anatrozole**

Anatrozole also known as arimidex is an aromatase inhibitor that lowers estrogen levels and is used as a treatment in estrogen positive breast cancer patients post-surgery. Results of the ATAC trial of 9399 women indicated that those only on arimidex had better clinical outcomes including vascular events and gynecological problems compared to the tamoxifen group with no differences seen in cognitive outcomes [25,32]. There data suggests that arimidex was the preferred initial treatment by women treated for breast cancer [32]. However, other studies indicate that women on arimidex treatment had greater cognitive decline than tamoxifen treatment in verbal and visual memory (Table 1) [33,34]. At this point no effects have been seen on cholesterol, lipoproteins or apolipoprotein levels in both animal models and clinical studies (Table 1) [35,36]. Although, there are not alternations in lipids levels, the cognitive side effects are of concern with this medication and should be examined with respect to *ApoE* genotype.

#### **3.3. Letrozole**

Letrozole, a potent aromatase interfering with adrenal steroid biosynthesis, has also been assessed as a replacement for tamoxifen or as secondary maintenance treatment after tamoxifen as part of the Breast International Group (BIG 1-98) trial [37,38]. In the BIG 1-98 study, better overall cognitive outcomes were seen in women on letrozole treatment compared to those on a tamoxifen treatment (Table 1) [37,38]. In the tamoxifen only group, increased endometrial cancer and vaginal bleeding where found [38]. Participants given letrozole only did displayed more incidences of skeletal and cardiac events and hypercholesterolemia compared to the tamoxifen group [37]. After letrozole treatment, unfavorable effects have been seen including increased serum total cholesterol, LDL and ApoB with atherogenic ratio risk of total cholesterol/HDL and LDL/HDL levels (Table 1) [39]. Therefore, the better cognitive outcomes may not out-weigh negative effects on lipoprotein levels, particularly in those with or at risk for hypertension and/or diabetes. The potential confounding factor of *ApoE* genotype has not been reported.

#### **3.4. Exemestane**

258 Lipoproteins – Role in Health and Diseases

drugs should be seriously considered.

understand the mechanistic relationship.

examined with respect to *ApoE* genotype.

**3.2. Anatrozole**

**3.3. Letrozole**

[28]. These studies were conducted when women were still in treatment, leaving the question of potential long-term effects. There is a study that examined women who used tamoxifen for <4 years compared to >6 years of exposure. The study found that the current exposure led to greater memory deficits compared to non-users (Table 1) [29]. This suggests that while on therapy, patients may have acute memory impairments and that alternative

In breast cancer survivors undergoing tamoxifen treatment, their total cholesterol, VLDL, high density lipoproteins (HDL) and Apolipoprotein B (ApoB) protein levels have been shown to decrease in both *ApoE4* carriers and non-*ApoE4* carriers (Table 1) [30,31]. Breast cancer patients who are *ApoE4* carriers have higher plasma triglyceride levels and altered ApoA-1/ApoB ratio. Both are risk factors for cardiovascular events, after tamoxifen treatment (Table 1) [30,31]. In non-*ApoE4* carriers, there were lower levels of lipoprotein (a) after treatment, but no effect on triglycerides or the ApoA-1/ApoB ratio (Table 1). This suggests that non-*ApoE4* carriers have a more positive response to tamoxifen with respect to lipid profiles and risk for cardiovascular complications [31]. Considering tamoxifen and *ApoE4* both have a deleterious effect on cognition and tamoxifen has an *ApoE* genotype dependent effect on lipid profiles, suggest further investigations are warranted to

Anatrozole also known as arimidex is an aromatase inhibitor that lowers estrogen levels and is used as a treatment in estrogen positive breast cancer patients post-surgery. Results of the ATAC trial of 9399 women indicated that those only on arimidex had better clinical outcomes including vascular events and gynecological problems compared to the tamoxifen group with no differences seen in cognitive outcomes [25,32]. There data suggests that arimidex was the preferred initial treatment by women treated for breast cancer [32]. However, other studies indicate that women on arimidex treatment had greater cognitive decline than tamoxifen treatment in verbal and visual memory (Table 1) [33,34]. At this point no effects have been seen on cholesterol, lipoproteins or apolipoprotein levels in both animal models and clinical studies (Table 1) [35,36]. Although, there are not alternations in lipids levels, the cognitive side effects are of concern with this medication and should be

Letrozole, a potent aromatase interfering with adrenal steroid biosynthesis, has also been assessed as a replacement for tamoxifen or as secondary maintenance treatment after tamoxifen as part of the Breast International Group (BIG 1-98) trial [37,38]. In the BIG 1-98 study, better overall cognitive outcomes were seen in women on letrozole treatment compared to those on a tamoxifen treatment (Table 1) [37,38]. In the tamoxifen only group, increased endometrial cancer and vaginal bleeding where found [38]. Participants given letrozole only did displayed more incidences of skeletal and cardiac events and There is another alternative for estrogen suppression therapy exemestane, an aromatase inhibitor, general used after tamoxifen is not working in post-menopausal women. Exemestane has been examined as part of the randomized Tamoxifen and Exemestane Adjunctive Multinational (TEAM) trial [24]. The preliminary data from the TEAM trial found that tamoxifen is associated with lower verbal and executive function, while exemestane did not seem to alter cognitive performance levels (Table 1) [24]. Analysis of data from 72 patients as part of the EORTC trail 10958 indicates that treatment with exemestane resulted in reduced triglyceride levels and tamoxifen treatment increased triglyceride levels [40]. All other lipid parameters including HDL, ApoA-1, ApoB or Lip (a) levels at 8, 24 and 48 weeks were unchanged by either treatment [40]. Further studies will be needed to determine if stable cognitive performance and lipid levels are effect of *ApoE* genotype.

#### **3.5. Raloxifene**

Raloxifene, also a SERM and is used as a hormone replacement therapy, has the positive effects of estrogen on the skeletal system and is an antagonist of estrogen in breast or endometrial tissues [41]. Raloxifene treatment also appears to be less detrimental to cognitive function assessed by Modified Mini-Mental State (3MS) compared to tamoxifen [42]. Used to prevent osteoporosis, it has been shown that after three years of treatment raloxifene did not affect overall cognitive scores [43]. The Multiple Outcomes of Raloxifene Evaluation (MORE) study of 7478 women, reported finding that raloxifene treatment lowered the risk of cognitive decline in word list recall test and there was no overall effect on cognitive function (Table 1) [44]. A subset of the National Surgical Adjuvant Breast and Bowel Project (NSABP) Study of Tamoxifen and Raloxifene (STAR), the CoSTAR study for women at high risk for breast cancer did not find any cognitive effects of either drug (Table 1) [45]. Together the evidence supports that raloxifene treatment does not impair cognitive function the way that tamoxifen treatment and in fact may even lower the risk of cognitive decline [34].

With respect to lipid and lipoprotein levels post hoc analysis of 2659 women in the MORE study, found that raloxifene treatment in women with or without high triglycerides lead to reduced cholesterol levels with healthier lipoprotein parameters (Table 1) [46]. Studies in Greek women, found that LDL cholesterol levels were lower in women treated with raloxifene [47]. Raloxifene also appears to raise HDL levels and ApoA-1 while decreasing ApoB protein levels and improving ratios of total cholesterol to lipoproteins (Table 1). After one year of treatment with raloxifene women had reduced fat mass and trunk and central regions along with decreased adiposity in their truck and abdominal regions (Table 1) [48]. Overall, raloxifene treatment improves cholesterol health and alters fat distribution in a positive manner to help prevent obesity, making it a better candidate for overall health compared to tamoxifen. Its effects in relation to *ApoE* genotype have not been reported.

#### **3.6. Estradiol**

In post-menopausal estradiol (also known as 17β-estradiol or oestradiol) treatment is used for estrogen replacement therapy.  Other non-randomized studies in women with surgically induced amenorrhea or those with AD indicate that estrogen replacement treatment may help to improve or minimize cognitive deficits [50]. Even in men those given estradiol performed better on visual memory after treatment (Table 1) [51]. These results are consistent with improved memory in mice given other replacement estrogens treatments [52]. Over half of randomized clinical studies find significant improvements in cognition and attention after estrogen replacement therapy (Table 1) [53]. Estradiol has been shown to increase levels of ApoE in the brain, proposed to be beneficial for neuronal reorganization and repair [7]. In a health study of 3,393 women, results suggest that estrogen replacement reduces the risk of agerelated cognitive decline in non-*ApoE4* women, but not in *ApoE4* carriers (Table 1) [54]. Another study with 181 post-menopausal women, also found the best learning and memory performance after estrogen replacement is seen in non-*ApoE4* carriers [55]. This suggests that knowing *ApoE* genotype may be helpful to assess potential response to estrogen replacement therapy.

estrogen replacement therapy.

Tamoxifen Estrogen receptor

function

function

Anatrozole Aromatase inhibitor

**Treatment Function Effects on**

modulator (SERM) lowers estrogen

lowers estrogen

been reported.

**3.6. Estradiol**

raloxifene [47]. Raloxifene also appears to raise HDL levels and ApoA-1 while decreasing ApoB protein levels and improving ratios of total cholesterol to lipoproteins (Table 1). After one year of treatment with raloxifene women had reduced fat mass and trunk and central regions along with decreased adiposity in their truck and abdominal regions (Table 1) [48]. Overall, raloxifene treatment improves cholesterol health and alters fat distribution in a positive manner to help prevent obesity, making it a better candidate for overall health compared to tamoxifen. Its effects in relation to *ApoE* genotype have not

In post-menopausal estradiol (also known as 17β-estradiol or oestradiol) treatment is used for estrogen replacement therapy.  Other non-randomized studies in women with surgically induced amenorrhea or those with AD indicate that estrogen replacement treatment may help to improve or minimize cognitive deficits [50]. Even in men those given estradiol performed better on visual memory after treatment (Table 1) [51]. These results are consistent with improved memory in mice given other replacement estrogens treatments [52]. Over half of randomized clinical studies find significant improvements in cognition and attention after estrogen replacement therapy (Table 1) [53]. Estradiol has been shown to increase levels of ApoE in the brain, proposed to be beneficial for neuronal reorganization and repair [7]. In a health study of 3,393 women, results suggest that estrogen replacement reduces the risk of agerelated cognitive decline in non-*ApoE4* women, but not in *ApoE4* carriers (Table 1) [54]. Another study with 181 post-menopausal women, also found the best learning and memory performance after estrogen replacement is seen in non-*ApoE4* carriers [55]. This suggests that knowing *ApoE* genotype may be helpful to assess potential response to

**Lipids/Apolipoproteins**

levels.

Decreased total cholesterol, VLDL, HDL and ApoB protein

No effects seen in rodent or

clinical studies.

**Effects on Cognition**

Reduced verbal and visual memory compared to tamoxifen. Deficits

Leads to impairments in visual memory, verbal working memory and visuospatial ability.

**Table 1.** Hormone treatments effects of lipids/apolipoproteins levels and cognition.

#### **3.7. Tibolone**

Tibolone is another drug used in hormonal replacement therapy having estrogenic, progestogenic, and androgenic effects. Long-term treatment does appear to decrease anxiety, improve semantic memory and overall quality of life; however, one study reported that those in treatment did score worse on attention task compared to women not on treatment (Table 1) [56,57]. Tibolone appears to be the most beneficial with respect to reducing total cholesterol, triglyercide, HDL and ApoA-1 levels compared to raloxifene and estradiol (Table 1) [47,58-60]. This suggests that hormone replacement therapy with medications such as tibolone in post-menopausal women are beneficial to cognitive health and lipid profiles.

### **3.8. Cetrorelix**

Cetrorelix an antagonist of hypothalamic luteinizing hormone-releasing hormone (LHRH), is used in treatment of prostate carcinoma, benign prostatic hyperplasia, and ovarian cancer to reduce gonadotrophins and sex steroids [61]. In mice, a study suggests that it is anxiolytic, anti-depressive and able to correct beta-amyloid 25-35 associated memory consolidation impairments (Table 1) [61]. Injection of cetrorelix into *ApoE* deficient mice (*ApoE-/-*) mice suggests that the associated suppression of testosterone leads to increased atherosclerosis despite lower cholesterol levels in the male mice [62]. In female *ApoE-/-* mice, the reduction in testosterone also leads to reduction in estradiol, insulin and HDL levels without effects on atherosclerosis [62]. In a pilot study conducted in men, treatment with cetrorelix resulted in increased ApoA-1, HDL, insulin and leptin consistently (Table 1) [63]. Therefore, when this drug is used within a chemotherapy treatment regiment it is important to carefully monitor lipid levels. Additional studies are needed to examine if *ApoE* genotype has any effect on response and potential long-term cognitive side effects of this drug.

#### **4. Recommendations**