A nascent polypeptide chain achieves its native structure with the help of folding machinery present inside the cells that includes molecular chaperones and other associated proteins. However, many of these proteins are not able to fold properly and are needed to be cleared from the cell by the cellular protein quality control system that includes mainly ubiquitin proteasome system (UPS) and autophagy. These two systems either work independently or in collaboration with each other (Goldberg, 2003; Korolchuk et al., 2010). Molecular chaperones are the supervisors of newly synthesized polypeptides in the crowded cellular milieu. The guidance of chaperones assists immature polypeptides to acquire a functional macromolecular structure and move towards appropriate cellular localization to perform pre-defined functions (Ellis and Hemmingsen, 1989; Hartl, 1996). Two well-studied chaperones are heat shock proteins, Hsp70 and Hsp90. Heat shock proteins function as stress-induced proteins to monitor changes in the molecular environment of the cell (Nollen and Morimoto, 2002). The chaperones not only assist new proteins in achieving the functional activity by proper folding, but also play crucial roles in removal of aberrant proteins, either by UPS or by autophagy pathway (Hartl et al., 2011).

Chaperones participate in protein quality control processes of the cell by forming complexes with E3 ubiquitin ligases, ubiquitin molecules, various accessory co-chaperones (Hsp40, BAG-1, BAG-3, etc.) and other proteins to tag misfolded or improperly folded proteins for their removal from the cells (Edkins, 2015). UPS involves ubiquitin-dependent degradation of various misfolded proteins (Ciechanover, 1994; Hochstrasser, 1996). UPS consists of three enzymes E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases. Other than these enzymes a small 76 amino acid long ubiquitin protein, and 26S proteasome, the proteolytic machinery of the cell with different types of protease activities, are other major components of the UPS (Hershko, 1998). As shown in Figure 1A, ubiquitination of a particular protein, with the help of successive activities of E1, E2 and E3 enzymes generates a degradation signal onto a protein, for its translocation to the 26S proteasome interior, where the catalytic sites of proteasome cleave polypeptide into smaller oligopeptides. The specificity in the functioning of UPS comes from E3 ubiquitin ligase enzymes, which recognize misfolded proteins and continue their elimination through catalytic activities of 26S proteasome (Pickart, 2001).

Autophagy is another widely explored pathway that helps in clearance of the cytoplasmic bulk of misfolded proteins, by using lysosomal proteolysis (Seglen and Bohley, 1992). The cytoplasmic bulk includes aggregates, oligomers, protein complexes, misfolded soluble proteins and sometimes cellular organelles. Autophagic degradation begins with the formation of the double-layered membrane, the phagophore, that is designated as autophagosome when wrapped around autophagic substrates. The tag of autophagosomal markers, like p62 helps in the transportation of autophagosome to lysosome for final degradation by lysosomal hydrolases (Korolchuk et al., 2010). Chaperone-assisted selective autophagy (CASA) and chaperone-mediated autophagy (CMA) are examples of two types of autophagy, where chaperones are involved in protein degradation, using this lysosomal pathway (Kaushik and Cuervo, 2012). Chaperone-assisted proteasomal degradation (CAP) elucidates another mechanism where chaperones steer misfolded proteins towards the ubiquitin-mediated proteasomal degradation (Kettern et al., 2010). Chaperones are also responsible for the crosstalk between these two pathways of protein degradation (Park and Cuervo, 2013).

C-terminus of Hsc70-interacting protein (CHIP) is profoundly involved in most of the above-described quality control pathways (Kettern et al., 2010). Previously, we have shown various cellular strategies embraced by E3 ubiquitin ligases in the removal of aberrant cellular proteins. Few E3 ubiquitin ligases like CHIP, cullin5, E6-AP, etc. function in association with various chaperones like Hsp70, Hsp90, etc. to maintain the quality of cellular proteins (Chhangani et al., 2013). CHIP is one of the most extensively studied E3 ubiquitin ligase associated with chaperones (Murata et al., 2001). E3 ubiquitin ligase activity of CHIP is because of its evolutionarily conserved U-box domain, which is a modified form of the Really Interesting New Gene (RING) domain which also contains a metal chelating region (Hatakeyama et al., 2001; Jiang et al., 2001). Cytoplasmic 35 kDa protein CHIP was firstly recognized as a chaperone interacting protein because of its tetratricopeptide repeat containing (TPR) domain, which is similar to Hsp70-Hsp90 organizing protein (Hop) and Hsc70 interacting protein (Hip; Ballinger et al., 1999). Negative regulation of a substrate binding cycle of Hsc70-Hsp70 by CHIP reveals its dual role as co-chaperone as well as E3 ubiquitin ligase for protein quality control in cells (Connell et al., 2001). CHIP has also been shown as a connecting link between chaperones and proteasome system (McDonough and Patterson, 2003).

The involvement of CHIP in various physiological functions defines its worth in the maintenance of cellular homeostasis; earlier findings have described its roles in a number of pathways and diseases (Paul and Ghosh, 2015). However, its exceptional significance was found in an array of neurodegenerative disorders with greater extent (Dickey et al., 2007). Another potential reason of extensive research on CHIP is its association with a wide range of diseases, such as cardiac disorders, muscular disorders and different types of cancers (Adachi et al., 2007; Willis et al., 2008; Cao et al., 2016). In this review, we first describe structural aspects and involvement of CHIP in various quality control pathways, and then we elaborate its liaison in various diseases, with a special focus on neurodegeneration and associated disorders. Loss of neurons is more crucial than the loss of any other cell in the body, as the possibility of recovery or replacement is negligible in this case (Currais et al., 2009). Understanding and analyzing available data about molecules like CHIP, that are directly linked to multiple neurodegenerative diseases, will provide hope for the development of preventive measures for these pathologies. We, here, try to compile a detailed and descriptive analysis of CHIP and its association with neurodegenerative disorders, to focus on its wide scope in future drug discovery.

The CHIP protein in humans is encoded by STIP1 Homology and U-Box Containing gene 1 (STUB1), with the cytogenetic band at location p13.3 on chromosome number 16. The pre-mRNA consists of seven exons and six introns within 2760 base pairs of the CHIP/STUB1 gene. As represented in Figure 1B, human CHIP protein is of 303 amino acids in length (Ballinger et al., 1999; Shi et al., 2013). The first identification of CHIP antigen was done in chronic lymphocytic leukemia patients, using serologic identification by recombinant expression cloning (SEREX) method (Krackhardt et al., 2002). The two isoforms produced by alternate splicing are of 303 and 231 amino acid long sequences having NCBI accession: NP_005852.2 and NP_001280126.1 respectively, but no experimental confirmation for the second isoform is available.

Till date, more than 600 E3 ubiquitin ligases of different domains, properties and activities have been identified, and search is still on (Li W. et al., 2008). CHIP is important because of its functional relevance in overall working and regulation of the cell proteins. CHIP orchestrates regulation of cellular proteins from folding to degradation by various mechanisms, including a coordinated function with molecular chaperones to refold the unfolded protein species, as well as a well-directed degradation of those, which are beyond the refolding range, via proteasome or autophagy. So, to accomplish such a wide range of responsibilities, it should be a multifaceted molecule (Connell et al., 2001). To perform this task, CHIP has evolved various ways to ubiquitinate its substrates by interaction with different E2 conjugating enzymes that form a variety of signals, according to which it decides the fate of substrates. E2s are the family of enzymes, having highly conserved ubiquitin conjugating domain. These enzymes, in combination with E3s, play an important role in determining the fate of substrate proteins, by selecting a lysine residue, on which ubiquitin moiety must be added. NMR studies provide evidence that E2 enzymes require a well conserved S-P-A motif in loop seven, which is found in many E2 enzymes, to recognize and bind with CHIP. The specificity that determines the binding of UbcH5 and Ubc13-Uev1a, the E2 ubiquitin-conjugating enzymes, with CHIP E3 ubiquitin ligase is their S-P-A motif. It also interacts with Ube2e2, the class III E2 enzyme because of this conserved S-P-A motif, providing the significance of this motif in such kind of interactions between U-box containing E3 ligases and associated E2 enzymes (Xu et al., 2008).

The mechanism of polyubiquitination by binding of Ubc13-Uev1a and UbcH5 with CHIP differ from each other as Ubc13-Uev1a association causes Lys⁶³-linked polyubiquitination, whereas ubiquitination with UbcH5 does not have any specificity of a lysine residue and the polyubiquitin chain remains linked to CHIP (Windheim et al., 2008). It has been shown in another finding that interaction with UbcH5 causes Lys⁴⁸-linked ubiquitination and it leads to degradation of proteins (Murata et al., 2003; Schulman and Chen, 2005). An in vitro study has shown that CHIP, with the aid of UbcH5 and UbcH6, can efficiently ubiquitinate interferon regulatory factor 1 (IRF1), giving an idea of its modulation, under stress conditions (Narayan et al., 2011). Similarly, another study based on CHIP U-box domain showed that eight E2’s (Ube2D1, Ube2D2, Ube2D3, Ube2E1, Ube2E2, Ube2E3, Ube2N and Ube2W) can specifically interact with this domain (Soss et al., 2011). As evident from the above studies, CHIP interacts and cooperates with different E2’s. However, the basis of interaction towards a particular E2, thereby, producing different ubiquitin signals, is highly complex and still much work is needed to be done to understand this mechanism.

CHIP was first identified as a cytoplasmic protein, but in later years, its functional role in the nucleus was also well established (Ballinger et al., 1999). The localization studies of CHIP with cystic-fibrosis transmembrane-conductance regulator (CFTR) proposed its colocalization at the endoplasmic reticulum membrane, to degrade it by the proteasome (Meacham et al., 2001). The role of CHIP in the cytoplasmic quality control system, later confirms its localization in the cytoplasm (Dai et al., 2005). It was found that CHIP is partly localized in the nucleus also, to perform functions like activation of heat shock factor 1 (HSF1; Dai et al., 2003). The cytoplasmic CHIP does not only take care of cytoplasmic proteins, but is also involved in the ubiquitination and degradation of mitochondrial proteins like leucine-rich repeat kinase 2 (LRRK2), a mutation in which has largely been implicated in the onset of many cases of familial Parkinson’s disease (Ding and Goldberg, 2009). CHIP also ubiquitinates and degrades substrates like microtubule-associated tau and β-amyloid precursor protein (β-APP), which is localized in the Golgi complex (Kumar et al., 2007; Wang and Mandelkow, 2012). Hence, enormous data presented by various studies showed that function of CHIP is not confined to any special cellular location, but, it plays significant roles in the regulation of a variety of substrates, present in different cellular locations and compartments. In Figure 3, we depict the subcellular distribution of CHIP with few of its substrate proteins to evince its cellular localization and functional potency. As mentioned earlier, CHIP is involved in both pathways of protein quality control: UPS and autophagy. The subsections below describe and categorize substrates of CHIP, in accordance with their degradative mechanisms. Most of its substrates have found to be a proteasomal target, whereas some of them are degraded through the lysosomal pathway. Interestingly, few of them viz. inositol-requiring enzyme 1 (IRE1) are only modified functionally, instead of being degraded by any of these degradation pathways (Zhu et al., 2014).

In the above section, we have described the role of CHIP in quality control pathways, to maintain healthy cellular proteome. It is evident from earlier research works, done by different groups that most of the neurodegenerative diseases start developing with few common pathological hallmarks (Jellinger, 2001). Extra- or intracellular stresses and simultaneously, insufficient functioning of various arms of cellular protein quality control machinery may lead to structural modifications of some class of cellular proteins, which may form misfolded oligomers. In many cases, they start accumulating and forming inclusion bodies by entangling with other functional cellular proteins, as evident from microscopic analyses of different diseased brain tissues (Irvine et al., 2008). Such kinds of toxic proteinaceous species may lead to an array of neurological disorders e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS) and Lafora disease, etc. (Dickey et al., 2007).

An interesting study in model organisms for identification of common genetic modifiers of neurodegenerative diseases has also shown the importance of CHIP (Chen et al., 2012). The gene expression analysis data of SAMP8 mouse model of Alzheimer’s disease by cDNA microarray technique, form a protein network where CHIP represents an important node, which signifies its influence on the disease (Cheng et al., 2013). In Figure 5, we summarized the roles of CHIP in different neurodegenerative disorders, where we have shown its association with the misfolded proteins. Here, in this section of the review, we are focusing on various roles attributed to E3 ubiquitin ligase like activities of CHIP, in various neurodegenerative diseases.

CHIP is an E3 ubiquitin ligase that has the capability to regulate its substrate proteins, right from their folding, and up to the stages of its degradation inside cells. It associates with different chaperone complexes to monitor the folding processes, as well as different protein degradation pathways, such as proteasome and autophagy. It presents a direct connecting link between these different arms of the protein quality control system: molecular chaperones, UPS, and autophagy, as described in the above sections. Apart from playing crucial roles in protein structural modifications by its co-chaperone activity, CHIP also takes part in the development of organisms. We, here, try to emphasize few crucial roles of this E3 ubiquitin ligase in development processes. CHIP substrate Runx2 is involved in regulation of osteoblast differentiation. CHIP co-chaperone domain TPR and C-terminus of Runx2 interacts with each other and results in ubiquitination and degradation of Runx2, which suppresses differentiation of preosteoblasts into osteoblasts. It could probably be due to the repressed transcriptional activity of Runx2. On the other hand, this inhibition of Runx2 by CHIP results in increased rate of adipogenesis (Li X. et al., 2008).

Another study showed the negative effect of the CHIP knockout on total body weight of mice. The study points out its role in the overall aging process of the organism. CHIP knockout resulted in increased rate of atrophy, which was measured by decreased size of the thymus, testis and skeletal muscles and the cardiac hypertrophy also signifies the increased rate of aging. Histological considerations were also taken into account, apart from these anatomical studies. It was found that CHIP knockout has a universal effect on the dermis, resulting in thinner dermis layers as compared to control, as well as the subcutaneous deposits of fat, which was also reduced significantly. Bone mineral density was also decreased with symptoms of kyphosis (Min et al., 2008). CHIP has also been found to be involved in early neuronal development stages. The study conducted by Yang et al. (2013) shows that CHIP regulates the level of katanin-p60 protein, which is required for the proper axonal growth in early developmental stages. CHIP antagonizes the effects of a deubiquitylating enzyme, USP47, by ubiquitinating and destabilizing katanin-p60, and hence presents an important role in axonal growth (Yang et al., 2013). Another study showed abnormal motor disturbances with altered phenotypic and behavioral functions in CHIP heterozygous mice (McLaughlin et al., 2012). CHIP may also have some indirect regulatory roles over various neurodevelopmental processes through its large number of substrates, where it regulates various proteins which are directly involved in brain development processes. For example, one of its substrates, PTEN is a key signaling molecule that have direct role in various developmental processes like neuronal differentiation and neurite growth (Li et al., 2003; Hsia et al., 2014). The cellular level of PTEN is largely regulated by its proteasomal degradation, governed by various E3 ubiquitin ligases, including CHIP (Ahmed et al., 2012). Cytoskeleton of large neurons are majorly built by cytoskeletal proteins, neurofilaments. Evidence shows that CHIP has direct involvement in proteasomal degradation of neurofilaments medium subunit of 95 kDa, which are highly damaged and decreased in the less understood mechanism of 2, 5-hexanedione mediated neuropathology. Therefore, involvement of CHIP-mediated proteasomal degradation presents a regulatory mechanism to maintain the levels of neurofilament proteins (Wang Q. et al., 2011; Dale and Garcia, 2012). Although these studies indicate possibilities of CHIP involvement in various developmental processes; a lack of function of which may result in severe developmental abnormalities. These reports give us some clues to further investigate its roles in more details.

Involvement of CHIP in the regulation of aging processes is also very crucial and yet to be established completely. Apart from activation of HSF1, CHIP also affects the activity and stability of the sirtuin family of proteins, which importantly regulate DNA repair mechanisms and maintain cellular homeostasis, by providing required genomic integrity. It was observed that in mouse embryonic fibroblast and human embryonic kidney cells, CHIP knockdown causes post-translational decay of Sirtuin (SirT6) protein levels in the cells. Ubiquitination of SirT6 at K170 position by CHIP E3 ubiquitin ligase stabilizes the protein, which is rather degraded by some unknown E3 ubiquitin ligase. Such kind of positive regulation of SirT6 by CHIP provides a novel example where these two arms of DNA and protein quality maintenance systems cooperatively regulate homeostasis and aging processes (Ronnebaum et al., 2013). Exome sequencing analysis in a family affected with ataxia, found the loss of function mutation in CHIP resulting in hypogonadism and ataxia like symptoms. The reported Thr346Met mutation resulted in Gordon Holmes Syndrome and other accelerated aging related changes which may probably be a result of protein instability or impaired substrate binding (Heimdal et al., 2014; Shi et al., 2014). Therefore, apart from development, aging is another major area of research, where detailed reports stating direct roles of CHIP are still missing, which gives us an opportunity to dig out further, the possibilities of CHIP being targeted as a candidate for anti-aging strategies.

Apart from the above described roles of CHIP in neurodegeneration, development and aging, strong evidence has indicated the importance of CHIP in diseases, such as cancer, cardiac, muscular disorders, etc. A study in past has elucidated the potential of CHIP as a biomarker and therapeutic target in various human cancers (Sun et al., 2014). The strategies have been adapted to target E1 enzymes, chaperones, histone deacetylases, proteasomal subunits and E3 ubiquitin ligases, like mouse double minute 2 homolog (MDM2), anaphase-promoting complex (APC), etc., to treat various types of cancers with varying success (Eldridge and O’brien, 2010; Shen et al., 2013; Weathington and Mallampalli, 2014). As in renal cell carcinoma and gastric cancer, decreased CHIP expression has shown potential to serve as a potent biomarker of angiogenesis (Wang et al., 2013b; Sun et al., 2014). Similarly, in esophageal squamous cell carcinoma patients, elevated CHIP expression levels in metastatic lymph node have shown a high prognostic value (Wen et al., 2013). CHIP’s ability to downregulate ErbB2 makes it a potential indicator for breast cancer patients’ survival (Jan et al., 2011). Besides being extensively studied for prognostic ability, CHIP has also been induced with the help of certain small molecules.

Trehalose, a disaccharide, improves the deteriorating changes taking place in patients with ataxia due to a genetic mutation in STUB1/CHIP. Trehalose, apart from activation of autophagy, also increases the levels of CHIP and Hsp70 (Casarejos et al., 2014). Another molecule quercetin, a flavonoid present in fruits and vegetables, is a chemopreventive agent in cancer; shows its beneficiary effects by down regulating ErbB2 and upregulating its E3 ubiquitin ligase CHIP (Jeong et al., 2008). Another interesting example of using small natural molecule is gambogic acid, a xanthonoid, obtained from tree Garcinia hanburyi that inhibits the interaction of Hsp90 and mutant p53 to make it available for CHIP-mediated proteasomal degradation in cancer cells (Wang J. et al., 2011). Similarly, curcumin has been shown to induce CHIP-ErbB2 interaction, thereby reducing ErbB2 levels, a strategy shown to have an enormous potential in controlling metastasis (Jung et al., 2007). Besides these, a recent study also showed a protective effect of CHIP against ER stress-mediated cell death in the central nervous system, by preventing upregulation of p53 pathway and regulating unfolded protein response pathways (Cabral Miranda et al., 2015). Also, upregulating CHIP activity with Hsp90 inhibitors like 17-N-allylamino-17-demethoxygeldanamycin, for anticancer therapy could be an effective strategy (Paul et al., 2013).

Deubiquitinases (DUBs) are special class of proteases which can release ubiquitin from substrate proteins by breaking the peptide bonds in between substrate and ubiquitin molecules. Regulating DUBs activities is assumed to have some possible therapeutic benefits. Ataxin-3, one of the most studied DUB, found to have a direct interaction with CHIP along with an E2 enzyme Ube2W in degradation of its substrates. An important mechanism of regulation of DUB ataxin-3 by its ubiquitination is also present inside the cells. CHIP has shown to have the capacity to ubiquitinate ataxin-3 and in turn may have a regulatory effect on this neuroprotective DUB enzyme (Scaglione et al., 2011; Tsou et al., 2013). A different therapeutic strategy was proposed to enhance the efficacy of anticancer drugs, through CHIP, by modulating CYP3A4 functioning (Peer et al., 2011). CHIP also has a pivotal role in cardiac disorders, where it provides cardioprotection by degrading inducible cAMP early repressor in complex with extracellular stress-regulated kinase 5 (Woo et al., 2010). All the above described roles of CHIP, in various disorders elucidate its significance for future research to develop advanced strategies to control these pathological disorders. Although, CHIP is a potential molecule for effectively targeting multiple cellular targets involved in several diseases, still success in devising any notable strategy to treat any of these diseases is not reported so far. So, a precise focus on some key questions, which are yet to be answered, must be given and more conceptual understanding of its functioning inside the cell should be developed.

Many years of research has established CHIP, as a unique molecule, which has a large number of interacting partners. An interaction network of such a wide variety of substrates enables this E3 ligase enzyme to take part, directly or indirectly, in an array of cellular functions including metabolism, signaling, proliferation and apoptosis. The structure of CHIP, consisting of both TPR and U-box domain, is also a crucial factor in deciding its functional capabilities. Performing the roles of co-chaperone and E3 ligase simultaneously needs enormous molecular capabilities, and its advantageous structure provides it an extra edge in performing these dual functions. It is still a matter of investigation how exactly these decisions are being made by CHIP, inside the cells. A major question yet to be answered is, how CHIP, in combination with other chaperones recognizes the abnormal and to be degraded substrates inside the cytoplasm. Further work in this direction will indeed aid in understanding the possible ways of identifying cellular proteins on the basis of their structural and physiological features. Several studies have been done to explore the methods of discriminating the abnormal proteins from the healthy ones, whether they are to be modified, modulated or degraded. But, a clear answer is still far from our current understanding. Major focus of research over CHIP has been given to its roles in signaling, apoptosis and cell proliferation. But, a line of evidence clearly indicates that this molecule has equally important contribution in maintaining the cellular homeostasis and providing protection from various stresses, which are major factors causing protein aggregation and related neurodegenerative diseases. Few possible developmental and aging-related roles of CHIP has also been observed; still a lot of research is needed to be done to understand the physiological importance of CHIP in neurodevelopmental and aging processes.

Maintaining a healthy proteome is a prerequisite of a living cell. To achieve this, cells are evolved with chaperone machinery and quality control pathways that maintain proteins into proper conformation and protect cells from toxicity generated in various pathological conditions (Yerbury et al., 2016). CHIP represents a protein which links all these pathways and machinery together. It not only helps chaperones to fold proteins, but also directs misfolded proteinaceous species for their proteasomal or lysosomal degradation. Interestingly, CHIP also regulates chaperones by ubiquitination and to more surprise, it also has self-ubiquitination property, which makes it capable of regulating its own turnover (Murata et al., 2001). Despite the potential that CHIP and other E3 ubiquitin ligases have, as therapeutic agents, more data is required, and further work is needed to be done, to understand the extent of E3 ubiquitin ligases in controlling various cellular processes. Chaperone-independent role of CHIP is still needed to be fully established, which can unveil its various other properties. How to achieve specificity, in the case of CHIP, is another criterion that should be worked upon, if this multifaceted ligase has to be used for therapeutic benefits. Being a central molecule in various processes, merely overexpressing it will also affect other pathways and substrates that could prove this strategy detrimental in place of being beneficial. So, besides looking for pathways and substrates, properties and strategies, that could increase specificity for intended substrate will undoubtedly open a new area for treating diseases that are difficult to handle, with our current treatment strategies.

Although, enough reports have been made to establish protective roles of E3 ubiquitin ligases in various neurodegenerative diseases, still no notable success has been registered so far to treat these life-threatening neuronal disorders. In one of our previous review articles, we have selected few such E3 ligases which have shown cellular protection against these pathological conditions (Upadhyay et al., 2015). CHIP, with an array of protein quality control functions, including regulation of chaperones and autophagic pathways, posits it as one prominent candidate, which has a huge potential to be targeted in the future therapeutic models of neurodegenerative diseases. The ability of CHIP to interact with numerous proteins and participation in multiple cellular pathways makes it one of the most interesting E3 ubiquitin ligase. CHIP, with described functions, bestows to be a possible future therapeutic target which could be exploited by the drug industry in the future. With such a significant contribution in cellular homeostasis, CHIP needs further attention of the scientific community to think and devise strategies to regulate multiple pathways of cellular importance by using this molecule. Focus on specific details and elaborated knowledge of underlying mechanisms will allow us to use CHIP as a therapeutic molecule in future.

VJ, AU, AA and RM, executed complete drawing of figures, AK provided critical inputs. AM formulated the entire concept of manuscript and designed the initial draft of figures. All authors reviewed the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.