PEX5 translocation into and out of peroxisomes drives matrix protein import.

Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal enzymes are imported from the cytosol by the receptor PEX5, which interacts with a docking complex in the peroxisomal membrane and then returns to the cytosol after monoubiquitination by a membrane-embedded ubiquitin ligase. The mechanism by which PEX5 shuttles between cytosol and peroxisomes and releases cargo inside the lumen is unclear. Here, we use Xenopus egg extract to demonstrate that PEX5 accompanies cargo completely into the lumen, utilizing WxxxF/Y motifs near its N terminus that bind a lumenal domain of the docking complex. PEX5 recycling is initiated by an amphipathic helix that binds to the lumenal side of the ubiquitin ligase. The N terminus then emerges in the cytosol for monoubiquitination. Finally, PEX5 is extracted from the lumen, resulting in the unfolding of the receptor and cargo release. Our results reveal the unique mechanism by which PEX5 ferries proteins into peroxisomes.

Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris.

Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on ß-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.

Defining upstream enhancing and inhibiting sequence patterns for plant peroxisome targeting signal type 1 using large-scale in silico and in vivo analyses.

Peroxisomes are universal eukaryotic organelles essential to plants and animals. Most peroxisomal matrix proteins carry peroxisome targeting signal type 1 (PTS1), a C-terminal tripeptide. Studies from various kingdoms have revealed influences from sequence upstream of the tripeptide on peroxisome targeting, supporting the view that positive charges in the upstream region are the major enhancing elements. However, a systematic approach to better define the upstream elements influencing PTS1 targeting capability is needed. Here, we used protein sequences from 177 plant genomes to perform large-scale and in-depth analysis of the PTS1 domain, which includes the PTS1 tripeptide and upstream sequence elements. We identified and verified 12 low-frequency PTS1 tripeptides and revealed upstream enhancing and inhibiting sequence patterns for peroxisome targeting, which were subsequently validated in vivo. Follow-up analysis revealed that nonpolar and acidic residues have relatively strong enhancing and inhibiting effects, respectively, on peroxisome targeting. However, in contrast to the previous understanding, positive charges alone do not show the anticipated enhancing effect and that both the position and property of the residues within these patterns are important for peroxisome targeting. We further demonstrated that the three residues immediately upstream of the tripeptide are the core influencers, with a 'basic-nonpolar-basic' pattern serving as a strong and universal enhancing pattern for peroxisome targeting. These findings have significantly advanced our knowledge of the PTS1 domain in plants and likely other eukaryotic species as well. The principles and strategies employed in the present study may also be applied to deciphering auxiliary targeting signals for other organelles.

Determining the targeting specificity of the selective peroxisomal targeting factor Pex9.

Accurate and regulated protein targeting is crucial for cellular function and proteostasis. In the yeast Saccharomyces cerevisiae, peroxisomal matrix proteins, which harboring a Peroxisomal Targeting Signal 1 (PTS1), can utilize two paralog targeting factors, Pex5 and Pex9, to target correctly. While both proteins are similar and recognize PTS1 signals, Pex9 targets only a subset of Pex5 cargo proteins. However, what defines this substrate selectivity remains uncovered. Here, we used unbiased screens alongside directed experiments to identify the properties underlying Pex9 targeting specificity. We find that the specificity of Pex9 is largely determined by the hydrophobic nature of the amino acid preceding the PTS1 tripeptide of its cargos. This is explained by structural modeling of the PTS1-binding cavities of the two factors showing differences in their surface hydrophobicity. Our work outlines the mechanism by which targeting specificity is achieved, enabling dynamic rewiring of the peroxisomal proteome in changing metabolic needs.

Multi-targeted trehalose-6-phosphate phosphatase I harbors a novel peroxisomal targeting signal 1 and is essential for flowering and development.

MAIN CONCLUSION: This work reveals information about new peroxisomal targeting signals type 1 and identifies trehalose-6-phosphate phosphatase I as multitargeted and is implicated in plant development, reproduction, and stress response. A putative, non-canonical peroxisomal targeting signal type 1 (PTS1) Pro-Arg-Met > was identified in the extreme C-terminus of trehalose-6-phosphate phosphatase (TPP)I. TPP catalyzes the final step of trehalose synthesis, and the enzyme was previously characterized to be nuclear only (Krasensky et al. in Antioxid Redox Signal 21(9):1289-1304, 2014). Here we show that the TPPI C-terminal decapeptide ending with Pro-Arg-Met > or Pro-Lys-Met > can indeed function as a PTS1. Upon transient expression in two plant expression systems, the free C- or N-terminal end led to the full-length TPPI targeting to peroxisomes and plastids, respectively. The nucleus and nucleolus targeting of the full-length TPPI was observed in both cases. The homozygous T-DNA insertion line of TPPI showed a pleiotropic phenotype including smaller leaves, shorter roots, delayed flowering, hypersensitivity to salt, and a sucrose dependent seedling development. Our results identify novel PTS1s, and TPPI as a protein multi-targeted to peroxisomes, plastids, nucleus, and nucleolus. Altogether our findings implicate an essential role for TPPI in development, reproduction, and cell signaling.

Predicting Peroxisomal Targeting Signals to Elucidate the Peroxisomal Proteome of Mammals.

Peroxisomes harbor a plethora of proteins, but the peroxisomal proteome as the entirety of all peroxisomal proteins is still unknown for mammalian species. Computational algorithms can be used to predict the subcellular localization of proteins based on their amino acid sequence and this method has been amply used to forecast the intracellular fate of individual proteins. However, when applying such algorithms systematically to all proteins of an organism the prediction of its peroxisomal proteome in silico should be possible. Therefore, a reliable detection of peroxisomal targeting signals (PTS ) acting as postal codes for the intracellular distribution of the encoding protein is crucial. Peroxisomal proteins can utilize different routes to reach their destination depending on the type of PTS. Accordingly, independent prediction algorithms have been developed for each type of PTS, but only those for type-1 motifs (PTS1) have so far reached a satisfying predictive performance. This is partially due to the low number of peroxisomal proteins limiting the power of statistical analyses and partially due to specific properties of peroxisomal protein import, which render functional PTS motifs inactive in specific contexts. Moreover, the prediction of the peroxisomal proteome is limited by the high number of proteins encoded in mammalian genomes, which causes numerous false positive predictions even when using reliable algorithms and buries the few yet unidentified peroxisomal proteins. Thus, the application of prediction algorithms to identify all peroxisomal proteins is currently ineffective as stand-alone method, but can display its full potential when combined with other methods.

The peroxisomal import receptor PEX5 functions as a stress sensor, retaining catalase in the cytosol in times of oxidative stress.

Accumulating evidence indicates that peroxisome functioning, catalase localization, and cellular oxidative balance are intimately interconnected. Nevertheless, it remains largely unclear why modest increases in the cellular redox state especially interfere with the subcellular localization of catalase, the most abundant peroxisomal antioxidant enzyme. This study aimed at gaining more insight into this phenomenon. Therefore, we first established a simple and powerful approach to study peroxisomal protein import and protein-protein interactions in living cells in response to changes in redox state. By employing this approach, we confirm and extend previous observations that Cys-11 of human PEX5, the shuttling import receptor for peroxisomal matrix proteins containing a C-terminal peroxisomal targeting signal (PTS1), functions as a redox switch that modulates the protein's activity in response to intracellular oxidative stress. In addition, we show that oxidative stress affects the import of catalase, a non-canonical PTS1-containing protein, more than the import of a reporter protein containing a canonical PTS1. Furthermore, we demonstrate that changes in the local redox state do not affect PEX5-substrate binding and that human PEX5 does not oligomerize in cellulo, not even when the cells are exposed to oxidative stress. Finally, we present evidence that catalase retained in the cytosol can protect against H2O2-mediated redox changes in a manner that peroxisomally targeted catalase does not. Together, these findings lend credit to the idea that inefficient catalase import, when coupled with the role of PEX5 as a redox-regulated import receptor, constitutes a cellular defense mechanism to combat oxidative insults of extra-peroxisomal origin.

Identification of New Fungal Peroxisomal Matrix Proteins and Revision of the PTS1 Consensus.

The peroxisomal targeting signal type 1 (PTS1) is a seemingly simple peptide sequence at the C-terminal end of most peroxisomal matrix proteins. PTS1 can be described as a tripeptide with the consensus motif [S/A/C] [K/R/H] L. However, this description is neither necessary nor sufficient. It does not cover all cases of PTS1 proteins, and some proteins in accordance with this consensus do not target to the peroxisome. In order to find new PTS proteins in yeast and to arrive at a more complete description of the PTS1 consensus motif, we developed a machine learning approach that involves orthologue expansion of the set of known peroxisomal proteins. We performed a genome-wide in silico screen, characterised several PTS1-containing peptides and identified two new peroxisomal matrix proteins, which we named Pxp1 (Yel020c) and Pxp2 (Yjr111c). Based on these in silico and in vivo analyses, we revised the yeast PTS1 consensus which now includes all known PTS1 proteins.

Pex9p is a new yeast peroxisomal import receptor for PTS1-containing proteins.

Peroxisomal proteins carrying a type 1 peroxisomal targeting signal (PTS1) are recognized by the well-conserved cycling import receptor Pex5p. The yeast YMR018W gene encodes a Pex5p paralog and newly identified peroxin that is involved in peroxisomal import of a subset of matrix proteins. The new peroxin was designated Pex9p, and it interacts with the docking protein Pex14p and a subclass of PTS1-containing peroxisomal matrix enzymes. Unlike Pex5p, Pex9p is not expressed in glucose- or ethanol-grown cells, but it is strongly induced by oleate. Under these conditions, Pex9p acts as a cytosolic and membrane-bound peroxisome import receptor for both malate synthase isoenzymes, Mls1p and Mls2p. The inducible Pex9p-dependent import pathway provides a mechanism for the oleate-inducible peroxisomal targeting of malate synthases. The existence of two distinct PTS1 receptors, in addition to two PTS2-dependent import routes, contributes to the adaptive metabolic capacity of peroxisomes in response to environmental changes and underlines the role of peroxisomes as multi-purpose organelles. The identification of different import routes into peroxisomes contributes to the molecular understanding of how regulated protein targeting can alter the function of organelles according to cellular needs.

SKLPT imaging: Efficient in vivo pre-evaluation of genome-editing modules using fluorescent protein with peroxisome targeting signal.

Numerous studies have used genome-editing modules such as CRISPR-Cas9 for site-directed mutagenesis; however, evaluation of the efficiency of these modules remains a time-consuming process. Here, we report the development of SKL-mediated Peroxisome Targeting Imaging (SKLPT imaging), an efficient in vivo pre-evaluation method based on the change in subcellular localization of a fluorescent protein. In this method, frameshifts resulting from successful editing cause the fusion of green fluorescent protein to the peroxisome localization signal Serine-Lysine-Leucine (SKL). Using SKLPT imaging, we pre-evaluated three CRISPR-Cas9 modules in vivo at the single-cell level, and then efficiently mutagenized the liverwort (Marchantia polymorpha) genome using a high-efficiency module.

The first minutes in the life of a peroxisomal matrix protein.

In the field of intracellular protein sorting, peroxisomes are most famous by their capacity to import oligomeric proteins. The data supporting this remarkable property are abundant and, understandably, have inspired a variety of hypothetical models on how newly synthesized (cytosolic) proteins reach the peroxisome matrix. However, there is also accumulating evidence suggesting that many peroxisomal oligomeric proteins actually arrive at the peroxisome still as monomers. In support of this idea, recent data suggest that PEX5, the shuttling receptor for peroxisomal matrix proteins, is also a chaperone/holdase, binding newly synthesized peroxisomal proteins in the cytosol and blocking their oligomerization. Here we review the data behind these two different perspectives and discuss their mechanistic implications on this protein sorting pathway.

Structural biology of the import pathways of peroxisomal matrix proteins.

The peroxisomal proteins (peroxins) that mediate the import of peroxisomal matrix proteins have been identified. Recently, the purification of a functional peroxisomal translocon has been reported. However, the molecular details of the import pathways and the mechanisms by which the cargo is translocated into the lumen of the organelle are still poorly understood. Structural studies have begun to provide insight into molecular mechanisms of peroxisomal import pathways for cargo proteins that harbor peroxisomal targeting signals, PTS1 and PTS2, at their C- and N-termini, respectively. So far structures have been reported for binary or tertiary protein-protein interfaces, and highlight the role of intrinsically disordered regions for these interactions. Here, we provide an overview of the currently available structural biology of peroxisomal import pathways. Current challenges and future perspectives of the structural biology of peroxisomal protein translocation are discussed.

Alternative splicing affects the targeting sequence of peroxisome proteins in Arabidopsis.

KEY MESSAGE: A systematic analysis of the Arabidopsis genome in combination with localization experiments indicates that alternative splicing affects the peroxisomal targeting sequence of at least 71 genes in Arabidopsis. Peroxisomes are ubiquitous eukaryotic cellular organelles that play a key role in diverse metabolic functions. All peroxisome proteins are encoded by nuclear genes and target to peroxisomes mainly through two types of targeting signals: peroxisomal targeting signal type 1 (PTS1) and PTS2. Alternative splicing (AS) is a process occurring in all eukaryotes by which a single pre-mRNA can generate multiple mRNA variants, often encoding proteins with functional differences. However, the effects of AS on the PTS1 or PTS2 and the targeting of the protein were rarely studied, especially in plants. Here, we systematically analyzed the genome of Arabidopsis, and found that the C-terminal targeting sequence PTS1 of 66 genes and the N-terminal targeting sequence PTS2 of 5 genes are affected by AS. Experimental determination of the targeting of selected protein isoforms further demonstrated that AS at both the 5' and 3' region of a gene can affect the inclusion of PTS2 and PTS1, respectively. This work underscores the importance of AS on the global regulation of peroxisome protein targeting.

PEX5, the shuttling import receptor for peroxisomal matrix proteins, is a redox-sensitive protein.

Peroxisome maintenance depends on the import of nuclear-encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C-terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N-terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibroblasts is a redox-sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination-deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed.

Towards understanding peroxisomal phosphoregulation in Arabidopsis thaliana.

MAIN CONCLUSION: This work identifies new protein phosphatases and phosphatase-related proteins targeting peroxisomes, and raises the question of a novel protein import pathway from ER to peroxisomes involving peroxisomal targeting signal type 1 (PTS1) Plant peroxisomes are essential for several processes, for example lipid metabolism, free radical detoxification, development, and stress-related functions. Although research on peroxisomes has been intensified, reversible phosphorylation as a control mechanism in peroxisomes is barely studied. Therefore, it is crucial to identify all peroxisomal proteins involved in phosphoregulation. We here started with protein phosphatases, and searched the Arabidopsis thaliana genome for phosphatase-related proteins with putative peroxisomal targeting signals (PTS). Five potential peroxisomal candidates were detected, from which four were confirmed to target peroxisomes or have a functional PTS. The highly conserved Ser-Ser-Met> was validated for two protein phosphatase 2C (PP2C) family members (POL like phosphatases, PLL2 and PLL3) as a functional peroxisomal targeting signal type 1 (PTS1). Full-length PLL2 and PLL3 fused with a reporter protein targeted peroxisomes in two plant expression systems. A putative protein phosphatase, purple acid phosphatase 7 (PAP7), was found to be dually targeted to ER and peroxisomes and experiments indicated a possible trafficking to peroxisomes via the ER depending on peroxisomal PTS1. In addition, a protein phosphatase 2A regulator (TIP41) was validated to harbor a functional PTS1 (Ser-Lys-Val>), but the full-length protein targeted cytosol and nucleus. Reverse genetics indicated a role for TIP41 in senescence signaling. Mass spectrometry of whole seedlings and isolated peroxisomes was employed, and identified new putative phosphorylated peroxisomal proteins. Previously, only one protein phosphatase, belonging to the phospho-protein phosphatase (PPP) family, was identified as a peroxisomal protein. The present work implies that members of two other main protein phosphatase families, i.e. PP2C and PAP, are also targeting peroxisomes.

Redox-regulated cargo binding and release by the peroxisomal targeting signal receptor, Pex5.

In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1-110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle.

A cargo-centered perspective on the PEX5 receptor-mediated peroxisomal protein import pathway.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by PEX5, the peroxisomal shuttling receptor. The pathway followed by PEX5 during this process is known with reasonable detail. After recognizing cargo proteins in the cytosol, the receptor interacts with the peroxisomal docking/translocation machinery, where it gets inserted; PEX5 is then monoubiquitinated, extracted back to the cytosol and, finally, deubiquitinated. However, despite this information, the exact step of this pathway where cargo proteins are translocated across the organelle membrane is still ill-defined. In this work, we used an in vitro import system to characterize the translocation mechanism of a matrix protein possessing a type 1 targeting signal. Our results suggest that translocation of proteins across the organelle membrane occurs downstream of a reversible docking step and upstream of the first cytosolic ATP-dependent step (i.e. before ubiquitination of PEX5), concomitantly with the insertion of the receptor into the docking/translocation machinery.

Identification of the kinesin KifC3 as a new player for positioning of peroxisomes and other organelles in mammalian cells.

The attachment of organelles to the cytoskeleton and directed organelle transport is essential for cellular morphology and function. In contrast to other cell organelles like the endoplasmic reticulum or the Golgi apparatus, peroxisomes are evenly distributed in the cytoplasm, which is achieved by binding of peroxisomes to microtubules and their bidirectional transport by the microtubule motor proteins kinesin-1 (Kif5) and cytoplasmic dynein. KifC3, belonging to the group of C-terminal kinesins, has been identified to interact with the human peroxin PEX1 in a yeast two-hybrid screen. We investigated the potential involvement of KifC3 in peroxisomal transport. Interaction of KifC3 and the AAA-protein (ATPase associated with various cellular activities) PEX1 was confirmed by in vivo colocalization and by coimmunoprecipitation from cell lysates. Furthermore, knockdown of KifC3 using RNAi resulted in an increase of cells with perinuclear-clustered peroxisomes, indicating enhanced minus-end directed motility of peroxisomes. The occurrence of this peroxisomal phenotype was cell cycle phase independent, while microtubules were essential for phenotype formation. We conclude that KifC3 may play a regulatory role in minus-end directed peroxisomal transport for example by blocking the motor function of dynein at peroxisomes. Knockdown of KifC3 would then lead to increased minus-end directed peroxisomal transport and cause the observed peroxisomal clustering at the microtubule-organizing center.

Identification of Auxiliary Organellar Targeting Signals for Plant Peroxisomes Using Bioinformatic Analysis of Large Protein Sequence Datasets Followed by Experimental Validation.

Eukaryotic cells are compartmentalized by membrane-bounded organelles to ensure that specific biochemical reactions and cellular functions occur in a spatially restricted manner. The subcellular localization of proteins is largely determined by their intrinsic targeting signals, which are mainly constituted by short peptides. A complete organelle targeting signal may contain a core signal (CoreS) as well as auxiliary signals (AuxiS). However, the AuxiS is often not as well characterized as the CoreS. Peroxisomes house many key steps in photorespiration, besides other crucial functions in plants. Peroxisome targeting signal type 1 (PTS1), which is carried by most peroxisome matrix proteins, was initially recognized as a C-terminal tripeptide with a "canonical" consensus of [S/A]-[K/R]-[L/M]. Many studies have shown the existence of auxiliary targeting signals upstream of PTS1, but systematic characterizations are lacking. Here, we designed an analytical strategy to characterize the auxiliary targeting signals for plant peroxisomes using large datasets and statistics followed by experimental validations. This method may also be applied to deciphering the auxiliary targeting signals for other organelles, whose organellar targeting depends on a core peptide with assistance from a nearby auxiliary signal.

Assay of Reactive Oxygen/Nitrogen Species (ROS/RNS) in Arabidopsis Peroxisomes Through Fluorescent Protein Containing a Type 1 Peroxisomal Targeting Signal (PTS1).

Plant peroxisomes have an active nitro-oxidative metabolism. However, the assay of reactive oxygen and nitrogen species (ROS/RNS) could be a challenge since the purification of peroxisomes is technically a high time-consuming approach that needs to be optimized for each tissue/organ (root, leaf, fruit) and plant species. Arabidopsis thaliana, as a model plant for biochemical and molecular studies, has become a useful tool to study the basic metabolism, including also that of ROS/RNS. The combination of specific fluorescent probes with Arabidopsis plants expressing a fluorescent protein containing a type 1 peroxisomal targeting signal (PTS1) is a powerful tool to address the profile of ROS/RNS in peroxisomes by confocal laser scanning microscope (CLSM). This chapter provides a detailed description to detect the content and distribution of ROS and RNS in Arabidopsis peroxisomes, together with a critical analysis of their potentialities and limitations, since these approaches require appropriate controls to corroborate the obtained data.