Effects of Argentilactone on the Transcriptional Profile, Cell Wall and Oxidative Stress of Paracoccidioides spp.

Paracoccidioides spp., a dimorphic pathogenic fungus, is the etiologic agent of paracoccidioidomycosis (PCM). PCM is an endemic disease that affects at least 10 million people in Latin America, causing severe public health problems. The drugs used against pathogenic fungi have various side effects and limited efficacy; therefore, there is an inevitable and urgent medical need for the development of new antifungal drugs. In the present study, we evaluated the transcriptional profile of Paracoccidioides lutzii exposed to argentilactone, a constituent of the essential oil of Hyptis ovalifolia. A total of 1,058 genes were identified, of which 208 were up-regulated and 850 were down-regulated. Cell rescue, defense and virulence, with a total of 26 genes, was a functional category with a large number of genes induced, including heat shock protein 90 (hsp90), cytochrome c peroxidase (ccp), the hemoglobin ligand RBT5 (rbt5) and superoxide dismutase (sod). Quantitative real-time PCR revealed an increase in the expression level of all of those genes. An enzymatic assay showed a significant increase in SOD activity. The reduced growth of Pbhsp90-aRNA, Pbccp-aRNA, Pbsod-aRNA and Pbrbt5-aRNA isolates in the presence of argentilactone indicates the importance of these genes in the response of Paracoccidioides spp. to argentilactone. The response of the P. lutzii cell wall to argentilactone treatment was also evaluated. The results showed that argentilactone caused a decrease in the levels of polymers in the cell wall. These results suggest that argentilactone is a potential candidate for antifungal therapy.

Data Availability Statement: All relevant data are within the paper and its Supporting Information files.

Introduction
The genus Paracoccidioides, which comprises the species lutzii and brasiliensis, is the etiological agent of paracoccidioidomycosis (PCM), an important systemic mycosis in Latin America. The inhalation of mycelia fragments, infectious forms of the fungus, is the common route of infection that primarily affects the lungs [1]. PCM has been reported to affect individuals from northern Argentina to southern Mexico, with prevalence in Brazil, Colombia, Venezuela and Argentina. The most cases of PCM occur in men, rural workers and individuals between 30-50 years of age, although it affects individuals at any age [2]. In Brazil, PCM is responsible for over 50% of deaths caused by fungal infections [3].
Fungal infections are a serious threat to public health due to their association with high rates of morbidity and mortality [4]. Despite the existence of potent antifungal agents, the development of antifungal resistance by the fungal species, as well as cytotoxicity and collateral effects, has limited the use of current antifungals [5]. In addition, PCM treatment is a slow process that extends over months or years depending on the severity of the disease and the site of injury [6]. Given these facts, it is important to search for and identify novel antifungals.
In our previous work, we have investigated the antifungal potential of argentilactone and its semi-synthetic derivatives on P. lutzii [19]. Argentilactone and the tetrahydro derivative inhibited native and recombinant isocitrate lyase from P. lutzii in the presence of glucose and acetate. Additionally, argentilactone and the tetrahydro derivative exhibited inhibitory activity against P. lutzii yeast cells and dose-dependently interfered with the dimorphic transition from the mycelium to the yeast phase. Argentilactone interfered with the viability of Paracoccidioides spp., but was not toxic to MRC5 cells at the IC 50  showed that argentilactone and reduced argentilactone bound to the catalytic site of PbICL, and the amino acids involved in their binding were identified. The data obtained indicate that argentilactone is a potential candidate for antifungal therapy.
In this study, we investigated the transcriptional profile of P. lutzii yeast cells grown in the presence of argentilactone using the Illumina/Hiseq™2000 platform (Illumina, San Diego, CA, USA). A total of 1,058 genes were identified, of which 208 were up-regulated and 850 were down-regulated in response to argentilactone treatment. The genes identified were classified by the biological function of the encoded protein products. The main categories identified in the up-regulated genes were metabolism, cell rescue, defense and virulence, energy and cell cycle and DNA processing. The down-regulated gene categories were related to metabolism, transcription, protein fate and cell cycling and DNA processing.
The viability of P. lutzii cells grown in the absence or presence of 9 μg/mL argentilactone was determined using the trypan blue method [22], in which viable and non-viable cells are counted in a Neubauer chamber. Except for the transcriptional profiling of P. lutzii yeast cells, all experiments were done using yeast cells from three different seedings from different days.

Extraction and quantification of RNA
All procedures for extraction and manipulation of total RNA were performed in RNAse-free conditions. Total RNA from Paracoccidioides spp. yeast cells not treated or treated with 9 μg/ mL argentilactone for 6 h at 36°C in MMcM liquid medium was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the supplier's instructions. Each experiment (not treated and treated) was performed in triplicate and pooled. The mRNA was purified using the GenElute mRNA kit (Sigma Aldrich, St. Louis, MO, USA). Total RNA was quantified on a NanoDrop 8000 Spectrophotometer and stored at -80°C. Total RNA integrity was visualized using an agarose gel. the P. lutzii genome (http://www.broadinstitute.org/annotation/genome/paracoccidioides_ brasiliensis/Multiome.html) using the Bowtie 2 tool. Mapped read data were analyzed by the DEGseq package. Each read was allowed to align to just one site of the genome, and the reads were counted. The default parameters were used to perform the alignment. The number of mismatches allowed in seed alignment (-N) was 0, and the length of each seed (-L) was 20. The fold change selection method was used for differentially expressed gene selection using Fisher's exact test, and a p-value of 0.001 was considered to select the genes. From the selected genes, a 1.5-fold change cut-off was considered. Genes with log 2 (fold change) higher than 0.58 or less than -0.58 were selected and classified as up-and down-regulated genes, respectively. Genes' identifications and annotations were determined from the P. lutzii genome database (http:// www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html). The biological processes were obtained using the Pedant on MIPS (http://pedant.helmholtzmuenchen.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_r48325_Par_brasi_ Pb01), which provides a tool to browse and search the functional categories (FunCat) of proteins. Additionally, hypothetical proteins were annotated using the Blast program (https:// blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_ LOC=blasthome).

Gene expression analysis by quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. The RNA was reverse transcribed using the high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA). The cDNA was quantified by qRT-PCR using a SYBR green PCR master mix (Applied Biosystems Step One Plus PCR System). α-tubulin was used as endogenous control for data normalization, and its amplification was presented as relative expression in comparison to that of the experimental samples, whose value was set to 1. Data were expressed as the mean ± standard deviation of the biological triplicates of independent experiments. Standard curves were generated by diluting the cDNA solution 1:5. Relative expression levels of genes of interest were calculated using the standard curve method for relative quantification. Statistical comparisons were performed using Student's t test and pvalues < 0.05 were considered statistically significant. The specific sense and antisense primers are listed in S1 Table. Argentilactone-regulated transcripts were selected for qRT-PCR validation assays.

Protein extraction
The protein extraction was performed with cells grown in the presence and absence of argentilactone. After incubation with 9 μg/mL argentilactone for 6 h in MMcM liquid medium, the cells were centrifuged at 10,000 x g for 15 min at 4°C and protein was extracted using extraction buffer (20 mM Tris-HCl pH 8.8; 2 mM CaCl 2 ) containing a mixture of protease inhibitors (GE Healthcare). After the addition of glass beads (0.45 mm), the cells were lysed in a bead-beater, followed by centrifugation at 10,000 x g for 15 min at 4°C. The supernatant was collected, and the protein concentrations were determined using Bradford reagent (Sigma-Aldrich) [27].

Assay of superoxide dismutase enzymatic activity
The superoxide dismutase activity was quantified using an SOD assay kit (Sigma-Aldrich) that determine to production of formazan dye upon reduction with O 2 by colorimetric detection at 440 nm. The levels of SOD activity were quantified using 1 μg of total protein extract. Enzyme activity data were plotted as the mean of three independent experiments. The statistical analysis was performed using Student's t-test and samples with a p-value < 0.05 were considered statistically significant.

Fluorescence microscopy
Fluorescence microscopy assays were performed as previously described [28]. Calcofluor White (CFW) and Congo Red (CR) (Sigma-Aldrich) were used to label the cell wall of P. lutzii yeast cells. A total of 10 6 cells/mL P. lutzii yeast cells were inoculated in Fava Netto's liquid medium and grown for 3 days with agitation at 150 rpm. Afterwards, the cultures were incubated in MMcM liquid medium overnight at 36°C with shaking for 16 h. The cells were centrifuged at 5,000 x g for 5 min and then transferred to MMcM liquid medium containing 9 μg/mL argentilactone for 6 h. Control cells were incubated in MMcM liquid medium without argentilactone. Cells were fixed in 100% methanol at -80°C for 20 min, and then at -20°C for 20 min, and finally they were washed and centrifuged. The collected cells were stained with 100 μg/mL CR and CFW in PBS for 15 min and washed with 1x PBS. The samples were analyzed under a fluorescence microscope at 345 nm and 500 nm for CR and CFW, respectively (Zeiss Axiocam MRc-Scope A1, Carl Zeiss, Jena, Germany).

Ethanol dosing
The level of ethanol was measured as previously described [23] using an enzymatic detection kit UV-test for ethanol (RBiopharm, Darmstadt, Germany). A total of 1 x 10 6 cells were grown in the presence and absence of the argentilactone. Afterwards, the protein extract was obtained after cell lysis using glass beads and a bead beater apparatus (BioSpec, Oklahoma, USA) in 5 cycles of 30 sec. The cell lysate was subjected to centrifugation for 15 min, at 4°C, at 10,000 × g. The enzyme assay was performed in triplicate using the supernatant according to the manufacturer's instructions.

Mitochondrial membrane potential measurement
The mitochondrial membrane was monitored using the rhodamine 123 fluorescent dye. A total of 10 6 cells/mL were treated with 9 μg/mL argentilactone for 6 h. After treatment, the cells were centrifuged and incubated with 20 μM rhodamine 123 for 20 min at room temperature. Afterwards, the cells were washed 2 times with 1X PBS and resuspended in 1 mL PBS for analysis using a guava easyCyte flow cytometer with excitation and emission wavelengths of 488 and 530 nm, respectively.

Transcriptional response pattern of Paracoccidioides lutzii to argentilactone
Next-generation sequencing was used to produce the transcriptional profile. Approximately 46 million reads of 100-bp single-end sequences were obtained. The data were submitted in the ncbi.nlm.nih.gov, generating access number SRP064389. The reads were mapped using the reference genome of the P. lutzii genome database (http://www.broadinstitute.org/annotation/ genome/paracoccidioides_brasiliensis/MultiHome.html) and were analyzed using the DEGseq package. For the global analysis, plotting graphs were prepared. The number of reads counted for each transcript in the presence or absence of argentilactone was represented by scattered dots. The transcripts are represented by dots, which could represent a different number of reads in each condition (S1A Fig). The statistical test was applied to identify differentially expressed transcripts, represented by red dots (S1B Fig).
To determine the up-and down-regulated transcripts (S2 and S3 Tables, respectively), a cut-off of 1.5-fold change was used, resulting in 1,058 differentially expressed transcripts in P. lutzii yeast cells. A biological process classification was performed to gain a general understanding of the functional categories affected by argentilactone. A total of 54% (567 transcripts) were represented by proteins of unknown function ( Fig 1A). After 6 h of exposure to argentilactone, transcripts associated with metabolism (23.6%) were the most represented. Other groups were also regulated, such as transcription (12.6%), cell rescue, defense and virulence (11.2%) and cell cycle and DNA processing (11%) (Fig 1B). The genes related to metabolism (20%), transcription (15.8%), protein fate (12.3%), cell cycle and DNA processing (11.7%) and cellular transport, transport facilities and transport routes (11.2%) ( Fig 1C) were down-regulated. The up-regulated genes were mainly related to metabolism (32.4%), cell rescue, defense and virulence (18.3%), cell cycle and DNA processing (9.2%), energy (9.2%) and protein fate (5.6%) (Fig 1D).

Argentilactone seems interferes with urea synthesis and excretion in Paracoccidioides lutzii
From the transcriptional data obtained (S2 and S3 Tables), we could infer that, we could infer that P. lutzii down-regulated glutamine synthase, which catalyzes the condensation of glutamate and ammonia to glutamine, and glutamate dehydrogenase, which converts glutamate to α-ketoglutarate, and vice versa. In addition, two glutamate-1-semialdehyde 2,1-aminomutase, which converts glutamate 1-semialdehyde to 5-aminolevulinate, was down-regulated. Glutamate 1-semialdehyde is a molecule formed from glutamate and is a precursor to ornithine and proline. The cofactor of glutamate-1-semialdehyde 2,1-aminomutase is pyridoxal phosphate, which is produced from pyridoxal by pyridoxine kinase, was also down-regulated. On the other hand, in the presence of argentilactone, P. lutzii seems to use the amide group of xanthine, a purine base, in the synthesis of uric acid, which is excreted and metabolized into allantoic acid, allantoin and urea through an amphibian-like uricolytic pathway [29], as xanthine dehydrogenase, uricase and allantoinase were up-regulated. The production of putrescine and polyamines from ornithine is absent, as ornithine decarboxylase is down-regulated.

Effect of argentilactone on the metabolism of Paracoccidioides lutzii
Glycolysis is induced in P. lutzii in the presence of argentilactone, as class II aldolase and glyceraldehyde-3-phosphate dehydrogenase are up-regulated. It is noteworthy that several transporters, including sugar and amino acid transporters, were down-regulated. Although alcoholic fermentation is up-regulated in P. lutzii yeast cells [30], the presence of argentilactone repressed it because two alcohol dehydrogenases are down-regulated. The ethanol dosage assay confirmed these data, as ethanol is reduced in the presence of argentilactone (Fig 2).
To investigate the chitin and glucan levels in the cell wall, the cells were stained with CFW and CR and visualized by fluorescence microscopy. The fluorochrome CR and CFW interact with polysaccharides of cell wall [34] exhibiting strong affinity for chains of chitin [35,36]. P. lutzii yeast cells after treatment with argentilactone showed a decrease in fluorescence, indicating a reduction in the levels of this polymer (Fig 3), corroborating the transcriptional data.
The growth of hsp90, sod, ccp and rbt5 silenced mutants was evaluated in the presence of argentilactone (Fig 5). Due the difficulty in obtaining mutants in P. lutzii, the mutants used here were obtained to P. brasiliensis from studies performed previously (14,24,25,26). Pb339, Pb60855, Pb339EV, Pb60855EV, Pbsod-aRNA, Pbhsp90-aRNA, Pbccp-aRNA and Pbrbt5-aRNA were grown for 6 days in the presence of 4.5, 9, 18 and 36 μg/mL argentilactone. All mutants presented   genes in Paracoccidoides spp. yeast cells grown in MMcM liquid medium with or without argentilactone were analyzed. The data were normalized using the constitutive gene encoding α-tubulin as the endogenous control and are presented as relative expression in comparison to the experimental control cells, whose value was set to 1. Data are expressed as the mean ± standard deviation of the triplicates of independent experiments. *, significantly different from the control at a p-value of < 0.05. greater sensitivity to argentilactone compared to wild-type cells and cells containing the empty vector. Argentilactone inhibits Pb60855 and Pb60855EV cells in the presence of 18 μg/mL of the compound, as a dose of 36 μg/mL was necessary to affect the cell growth of Pb339 and Pb339EV. For the silenced mutants Pbsod-aRNA, Pbhsp90-aRNA, Pbccp-aRNA and Pbrbt5-aRNA, 9 μg/mL argentilactone was sufficient to inhibit the growth of the Pbhsp90-aRNA-silenced mutant. These results suggest that overexpression of the hsp90, sod, ccp and rbt5 transcripts are important for the response of Paracoccidioides spp. to argentilactone and that the silencing of these genes directly influences the growth of the fungus in the presence of argentilactone.
Argentilactone promotes the induction of oxidative stress and dysfunction of the mitochondrial membrane potential Due to the induction of genes related to oxidative stress, SOD enzymatic activity was investigated. The enzymatic activity was measured after growing P. lutzii for 6 h in the presence or absence of argentilactone. A significant increase of enzymatic activity was observed in the presence of argentilactone (Fig 6).
The ability of argentilactone to cause damage to the mitochondrial membrane potential was compared to antimycin A, a potent inhibitor of the electron transport chain. The mitochondrial membrane was monitored using rhodamine 123, which decreases its fluorescence when there is depolarization of the mitochondrial membrane potential. Flow cytometry data showed that argentilactone exerted a similar effect on cells as antimycin A. In the presence of argentilactone, the intracellular fluorescence due to rhodamine 123 was decreased (Fig 7).

Discussion
The objective of this study was to analyze the transcriptional profile of P. lutzii in the presence of argentilactone. The regulation of transcripts and proteins from metabolic pathways by antifungals has been described for several microorganisms, including the antifungal itraconazole, which acts in a similar pathway as ergosterol [37,38,39]. The concentration of argentilactone utilized in the experiments affected global gene expression in P. lutzii. Here, argentilactone affected the metabolism of P. lutzii, as alcoholic fermentation was down-regulated, and glycolysis, β-oxidation and the methylcitrate cycle were up-regulated. In addition, argentilactone interfered with urea synthesis and excretion in P. lutzii. In treatment with argentilactone, the fungus seems to use the amphibian-like uricolytic pathway to excrete and metabolize amide groups. The utilization of different pathways to reach the destination of the amide groups has been reported in organisms. In Aedes aegypti, urea synthesis and excretion are regulated by a unique cross-talk mechanism, which is one of the biochemical mechanisms responsible for the success of the infection [40].
Chitin synthases are enzymes that play an important role in cellular integrity, growth and virulence of human fungal pathogens, including Candida albicans and Cryptococcus neoformans [41,42]. It has been observed that high ergosterol levels can inhibit chitin synthases, whereas C. albicans mutants with low ergosterol content showed increased levels of chitin synthesis [43]. Here, an ergosterol biosynthesis protein and sterol desaturase transcripts were upregulated, and several transcripts related to the synthesis of chitin and glucan were down-regulated. In addition, the polymer levels of the cell wall of P. lutzii were decreased when treated with argentilactone. Studies have demonstrated the importance of genes related to cell wall biosynthesis in Paracoccidioides spp., including chitin synthase genes [9,[44][45][46][47][48].
We assumed that central carbon metabolism is inhibited by argentilactone, as we found a profile similar to the effects of pyrazole (an alcohol dehydrogenase inhibitor), which reduced the ethanol yield in Saccharomyces cerevisiae [49].
hsp90, a molecular chaperone that plays an important role in the assembly and regulation of several signaling systems in eukaryotes [50], is a highly conserved protein and represents about 2% of all cellular proteins in cells [51]. Furthermore, hsp90 is required for viability under stress conditions in eukaryotes [52] and plays an important role in fungi, including the physiology of Effects of Argentilactone in Paracoccidioides spp. P. lutzii [23], indicating its utility as a potential antifungal target [53,54]. In P. lutzii, the heat shock response is associated with pathogenesis because the change in temperature is responsible for dimorphic transition, which is essential to establish infection. hsp90 regulates the proliferation and adaptation of P. lutzii to different environmental conditions [23,55]. Here, transcriptional and qRT-PCR date showed that hsp90 was up-regulated in the presence of argentilactone. The plate sensitivity test using the mutant P. brasiliensis Pbhsp90-aRNA showed that hsp90 was recruited by P. brasiliensis growth in the presence of argentilactone.
Many organisms present a ROS defense system generated by aerobic respiration and substrate oxidation [56]. SOD is required for P. lutzii growth in the presence of argentilactone, as its activity was increased in this condition. Increased levels of SOD activity may represent a primary antioxidant defense against ROS in P. lutzii [57]. In addition, the P. brasiliensis mutant Pbsod-aRNA exhibited decreased growth in the presence of this compound, as found in C. neoformans [58]. Argentilactone seems to stimulate oxidative stress in Paracoccidioides spp. Therefore, the fungus seems to induce antioxidant enzymes, such as SOD, which act to reduce ROS, aiming to prevent cell damage. The induction of hsp90 and sod suggests that the primary heat shock stress could induce subsequent oxidative stress due to oxygen availability. Similar data have been previously described in S. cerevisiae [59,60]. Argentilactone seems to be acting on one of the main sources of ROS, the electron transport chain, as several transcripts related to electron transport, such as NADPH-adrenodoxin oxidoreductase, NADH-cytochrome b5 reductase and the indirectly related ccp, were up-regulated.
Peroxidases are enzymes that use various electron donors to reduce H 2 O 2 to H 2 O. In C. neoformans, the growth of a ccp mutant was affected by the presence of H 2 O 2 [58]. CCP catalyzes the reduction of hydrogen peroxide using cytochrome c as an electron donor. In S. cerevisiae, the increased expression of ccp seems to be caused by an increase of ROS produced during aerobic growth, thus confirming the biological role of this enzyme in cellular detoxification and the elimination of hydrogen peroxide [60]. We suggest here that the cellular oxidative stress caused by argentilactone results in an overexpression of sod, which is responsible for catalyzing the dismutation of superoxide into H 2 O 2 . The presence of H 2 O 2 would induce the increased expression of ccp in an attempt to reduce hydrogen peroxide. Here, the increase in the ccp transcript level demonstrated in the transcriptional and qRT-PCR data and the decreased growth of Paracoccidioides Pbrbt5-aRNA in the presence of argentilactone show that ccp is required for the fungus in this condition.
ROS are universal products of aerobic metabolism, which can also be produced under stressful conditions. In eukaryotic cells, mitochondria are the main source of ROS [61]. Changes in the proton gradient resulting from the inhibition of electron transport can result in the production of reactive oxygen species (ROS) [61,62]. Thus, we assumed that argentilactone could have been acting on mitochondria, and our results confirmed this hypothesis.
In Coccidioides immitis [63] and S. cerevisiae [64], RBT5 is an antigenic protein anchored to the cell wall. rbt5 is important for the morphogenesis of the cell wall of C. albicans [65] and Coccidioides during infection [66]. In P. lutzii, rbt5 is a major surface antigen [67]. Here, the increase in the rbt5 transcript level exhibited in the transcriptional and qRT-PCR data and the decrease of Pbrbt5-aRNA growth in the presence of argentilactone suggest that rbt5 is required for the fungus in this condition. rbt5 could be increased in the presence of argentilactone in an attempt to maintain the morphogenesis of the cell wall, as several transcripts related to the biosynthesis of the cell wall, mainly to chitin biosynthesis, were down-regulated. Fluorescence microscopy data showing that the level of chitin and glucan polymers in the cell wall of P. lutzii yeast cells was lower in the presence than in the absence of argentilactone corroborate these results. Genes important to the maintenance and integrity of the cell wall were described to be down-regulated in fungi exposed to amphotericin B [68,69] or to investigatory antifungals, such as artemisinin [70] or oenothein B, an anti-Paracoccidioides [28]. In Aspergillus niger and S. cerevisiae, the transcription factor RlmA is required for the up-regulation of cell wall stress-induced genes [71] leading to increased chitin content. Here, RlmA is down-regulated, corroborating the down-regulation of genes related to chitin synthesis.
The considerable number of transcription factors regulated in the presence of argentilactone suggests a complex regulatory mechanism. Conversely, the high percentage of unclassified proteins indicates that additional studies must be performed to elucidate the mode of action of argentilactone on Paracoccidioides spp.
One of the limitations of the study was that all experiments were not performed on P. lutzii and P. brasiliensis due to the difficulty in obtaining mutants of P. lutzii. This analysis should be attempted in the future.

Conclusion
Argentilactone seems to be able to penetrate into Paracoccidioides spp. yeast cells and modulate cellular targets. Argentilactone seems to induce oxidative stress and interfere with the biosynthesis of the Paracoccidioides spp. cell wall. Given the overall stress caused by argentilactone in Paracoccidioides spp., other studies must be performed to better elucidate the mode of action of argentilactone in Paracoccidioides spp. The expression levels of hsp90, ccp, sod and rbt5 genes in Paracoccidoides brasiliensis. yeast cells grown in MMcM liquid medium with or without argentilactone were analyzed. The data were normalized using the constitutive gene encoding α-tubulin as the endogenous control and are presented as relative expression in comparison to the experimental control cells, whose value was set to 1. Data are expressed as the mean ± standard deviation of the triplicates of independent experiments. Ã , significantly different from the control at a pvalue of < 0.05. (TIF) S1 Table. Gene-specific primers used for qRT-PCR assays.