Multi‐Omics Analysis of Experimentally Evolved Candida auris Isolates Reveals Modulation of Sterols, Sphingolipids, and Oxidative Stress in Acquired Amphotericin B Resistance

May combine two claims.

A1.1 and B1.1 exhibited MIC50 values of 2 μg/mL and 4 μg/mL, respectively, for AmB after passage on drug-free medium.

“The adapted strains A1.1 and B1.1 exhibited MIC50 values of 2 µg mL-1 and 4 µg mL-1, respectively, for AmB (Figure 1d).”

May combine two claims.

There were no common upregulated genes in both adapted strains, but there were seven common downregulated genes.

“Notably, there were no common upregulated genes found in both the adapted strains, but there were seven common downregulated genes in these strains”

May combine two claims.

The major GlcCer contributing species was GlcCer (d19:2/18:0(2OH)), and its precursor Cer (d19:2/18:0(2(OH)) accumulated in B1.1.

“The major GlcCer contributing species was GlcCer (d19:2/18:0(2OH)), and its precursor Cer (d19:2/18:0(2(OH)) which accumulated in the B1.1-adapted strain”

May combine two claims.

ROS levels were elevated in A1.1/aox2Δ cells, but no comparable increase was observed in B1.1/aox2Δ mutants.

“Consistent with this, ROS levels were elevated in A1.1/aox2Δ cells, though no comparable increase was observed in B1.1/aox2Δ mutants”

May combine two claims.

The total number of DEGs in A1.1 and B1.1 were 594 and 83, respectively; upregulated genes were 383 and 42, and downregulated genes were 211 and 41.

“The total number of DEGs in A1.1 and B1.1 were 594 and 83, respectively. Among these, the upregulated genes were 383 and 42 in A1.1 and B1.1, and 211 and 41 were downregulated in A1.1 and B1.1, respectively”

May combine two claims.

Lipid analysis of B1.1 could only quantify IPCs and observed their reduction in the adapted strain.

“Our lipid analysis of the B1.1 adapted strain could only quantify Inositol phosphoryl ceramide (IPCs) and observed their reduction in the adapted strain”

May combine two claims.

Quantitative analysis of CW components revealed that the level of glucosamine was significantly elevated in B1.1, aligning with increased susceptibility towards CFW.

“Quantitative analysis of CW components (Figures 9c, 9d, and 9e) revealed that the level of glucosamine (a precursor of chitin biosynthesis) was significantly elevated in the B1.1 adapted strain, which also aligns well with our phenotypic analysis where cells showed increased susceptibility towards CFW”

May combine two claims.

In A1.1, UPC2S332R, RTG3S101T, GNP2D381Y, and CDR1Y79* appeared from generation 40 onwards; OPT1K217I and HRD3K321* emerged only after generation 80.

“in the A1.1 adapted strain, where increased MIC50 value towards AmB was recorded from generation 50 onwards, out of six SNPs, UPC2S332R, RTG3S101T, GNP2D381Y, and CDR1Y79* appeared from generation 40 onwards. The only exceptions were those of OPT1K217I and HRD3K321*, which emerged only after generation 80”

May combine two claims.

AOX2 knockout strains failed to develop AmB resistance during experimental evolution, unlike wild-type strains.

“Unlike the wild-type strains, which successfully evolved AmB resistance, the AOX2 knockout strains failed to develop resistance, indicating a direct role for AOX2 in facilitating adaptation to AmB”

May combine two claims.

qRT-PCR confirmed AOX2 upregulation: 5-6 log2-fold in A1.1 and 0.8-0.9 log2-fold in B1.1.

“This was further confirmed by qRT-PCR, where the upregulation of AOX2 in A1.1 adapted strain was a 5-6 log2-fold increase, and in B1.1 adapted strain, it was a 0.8-0.9 log2-fold increase”

May combine two claims.

B1.1 has upregulation of ergosterol biosynthesis pathway genes, increased sensitivity to cell wall-perturbing agents, and altered expression of cell wall-related genes.

“For instance, in the B1.1 adapted strain, there is upregulation of ergosterol biosynthesis pathway genes, increased sensitivity to cell wall-perturbing agents, and altered expression of cell wall-related genes”

May combine two claims.

A1.1 and B1.1 exhibited cross-resistance to nystatin and natamycin but no cross-resistance to azoles.

“The A1.1 and B1.1 adapted strains, when tested for acquired resistance to other antifungals, were found to exhibit cross-resistance to other polyenes, nystatin, and natamycin. However, no cross-resistance to azoles was observed”

May combine two claims.

A1.1 showed a 2-fold increase in MIC50 for fluconazole, voriconazole, and clotrimazole compared to wt_A; B1.1 showed minimal changes for most azoles except a 2-fold MIC50 increase for fluconazole and ketoconazole.

“A1.1 stayed within the susceptibility range but showed a 2-fold increase in MIC50 for fluconazole, voriconazole, and clotrimazole compared to wt_A. B1.1 showed minimal changes for most azoles, except for a comparable 2-fold MIC50 increase for fluconazole and ketoconazole”

May combine two claims.

WGS of A1.1 and B1.1 revealed six SNPs in A1.1 (missense and nonsense) and three missense mutations in B1.1.

“WGS of A1.1 and B1.1 adapted strains revealed six SNPs, including missense and nonsense mutations in the A1.1-adapted strain and three missense mutations in the B1.1-adapted strain”

May combine two claims.

GO enrichment revealed elevated B9J08_001930 (orthologue of AOX2) and downregulation of HSX11 were most noteworthy.

“Gene Ontology enrichment analysis revealed that among the DEGs linked to various cellular processes, an elevated level of an uncharacterized gene, B9J08_001930, which is an orthologue of the AOX2 gene in C. albicans and encodes an alternative oxidase AOX2, and down-regulation of HSX11 were most noteworthy”

May combine two claims.

A1.1 exhibited resistance to nystatin and natamycin similar to that of AmB, while B1.1 showed slightly higher resistance to nystatin than to natamycin.

“While A1.1 exhibited resistance to nystatin and natamycin similar to that of AmB, B1.1 showed slightly higher resistance to nystatin than to natamycin”

May combine two claims.

AOX2 log2-fold change was 8.8 in A1.1 and 1.3 in B1.1 compared to their respective wild-type progenitors.

“The log2-fold change value in the A1.1 adaptor was 8.8 log2-fold, and B1.1 showed a more modest 1.3 log2-fold increase compared to their respective wild-type progenitors.”

May combine two claims.

In B1.1, ERG6K371N and GEA2E1229K emerged by generation 60 onwards, while PRP8T1561M was detected after generation 80.

“In the B1.1 adapted strain, two of the three SNPs (ERG6K371N and GEA2E1229K) emerged by generation 60 onwards, while PRP8T1561M was detected after generation 80”

May combine two claims.

B1.1 exhibited perturbations in sterol and SL metabolism, characterized by an SNP in ERG6K371N and significant downregulation of HSX11.

“Instead, they exhibited perturbations in sterol and SL metabolism, characterized by an SNP in the ERG6K371N gene and a significant downregulation of HSX11, a gene encoding glucosylceramide synthase.”

May combine two claims.

Strain B11220 is the type strain of Clade II and was re-confirmed as a Clade II isolate.

“Strain B11220 (CBS10913T) is the type strain of Clade II (Satoh et al., 2009; Muñoz et al., 2018b) and was re-confirmed as a Clade II isolate in our analysis”

• cites Satoh et al., 2009Not checked yet

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• cites Muñoz et al., 2018bNot checked yet

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May combine two claims.

wt_A had an MIC50 value of 0.5 μg/mL for AmB and wt_B had an MIC50 value of 0.5-1 μg/mL for AmB.

“Single colonies from strain wt_A (MIC50 value of 0.5 µg mL-1 for AmB) and strain wt_B (MIC50 value 0.5–1 µg mL-1 for AmB)”

May combine two claims.

Both strains displayed increased MIC50 values yet remained susceptible to azoles.

“Both strains displayed increased MIC50 values, yet remained susceptible to azoles.”

May combine two claims.

This resulted in a significant loss of GlcCer and an accumulation of its precursor αOHCer.

“This resulted in a significant loss of GlcCer and an accumulation of its precursor, αOHCer.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In other Candida species, mutations in ERG1, ERG2, and ERG6 genes have been linked to AmB resistance.

“In other Candida species, mutations in ERG1, ERG2, and ERG6 genes have also been linked to AmB resistance (Ahmad et al., 2019; Carolus et al., 2020).”

• cites Ahmad et al., 2019Not checked yet

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• cites Carolus et al., 2020Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Approximately 30% of C. auris isolates are resistant to amphotericin B.

“Almost all C. auris isolates are resistant to fluconazole, approximately 50% are resistant to voriconazole, 30% to amphotericin B (AmB), and 10% to echinocandins (Sekyere, 2017; Rybak et al., 2022b).”

• cites Sekyere, 2017Not checked yet

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• cites Rybak et al., 2022bNot checked yet

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May combine two claims.

Alterations in ergosterol biosynthesis can contribute to acquired AmB resistance in C. auris laboratory strains but are rarely seen in clinical isolates.

“Alterations in ergosterol biosynthesis can contribute to acquired AmB resistance in C. auris laboratory strains but are rarely seen in clinical isolates.”

May combine two claims.

Increased expression of ERG1, ERG2, and ERG6 is linked to AmB resistance, suggesting a compensatory response to AmB's interaction with ergosterol.

“Additionally, increased expression of ERG1, ERG2, and ERG6 has been linked to AmB resistance, suggesting a compensatory response to counteract the disruption caused by AmB's interaction with ergosterol (Young et al., 2003; Vincent et al., 2013; Muñoz et al., 2018a; Ahmad et al., 2019; Silva et al., 2020).”

• cites Young et al., 2003Not checked yet

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• cites Vincent et al., 2013Not checked yet

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• cites Muñoz et al., 2018aNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

AmB forms sponges in the cell wall that remove ergosterol from the cell membrane, leading to its destabilization.

“The AmB forms sponges in the cell wall that remove ergosterol from the cell membrane, leading to its destabilization. (Anderson et al., 2014).”

• cites Anderson et al., 2014Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

A truncated 164 amino acid Erg6 protein conferred AmB resistance.

“Additionally, Kordalewska et al., 2021 reported that a truncated 164 amino acid Erg6 protein conferred AmB resistance.”

• cites Kordalewska et al., 2021Not checked yet

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Classified as probably not a substantial claim.

AmB exerts antifungal effects by extracting ergosterol from the fungal cell membrane.

“Polyenes such as AmB exert their antifungal effects by extracting ergosterol from the fungal cell membrane (Lewandowska et al., 2021).”

• cites Lewandowska et al., 2021Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

DNA damage checkpoint proteins such as Mec3 and transcription factor Flo8 have been suggested to play a role in AmB resistance in C. auris.

“Beyond sterol biosynthesis, DNA damage checkpoint proteins, such as Mec3, and the transcription factor Flo8, which positively regulates ERG11 expression, have also been suggested to play a role in AmB resistance in C. auris (Escandón et al., 2019; Carolus et al., 2021b).”

• cites Escandón et al., 2019Not checked yet

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• cites Carolus et al., 2021bNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Mutations in NCP1, ERG11, ERG3, HMG1, ERG10, and ERG12 have been linked to AmB resistance in C. auris.

“Mutations in, NCP1, ERG11, ERG3, HMG1, ERG10, and ERG12, have been linked to AmB resistance in C. auris (Carolus et al., 2024)”

• cites Carolus et al., 2024Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Clinically acquired AmB resistance was initially caused by a frameshift mutation in ERG6 leading to premature truncation of Erg6, causing high resistance of C. auris to AmB.

“Clinically acquired AmB resistance was initially caused by a frameshift mutation in the ERG6 gene, leading to premature truncation of Erg6 in patients, causing high resistance of C. auris to AmB (Rybak et al., 2022a).”

• cites Rybak et al., 2022aNot checked yet

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May combine two claims.

Such expression patterns suggest differences in the regulatory response of the CW biosynthesis pathway between the two adapted strains, potentially suggesting distinct adaptive mechanisms.

“Such expression patterns suggest differences in the regulatory response of the CW biosynthesis pathway between the two adapted strains, potentially suggesting distinct adaptive mechanisms.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Rtg3-mediated regulation of AOX2 appears conserved, consistent with a previous study by Liu et al., 2023.

“Notably, Rtg3-mediated regulation of AOX2 appears to be conserved, consistent with a previous study by Liu et al., 2023 demonstrating that Rtg3 regulates AOX2 promoter activity, impacting C. albicans virulence in a mouse model.”

• cites Liu et al., 2023Not checked yet

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May combine two claims.

AmB resistance in B1.1 results from a combination of factors, including enhanced sterol biosynthesis, compromised cell wall integrity, and reduced GlcCer levels due to HSX11 downregulation.

“Taken together, these findings suggest that AmB resistance in the B1.1 strain results from a combination of factors, including enhanced sterol biosynthesis, compromised cell wall integrity, and reduced GlcCer levels due to HSX11 downregulation.”

Classified as probably not a substantial claim. May combine two claims.

CFW binds to chitin in the fungal cell wall, and CR binds to β-glucans.

“CFW binds to the chitin in the fungal cell wall, and CR binds to β-glucans.”

May combine two claims.

The presence of the ERG6 SNP leads to a partial loss-of-function mutation, as confirmed by the altered sterol profile of B1.1.

“Moreover, the presence of an ERG6 SNP leads to a partial loss-of-function mutation, as confirmed by the altered sterol profile of B1.1.”

Classified as probably not a substantial claim.

Hsx11 catalyzes transfer of UDP-linked glucose to the sphingoid backbone of ceramide precursors.

“Hsx11 catalyzes the transfer of UDP-linked glucose to the sphingoid backbone of ceramide precursors (Ali et al., 2024).”

• cites Ali et al., 2024Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Expression levels of the mentioned CW-related genes were validated by qRT-PCR.

“The expression levels of the mentioned CW-related genes were validated by qRT-PCR”

Probably not a substantial claim: may not pass S3 (load-bearing).

The role of ERG6 in mediating AmB resistance has been well documented.

“The role of ERG6 in mediating AmB resistance has been well documented in previous studies (Rybak et al., 2022a; Carolus et al., 2024).”

• cites Rybak et al., 2022aNot checked yet

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• cites Carolus et al., 2024Not checked yet

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May combine two claims.

ERG6K371N may represent a partial loss-of-function mutation, leading to buildup of cholesta-5,7,24-trienol.

“These findings suggest that ERG6K371N may represent a partial loss-of-function mutation, leading to the buildup of this intermediate sterol.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Mesa-Arango et al., 2016 reported increased levels of β-glucans in AmB resistant isolates of C. tropicalis, which also exhibited susceptibility towards Congo Red and impacted immune response.

“A study by Mesa-Arango et al., 2016 had reported increased levels of β-glucans in AmB resistant isolates of C. tropicalis. These isolates also exhibited susceptibility towards Congo Red and impacted immune response elicited by the host.”

• cites Mesa-Arango et al., 2016Not checked yet

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May combine two claims.

UPC2S332R is upstream of the hotspot of gain-of-function mutations and has not been reported previously.

“this mutation, UPC2S332R, is upstream of the hotspot of gain-of-function mutations (Flowers et al., 2012) and has not been reported previously.”

• cites Flowers et al., 2012Not checked yet

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May combine two claims.

Glucosyl derivatives are the main complex SLs found in C. auris, indicating that the GlcCer branch of the SL pathway is active in this species.

“Based on our earlier analysis, glucosyl derivatives are the main complex SLs found in C. auris, indicating that the GlcCer branch of the SL pathway is active in this species (Kumar et al., 2021).”

• cites Kumar et al., 2021Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The observation of increased susceptibility and ergosterol could plausibly be a result of underlying genetic heterogeneity between SNP-reverted adapted strains and wt_A.

“While the observation is intriguing, this could plausibly be a result of the underlying genetic heterogeneity between the SNP-reverted adapted strains (A1.1/upc2_c1–c6) and the original wild-type strain (wt_A).”

May combine two claims.

AOX2 appears important for developing resistance, likely due to its role in managing oxidative stress, a known factor in AmB's fungicidal action.

“AOX2 appears to be important for developing resistance, likely due to its role in managing oxidative stress, a known factor in AmB's fungicidal action (Liu et al., 2005).”

• cites Liu et al., 2005Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

It is possible that the distinct genetic background of wt_B did not fully replicate the context of B1.1, potentially masking the impact of HSX11 deletion.

“It is also possible that the distinct genetic background of wt_B did not fully replicate the context of B1.1, potentially masking the impact of HSX11 deletion on AmB susceptibility.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Seo et al., 1999 reported altered CW composition in response to AmB resistance in Aspergillus flavus.

“Another study (Seo et al., 1999) reported altered CW composition in response to AmB resistance in Aspergillus flavus.”

• cites Seo et al., 1999Not checked yet

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May combine two claims.

These findings highlight the critical role of AOX2 in AmB resistance not only in the adapted strains but also in the clinical isolate of clade I.

“These findings highlight the critical role of AOX2 in AmB resistance not only in the adapted strains but also in the clinical isolate of clade I.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Gene expression was normalised with the housekeeping genes CauACT1, CauLSC2, and CauUBC4.

“following normalisation with the housekeeping genes CauACT1, CauLSC2, and CauUBC4.”

• AssumptionRests on an assumption: The genes CauACT1, CauLSC2, and CauUBC4 are suitable housekeeping genes for normalizing qRT-PCR expression in C. auris under the studied conditions.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Aspergillus terreus, which is intrinsically resistant to AmB, has normal levels of ergosterol in its cell membrane and exhibits increased catalase levels, suggesting a catalase-based resistance mechanism.

“For example, Aspergillus terreus, which is intrinsically resistant to AmB, has normal levels of ergosterol in its cell membrane and exhibits increased catalase levels, suggesting a catalase-based resistance mechanism that mitigates AmB-induced oxidative stress, preventing cell damage (Blatzer et al., 2015; Posch et al., 2018).”

• cites Blatzer et al., 2015Not checked yet

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• cites Posch et al., 2018Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Support values for the phylogenetic tree were calculated from 1000 bootstrap replicates.

“The final tree was obtained using midpoint rooting with support values calculated from 1000 bootstrap replicates.”

May combine two claims.

While a direct role of UPC2 in impacting AmB resistance is evident in A1.1, a different mechanism involving sterol metabolism is observed in B1.1.

“While a direct role of UPC2 in impacting AmB resistance is evident in the A1.1 adapted strain, a different mechanism involving sterol metabolism is observed in the adapted strain B1.1.”

May combine two claims.

These observations suggest that a full loss of function of ERG6 leads to fitness trade-offs that might be mitigated by a partial loss-of-function mutation.

“These observations suggest that a full loss of function of ERG6 leads to fitness trade-offs that might be mitigated by a partial loss-of-function mutation.”

Classified as probably not a substantial claim.

Only variants that passed the filters of all three variant callers were considered high-confidence for each sample.

“Only those variants that passed the filters of all three callers were considered as high-confidence per each sample.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Sterol extraction and analysis of each strain were performed in biological triplicate.

“Sterol extraction and analysis of each strain were performed in biological triplicate.”

May combine two claims.

AOX2-deleted strains could not develop resistance to AmB, underscoring the necessity of a functional AOX2 for sustained resistance.

“Unlike their progenitors, the AOX2 knockouts could not develop resistance to AmB, underscoring the necessity of a functional AOX2 for sustained resistance.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A partial loss-of-function point mutation in ERG6E329* in an isolate of C. auris belonging to Clade IV has been previously reported.

“A partial loss-of-function point mutation in ERG6E329* in an isolate of C. auris belonging to Clade IV has been previously reported (Carolus et al., 2024).”

• cites Carolus et al., 2024Not checked yet

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Classified as probably not a substantial claim.

MIC50 was determined using the broth dilution assay following Clinical and Laboratory Standards Institute (CLSI) guidelines to assess susceptibility to amphotericin B.

“MIC50 was determined using the broth dilution assay (BDA) following Clinical and Laboratory Standards Institute (CLSI) guidelines to assess the susceptibility to AMB (amphotericin B; Merck).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Technical triplicates and biological duplicates were used for qRT-PCR.

“Technical triplicates and biological duplicates were used for qRT-PCR.”

May combine two claims.

Despite ERG6K371N leading to accumulation of a sterol intermediate, significant levels of ergosterol were observed, likely because the mutation results in a partially active protein.

“Despite this mutation leading to the accumulation of sterol intermediate, significant levels of ergosterol were also observed, likely because the mutation results in a partially active protein.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Internal standard normalisation was used for quantification.

“Internal standard normalisation was used for quantification.”

May combine two claims.

While deletion of AOX2 resulted in similar phenotypes in both A1.1 and B1.1, transcriptional differences indicate nuanced resistance mechanisms.

“While the deletion of AOX2 resulted in similar phenotypes in both A1.1 and B1.1 adapted strains, transcriptional differences between the strains indicate nuanced resistance mechanisms that warrant further investigation.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For every analysis, three replicates of every sample were utilised.

“For every analysis, three replicates of every sample were utilised.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Normalizing lipid contents to total protein is done routinely as a measure.

“This is done routinely as a measure while normalizing lipid contents (Shahi et al., 2020; Kumar et al., 2021; Ali et al., 2024).”

• cites Shahi et al., 2020Not checked yet

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• cites Kumar et al., 2021Not checked yet

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• cites Ali et al., 2024Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Liu et al., 2005 showed that C. albicans AOX2 and other stress response genes were upregulated in response to AmB exposure.

“Another study (Liu et al., 2005) showed that C. albicans AOX2 and other stress response genes were upregulated in response to AmB exposure.”

• cites Liu et al., 2005Not checked yet

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Classified as probably not a substantial claim.

Lipid content in each replicate was normalized to the total protein of that replicate to maintain homogeneity across replicates and samples.

“The lipid content(ng/ml) in each replicate was then normalized to the total protein(mg/ml) of that replicate. This was done to maintain homogeneity across replicates and samples.”

May combine two claims.

While UPC2, RTG3, and AOX2 play a central role in A1.1, lipid metabolism, ergosterol pathway, and compromised CWI are more prominent in B1.1.

“While UPC2, RTG3, and AOX2 play a central role in A1.1, lipid metabolism, ergosterol pathway, and compromised CWI are more prominent in B1.1.”

Classified as probably not a substantial claim.

Sterols were identified by matching spectra to GC-MS libraries described in Müller et al., 2017 and NIST/EPA/NIH version 2.

“The spectra were matched to GC-MS libraries described in (Müller et al., 2017), and NIST/EPA/NIH version 2.”

• cites Müller et al., 2017Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Previous studies indicated that AOX2 is involved in mitochondrial alternative respiration, mycelial development, and virulence in Candida species.

“Previous studies (Li et al., 2011; Duvenage et al., 2019) have also indicated that AOX2 is involved in mitochondrial alternative respiration, mycelial development, and virulence in Candida species.”

• cites Li et al., 2011Not checked yet

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• cites Duvenage et al., 2019Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Approximately 5×10^8 cells were harvested with three biological replicates for lipid extraction.

“Approximately 5×108 cells were harvested by centrifugation at 4000xg for 5 min with three biological replicates and three biological replicates.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Additional filters (read coverage below 30 and minimum allele frequency below 0.9) were applied to remove low-quality variants from variant calling results.

“Additional filters (read coverage below 30 and a minimum allele frequency (--min_AF) below 0.9) were applied to remove low-quality variants from the results of the variants calling.”

May combine two claims.

Both transcription factors UPC2 and RTG3 regulate AOX2 transcription.

“Interestingly, both the transcription factors, UPC2 and RTG3, also regulate AOX2 transcription.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Hypoxia can protect protoplasts of C. albicans from AmB, and enzymes like catalase and superoxide dismutase can prevent AmB-induced cell lysis.

“Several studies have shown that hypoxia can protect the protoplasts of C. albicans from AmB, and enzymes like catalase and superoxide dismutase (SOD) can prevent AmB-induced cell lysis.”

May combine two claims.

Our findings demonstrate the complex processes contributing to AmB resistance and show that the two isolates adapted to AmB through distinct pathways.

“Our findings demonstrate the complex processes contributing to AmB resistance and show that the two isolates adapted to AmB through distinct pathways.”

May combine two claims.

The study highlighted the impact of UPC2 and RTG3 in regulating AOX2 expression and impacting AmB susceptibility.

“The study also highlighted the impact of UPC2 and RTG3 in regulating AOX2 expression and impacting AmB susceptibility.”