Natural mismatch repair mutations mediate phenotypic diversity and drug resistance in Cryptococcus deuterogattii

May combine two claims.

This single base deletion occurs within an early exon of MSH2 and results in a nonsense mutation, with both an out of frame message and an early stop codon present in the predicted transcript.

“This single base deletion occurs within an early exon of MSH2 and results in a nonsense mutation, with both an out of frame message and an early stop codon present in the predicted transcript (Figure 1C).”

Classified as probably not a substantial claim.

The VGIIa-like sublineage is composed of three isolates: NIH444, ICB107, and CBS7750.

“The VGIIa-like sublineage is composed of three isolates: NIH444, a clinical isolate from Seattle in 1975, well before the start of the outbreak; ICB107, a clinical isolate from Brazil in 1981; and CBS7750, an environmental isolate from California in 1990.”

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

The longest homopolymer run in the URA5 locus contains only four bases, which is predicted to be relatively stable and among the coding genes with relatively shorter homopolymer runs.

“The longest homopolymer run in the URA5 locus contains only four bases, which is predicted to be both relatively stable and among the coding genes with relatively shorter homopolymer runs (Figure 3C).”

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

A spontaneous ade2 mutant was fortuitously identified based on its classic red pigmentation during growth of the msh2Δ::NEO strains.

“During growth of the msh2Δ::NEO strains a spontaneous ade2 mutant was fortuitously identified based on its classic red pigmentation (Figure 6A).”

May combine two claims.

All 14 progeny demonstrated unique SNP profiles and only one SNP demonstrated inheritance congruent with the hypermutator phenotype: a SNP on scaffold 3 that was linked to the msh2 del131 indel (~175 kb).

“All 14 progeny demonstrated unique SNP profiles and only one SNP demonstrated inheritance congruent with the hypermutator phenotype: a SNP on scaffold 3 that was linked to the msh2 del131 indel (~175 kb) (Figure 5E).”

May combine two claims.

One white derivative had reverted the original mutation, while the second had eliminated production of the red intermediate but had not reverted the original ade2 mutation.

“One white derivative had reverted the original mutation (RBB23), while the second had eliminated production of the red intermediate but had not reverted the original ade2 mutation (RBB24).”

May combine two claims.

R265 primarily acquired resistance through substitutions, while NIH444 almost exclusively underwent single base additions or single base deletions, all within the 7C homopolymer run.

“while R265 still primarily acquired resistance through substitutions, NIH444 now almost exclusively underwent single base additions or single base deletions, all within the 7C homopolymer run (R265 vs NIH444, p=0.0003, Fisher's Exact Test).”

May combine two claims.

In multiple replicates the hypermutator rapidly acquired resistance and overtook the entire co-culture.

“In multiple replicates the hypermutator rapidly acquired resistance and overtook the entire co-culture.”

May combine two claims.

All of the progeny from this mating had an unusually high rate of aneuploidy with 8 of the 14 progeny aneuploid for at least one scaffold/chromosome.

“all of the progeny from this mating had an unusually high rate of aneuploidy with 8 of the 14 progeny aneuploid for at least one scaffold/chromosome (Figure 5—source data 1).”

May combine two claims.

Five of the msh2 del131 strains displayed the predicted hypermutator phenotype, while two spores (#4 and 5) appeared wildtype despite inheritance of a defective copy of MSH2.

“While five of the msh2 del131 strains displayed the predicted hypermutator phenotype, surprisingly two of the spores (#4 and 5) appeared wildtype despite inheritance of a defective copy of MSH2.”

May combine two claims.

Direct reversion to functional adenine biosynthesis was more common than additional inactivating mutations, with 37/42 (88%) white colonies demonstrating adenine prototrophy.

“direct reversion to functional adenine biosynthesis was more common than additional inactivating mutations, with 37/42 (88%) white colonies demonstrating adenine prototrophy (Figure 6E).”

May combine two claims.

Shifts in homopolymer runs in the R272 branch account for only 0.84% of the variants, while in ICB107 they account for 45.3%, exceeding even the proportion of SNPs.

“While shifts in homopolymer runs in the R272 branch account for only 0.84% of the variants, in ICB107 they account for 45.3%, exceeding even the proportion of SNPs.”

May combine two claims.

The VGIIa-like hypermutator is derived and not ancestral to the Pacific Northwest outbreak.

“The VGIIa-like hypermutator is derived and not ancestral to the Pacific Northwest outbreak.”

May combine two claims.

Whole genome sequencing confirmed that both segregants carried an extra copy of one chromosome, suggesting that a 1N + 1 aneuploidy was causing temperature sensitivity.

“Whole genome sequencing confirmed that both segregants carried an extra copy of one chromosome, suggesting that a 1N + 1 aneuploidy was causing temperature sensitivity (Figure 5B).”

May combine two claims.

Longer base runs appear to be less stable than shorter runs, as approximately 24% of the mutations occur within the context of a nine base homopolymer run.

“it is apparent that longer base runs appear to be less stable than shorter runs, as approximately 24% of the mutations occur within the context of a nine base homopolymer run (Figure 3C).”

May combine two claims.

These indels resulted in nonsense mutations and resistance to both FK506 and rapamycin.

“These indels resulted in nonsense mutations and resistance to both FK506 and rapamycin.”

May combine two claims.

While a high rate of transitions as compared to transversions was maintained in the hypermutator branch, there was a slight reduction in the frequency of A->G and T->C mutations relative to C->T and G->A mutations.

“While a high rate of transitions as compared to transversions was maintained in the hypermutator branch, there was a slight reduction in the frequency of A->G and T->C mutations relative to C->T and G->A mutations (Figure 3A).”

May combine two claims.

These hypermutator strains were isolated over a period of fifteen years from two different continents and include both clinical and environmental strains.

“These strains were isolated over a period of fifteen years from two different continents and include both clinical and environmental strains.”

May combine two claims.

Growth defects were observed for the msh2 mutants at either 30°C or 37°C, suggesting that the msh2 mutation is deleterious under rich growth conditions.

“Instead, growth defects were observed for the msh2 mutants at either 30°C or 37°C, suggesting that the msh2 mutation is deleterious under rich growth conditions (Figure 7C).”

May combine two claims.

The mutation spectrum was similar to that in S. cerevisiae, as homopolymer run indels were responsible almost exclusively for mutations within run-containing genes in msh2 mutants, but not in wildtype strains.

“The mutation spectrum was also similar, as homopolymer run indels were responsible almost exclusively for mutations within run-containing genes in msh2 mutants, but not in wildtype strains as previously reported in S. cerevisiae (Tran et al., 1997).”

• cites Tran et al., 1997Not checked yet

  Not checked in the free run.

May combine two claims.

Hypermutators can allow both inactivating mutations in genes, and reversion of those mutations to the wildtype.

“We also showed that hypermutators can allow both inactivating mutations in genes, and reversion of those mutations to the wildtype.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Mismatch repair-defective mutator strains are characterized by particularly high rates of slippage within homopolymer runs, and these occur at run lengths that are common within fungal genomes.

“Mismatch repair-defective mutator strains are characterized by particularly high rates of slippage within homopolymer runs, and these occur at run lengths that are common within fungal genomes.”

May combine two claims.

The most severe temperature defect was observed in ICB107, a strain isolated from a patient in Brazil and also the strain with the largest number of mutations separating it from the VGIIa group, which would support the second hypothesis.

“The most severe temperature defect was observed in ICB107, a strain isolated from a patient in Brazil and also the strain with the largest number of mutations separating it from the VGIIa group, which would support the second hypothesis.”

May combine two claims.

The FRR1 gene encodes the FKBP12 homolog responsible for the antifungal action of both FK506 and rapamycin and has a run of seven cytosines (7C) within its coding region.

“the FRR1 gene that encodes the FKBP12 homolog responsible for the antifungal action of both FK506 and rapamycin has a run of seven cytosines (7C) within its coding region.”

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

The msh2 defect results in dramatic phenotypic diversity from inactivation and possibly also activation or reactivation of genes.

“We predict that this results in dramatic phenotypic diversity from inactivation and possibly also activation or reactivation of genes.”

Classified as probably not a substantial claim.

FRR1 is an appropriate locus to test the effect of mismatch repair mutants on homopolymers containing genes.

“suggesting that FRR1 is an appropriate locus to test the effect of mismatch repair mutants on homopolymers containing genes (Figure 3C).”

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

Mating could reintroduce a functional MSH2 allele.

“Likewise, mating could also reintroduce a functional MSH2 allele.”

May combine two claims.

Antifungal treatment and transitions from environment to host are likely to provide opportunities for adaptive mutation and favor mutator alleles.

“Antifungal treatment and transitions from environment to host are likely to provide opportunities for adaptive mutation and favor mutator alleles.”

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

This may represent an unselected drug resistance phenotype potentiated by the msh2 defect.

“This may represent an unselected drug resistance phenotype potentiated by the msh2 defect.”

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

The majority of 5-FOA resistance in Cryptococcus neoformans is acquired through mutation of URA5.

“The majority of 5-FOA resistance in Cryptococcus neoformans is acquired through mutation of URA5 (Kwon-Chung et al., 1992)”

• cites Kwon-Chung et al., 1992Not checked yet

  Not checked in the free run.

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

Elevated mutation rates are a common adaptive mechanism in bacteria, but are typically thought of as transient states that allow beneficial mutations in the short term but are selected against in the long term.

“Elevated mutations rates are a common adaptive mechanism in bacteria, but are typically thought of as transient states that allow beneficial mutations in the short term but are selected against in the long term (Wielgoss et al., 2013).”

• cites Wielgoss et al., 2013Not checked yet

  Not checked in the free run.

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

In the rare cases where the incompatible arrangement is found in nature, additional suppressors have arisen to restore wildtype mutation rate, suggesting that hypermutator phenotypes may not be tolerated over long periods of time.

“However, in the rare cases where the incompatible arrangement is found in nature, additional suppressors have arisen to restore wildtype mutation rate, suggesting that hypermutator phenotypes may not be tolerated over long periods of time (Bui et al., 2017; Skelly et al., 2017).”

• cites Bui et al., 2017Not checked yet

  Not checked in the free run.

• cites Skelly et al., 2017Not checked yet

  Not checked in the free run.

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

Bacteria solve the problem of transient mutator states through horizontal gene transfer of genes from the mismatch repair pathway, allowing the initial beneficial mutation to be separated from the deleterious mutator allele.

“Bacteria solve this problem through horizontal gene transfer of genes from the mismatch repair pathway, allowing the initial beneficial mutation to be separated from the deleterious mutator allele (Denamur et al., 2000).”

• cites Denamur et al., 2000Not checked yet

  Not checked in the free run.

May combine two claims.

Hypermutator phenotypes are not limited to prokaryotes, but may represent a frequent avenue to evolutionary change and phenotypic diversity in eukaryotic microbes.

“This work suggests that hypermutator phenotypes are not limited to prokaryotes, but may represent a frequent avenue to evolutionary change and phenotypic diversity in eukaryotic microbes as well.”

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

In S. cerevisiae, incompatible alleles of PMS1 and MLH1 result in elevated mutation rates if present in combination.

“In S. cerevisiae, incompatible alleles of PMS1 and MLH1 result in elevated mutation rates if present in combination (Heck et al., 2006; Bui et al., 2015).”

• cites Heck et al., 2006Not checked yet

  Not checked in the free run.

• cites Bui et al., 2015Not checked yet

  Not checked in the free run.

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

In bacteria, the hypermutator state is often transient and wildtype mismatch repair is often restored via horizontal gene transfer.

“In bacteria, the hypermutator state is often transient and wildtype mismatch repair is often restored via horizontal gene transfer (Denamur et al., 2000).”

• cites Denamur et al., 2000Not checked yet

  Not checked in the free run.

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

In this model, thermotolerance could be selected by high ambient temperatures in a non-host environment.

“In this model, thermotolerance could be selected by high ambient temperatures in a non-host environment (Casadevall et al., 2003).”

• cites Casadevall et al., 2003Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing). May combine two claims.

As the VGIIa outbreak strains continue to adapt to the Pacific Northwest, they will gradually lose virulence potential, like the VGIIa-like lineage, making the outbreak self-limiting over time.

“This could suggest that as the VGIIa outbreak strains continue to adapt to the Pacific Northwest, they will gradually lose virulence potential, like the VGIIa-like lineage, making the outbreak self-limiting over time.”

May combine two claims.

This is congruent with the fact that NIH444 has the shortest divergence from the VGIIa clonal group, while CBS7750 and ICB107 have much longer branches, and is also congruent with the high temperature growth defects we observed.

“This is congruent with the fact that NIH444 has the shortest divergence from the VGIIa clonal group, while CBS7750 and ICB107 have much longer branches, and is also congruent with the high temperature growth defects we observed.”

May combine two claims.

A contributing factor is likely the highly biased mating type distribution in C. deuterogattii, as the vast majority of C. deuterogattii isolates are MATα, with only a handful of MATa isolates described globally.

“A contibuting factor is likely the highly biased mating type distribution in C. deuterogattii, as the vast majority of C. deuterogattii isolates are MATα, with only a handful of MATa isolates described globally (Byrnes et al., 2010).”

• cites Byrnes et al., 2010Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

An alternate hypothesis is that ploidy may play a role in the success of mutator strains.

“An alternate hypothesis is that ploidy may play a role in the success of mutator strains.”

• AssumptionRests on an assumption: C. deuterogattii, C. neoformans, and C. glabrata exist primarily as haploids in nature, while S. cerevisiae is predominantly a diploid.

May combine two claims.

C. deuterogattii is characterized by long periods of mitotic clonal expansion and only intermittent sexual crosses.

“We previously described C. deuterogattii as a species characterized by long periods of mitotic clonal expansion and only intermittent sexual crosses (Billmyre et al., 2014).”

• cites Billmyre et al., 2014Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Moving from a tropical/sub-tropical environment in South America to the more temperate climate of the Pacific Northwest may relieve this high temperature selection.

“Moving from a tropical/sub-tropical environment in South America to the more temperate climate of the Pacific Northwest may relieve this high temperature selection.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Elevated mutation rates may be selected for by Red Queen interactions with a host, meaning that pathogens like C. glabrata that grow only in association with their hosts would experience higher mutation rates than facultative pathogens like C. deuterogattii, or effectively nonpathogenic yeasts like S. cerevisiae.

“Elevated mutation rates may be selected for by Red Queen interactions with a host, meaning that pathogens like C. glabrata that grow only in association with their hosts would experience higher mutation rates than facultative pathogens like C. deuterogattii, or effectively nonpathogenic yeasts like S. cerevisiae.”

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

This unisexual cycle can result in both de novo variation, but also recombination and admixture at similar levels to that observed in typical bisexual crosses.

“This unisexual cycle can result in both de novo variation, but also recombination and admixture at similar levels to that observed in typical bisexual crosses (Ni et al., 2013; Lin et al., 2005; Sun et al., 2014).”

• cites Ni et al., 2013Not checked yet

  Not checked in the free run.

• cites Lin et al., 2005Not checked yet

  Not checked in the free run.

• cites Sun et al., 2014Not checked yet

  Not checked in the free run.

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

A putative recurrent infection of Cryptococcus neoformans was recently demonstrated to contain nonsense mutations in MSH2, MSH5, and RAD5, predicted to result in a mutator phenotype.

“In fact, a putative recurrent infection of Cryptococcus neoformans was recently demonstrated to contain nonsense mutations in MSH2, MSH5, and RAD5, predicted to result in a mutator phenotype (Rhodes et al., 2017).”

• cites Rhodes et al., 2017Not checked yet

  Not checked in the free run.

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

Two independent transformations of independent overnight cultures of R265 were carried out to isolate independent mutants.

“Two independent transformations of independent overnight cultures of R265 were carried out to isolate independent mutants.”

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

Boyce et al. observed loss of ability to grow at high temperatures in one of three replicates of msh2 mutants in an experimental evolution assay, like that observed here in the VGIIa-like hypermutator clade.

“Interestingly, this study also observed loss of ability to grow at high temperatures in one of three replicates of msh2 mutants in an experimental evolution assay, like that observed here in the VGIIa-like hypermutator clade.”

• cites Boyce et al., 2017 sourceNot checked yet

  Not checked in the free run.

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

Many pathogenic fungi are thought to be 'accidental' pathogens where virulence factors are selected for by other environmental factors.

“However, many pathogenic fungi are thought to be ‘accidental’ pathogens where virulence factors are selected for by other environmental factors.”

• cites Casadevall et al., 2003Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Diploid strains are buffered from the effects of loss of function mutations like those observed in homopolymer runs in this study, which may reduce the supply of large effect beneficial mutations for mutator alleles to hitchhike upon in diploids.

“Diploid strains are buffered from the effects of loss of function mutations like those observed in homopolymer runs in this study, which may reduce the supply of large effect beneficial mutations for mutator alleles to hitchhike upon in diploids.”

• AssumptionRests on an assumption: C. deuterogattii, C. neoformans, and C. glabrata exist primarily as haploids in nature, while S. cerevisiae is predominantly a diploid.

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

This could elevate linkage between mutator alleles and the beneficial mutations they elicit, but also eliminate the ability to separate those beneficial mutations from additional deleterious alleles.

“This could elevate linkage between mutator alleles and the beneficial mutations they elicit, but also eliminate the ability to separate those beneficial mutations from additional deleterious alleles.”

Classified as probably not a substantial claim.

Evolutionary theory predicts that the admixture provided by sex in a population nullifies the ability of mutator alleles to hitchhike to high frequency.

“Evolutionary theory predicts that the admixture provided by sex in a population nullifies the ability of mutator alleles to hitchhike to high frequency (Tenaillon et al., 2000).”

• cites Tenaillon et al., 2000Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

This paradigm could also operate in fungi, with sexual recombination replacing direct DNA transfer.

“This paradigm could also operate in fungi, with sexual recombination replacing direct DNA transfer.”

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

This difference could reflect differences between DNA repair in C. neoformans and C. deuterogattii.

“This could reflect differences between DNA repair in C. neoformans and C. deuterogattii.”

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

While these loci are unstable even in wildtype populations, defects in the MMR pathway could enhance this instability and result in increased diversity.

“While these loci are unstable even in wildtype populations, defects in the MMR pathway could enhance this instability and result in increased diversity.”

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

In Cryptococcus deneoformans, which shares a similar biased mating type distribution, this has resulted in the development of a unisexual α-α sexual cycle, dispensing with the obligate need for a MATa partner.

“In Cryptococcus deneoformans, which shares a similar biased mating type distribution, this has resulted in the development of a unisexual α-α sexual cycle, dispensing with the obligate need for a MATa partner (Lin et al., 2005).”

• cites Lin et al., 2005Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based). May combine two claims.

The authors suspect that these episodes are common throughout the evolution of eukaryotic microbes and may be even more common among pathogenic microbes, reflecting their natural history as well as the result of Red Queen host-pathogen conflicts.

“We suspect that these episodes are common throughout the evolution of eukaryotic microbes and may be even more common among pathogenic microbes, reflecting their natural history as well as the result of Red Queen host-pathogen conflicts.”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

This transition is highly important for survival in the face of environmental threats such as amoeba, but compromises pathways responsible for high temperature growth, suggesting that oscillation between RAM+ and RAM- states may allow populations to survive as conditions change.

“This transition is highly important for survival in the face of environmental threats such as amoeba, but compromises pathways responsible for high temperature growth, suggesting that oscillation between RAM+ and RAM- states may allow populations to survive as conditions change.”

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

msh2 mutants of C. neoformans were previously reported to exhibit a modest growth advantage in mouse lungs in pooled signature tagged mutant experiments.

“This is interesting, at least in part, because msh2 mutants of C. neoformans were previously reported to exhibit a modest growth advantage in mouse lungs in pooled signature tagged mutant experiments (Liu et al., 2008).”

• cites Liu et al., 2008 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

These strains may have been able to persist in the Pacific Northwest in spite of the high mutation load because of a bottleneck effect during their introduction.

“These strains may have been able to persist in the Pacific Northwest in spite of the high mutation load because of a bottleneck effect during their introduction.”

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

In C. neoformans, a mutator phenotype has been previously described in a lab-passaged strain notable for frequent mutations in the RAM pathway, which enables a dimorphic transition between pseudohyphal and yeast phase growth, although the molecular basis of the elevated mutation rate is unknown.

“In C. neoformans, a mutator phenotype has been previously described in a lab-passaged strain notable for frequent mutations in the RAM pathway, which enables a dimorphic transition between pseudohyphal and yeast phase growth, although the molecular basis of the elevated mutation rate is unknown (Magditch et al., 2012).”

• cites Magditch et al., 2012Not checked yet

  Not checked in the free run.

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

Transformants were checked for tandem insertions using primers JOHE40500/JOHE40501 and a strain with only a single insertion was chosen and employed for the competition assay.

“Transformants were checked for tandem insertions using primers JOHE40500/JOHE40501 and a strain with only a single insertion was chosen and employed for the competition assay.”

May combine two claims.

Loss of virulence in the VGIIa-like strains may be a consequence of independent private mutations, i.e. each VGIIa-like strain is avirulent for its own reason.

“In addition, our results suggest that loss of virulence in the VGIIa-like strains may be a consequence of independent private mutations, i.e. each VGIIa-like strain is avirulent for its own reason.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Variation could be important in host-pathogen interactions as well.

“In addition, variation could be important in host-pathogen interactions as well.”

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

Past work in S. cerevisiae suggests that diploids adapt more quickly in msh2 mutant backgrounds, rather than less, suggesting that ploidy may play the opposite role, at least in S. cerevisiae.

“However, past work in S. cerevisiae suggests that diploids adapt more quickly in msh2 mutant backgrounds, rather than less, suggesting that ploidy may play the opposite role, at least in S. cerevisiae (Thompson et al., 2006).”

• cites Thompson et al., 2006 sourceNot checked yet

  Not checked in the free run.

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

In previous work, CBS7750 and ICB107 have the most substantial virulence defects, while NIH444 has a more modest defect.

“In previous work, CBS7750 and ICB107 have the most substantial virulence defects, while NIH444 has a more modest defect (Byrnes et al., 2010).”

• cites Byrnes et al., 2010Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Fungal contingency loci could be critical for responses to antifungal agents, both in the environment and the clinical setting.

“Fungal contingency loci could be critical for responses to antifungal agents, both in the environment and the clinical setting.”

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

Boyce et al. also observed changes in stability of homopolymer runs, similar to those described here but in a second homopolymer-containing locus.

“They also observed changes in stability of homopolymer runs, similar to those described here but in a second homopolymer-containing locus.”

• cites Boyce et al., 2017 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The loss of ability to grow under thermal stress observed in the VGIIa-like strains may be adaptive for the majority of their growth conditions in the environment, but also render them relatively avirulent in mammals.

“Consequently, the loss of ability to grow under thermal stress observed in the VGIIa-like strains may be adaptive for the majority of their growth conditions in the environment, but also render them relatively avirulent in mammals.”

• AssumptionRests on an assumption: The ability to grow at high temperatures is a critical component of virulence for fungal pathogens.

May combine two claims.

This provides further evidence that microbial lifestyle may be affecting the frequency of mutator strains, with pathogens being more likely to evolve elevated mutation rate, perhaps in response to drug or host challenges.

“This provides further evidence that microbial lifestyle may be affecting the frequency of mutator strains, with pathogens being more likely to evolve elevated mutation rate, perhaps in response to drug or host challenges.”

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

All animal work was performed according to the guidelines of NIH and Duke University IACUC under protocol number A245-13-09.

“All of the animal work was performed according to the guidelines of NIH and Duke University Institutional Animal Care and Use Committee (IACUC) under protocol number A245-13-09.”

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

C. deuterogattii is not an obligate pathogen and can cycle between an environmental lifestyle and an infectious lifestyle as an 'accidental pathogen'.

“C. deuterogatttii is not an obligate pathogen and can cycle between an environmental lifestyle and an infectious lifestyle as an ‘accidental pathogen’ (Springer et al., 2012).”

• cites Springer et al., 2012Not checked yet

  Not checked in the free run.

May combine two claims.

The mutator strains are better able to adapt to growth in the mouse lung, but not in a way that correlates directly with virulence in a dissemination and survival model of virulence.

“This suggests that the mutator strains are better able to adapt to growth in the mouse lung, but not in a way that correlates directly with virulence in a dissemination and survival model of virulence.”

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

Boyce et al. obtained similar results in C. neoformans to those reported here, with msh2Δ and mlh1Δ mutants appearing neutral in a murine infection model, and pms1Δ mutants even relatively less virulent.

“Boyce et al. obtained similar results in C. neoformans to those reported here, with msh2Δ and mlh1Δ mutants appearing neutral in a murine infection model, and pms1Δ mutants even relatively less virulent (Boyce et al., 2017).”

• cites Boyce et al., 2017 sourceNot checked yet

  Not checked in the free run.

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

The widespread availability of whole genome sequencing could now allow changes in mutation spectrum to be used to detect episodic hypermutation throughout entire populations of microbes.

“The widespread availability of whole genome sequencing could now allow changes in mutation spectrum to be used to detect episodic hypermutation throughout entire populations of microbes.”

May combine two claims.

The VGIIa-like lineage is a successful and relatively long-lived hypermutator lineage, capable of both disseminating over a large area and persisting in the environment.

“This is distinct from the mutators identified in C. glabrata, and suggests that the VGIIa-like lineage is a successful and relatively long-lived hypermutator lineage, capable of both disseminating over a large area and persisting in the environment.”

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

A second recent study identified two other hypermutator strains from clinical isolates of C. neoformans.

“In addition, a second recent study also identified two other hypermutator strains from clinical isolates of C. neoformans as well (Boyce et al., 2017).”

• cites Boyce et al., 2017 sourceNot checked yet

  Not checked in the free run.

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

In S. cerevisiae, tandem repeats are enriched within cell surface genes, which could enable alteration of antigenic diversity during pathogenic interactions.

“For example, in S. cerevisiae, tandem repeats are enriched within cell surface genes, which could enable alteration of antigenic diversity during pathogenic interactions (Verstrepen et al., 2005).”

• cites Verstrepen et al., 2005 sourceNot checked yet

  Not checked in the free run.

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

Boyce et al. examined changes in mutation rate and spectrum with similar results to those presented here, although with a much larger induction of mutation rate in URA5 than reported here (around 200 fold vs 6.8 fold).

“Boyce et al. went on to examine changes in mutation rate and spectrum with similar results to those presented here, although with a much larger induction of mutation rate in URA5 than reported here (around 200 fold vs 6.8 fold).”

• cites Boyce et al., 2017 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The VGIIa-like lineage is derived and may represent either an alternative pathway of adaptation or potentially an accelerated stage of the evolutionary trajectory of the outbreak.

“Rather, the VGIIa-like lineage is derived and may represent either an alternative pathway of adaptation or potentially an accelerated stage of the evolutionary trajectory of the outbreak.”

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based).

Alternatively, the defects could simply reflect a decrease in viability caused by a long period of growth with an elevated mutation rate, resulting in mutational meltdown.

“Alternatively, the defects could simply reflect a decrease in viability caused by a long period of growth with an elevated mutation rate, resulting in mutational meltdown.”

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

These events are difficult to detect if the hypermutator allele is purged from the population by selection after it is separated from its linked beneficial allele.

“However, these events are difficult to detect if the hypermutator allele is purged from the population by selection after it is separated from its linked beneficial allele.”

Classified as probably not a substantial claim.

As strains are introduced to new locales, they may need to survive and adapt via mitotic growth without sexual crosses for long periods of time.

“Consequently, as strains are introduced to new locales, they may need to survive and adapt via mitotic growth without sexual crosses for long periods of time.”

• AssumptionRests on an assumption: No laboratory unisexual cycle has been observed in C. deuterogattii to this point.

Classified as probably not a substantial claim.

Gene replacement was confirmed using in-gene, 5' junction, 3' junction, and spanning PCRs.

“Gene replacement was confirmed using in-gene, 5' junction, 3' junction, and spanning PCRs.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

GATK's VariantFiltration module was used to hard filter variants as suggested using the following expressions: 'QD <2.0 || FS >60.0 || MQ <40.0 ' for SNPs and 'QD <2.0 || FS >200.0' for indels.

“GATK's VariantFiltration module was used to hard filter variants as suggested using the following expressions: ‘QD <2.0 || FS >60.0 || MQ <40.0”

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

Variants resulting from erroneous calls in repetitive regions were manually removed.

“Variants resulting from erroneous calls in repetitive regions were manually removed.”