ABSTRACT Filamentation, a central virulence trait of the fungal pathogen Candida albicans, is required for host cell damage, tissue invasion, and biofilm formation. The transcription factor Bcr1 was first found to be required for biofilm formation but not filamentation. Subsequent studies revealed a negative role for Bcr1 in filamentation in opaque cells, a cell type that is required for mating, under hypoxic conditions, and in a wor2 Δ/Δ mutant background. Here we characterize a new context in which Bcr1 negatively regulates filamentation and present its associated gene expression impact. We compared the wild-type and bcr1 Δ/Δ mutant cells in the SC5314 reference strain background at 30°C in glucose media, conditions that do not induce filamentation. The bcr1 Δ/Δ mutant displayed increased invasive growth in a solid medium, and some filamentation ability in a liquid medium. The bcr1 Δ/Δ effect on filamentation was augmented by overexpression of hyphal cyclin gene HGC1, which caused pseudohyphal growth in wild-type, efg1 Δ/Δ, or brg1 Δ/Δ strains, yet caused hyphal growth in the bcr1 Δ/Δ strain. RNA-sequencing (RNA-seq) analysis of 30°C cells shows that the bcr1 Δ/Δ mutant has elevated expression of two genes that drive filamentation: HGC1 and UME6. In published data these same two genes have decreased expression in the bcr1 Δ/Δ mutant at 37°C, the temperature at which biofilm formation is typically assayed. A growing cadre of biofilm/hyphal regulators can have both positive and negative effects on hypha-associated gene expression, including Bcr1, Efg1, Ndt80, and Nrg1. IMPORTANCE Production of filamentous cells is a central virulence trait of the fungal pathogen Candida albicans . Evidence here shows that the transcription factor Bcr1 is a negative regulator of filamentation at low temperature (30°C). Bcr1 affects expression of two drivers of filamentation, the genes HGC1 and UME6. Surprisingly, Bcr1 inhibits their expression at 30°C but stimulates their expression at 37°C. A growing number of filamentation regulators seem to function as both positive and negative regulators of filamentation, depending upon genetic and environmental contexts.

Biofilm formation by the fungus Candida albicans is a central virulence trait that enables colonization of implanted medical devices and mucosal surfaces. Biofilm formation reflects a complex regulatory network, and depends upon multiple master regulators that include transcription factors Efg1 and Ndt80. It is well established that efg1Δ/Δ and ndt80Δ/Δ single gene mutants are defective in biofilm formation. We report here that an efg1Δ/Δ ndt80Δ/Δ double mutant of reference strain SC5314 is able to form a robust biofilm in vitro and in vivo. We refer to the efg1Δ/Δ ndt80Δ/Δ biofilm as an emergent biofilm because this phenotype could not have been predicted from the phenotypes of efg1Δ/Δ or ndt80Δ/Δ single gene mutants. In four additional strain backgrounds, efg1Δ/Δ ndt80Δ/Δ mutants do not form biofilms, but in all strain backgrounds the efg1Δ/Δ ndt80Δ/Δ mutants can form filamentous cells, which are components of biofilms. Emergent biofilm formation is especially pronounced in YPD + FBS medium at 30°C, and RNA-seq under those conditions reveals altered expression in the efg1Δ/Δ ndt80Δ/Δ double mutant of biofilm-related genes: upregulation of BCR1, UME6, and HGC1, and downregulation of ALS3, BRG1, and HWP1. These gene expression changes suggest that the emergent biofilm program is partially distinct from the conventional biofilm program. This inference is supported by functional analysis: emergent biofilm formation is independent of Brg1, Rob1, Tec1, and Wor3, all of which have positive roles in conventional biofilm formation. Emergent biofilm formation depends upon the hyphal cyclin Hgc1, the biofilm transcription factors Bcr1 and Ume6, and the Bcr1/Ume6-activated adhesin gene FLO9. The seemingly simple emergent biofilm program may represent a primordial surface colonization strategy.

The microbiome-associated fungus Candida albicans is an opportunistic pathogen. Virulence traits include its ability to produce biofilm, a surface-associated growth form that persists on mucosae and implanted medical devices. C. albicans clinical isolates vary in ability to produce biofilm and the constituent filamentous cell types. Here, we focus on two transcription factors that promote filamentation and biofilm formation, Ndt80 and Ume6. We address two questions. First, how variable is the impact of Ndt80 among C. albicans strains? Second, what is the genetic interaction between NDT80 and UME6? We find that Ndt80 is required for filamentation and biofilm formation in five clinical isolates in addition to the reference strain SC5314, where Ndt80 function has been well established. RNA-sequencing (RNA-seq) data indicate that UME6 RNA levels are reduced in an ndt80Δ/Δ mutant, possibly a result of altered RME1 and WOR1 expression, both of which control UME6. Increased expression of UME6 in ndt80Δ/Δ mutants of three strain backgrounds restores filamentation and biofilm formation, though RNA-seq assays indicate that it does not suppress the overall ndt80Δ/Δ gene expression defect. Ndt80 has an additional role in promoting tolerance to the antifungal drug fluconazole, an inhibitor of ergosterol synthesis. This ndt80Δ/Δ phenotype varies considerably among clinical isolates. In three strains tested, increased expression of UME6 in ndt80Δ/Δ mutants enhances their susceptibility to fluconazole. Therefore, our results show an unexpected relationship between Ume6 expression and azole drug sensitivity. To our knowledge, Ume6 has previously been understood to function only in filamentation, biofilm formation, and related processes.IMPORTANCEOur focus is the fungal pathogen Candida albicans. Two traits, biofilm/hypha formation and azole resistance, are major drivers of its infection ability. We examine the roles of two biofilm transcriptional regulators, Ndt80 and Ume6, in several C. albicans clinical isolates. Prior studies in one strain background (SC5314) indicated that Ndt80 controls both biofilm/hypha formation and azole drug susceptibility and that Ume6 controls biofilm/hypha formation. The four new findings here are that (i) Ndt80 effects on fluconazole sensitivity vary considerably with strain background; (ii) Ndt80 is required for filamentation and biofilm formation in multiple clinical isolates; (iii) the Ndt80 target Ume6 contributes to Ndt80 control of filamentation and biofilm formation in multiple clinical isolates; and (iv) Ume6 influences fluconazole vulnerability, the first Ume6 function to our knowledge that is unrelated to filamentation.

Candida albicans is an opportunistic fungal pathogen and a component of the human microbiome. C. albicans virulence traits include biofilm production, which is governed by a large transcriptional network. Mutations of some biofilm regulators cause the same severe biofilm-defective phenotype in multiple clinical isolates. Mutations of others, such as Wor3, Bcr1, Ndt80, and Ume6, have mild or variable phenotypes among clinical isolates. We hypothesized that Wor3 may share functions with another variable-phenotype biofilm regulator. This hypothesis predicts that a double mutant lacking Wor3 and the shared-function regulator will have a severe biofilm defect in all clinical isolates. We observed that a wor3Δ/Δ bcr1Δ/Δ double mutant has a severe biofilm defect in vitro in 5 strain backgrounds tested. It also has a severe oral biofilm defect in a mouse oropharyngeal candidiasis model in the SC5314 strain background. RNA-seq data indicate that 5 genes encoding cell surface/secreted proteins are upregulated in wor3Δ/Δ, bcr1Δ/Δ, and wor3Δ/Δ bcr1Δ/Δ strains: CWH8, DAG7, JEN2, PGA6, and YWP1. Deletion mutations of CWH8, DAG7, PGA6, or YWP1 enable biofilm formation in vitro in an SC5314-derived wor3Δ/Δ bcr1Δ/Δ strain, and deletion of YWP1 enables biofilm formation in vitro in wor3Δ/Δ bcr1Δ/Δ strains from 4 other genetic backgrounds. YWP1 has been shown to have antibiofilm activity previously, but CWH8, DAG7, and PGA6 are newly described antibiofilm genes. Our study illustrates the value of strain variation considerations for gene function analysis and the importance of repression targets of biofilm regulators. In addition, our results expand the number of antibiofilm genes.

Biofilm formation is vital for the survival and pathogenicity of the fungus Candida albicans. Expression of biofilm-promoting genes is coordinated by a transcription factor network that governs the yeast-filament transition and other processes. A second cell type transition, the white-opaque transition, is coordinated by its own transcription factor network. Initial studies suggested that the 2 transcriptional networks have a mutually exclusive relationship, driven in part by reciprocal repression of biofilm regulator Efg1 and white-opaque regulator Wor1. However, recent studies have shown that biofilm regulators and white-opaque regulators can promote one another's function in many situations. Here, we test the function of white-opaque regulator Wor2 in biofilm formation. We find that Wor2 has a functional relationship with biofilm regulator Bcr1. We characterized the phenotype of bcr1Δ/Δ wor2Δ/Δ mutants in 5 strain backgrounds and conducted RNA-sequencing (RNA-seq) analysis in the SC5314 reference strain background. The combined Bcr1-Wor2 impact is unexpected: although Bcr1 is known as a positive regulator of biofilm formation and biofilm-related genes, the bcr1Δ/Δ wor2Δ/Δ mutants have increased biofilm or filamentation capacity, depending on the strain, and increased expression of biofilm-related genes. Those properties suggest that Wor2 and Bcr1 function together as negative regulators of biofilm formation. Our findings argue that Bcr1 can act as both a positive and negative regulator of downstream effector genes in the biofilm network and establish a new connection between the biofilm and white-opaque regulatory networks.

Candida albicans, a fungal commensal and pathogen, occupies diverse niches in the human host. Its broad metabolic repertoire is critical for its survival. The model yeast Saccharomyces cerevisiae provides a starting point for analysis of C. albicans physiology and regulatory circuitry, but there are many examples of rewired transcription factors that govern different processes in the two organisms. We focus here on Adr1, which in S. cerevisiae promotes alternative carbon source utilization and in C. albicans promotes ergosterol synthesis. We find that C. albicans Adr1 is also required for growth on citrate and compounds that feed into the citric acid cycle, like glutamate and malate. RNA-sequencing (RNA-seq) shows that predicted citrate metabolic genes, representing both the citric acid cycle and gluconeogenesis, are downregulated in an adr1Δ/Δ mutant. In fact, the three Adr1-dependent genes HGT17, MDH1, and PCK1 are required for growth on citrate, as indicated by deletion mutant phenotypes. The hyphal regulator EED1 has a negative role in citrate utilization, and an adr1Δ/Δ eed1Δ/Δ double mutant is defective for growth on citrate. This result argues that Adr1 acts downstream or independently of Eed1 to govern citrate utilization. C. albicans Adr1 is rewired compared to its S. cerevisiae ortholog to govern the ability to use citrate, which S. cerevisiae lacks, and potentially to respond to Eed1, for which S. cerevisiae lacks an ortholog.IMPORTANCECandida albicans is a major fungal pathogen of humans, and its ability to grow on a range of carbon sources is critical for pathogenicity. Here, we find that a known regulator of ergosterol synthesis, Adr1, is also required to use citrate as a carbon source. Adr1 acts downstream or independently of Eed1, a well-known regulator of hypha formation and citrate utilization.

Biofilms of the fungal pathogen Candida albicans can form on implanted medical devices and contribute to fungal virulence and are recalcitrant to antifungal therapy. The transcription factor Ume6 directs hyphal cell elongation and thus promotes biofilm formation in C. albicans. However, how exactly this key biofilm and virulence regulator functions has remained unclear. Here RNA sequencing and chromatin immunoprecipitation with sequencing data show that Ume6 binds to and activates multiple biofilm-relevant genes. Ume6-associated sequence motifs correspond to binding sites for biofilm master regulators Efg1 and Ndt80, and hypoxic response regulator Upc2. Co-immunoprecipitation assays show the existence of Ume6-Efg1, Ume6-Ndt80 and Ume6-Upc2 protein complexes. Promoter binding of Ume6 is partially dependent upon Efg1, Ndt80 or Upc2, as is Ume6 target gene activation, thus indicating that the protein complexes function to drive Ume6-target gene interaction. Ume6 therefore acts as a bridge that connects the hyphal morphogenesis and adherence genes that shape biofilm architecture and the hypoxic response genes required for growth in the low-oxygen biofilm environment. These findings are vital for our understanding of the pathobiology of C. albicans and could open the way to new treatment options.

The opportunistic human pathogen Candida albicans is an important cause of nosocomial infections, in large part because of its propensity to form biofilms on indwelling medical devices such as catheters. The formation of these biofilms is controlled by a complex transcriptional network and involves over a thousand genes, many of which are uncharacterized. We have investigated three genes (ORF19.4654, ORF19.7608, and PBR1), found only in C. albicans and closely related species, that are highly induced under biofilm conditions and encode small proteins with N-terminal signal sequences. Through the construction of fluorescent protein fusions, we have examined the location of the encoded proteins in both planktonic and biofilm cells. Orf19.4654-Scarlet and Pbr1-Scarlet were localized to the vacuole under both conditions. In contrast, the Orf19.7608-GFP fusion generated a punctate pattern only under biofilm conditions and was designated Ppp1 (Punctate Pattern Protein 1). The Ppp1-GFP puncta were similar in location, stability, and size to those formed by the eisosome subunit Sur7, but co-localization studies suggest that Ppp1 and Sur7 define separate elements. The PPP1 mutation does not cause a distinct phenotype under various stress conditions or in the presence of antifungals and does not impact biofilm formation and biomass. These data suggest that while the expression and cellular localization of Ppp1 appear controlled by conditions generating biofilms, and define a unique subcellular localization pattern, Ppp1 protein function is not essential for biofilm formation.

Hyphal growth is strongly associated with virulence in the human fungal pathogen Candida albicans. While hyphal transcriptional networks have been the subject of intense study, relatively little is known about post-transcriptional regulation. Previous work reported that P-Body (PB) factors Dhh1 and Edc3 were required for C. albicans virulence and filamentation, suggesting an essential role for post-transcriptional regulation of these processes. However, the molecular roles of these factors have not been determined. To further study the function of PB factors in filamentation, we generated homozygous deletions of DHH1 and EDC3 in diverse prototrophic clinical strains using transient CRISPR-Cas9. Homozygous DHH1 deletion strongly impaired growth, altered filamentation, and exhibited unusual colony morphology in response to heat stress in five strain backgrounds. Using RNA-seq, we found DHH1 deletion disrupts the regulation of thousands of genes under both yeast and hyphal growth conditions in SC5314 and P57055. This included upregulation of many stress response genes in the absence of external stress, similar to deletion of the S. cerevisiae DHH1 homolog. In contrast, we found EDC3 was not required for heat tolerance or filamentation in diverse strains. These results support a model in which DHH1, but not EDC3, represses hyphal stress response transcripts in yeast and remodels the transcriptome during filamentation. Our work supports distinct requirements for specific mRNA decay factors, bolstering evidence for post-transcriptional regulation of filamentation in C. albicans.

The fungal pathogen Candida albicans can infect diverse tissues, a reflection of its broad metabolic repertoire. The transcription factor Adr1 is required for utilization of several citric acid cycle intermediates that are found in tissue. Many Adr1-activated genes encode enzymes with well-defined roles in citrate metabolism or gluconeogenesis. Here, we focus on HGT17 (C4_01070W, orf19.4682), an Adr1-activated gene that encodes a possible citrate transporter. We provide two lines of evidence that HGT17 is a key functional target of Adr1. First, forced expression of HGT17 in an adr1Δ/Δ mutant improves growth on citrate as a carbon source. Second, hgt17Δ/Δ and adr1Δ/Δ mutants incubated in citrate medium present similar gene expression defects compared to the wild type. Noteworthy is down-regulation in both mutants of citric acid cycle genes, glycolysis/gluconeogenesis genes, and ergosterol synthesis genes. These common features may reflect a specific effect of citrate as an inducer of citric acid cycle enzymes or a global effect of carbon and energy limitation. In either case, the results argue that reduced HGT17 expression contributes substantially to the impact of an adr1Δ/Δ mutation on growth and gene expression.

Central carbon metabolism is vital for the proliferation of Candida albicans, a fungus that is prominent as a commensal and pathogen. Glycolytic genes are activated by overlapping activities of the transcription factors Tye7 and Gal4, as shown by studies in the SC5314 genetic background. However, regulatory relationships can vary among C. albicans isolates. Here, we analyzed Tye7- and Gal4-related phenotypes in five diverse clinical isolates of C. albicans. We tested growth properties and gene expression impact through Nanostring profiling and, for the two strains SC5314 and P87, RNA sequencing. Our results lead to three main conclusions. First, the functional redundancy of Tye7 and Gal4 for glycolytic gene activation is preserved among all strains tested. Second, at the gene expression level, strain P87 is an outlier with regard to tye7Δ/Δ impact, and strain SC5314 is an outlier with regard to gal4Δ/Δ impact. Third, while Gal4 is well known to be dispensable for induction of the GAL1, GAL7, and GAL10 galactose-specific metabolic genes, we find that gal4Δ/Δ mutants of several strains have a mild galactose fermentation defect, as assayed by growth on galactose with the respiration inhibitor antimycin A. Our findings indicate that even a central metabolic regulatory network is subject to strain variation and illustrates an unexpected genotype-phenotype relationship.The fungal commensal and pathogen Candida albicans rely upon metabolic flexibility to colonize and infect host niches. Central carbon metabolism is governed by two regulators, Tye7 and Gal4, as defined in the reference strain SC5314. Here, we have explored the impact of Tye7 and Gal4 on carbon utilization and gene expression across five diverse C. albicans clinical isolates. Novel aspects of this study are the finding that even a central metabolic regulatory network is subject to strain variation and the observation of an unexpected mutant phenotype.

Candida albicans is a prominent fungal pathogen that can infect the bloodstream and deep tissues. One key pathogenicity trait is the ability to transition between yeast and hyphal growth. Hyphae are critical for the formation of biofilms, which in turn enable device-associated infection. Among signals that drive hypha formation is the presence of hemin, an oxidized Fe(III)-containing heme derivative found in blood. In this study, we asked 4 questions. First, how uniform is the filamentation response to hemin among C. albicans strains? We tested 26 diverse isolates and found that the strength of a strain's filamentation response to hemin reflected its filamentation level in the absence of hemin. Second, does hemin induce biofilm formation? Hemin biofilm induction was evident in 5 out of 10 isolates tested, including most of the weaker biofilm formers tested. Third, what is the gene expression response to hemin? We compared RNA-seq data for type strain SC5314 grown in pH 5.5 minimal media with or without hemin. We also compared that response to SC5314 grown in pH 7.0 minimal media, where it undergoes well-studied pH-dependent filamentation. We found a common set of 72 genes with upregulated RNA levels in response to both signals, including many known hypha-associated genes. Surprisingly, overlap among those 72 genes with 2 recent consensus definitions of hypha-associated genes was limited to only 16 genes. Fourth, which regulators govern hemin-induced filamentation? A mutant survey indicated that the response depends upon filamentation regulators Efg1, Brg1, and Rim101, but not upon heme acquisition regulator Hap1 or its target genes HMX1, RBT5, PGA10, PGA7, and CSA2. These findings argue that hemin induces hypha formation independently of its utilization.

ABSTRACT Iron acquisition is critical for pathogens to proliferate during invasive infection, and the human fungal pathogen Candida albicans is no exception. The iron regulatory network, established in reference strain SC5314 and derivatives, includes the central player Sef1, a transcription factor that activates iron acquisition genes in response to iron limitation. Here, we explored potential variation in this network among five diverse C. albicans strains through mutant analysis, Nanostring gene expression profiling, and, for two strains, RNA-Seq. Our findings highlight four features that may inform future studies of natural variation and iron acquisition in this species. (i) Conformity: In all strains, major iron acquisition genes are upregulated during iron limitation, and a sef1 Δ/Δ mutation impairs that response and growth during iron limitation. (ii) Response variation: Some aspects of the iron limitation response vary among strains, notably the activation of hypha-associated genes. As this gene set is tied to tissue damage and virulence, variation may impact the progression of infection. (iii) Genotype-phenotype variation: The impact of a sef1 Δ/Δ mutation on cell wall integrity varies, and for the two strains examined the phenotype correlated with sef1 Δ/Δ impact on several cell wall integrity genes. (iv) Phenotype discovery: DNA repair genes were induced modestly by iron limitation in sef1 Δ/Δ mutants, with fold changes we would usually ignore. However, the response occurred in both strains tested and was reminiscent of a much stronger response described in Cryptococcus neoformans , a suggestion that it may have biological meaning. In fact, we observed that the iron limitation of a sef1 Δ/Δ mutant caused recessive phenotypes to emerge at two heterozygous loci. Overall, our results show that a network that is critical for pathogen proliferation presents variation outside of its core functions. IMPORTANCE A key virulence factor of Candida albicans is the ability to maintain iron homeostasis in the host where iron is scarce. We focused on a central iron regulator, SEF1 . We found that iron regulator Sef1 is required for growth, cell wall integrity, and genome integrity during iron limitation. The novel aspect of this work is the characterization of strain variation in a circuit that is required for survival in the host and the connection of iron acquisition to genome integrity in C. albicans .

Biofilm formation by the fungal pathogen Candida albicans is the basis for its ability to infect medical devices. The metabolic gene ERG251 has been identified as a target of biofilm transcriptional regulator Efg1, and here we report that ERG251 is required for biofilm formation but not conventional free-living planktonic growth. An erg251Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo catheter infection model. In both in vitro and in vivo biofilm contexts, cell number is reduced and hyphal length is limited. To determine whether the mutant defect is in growth or some other aspect of biofilm development, we examined planktonic cell features in a biofilm-like environment, which was approximated with sealed unshaken cultures. Under those conditions, the erg251Δ/Δ mutation causes defects in growth and hyphal extension. Overexpression in the erg251Δ/Δ mutant of the paralog ERG25, which is normally expressed more weakly than ERG251, partially improves biofilm formation and biofilm hyphal content, as well as growth and hyphal extension in a biofilm-like environment. GC-MS analysis shows that the erg251Δ/Δ mutation causes a defect in ergosterol accumulation when cells are cultivated under biofilm-like conditions, but not under conventional planktonic conditions. Overexpression of ERG25 in the erg251Δ/Δ mutant causes some increase in ergosterol levels. Finally, the hypersensitivity of efg1Δ/Δ mutants to the ergosterol inhibitor fluconazole is reversed by ERG251 overexpression, arguing that reduced ERG251 expression contributes to this efg1Δ/Δ phenotype. Our results indicate that ERG251 is required for biofilm formation because its high expression levels are necessary for ergosterol synthesis in a biofilm-like environment.

ABSTRACT Prominent virulence traits of Candida albicans include its ability to produce filamentous hyphal cells and grow as a biofilm. These traits are under control of numerous transcription factors (TFs), including Brg1 and Rme1. In the reference strain SC5314, a brg1 Δ/Δ mutant has reduced levels of biofilm/filament production; a brg1 Δ/Δ rme1 Δ/Δ double mutant has wild-type levels of biofilm/filament production. Here, we asked whether this suppression relationship is preserved in four additional strain backgrounds: P76067, P57055, P87, and P75010. These strains represent diverse clades and biofilm/filament production abilities. We find that a rme1 Δ/Δ mutation restores biofilm/filament production in a brg1 Δ/Δ mutant of P76067, but not in brg1 Δ/Δ mutants of P57055, P87, and P75010. We speculate that variation in activities of two functionally related TFs, Nrg1, and Ume6, may cause the strain-limited impact of the rme1 Δ/Δ mutation. IMPORTANCE Candida albicans is a widespread fungal pathogen. The regulatory circuitry underlying virulence traits is well studied in the reference strain background, but not in other clinical isolate backgrounds. Here, we describe a pronounced example of strain variation in the control of two prominent virulence traits, biofilm formation and filamentation.

Importance Medicare Advantage (MA) has grown significantly over the last decade; however, MA's performance for patients with serious conditions, such as cancer, remains unclear. Objective To compare resource use and care quality between MA and traditional Medicare (TM) beneficiaries undergoing cancer chemotherapy. Design, Setting, and ParticipantsThis cohort study used TM claims and MA encounter records from January 2015 to December 2019. Participants were MA and TM beneficiaries who initiated cancer chemotherapy between January 2016 and December 2019. Inverse probability of treatment weighting balanced characteristics between MA and TM beneficiaries, and regression estimation was used. The analysis was conducted between August 2023 and May 2024. Exposure Chemotherapy initiation after a 1-year washout period. Main Outcomes and Measures Resource use and care quality were measured during a 6-month period following chemotherapy initiation. Resource use was measured using standardized prices for services in both MA and TM, covering hospital inpatient services, outpatient care, Part D drugs, and hospice services. Chemotherapy utilization was examined for Part B chemotherapy, Part B supportive drugs, and Part D chemotherapy. Quality measures included chemotherapy-related emergency department (ED) visits and hospitalizations, avoidable ED visits, preventable hospitalizations during the 6-month episode, and survival days up to 18 months from chemotherapy initiation. Results The study comprised 96 501 MA enrollees contributing to 98 872 episodes (mean [SD] age, 72.9 [7.6] years; 55 859 [56.5%] female; 7371 [7.5%] Hispanic, 14 778 [14.9%] non-Hispanic Black, and 75 130 [75.0%] non-Hispanic White participants) and 206 274 TM beneficiaries, contributing 212 969 episodes (mean [SD] age, 72.7 [8.3] years; 121 263 [56.9%] female; 8356 [3.9%] Hispanic, 16 693 [7.8%] non-Hispanic Black, and 182 228 [85.6%] non-Hispanic White participants). Adjusted total resource use per enrollee during the 6-month episode was $8718 (95% CI, $8343 to $9094) lower in MA than TM ($62 599 vs $71 317). Part B chemotherapy resource use accounted for most of the difference in total resource use, with MA enrollees having $5032 (95% CI, $4772 to $5293) lower use than TM beneficiaries. Lower resource use for Part B chemotherapy in MA was associated with both fewer chemotherapy visits (-1.06 visits; 95% CI, -1.10 to -1.02 visits) and less expensive chemotherapy per visit (-$277; 95% CI, -$275 to -$179). Findings on quality were mixed, but importantly, survival did not differ between MA and TM patients who initiated chemotherapy. Conclusions and Relevance In this cohort study of Medicare beneficiaries with cancer undergoing chemotherapy, MA enrollment was associated with lower resource use but not shorter survival.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

5 Background: The healthcare industry spends more on lobbying than any other industry, with over $700 million spent in 2022. However, healthcare lobbying related specifically to cancer has not been characterized. In the current study, we analyze both overall health sector lobbying spending and oncology-related lobbying spending across both provider and patient organizations. Methods: We obtained lobbying data from OpenSecrets.org and the Federal Election Commission website. We categorized overall health sector lobbying spending as (1) Pharmaceuticals/Health Products, (2) Health Services/HMOs, (3) Hospitals/Nursing Homes, or (4) Health Professionals. We identified and categorized oncology-related lobbying organizations: (1) Oncology Physician Professional Organizations (OPOs, e.g., ASCO); (2) PPS-Exempt Cancer Hospitals; (3) Patient Advocacy Organizations; and (4) Provider Networks (e.g., US Oncology Network). We analyzed temporal trends in lobbying spending, in both overall dollar value (inflation-adjusted 2023 dollars) and in per-physician spending (using AAMC data for number of hematologists/oncologists). Significance was analyzed using a Mann-Kendall Trend Test. Results: Of overall health sector lobbying, Pharmaceuticals/Health Products had the greatest increase in lobbying spending from 2014 to 2022, with an increase from $294 million to $376 million (28%, p = 0.0006). In contrast, lobbying spending by health professionals did not change over this period, remaining at $96 million (p = 0.35). Regarding oncology-related lobbying, per-physician spending by OPOs and PPS-exempt cancer hospitals increased (Table). Overall, OPO lobbying has increased as a percentage of overall physician lobbying from 1.16% in 2014 to 3.76% in 2022 (not shown). Conclusions: While overall health sector lobbying spending by physicians/health professional lobbying has been relatively stable in recent years, lobbying spending by OPOs has increased. Continued efforts to understand the utility and value of lobbying in healthcare and across oncology are needed as the costs of care continue to rise. [Table: see text]

ABSTRACT Major Candida albicans virulence traits include its ability to make hyphae, to produce a biofilm, and to damage host cells. These traits depend upon expression of hypha-associated genes. A gene expression comparison among clinical isolates suggested that transcription factor Rme1 , established by previous studies to be a positive regulator of chlamydospore formation, may also be a negative regulator of hypha-associated genes. Engineered RME1 overexpression supported this hypothesis, but no relevant rme1 Δ/Δ mutant phenotype was detected. We reasoned that Rme1 may function within a specific regulatory pathway. This idea was supported by our finding that an rme1 Δ/Δ mutation relieves the need for biofilm regulator Brg1 in biofilm formation. The impact of the rme1 Δ/Δ mutation is most prominent under static or “biofilm-like” growth conditions. RNA sequencing (RNA-seq) of cells grown under biofilm-like conditions indicates that Brg1 activates hypha-associated genes indirectly via repression of RME1 : hypha-associated gene expression levels are substantially reduced in a brg1 Δ/Δ mutant and partially restored in a brg1 Δ/Δ rme1 Δ/Δ double mutant. An rme1 Δ/Δ mutation does not simply bypass Brg1, because iron homeostasis genes depend upon Brg1 regardless of Rme1. Rme1 thus connects Brg1 to the targets relevant to hypha and biofilm formation under biofilm growth conditions. IMPORTANCE Candida albicans is a major fungal pathogen of humans, and its ability to grow as a surface-associated biofilm on implanted devices is a common cause of infection. Here, we describe a new regulator of biofilm formation, RME1 , whose activity is most prominent under biofilm-like growth conditions.