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Background: Previous studies showed that although the risk of thyroid dysfunction [thyroid immune-related adverse events (irAEs)] induced by anti-programmed cell death-1 antibodies (PD-1-Ab) was as low as 2% to 7% in patients negative for anti-thyroid antibodies (ATAs) at baseline, it was much higher (30%-50%) in patients positive for ATAs. However, whether a similar increase occurs with combination therapy using PD-1-Ab plus anti-cytotoxic T-lymphocyte antigen-4 antibody (CTLA-4-Ab) is unknown., Methods: A total of 451 patients with malignancies treated with PD-1-Ab, CTLA-4-Ab, or a combination of PD-1-Ab and CTLA-4-Ab (PD-1/CTLA-4-Abs) were evaluated for ATAs at baseline and for thyroid function every 6 weeks for 24 weeks after treatment initiation and then observed until the last clinical visit., Results: Of the 451 patients, 51 developed thyroid irAEs after immunotherapy [41 of 416 (9.9%) treated with PD-1-Ab, 0 of 8 (0%) treated with CTLA-4-Ab, and 10 of 27 (37.0%) treated with PD-1/CTLA-4-Abs]. The cumulative incidence of thyroid irAEs was significantly higher in patients who were positive vs negative for ATAs at baseline after both PD-1-Ab [28/87 (32.2%) vs 13/329 (4.0%), P < 0.001] and PD-1/CTLA-4-Abs [6/10 (60.0%) vs 4/17 (23.5%), P < 0.05] treatments. The risk of thyroid irAEs induced by PD-1/CTLA-4Abs, which was significantly higher than that induced by PD-1-Ab, in patients negative for ATAs at baseline was not statistically different from that induced by PD-1-Ab in patients positive for ATAs at baseline., Conclusions: This study showed that the incidence of thyroid irAEs was high and not negligible after PD-1/CTLA-4-Abs treatment even in patients negative for ATAs at baseline., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Penicillium Marneffei: is a thermally dimorphic fungus that causes penicilliosis, and become the third-most-common opportunistic fungal infection in immunocompromised patients in Southeast Asia. Azoles and amphotericin B have been introduced for the treatment, however, it is important to investigate possible mechanisms of azole resistance for future treatment failure. We identified 177 putative MFS transporters and classified into 17 subfamilies. Among those, members of the Drug:H + antiporter 1 subfamily are known to confer resistance to antifungals. Out of 39 paralogs, three (encoded by PmMDR1, PmMDR2 , and PmMDR3 ) were heterologously overexpressed in S. cerevisiae AD∆ conferred resistance to various drugs and compounds including azoles, albeit to different degrees. Pm MDR1-expressing strain showed resistance to the broadest range of drugs, followed by the Pm MDR3, and Pm MDR2 conferred weak resistance to a limited range of drugs. We conclude that PmMDR1 and PmMDR3 , may be able to serve as multidrug efflux pumps.

Objective: The impact of 'standard full-staged radical surgery (SRS)' on overall survival (OS) in elderly patients with early-stage epithelial ovarian cancer (EOC) remains unclear. In the current study, we investigated the impact of SRS on OS in elderly patients with early-stage EOC in a multicentric analysis using a propensity score (PS)-matching technique., Methods: Between 1986 and 2017, 3227 patients with EOC were registered and accumulated by the Tokai Ovarian Tumor Study Group, consisting of 14 collaborating institutions, after a central pathological review. Among them, 204 elderly patients aged older than or equal to 65 years who had a stage I EOC were analyzed, including 72 patients who had received SRS (Group I) and 132 who had undergone non-SRS limited surgery (Group II). Oncologic outcomes were compared between the two groups using a PS-matching technique to adjust for various clinicopathologic risk factors., Results: The median follow-up duration of all surviving patients was 55.9 months. Consequently, 54 patients (26.5%) developed recurrence. In addition, 33 patients (16.2%) died of the disease. In the original cohort, the 5-year OS rates of Groups I and II were 95.8 and 82.3%, respectively. We identified a marginally significant difference between the two groups (Log-rank: P = 0.086). In the PS-matched cohort after adjustment for multiple clinicopathologic factors, there was no significant difference in OS between the two groups [OS (Group I vs. II), HR: 0.766 (95% CI: 0.271-2.165), P = 0.615]., Conclusions: After adjustment for clinicopathologic factors, non-SRS limited surgery may not worsen the oncologic outcome in elderly women with early-stage EOC. A large-scale clinical study is necessary to validate the findings., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The edible, nitrate assimilating, yeast Candida utilis is a commercial food additive, and it is a potentially useful host for heterologous protein expression. A number of ATP-binding cassette (ABC) transporters are multidrug efflux pumps that can cause multidrug resistance in opportunistic pathogens. In order to develop optimal novel antimicrobial agents it is imperative to understand the structure, function and expression of these transporters. With the ultimate aim of developing an alternative yeast host for the heterologous expression of eukaryotic membrane transporters, and to identify ABC transporters potentially associated with C. utilis multidrug resistance, we classified the entire repertoire of 30 C. utilis ABC proteins. We named the open reading frame most similar to the archetype multidrug efflux pump gene C. albicans CDR1 as CuCDR1 Overexpression of CuCDR1 in Saccharomyces cerevisiae ADΔ caused multidrug resistance similar to that of cells overexpressing CaCDR1 Unlike CaCdr1p, however, the C-terminally green fluorescent protein (GFP) tagged CuCdr1p-GFP was functionally impaired and did not properly localize to the plasma membrane. CuCdr1p function could be recovered however by adding a 15 amino acid linker -GAGGSAGGSGGAGAG- between CuCdr1p and the C-terminal GFP tag., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: A mechanism for the acquisition of high-level echinocandin resistance in Candida glabrata was investigated. FKS mutants were constructed to: determine whether clinically significant micafungin resistance requires a hot-spot mutation in FKS1 and a premature stop codon in FKS2, as was observed in a clinical isolate; select for variants with reduced susceptibility and locate mutations in FKS genes; and assess the roles of FKS1 and FKS2., Methods: A panel of FKS mutants was constructed using micafungin-susceptible parents by site-directed mutagenesis. Drug susceptibility, gene expression and glucan synthase activities were compared between mutants. Mutations acquired by selection were identified by DNA sequence analysis of FKS genes from selected variants. Single FKS deletants were constructed and their phenotypes examined., Results: Introduction of the hot-spot mutation in FKS1 alone conferred an intermediate reduction in susceptibility, and the premature stop codon in FKS2 alone had no effect on susceptibility, while severely reduced susceptibility equivalent to that of the clinical isolate required both mutations. Exposure of susceptible strains to micafungin yielded variants with an intermediate reduction in susceptibility that possessed a hot-spot mutation in FKS1. Further exposure to micafungin yielded variants with severely reduced susceptibility that acquired various single mutations in FKS2. The phenotypes of Δfks1 and Δfks2 mutants indicate that the two FKS genes are functionally redundant, while deletion of both FKS1 and FKS2 conferred synthetic lethality., Conclusions: In the laboratory mutants of C. glabrata, clinically significant reduced susceptibility to micafungin required single nucleotide changes in both FKS1 and FKS2, and both genes encoded β-1,3-glucan synthase catalytic subunits.

Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.

Mice have a strong ability to eliminate renal calcium oxalate crystals, and our previous examination indicated a susceptibility in which monocyte-macrophage interaction could participate in the phenomenon. To clarify the macrophage-related factors playing roles in the prevention of crystal formation in mouse kidneys, morphologic and expression studies based on microarray pathway analysis were performed. Eight-week-old male C57BL/6N mice were administered 80 mg/kg of glyoxylate by daily intraabdominal injection for 15 days, and the kidneys were extracted every 3 days for DNA microarray analysis. Based on the raw data of microarray analysis, pathway analyses of inflammatory response demonstrated macrophage activation through the increased expression of chemokine (C-X-C) ligand 1, fibronectin 1, and major histocompatability (MHC) class II. Association analysis of related gene expression values by quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated the high association of chemokine (C-C) ligand 2, CD44, colony-stimulating factor 1, fibronectin 1, matrix gla protein, secreted phosphoprotein 1, and transforming growth factor β1 (TGF-β1) with the amount of both renal crystals and F4/80, a macrophage marker. Immunohistochemically, interstitial macrophages increased during the experimental course, and CD44 and MHC class II were upregulated around crystal-formation sites. Ultrastructural observation of renal macrophages by transmission electron microscopy indicated interstitial macrophage migration with the phagocytosis of crystals. In conclusion, increased expression of inflammation-related genes of renal tubular cells induced by crystal formation and deposition could induce monocyte-macrophage migration and phagocytosis via the interaction of CD44 with osteopontin and fibronectin. Such crystal-removing ability of macrophages through phagocytosis and digestion might become a new target for the prevention of stone formation., (Copyright © 2010 American Society for Bone and Mineral Research.)

Objectives: To determine the mechanism of intermediate- and high-level echinocandin resistance, resulting from heterozygous and homozygous mutations in GSC1 (FKS1), in both laboratory-generated and clinical isolates of Candida albicans., Methods: The DNA sequences of the entire open reading frames of GSC1, GSL1 (FKS3) and RHO1, which may contribute to the beta-1,3-glucan synthase of a micafungin-susceptible strain and a resistant clinical isolate, were compared. A spontaneous heterozygous mutant isolated by selection for micafungin resistance, and a panel of laboratory-generated homozygous and heterozygous mutants that possessed combinations of the echinocandin-susceptible and -resistant alleles, or mutants with individual GSC1 alleles deleted, were used to compare levels of echinocandin resistance and inhibition of glucan synthase activity., Results: DNA sequence analysis identified a mutation, S645P, in both alleles of GSC1 from the clinical isolate. GSL1 had two homozygous amino acid changes and five non-synonymous nucleotide polymorphisms due to allelic variation. The predicted amino acid sequence of Rho1p was conserved between strains. Reconstruction of the heterozygous (S645/S645F) and homozygous (S645F/S645F) mutation showed that the homozygous mutation conferred a higher level of micafungin resistance (4 mg/L) than the heterozygous mutation (1 mg/L). Exposure of the heterozygous mutant to micafungin resulted in a loss of heterozygosity. Kinetic analysis of beta-1,3-glucan synthase activity showed that the homozygous and heterozygous mutations gave echinocandin susceptibility profiles that correlated with their MIC values., Conclusions: A homozygous hot-spot mutation in GSC1, caused by mutation in one allele and then loss of heterozygosity, is required for high-level echinocandin resistance in C. albicans. Both alleles of GSC1 contribute equally and independently to beta-1,3-glucan synthase activity.

Objectives: Information on the function of Candida albicans ATP-binding cassette (ABC) membrane transporter Cdr1p has come from studying the effect of gene inactivation in C. albicans and from heterologous Cdr1p expression in the yeast Saccharomyces cerevisiae. These approaches, however, give only an indirect indication of Cdr1p function in C. albicans itself. The objective of this study was to determine Cdr1p function in C. albicans by induced overexpression of Cdr1p in a C. albicans CDR1-deleted strain., Methods: The C. albicans CDR1 open reading frame was fused to the C. albicans HEX1 promoter and used to complement a CDR1-null mutant to create strain FL3. The effect of inducing the FL3 HEX1 promoter, by growth on medium containing N-acetylglucosamine (GlcNAc) as the carbon source, on CDR1 expression and drug susceptibility was determined., Results: C. albicans FL3 cells grown on medium containing GlcNAc overexpressed CDR1 mRNA and a 170 kDa plasma membrane protein that reacted with anti-Cdr1p antibodies. Overexpression of Cdr1p in C. albicans FL3 conferred resistance to structurally unrelated chemicals such as terbinafine, brefeldin A, cerulenin and nigericin as well as to azole antifungal agents, but not resistance to polyene antibiotics. FK506, ascomycin and ciclosporin A chemosensitized FL3 to fluconazole. FL3 cells grown on GlcNAc effluxed 5.3 times as much Cdr1p substrate rhodamine 6G, over a 10 min period, as FL3 cells grown on glucose, and this rhodamine 6G efflux was inhibited by including fluconazole in the assay., Conclusion: This study provides the first direct demonstration of Cdr1p pump activity in C. albicans.

Four Candida albicans isolates, TIMM 3163, TIMM 3164, TIMM 3165 and TIMM 3166, with reduced fluconazole susceptibility were obtained from three AIDS patients in Japan, and the mechanisms of their drug resistance were studied. All isolates showed lower levels of intracellular accumulation of fluconazole than ATCC 10231, a susceptible control strain of C. albicans. Increased amounts of CDR1 and CDR2 mRNA encoding putative ATP binding cassette (ABC) transporters were associated with the azole resistance of all TIMM isolates, apart from TIMM 3164. In addition, increased Cdr1p levels were immunodetected in the cell membrane fractions of all the TIMM strains except for TIMM 3164. Gene amplification was not responsible for CDR1 overexpression and there were no significant differences in the mRNA levels of CDR3 or CDR4 (ABC transporters) in the azole-susceptible and -resistant cells. CaMDR1 (a major facilitator superfamily) gene expression was not observed in any of the resistant isolates or the control strain. These results suggest that energy-dependent drug efflux associated with increased expression of CDR1 and CDR2 is involved in the fluconazole resistance mechanisms in two of the four isolates, TIMM 3165 and TIMM 3166. TIMM 3164 demonstrated energy-dependent drug efflux without overexpression of CDR1-4 or CaMDR1, indicating that some other pump may be operating. Despite showing low levels of drug efflux and overexpression of CDR1 and CDR2, efflux in TIMM 3163 was not energy dependent, suggesting that the expressed Cdr1p non-functional Cdr1p and that other resistance mechanisms may operate in this strain.

Expression of the Candida albicans vacuolar aspartic proteinase (APR1) and beta-N-acetylglucosaminidase (HEX1) genes was studied when carbon-starved cells of strains ATCC 10261 and A72 were induced to grow as yeast or as germ tube-forming cells. Amounts of APR1 mRNA were similar under yeast or germ tube growth conditions. However, more APR1 mRNA was present in cells grown at 28 degrees C than in cells grown at 37 degrees C. The Apr1 enzyme activity of cell-free extracts was not affected by cellular morphology, culture pH or growth temperature. Amounts of HEX1 mRNA were also higher in N-acetylglucosamine (GlcNAc)-induced cells grown at 28 degrees C than in cells grown at 37 degrees C. There was slightly more HEX1 mRNA in cells grown at pH 4.5 than in cells grown at pH 6.7. The beta-N-acetylglucosaminidase activities of GlcNAc-grown cells correlated with the amounts of HEX1 mRNA and were higher when cells were grown at a lower temperature and at a lower pH. Although a similar temperature- and pH-dependent pattern of HEX1 mRNA expression was seen in cells grown on glucose, the enzyme activities in cell-free extracts were all very low. These data indicate that the APR1 and HEX1 genes play no direct role in the dimorphic transition of C. albicans and that transcription of both genes appears to be temperature regulated when the cells are released from carbon starvation. The expression of HEX1 mRNA is in part under the control of culture pH and translation of HEX1 mRNA seems to be regulated by glucose.

Spinach ferredoxin was trinitrophenylated by reaction with 2,4,6-trinitrobenzenesulfonate. Four amino groups in the ferredoxin could be modified of the total of five amino groups. The trinitrophenylated ferredoxin formed a complex with ferredoxin-NADP+ reductase just as native ferredoxin did. The modified ferredoxin also retained the activity of electron transport in the cytochrome c photoreduction system of chloroplasts, but could neither donate electrons to ferredoxin-NADP+ reductase in the NADP+ photoreduction system, nor accept electrons from the reductase in the NADPH-cytochrome c reduction system in vitro. Furthermore, it lost the inhibitory effect against the NADPH-diaphorase activity of the reductase. These results suggest that the complex formation of ferredoxin with ferredoxin-NADP+ reductase is a phenomenon essentially independent of the function of electron transport between the two proteins.