R432 is a key residue for the multiple functions of Ndt80p in Candida albicans.

Ndt80p is an important transcription modulator to various stress-response genes in Candida albicans, the most common human fungal pathogen in systemic infections. We found that Ndt80p directly regulated its target genes, such as YHB1, via the mid-sporulation element (MSE). Furthermore, the ndt80(R432A) allele, with a reduced capability to bind MSE, failed to complement the defects caused by null mutations of NDT80. Thus, the R432 residue in the Ndt80p DNA-binding domain is involved in all tested functions, including cell separation, drug resistance, nitric oxide inactivation, germ tube formation, hyphal growth, and virulence. Hence, the importance of the R432 residue suggests a novel approach for designing new antifungal drugs by blocking the interaction between Ndt80p and its targets.

Fruits are vehicles of drug-resistant pathogenic Candida tropicalis.

IMPORTANCE: Of 123 identified isolates from the fruit surface, C. tropicalis was the most frequently found species, followed by Meyerozyma caribbica and Candida krusei. All three fluconazole-resistant C. tropicalis were non-susceptible to voriconazole and belonged to the same predominant genotype of azole-resistant C. tropicalis causing candidemia in patients in Taiwan. Our findings provide evidence that fruit should be washed before eaten not only to remove chemicals but also potential drug-resistant pathogenic microbes, especially for immunocompromised individuals. To keep precious treatment options in patients, we not only continuously implement antimicrobial stewardship in hospitals but also reducing/stopping the use of agricultural fungicide classes used in human medicine.

The fatty acid synthase inhibitor C75 differentially affects the adipogenic differentiation of multipotent cells and preadipocytes.

Previously, we revealed the dual enhancing effect of netoglitazone, an agonist of the peroxisome proliferator-activated receptor γ, on adipogenesis and osteoblastogenesis, and reported that fatty acid synthase (FASN) knockdown selectively repressed its pro-adipogenic effect. Here, we examined if a FASN inhibitor, C75, could selectively repress the pro-adipogenic effect of netoglitazone. Surprisingly, C75 promoted the adipogenic differentiation of multipotent C3H10T1/2 cells but inhibited 3T3-L1 preadipocytes. By identifying glycogen synthase kinase-3ß and intracellular cAMP levels as regulatory targets of C75, we ultimately found the differential expression of adenosine receptor 3 (AR3) and AR2a on these cells. Inhibition of AR3 on C3H10T1/2 and AR2a on 3T3-L1 inhibited the effects of C75 on the differentiation of these cells. Our findings imply that cell-type-specific AR expression might account for the differential adipogenic effects of C75.

Associations of Rap1 with Cell Wall Integrity, Biofilm Formation, and Virulence in Candida albicans.

Rap1 (repressor activator protein 1) is a multifunctional protein, playing important roles in telomeric and nontelomeric functions in many eukaryotes. Candida albicans Rap1 has been previously shown to be involved in telomeric regulation, but its other functions are still mostly unknown. In this study, we found that the deletion of the RAP1 gene altered cell wall properties, composition, and gene expression. In addition, deletion of RAP1 affected C. albicans biofilm formation and modulated phagocytosis and cytokine release by host immune cells. Finally, the RAP1 gene deletion mutant showed attenuation of C. albicans virulence in a Galleria mellonella infection model. Therefore, these findings provide new insights into Rap1 functions that are particularly relevant to pathogenesis and virulence of C. albicans. IMPORTANCE C. albicans is an important fungal pathogen of humans. The cell wall is the outermost layer of C. albicans and is important for commensalism and infection by this pathogen. Moreover, the cell wall is also an important target for antifungals. Studies of how C. albicans maintains its cell wall integrity are critical for a better understanding of fungal pathogenesis and virulence. This work focuses on exploring unknown functions of C. albicans Rap1 and reveals its contribution to cell wall integrity, biofilm formation, and virulence. Notably, these findings will also improve our general understanding of complex machinery to control pathogenesis and virulence of fungal pathogens.

Genetic relatedness among azole-resistant Candida tropicalis clinical strains in Taiwan from 2014 to 2018.

To monitor trends in the distribution of yeast species and the susceptibilities of these species to commonly prescribed antifungal drugs, we conduct the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) every 4 years. We found that 25 of 294 Candida tropicalis isolates from TSARY 2014 and 31 of 314 C. tropicalis isolates from TSARY 2018 were resistant to fluconazole. We determined the genetic relatedness among fluconazole-resistant C. tropicalis isolates by multilocus sequence typing (MLST). Among 174 C. tropicalis isolates, including all 56 fluconazole-resistant, all 26 susceptible-dose dependent and 92 selected fluconazole-susceptible isolates, 59 diploid sequence types (DSTs) were identified. We found that 22 of the 25 fluconazole-resistant C. tropicalis from TSARY 2014 and 29 of the 31 fluconazole-resistant C. tropicalis from TSARY 2018 were genetically related and belonged to the same cluster (clade 4). A combination of mutation and overexpression of ERG11, encoding the target of azole drugs, was the major mechanism contributing to drug resistance. Approximately two-thirds of reviewed patients infected or colonised by fluconazole-resistant C. tropicalis were azole-naïve. Furthermore, there was no evidence of patient-to-patient transmission. Because the clade 4 fluconazole-resistant C. tropicalis strain persists in Taiwan, it is important to identify the source of azole-resistant C. tropicalis to prevent the spread of this resistant strain.

Decorin inhibits the insulin-like growth factor I signaling in bone marrow mesenchymal stem cells of aged humans.

Aging impairs the IGF-I signaling of bone marrow mesenchymal stem cells (bmMSCs), but the mechanism is unclear. Here, we found that the ability to auto-phosphorylate IGF-I receptor (IGF-IR) in response to IGF-I was decreased in the bmMSCs of aged donors. Conversely, data showed that decorin (DCN) expression was prominently increased in aged bmMSCs, and that under IGF-I treatment, DCN knockdown in serum-starved aged bmMSCs potentiated their mitogenic activity and IGF-IR auto-phosphorylation, whereas DCN overexpression in serum-starved adult bmMSCs decreased both activities. Co-immunoprecipitation assays suggested that IGF-I and DCN bound to IGF-IR in a competitive manner. Online MethPrimer predicted 4 CpG islands (CGIs) in the introns of DCN gene. RT-qPCR and bisulfite sequencing showed that dimethyloxalylglycine, an inhibitor of DNA demethylation, increased DCN mRNA expression and CGI-I methylation in adult bmMSCs, whereas 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, decreased DCN mRNA expression and CGI-I methylation in aged bmMSCs, and ultimately enhanced the proliferation of serum-starved aged bmMSCs under IGF-I stimulation. Thus, IGF-IR could be the prime target of aging in down-regulating the IGF-I signaling of bmMSCs, where DCN could be a critical mediator.

Rep1p negatively regulating MDR1 efflux pump involved in drug resistance in Candida albicans.

Overexpression of MDR1 efflux pump is a major mechanism contributing to drug resistance in Candida albicans, the most common human fungal pathogen. To elucidate the regulatory pathway of drug resistance, we have identified a negative regulator of MDR1 and named it Regulator of Efflux Pump 1 (REP1). Overexpression of REP1 in Saccharomyces cerevisiae increased susceptibility to fluconazole. Furthermore, null mutations on REP1 decreased the susceptibility to antifungal drugs in C. albicans resulting from increased expression of MDR1 mRNA. Hence, Rep1p is involved in drug resistance by negatively regulating MDR1 in C. albicans.

Cooperative impact of thiazolidinedione and fatty acid synthase on human osteogenesis.

Previous, we found that the small molecules capable of inhibiting the expression and the pro-adipogenic activity of ZNF521 might improve the osteogenic performance of aging human bone marrow MSCs (bmMSCs), and that fatty acid synthase (FASN) was a critical effector of ZNF521's pro-adipogenic activity. Here, by characterizing the netoglitazone (MCC-555), one of the thiazolidinediones known as adipogenic enhancers, as an inhibitor of ZNF521 expression, we found that MCC-555 indeed also harbored pro-osteoblastic effect. Investigation revealed that MCC-555 might function as a GSK3ß inhibitor to promote osteoblastogenesis and bone formation. Importantly, combination of MCC-555 with FASN knockdown, but not with GW9662 (a PPARγ2 antagonist), blocked the pro-adipogenic but retained the pro-osteoblastic effect of MCC-555. Using a 3-dimentional culture system, we showed that MCC-555 facilitated the FASN-knockdown of aging human bmMSCs to form cell clusters in scaffolds, and to promote osteoblastic differentiation and biomineralization in cell clusters. These data indicated that MCC-555 promoted bmMSCs to produce bone-like tissues. Our data narrate a thiazolidinedione-based novel strategy to improve the osteogenic performance of aging bmMSCs to support the application of autologous aging bmMSCs in cell therapy and in producing bone-like tissues for repairing bone injury in the elderly.

Zinc finger protein ZFP36L1 promotes osteoblastic differentiation but represses adipogenic differentiation of mouse multipotent cells.

Zinc finger protein 36, C3H type-like 1 (ZFP36L1) is a member of the tristetraprolin (TTP) family and its role in the aging-related bone loss is currently unknown. We present evidence that ZFP36L1 expression in rat femurs and bone marrow mesenchymal stem cells (bmMSCs) was down-regulated with aging. ZFP36L1 knockdown decreased osteoblastic differentiation of MC3T3-E1 and C3H10T1/2 cells, and increased adipogenic differentiation of 3T3-L1 and C3H10T1/2 cells, whereas ZFP36L1 overexpression did the opposite. The finding that ZFP36L1 overexpression enhanced osteoblastic and repressed adipogenic differentiation was also corroborated by ex vivo experiments. Troglitazone prevented ZFP36L1 from inhibiting adipogenic differentiation, suggesting the significance of PPARγ2 repression in ZFP36L1's inhibitory effect on adipogenic differentiation. ZFP36L1 overexpression repressed the expression of Pparγ2 mRNA, but not the PPARγ promoter activity. Biotin pull-down and electrophoretic mobility-shift assays suggested that ZFP36L1 might interact with endogenous Pparγ2 mRNA by binding to its 3'UTR. The ZFP36L1-containing ribonucleoprotein complexes of ZFP36L1-overexpressing cells contained less Pparγ2 mRNA than those of control cells. In a luciferase reporter construct, replacement of the SV40 poly(A) fragment by the 3'UTR of Pparγ2 mRNA reduced the expression of luciferase transcripts in ZFP36L1-overexpressing cells. Examination of the kinetic expression of Pparγ2 mRNA after transcriptional blockage showed that ZFP36L1 might enhance the degradation of the transcripts. Together, these data imply that ZFP36L1 overexpression might repress adipogenesis at least by down-regulating PPARγ2 expression through post-transcriptional mechanisms. Thus, our findings support the notion that decrease of ZFP36L1 expression in bmMSCs with aging might contribute to the aging-related bone loss.

Overexpression of Insulin-Like Growth Factor 1 Enhanced the Osteogenic Capability of Aging Bone Marrow Mesenchymal Stem Cells.

Many studies have indicated that loss of the osteoblastogenic potential in bone marrow mesenchymal stem cells (bmMSCs) is the major component in the etiology of the aging-related bone deficit. But how the bmMSCs lose osteogenic capability in aging is unclear. Using 2-dimentional cultures, we examined the dose response of human bmMSCs, isolated from adult and aged donors, to exogenous insulin-like growth factor 1 (IGF-1), a growth factor regulating bone formation. The data showed that the mitogenic activity and the osteoblastogenic potential of bmMSCs in response to IGF-1 were impaired with aging, whereas higher doses of IGF-1 increased the proliferation rate and osteogenic potential of aging bmMSCs. Subsequently, we seeded IGF-1-overexpressing aging bmMSCs into calcium-alginate scaffolds and incubated in a bioreactor with constant perfusion for varying time periods to examine the effect of IGF-1 overexpression to the bone-forming capability of aging bmMSCs. We found that IGF-1 overexpression in aging bmMSCs facilitated the formation of cell clusters in scaffolds, increased the cell survival inside the cell clusters, induced the expression of osteoblast markers, and enhanced the biomineralization of cell clusters. These results indicated that IGF-1 overexpression enhanced cells' osteogenic capability. Thus, our data suggest that the aging-related loss of osteogenic potential in bmMSCs can be attributed in part to the impairment in bmMSCs' IGF-1 signaling, and support possible application of IGF-1-overexpressing autologous bmMSCs in repairing bone defect of the elderly and in producing bone graft materials for repairing large scale bone injury in the elderly.

The DNA-binding domain of CaNdt80p is required to activate CDR1 involved in drug resistance in Candida albicans.

CaNdt80p, the Candida albicans homologue of the Saccharomyces cerevisiae transcription factor ScNdt80p, has been identified as a positive regulator of CDR1, which encodes an efflux pump involved in drug resistance in C. albicans. To investigate the involvement of the putative DNA-binding domain of CaNdt80p in drug resistance, chimeras of CaNdt80p and ScNdt80p were constructed. Interestingly, the DNA-binding domain of ScNdt80p could functionally complement that of CaNdt80p to activate CDR1p-lacZ in S. cerevisiae. Consistently, CaNdt80p containing a mutation in the DNA-binding domain failed to activate CDR1p-lacZ in S. cerevisiae. Furthermore, a copy of CaNDT80 with the same mutation also failed to complement the drug-sensitive phenotype caused by a null mutation in C. albicans. Thus, the DNA-binding domain of CaNdt80p is critical for its function in drug resistance in C. albicans.

Zinc finger factor 521 enhances adipogenic differentiation of mouse multipotent cells and human bone marrow mesenchymal stem cells.

Previously, we found that ZNF521 expression was up-regulated with advancing age in human bone marrow mesenchymal stem cells (bmMSCs). Here, we investigated the regulatory role of ZNF521 in the differentiation of mouse C3H10T1/2 cells and human bmMSCs. Our data show that ZNF521 overexpression repressed osteoblastic differentiation of C3H10T1/2 cells, accompanied by a decrease in Runx2 expression and an increase in PPARγ2 expression. In contrast, ZNF521 overexpression enhanced adipogenic differentiation of C3H10T1/2 cells, concomitant with increased expression of PPARγ2, aP2, adiponectin and C/EBPδ. Chromatin immunoprecipitation followed by quantitative PCR analyses and luciferase reporter assays suggested that ZNF521 overexpression enhances PPARγ2 expression at the transcriptional level. The enhancing effect of ZNF521 overexpression on the adipogenic differentiation of C3H10T1/2 cells was also observed ex vivo. Finally, similar to those noted in C3H10T1/2 cells, ZNF521 overexpression in human bmMSCs was found to promote adipogenic differentiation in vitro and ex vivo, but repressed osteoblastic differentiation in vitro. ZNF521 knockdown significantly repressed adipogenic differentiation in vitro and ex vivo, but promoted osteoblastic differentiation in vitro. We propose that ZNF521 can function as a repressor of osteoblastic differentiation of bmMSCs while promoting adipogenesis, and that elevated ZNF521 expression might play a role in the age-related bone loss.

Cph1p negatively regulates MDR1 involved in drug resistance in Candida albicans.

The cph1/cph1 efg1/efg1 double mutant in Candida albicans is defective in filamentous growth and is avirulent in a mouse model. We previously reported that Efg1p but not Cph1p is involved in drug resistance by negatively regulating ERG3 in C. albicans. In the current study, we have found that overexpression of CPH1 in Saccharomyces cerevisiae increases susceptibility to the antifungal drug fluconazole. Furthermore, in C. albicans, null mutation of CPH1 increased the expression of MDR1 as well as decreased susceptibility to fluconazole and voriconazole but not to amphotericin B. These findings indicate that although Efg1p and Cph1p may have the same effects on virulence, they have opposite effects on drug resistance in C. albicans.

Polypyrimidine tract-binding protein induces p19(Ink4d) expression and inhibits the proliferation of H1299 cells.

The expression of polypyrimidine tract-binding protein (PTB) is up-regulated in many types of cancer. Here, we studied the role of PTB in the growth of non small cell lung cancer cells. Data showed that PTB overexpression inhibited the growth of H1299 cells at least by inhibiting DNA synthesis. Quantitative real-time PCR and Western blot analyses showed that PTB overexpression in H1299 cells specifically induced the expression of p19(Ink4d), an inhibitor of cyclin-dependent kinase 4. Repression of p19(Ink4d) expression partially rescued PTB-caused proliferation inhibition. PTB overexpression also inhibited the growth and induced the expression of p19(Ink4d) mRNA in A549 cells. However, Western blot analyses failed to detect the presence of p19(Ink4d) protein in A549 cells. To address how PTB induced p19(Ink4d) in H1299 cells, we showed that PTB might up-regulate the activity of p19(Ink4d) gene (CDKN2D) promoter. Besides, PTB lacking the RNA recognition motif 3 (RRM3) was less effective in growth inhibition and p19(Ink4d) induction, suggesting that RNA-binding activity of PTB plays an important role in p19(Ink4d) induction. However, immunoprecipitation of ribonuclearprotein complexes plus quantitative real-time PCR analyses showed that PTB might not bind p19(Ink4d) mRNA, suggesting that PTB overexpression might trigger the other RNA-binding protein(s) to bind p19(Ink4d) mRNA. Subsequently, RNA electrophoretic mobility-shift assays revealed a 300-base segment (designated as B2) within the 3'UTR of p19(Ink4d) mRNA, with which the cytoplasmic lysates of PTB-overexpressing cells formed more prominent complexes than did control cell lysates. Insertion of B2 into a reporter construct increased the expression of the chimeric luciferase transcripts in transfected PTB-overexpressing cells but not in control cells; conversely, overexpression of B2-containing reporter construct in PTB-overexpressing cells abolished the induction of p19(Ink4d) mRNA. In sum, we have shown that PTB plays as a negative regulator in H1299 cell proliferation at least by inducing p19(Ink4d) expression at transcriptional and post-transcriptional levels.

Gene expression profiling suggests a pathological role of human bone marrow-derived mesenchymal stem cells in aging-related skeletal diseases.

Aging is associated with bone loss and degenerative joint diseases, in which the aging of bone marrow-derived mesenchymal stem cell (bmMSC)[1] may play an important role. In this study, we analyzed the gene expression profiles of bmMSC from 14 donors between 36 and 74 years old, and obtained age-associated genes (in the background of osteoarthritis) and osteoarthritis-associated genes (in the background of old age). Pathway analysis of these genes suggests that alterations in glycobiology might play an important role in the aging of human bmMSC. On the other hand, antigen presentation and signaling of immune cells were the top pathways enriched by osteoarthritis-associated genes, suggesting that alteration in immunology of bmMSC might be involved in the pathogenesis of osteoarthritis. Most intriguingly, we found significant age-associated differential expression of HEXA, HEXB, CTSK, SULF1, ADAMTS5, SPP1, COL8A2, GPNMB, TNFAIP6, and RPL29; those genes have been implicated in the bone loss and the pathology of osteoporosis and osteoarthritis in aging. Collectively, our results suggest a pathological role of bmMSC in aging-related skeletal diseases, and suggest the possibility that alteration in the immunology of bmMSC might also play an important role in the etiology of adult-onset osteoarthritis.