Response to the letter 'Absent regulation of iron acquisition by the copper regulator Mac1 in A. fumigatus'.

The answer to the letter 'Absent regulation of iron acquisition by the copper regulator Mac1 in A. fumigatus' has been prepared. We explained our data and showed supplementary information to answer the questions. And we respect the results of other groups first and explain the differences from our results.

Bexarotene - a novel modulator of AURKA and the primary cilium in VHL-deficient cells.

Loss of the gene von Hippel-Lindau (VHL) is associated with loss of primary cilia and is causally linked to elevated levels of Aurora kinase A (AURKA). We developed an image-based high-throughput screening (HTS) assay using a dual-labeling image analysis strategy that identifies both the cilium and the basal body. By using this strategy, we screened small-molecule compounds for the targeted rescue of cilia defects associated with VHL deficiency with high accuracy and reproducibility. Bexarotene was identified and validated as a positive regulator of the primary cilium. Importantly, the inability of an alternative retinoid X receptor (RXR) agonist to rescue ciliogenesis, in contrast to bexarotene, suggested that multiple bexarotene-driven mechanisms were responsible for the rescue. We found that bexarotene decreased AURKA expression in VHL-deficient cells, thereby restoring the ability of these cells to ciliate in the absence of VHL Finally, bexarotene treatment reduced the propensity of subcutaneous lesions to develop into tumors in a mouse xenograft model of renal cell carcinoma (RCC), with a concomitant decrease in activated AURKA, highlighting the potential of bexarotene treatment as an intervention strategy in the clinic to manage renal cystogenesis associated with VHL deficiency and elevated AURKA expression.

The Role of Zinc in Copper Homeostasis of Aspergillus fumigatus.

Copper is an essential metal ion that performs many physiological functions in living organisms. Deletion of Afmac1, which is a copper-responsive transcriptional activator in A. fumigatus, results in a growth defect on aspergillus minimal medium (AMM). Interestingly, we found that zinc starvation suppressed the growth defect of the Δafmac1 strain on AMM. In addition, the growth defect of the Δafmac1 strain was recovered by copper supplementation or introduction of the CtrC gene into the Δafmac1 strain. However, chelation of copper by addition of BCS to AMM failed to recover the growth defect of the Δafmac1 strain. Through Northern blot analysis, we found that zinc starvation upregulated CtrC and CtrA2, which encode membrane copper transporters. Interestingly, we found that the conserved ZafA binding motif 5'-CAA(G)GGT-3' was present in the upstream region of CtrC and CtrA2 and that mutation of the binding motif led to failure of ZafA binding to the upstream region of CtrC and upregulation of CtrC expression under zinc starvation. Furthermore, the binding activity of ZafA to the upstream region of CtrC was inversely proportional to the zinc concentration, and copper inhibited the binding of ZafA to the upstream region of CtrC under a low zinc concentration. Taken together, these results suggest that ZafA upregulates copper metabolism by binding to the ZafA binding motif in the CtrC promoter region under low zinc concentration, thus regulating copper homeostasis. Furthermore, we found that copper and zinc interact in cells to maintain metal homeostasis.

A copper transcription factor, AfMac1, regulates both iron and copper homeostasis in the opportunistic fungal pathogen Aspergillus fumigatus.

Although iron and copper are co-ordinately regulated in living cells, the homeostatic effects of each of these metals on the other remain unknown. Here, we show the function of AfMac1, a transcriptional activator of the copper and iron regulons of Aspergillus fumigatus, on the interaction between iron and copper. In addition to the copper-specific AfMac1-binding motif 5'-TGTGCTCA-3' found in the promoter region of ctrC, the iron-specific AfMac1-binding motif 5'-AT(C/G)NN(A/T)T(A/C)-3' was identified in the iron regulon but not in the copper regulon by ChIP sequence analysis. Furthermore, mutation of the AfMac1-binding motif of sit1 eliminated AfMac1-mediated sit1 up-regulation. Interestingly, the regulation of gene expression in the iron regulon by AfMac1 was not affected by copper and vice versa AfMac1 localized to the nucleus under iron- or copper-depleted conditions, and AfMac1 was mostly detected in the cytoplasm under iron- or copper-replete conditions. Taken together, these results suggest that A. fumigatus independently regulates iron and copper homeostasis in a manner that involves AfMac1 and mutual interactions.

The Role of Zinc in Gliotoxin Biosynthesis of Aspergillus fumigatus.

Zinc performs diverse physiological functions, and virtually all living organisms require zinc as an essential trace element. To identify the detailed function of zinc in fungal pathogenicity, we carried out cDNA microarray analysis using the model system of Aspergillus fumigatus, a fungal pathogen. From microarray analysis, we found that the genes involved in gliotoxin biosynthesis were upregulated when zinc was depleted, and the microarray data were confirmed by northern blot analysis. In particular, zinc deficiency upregulated the expression of GliZ, which encodes a Zn2-Cys6 binuclear transcription factor that regulates the expression of the genes required for gliotoxin biosynthesis. The production of gliotoxin was decreased in a manner inversely proportional to the zinc concentration, and the same result was investigated in the absence of ZafA, which is a zinc-dependent transcription activator. Interestingly, we found two conserved ZafA-binding motifs, 5'-CAAGGT-3', in the upstream region of GliZ on the genome and discovered that deletion of the ZafA-binding motifs resulted in loss of ZafA-binding activity; gliotoxin production was decreased dramatically, as demonstrated with a GliZ deletion mutant. Furthermore, mutation of the ZafA-binding motifs resulted in an increase in the conidial killing activity of human macrophage and neutrophil cells, and virulence was decreased in a murine model. Finally, transcriptomic analysis revealed that the expression of ZafA and GliZ was upregulated during phagocytosis by macrophages. Taken together, these results suggest that zinc plays an important role in the pathogenicity of A. fumigatus by regulating gliotoxin production during the phagocytosis pathway to overcome the host defense system.

Efficacy of double inversion recovery magnetic resonance imaging for the evaluation of the synovium in the femoro-patellar joint without contrast enhancement.

OBJECTIVE: To investigate the efficacy of double inversion recovery (DIR) sequence for evaluating the synovium of the femoro-patellar joint without contrast enhancement (CE). METHODS: Two radiologists independently evaluated the axial DIR and CE T1-weighted fat-saturated (CET1FS) images of 33 knees for agreement; the visualisation and distribution of the synovium were evaluated using a four-point visual scaling system at each of the five levels of the femoro-patellar joint and the location of the thickest synovium. The maximal synovial thickness at each sequence was measured by consensus. RESULTS: The interobserver agreement was good (κ = 0.736) for the four-point scale, and was excellent for the location of the thickest synovium on DIR and CET1FS (κ = 0.955 and 0.954). The intersequential agreement for the area with the thickest synovium was also excellent (κ = 0.845 and κ = 0.828). The synovial thickness on each sequence showed excellent correlation (r = 0.872). CONCLUSION: The DIR showed as good a correlation as CET1FS for the evaluation of the synovium at the femoro-patellar joint. DIR may be a useful MR technique for evaluating the synovium without CE. KEY POINTS: • DIR can be useful for evaluating the synovium of the femoro-patellar joint. • Interobserver and intersequential agreements between DIR and CET1FS were good. • Mean thickness of the synovium was significantly different between two sequences.

Switchable electrorheological activity of polyacrylonitrile microspheres by thermal treatment: from negative to positive.

The study focuses on the effect of thermal deformation degree of polyacrylonitrile (PAN) particles on the electrorheological (ER) properties of their suspensions. The heat-treated PAN particles are manufactured as ER materials using a thermocatalytic processes. The molecular structures of ER materials are analyzed to confirm a stabilization or a carbonization degree. We categorized the prepared ER particles into three basic types according to their deformation degree: Thermal dried PAN, stabilized PAN, and pre-carbonized PAN. The raw, stabilized, and pre-carbonized PAN particle-dispersed suspensions showed positive ER properties. The ER properties are enhanced as the heat-treatment temperature increases due to improved dielectric property. However, the thermal dried PAN particle ER suspensions showed negative ER behavior though the particles have higher conductivity and dielectric constants than those of the host fluid, which is contrary to the conduction model. XRD results indicate that the ER materials could show contradictory ER behavior even if they have the same molecular structures due to their crystallinity. This discovery is expected to boost the development of both positive ER and negative ER suspensions based on carbonaceous ER materials.

Functional characterization of the copper transcription factor AfMac1 from Aspergillus fumigatus.

Although copper functions as a cofactor in many physiological processes, copper overload leads to harmful effects in living cells. Thus, copper homeostasis is tightly regulated. However, detailed copper metabolic pathways have not yet been identified in filamentous fungi. In this report, we investigated the copper transcription factor AfMac1 ( Aspergillus fumigatusMac1 homolog) and identified its regulatory mechanism in A. fumigatus AfMac1 has domains homologous to the DNA-binding and copper-binding domains of Mac1 from Saccharomyces cerevisiae, and AfMac1 efficiently complemented Mac1 in S. cerevisiae Expression of Afmac1 resulted in CTR1 up-regulation, and mutation of the DNA-binding domain of Afmac1 failed to activate CTR1 expression in S. cerevisiae The Afmac1 deletion strain of A. fumigatus failed to grow in copper-limited media, and its growth was restored by introducing ctrC We found that AfMac1 specifically bound to the promoter region of ctrC based on EMSA. The AfMac1-binding motif 5'-TGTGCTCA-3' was identified from the promoter region of ctrC, and the addition of mutant ctrC lacking the AfMac1-binding motif failed to up-regulate ctrC in A. fumigatus Furthermore, deletion of Afmac1 significantly reduced strain virulence and activated conidial killing activity by neutrophils and macrophages. Taken together, these results suggest that AfMac1 is a copper transcription factor that regulates cellular copper homeostasis in A. fumigatus.

Diagnostic accuracy of low-dose versus ultra-low-dose CT for lumbar disc disease and facet joint osteoarthritis in patients with low back pain with MRI correlation.

OBJECTIVE: To compare the image quality, radiation dose, and diagnostic performance between low-dose (LD) and ultra-low-dose (ULD) lumbar-spine (L-spine) CT with iterative reconstruction (IR) for patients with chronic low back pain (LBP). METHODS: In total, 260 patients with chronic LBP who underwent L-spine CT between November 2015 and September 2016 were prospectively enrolled. Of these, 143 underwent LD-CT with IR and 117 underwent ULD-CT with IR. The patients were divided according to their body mass index (BMI) into BMI1 (<22.9 kg/m2), BMI2 (23.0-24.9 kg/m2), and BMI3 (≥25 kg/m2) groups. Two blinded radiologists independently evaluated the signal-to-noise ratio (SNR), qualitative image quality, and final diagnoses (lumbar disc disease and facet joint osteoarthritis). L-spine MRIs interpreted by consensus were used as the reference standard. All data were statistically analyzed. RESULTS: ULD protocol showed significantly lower SNR for all patients (p < 0.001) except the vertebral bodies and lower qualitative image quality for BMI3 patients (p ≤ 0.033). There was no statistically significant difference between ULD (sensitivity, 95.1-98.1%; specificity, 92.5-98.7%; accuracy, 94.6-98.0%) and LD protocols (sensitivity, 95.6-100%; specificity, 95.5-98.9%; accuracy, 97.4-98.1%), (all p≥0.1) in the BMI1 and BMI2; while dose was 60-68% lower with the ULD protocol. Interobserver agreements were excellent or good with regard to image quality and final diagnoses. CONCLUSIONS: For the BM1 and BMI2 groups, ULD-CT provided an acceptable image quality and exhibited a diagnostic accuracy similar to that of LD-CT. These findings suggest that it is a useful diagnostic tool for patients with chronic LBP who exhibit a BMI of <25 kg/m2.

Effects of the bottom slope and guiding wall length on the performance of a vortex drop inlet.

Laboratory experiments were conducted to assess the performance of a vortex drop inlet with a spiral intake in subcritical and supercritical flow conditions. The water surface elevation at multiple locations was measured for different flowrates by varying the extent of the guiding wall and the longitudinal and radial bottom slopes. The measurements show that a steeper longitudinal bottom slope decreases the water surface elevation at the beginning of the intake, resulting in a transcritical flow in the intake structure. However, a steeper longitudinal bottom slope also causes the maximum water surface elevation to occur within the spiral intake. For an effective vortex drop inlet design, achieving a low water surface elevation throughout the entire spiral intake structure is required. Experimental results show that the two seemingly conflicting design criteria, namely, achieving a low water surface elevation in the approach channel and reducing the maximum water surface elevation in the intake structure, can be simultaneously achieved by adding a radial bottom slope.

Validity of Radiograph-Based Infrapatellar Fat Pad Opacity Grading for Assessing Knee Synovitis: Correlation With Contrast-Enhanced MRI.

OBJECTIVE: The purpose of this study was to determine the validity of infrapatellar fat pad (IPFP) opacity grading based on lateral knee radiography for assessing knee synovitis using correlation with contrast-enhanced (CE) MRI. MATERIALS AND METHODS: Retrospective reviews of radiographs and CE knee MR images from 79 patients were independently performed by two radiologists. They evaluated IPFP opacity alteration (IPFPCR) and joint effusion grades on lateral knee conventional radiographs, IPFP signal intensity alteration (IPFPMR) and joint effusion grades on CE MR images, and synovitis (SYNMR) grade in nine divided regions, three compartments (parapatellar, periligamentous, perimeniscal), and the whole knee on CE MR images. Correlations between radiographic grades and MRI assessments were evaluated using Spearman correlation tests, and the correlation coefficients (ρ) were compared. Interobserver agreement was evaluated using weighted kappa values. RESULTS: The IPFPCR grade was very highly correlated with the IPFPMR grade (ρ = 0.906, p < 0.001) and highly correlated with SYNMR grades from four regions (suprapatellar, infrapatellar, intercondylar, lateral parapatellar recess), the parapatellar compartment, and the whole knee (ρ = 0.614-0.740, all p < 0.001). The IPFPCR grade was moderately correlated with the SYNMR grades of the remaining five regions and two compartments (ρ = 0.457-0.547, all p < 0.001). The differences between correlation coefficients for SYNMR grades and the IPFPCR and IPFPMR grades were not statistically significant (p = 0.290-1.0). Interobserver reliabilities were excellent or good for IPFPCR, IPFPMR, and SYNMR grades (κ = 0.661-1.000). CONCLUSION: IPFPCR grade assessment enables valid evaluation and reporting of knee synovitis, especially in the parapatellar compartment and the whole knee.

Identification of ferrichrome- and ferrioxamine B-mediated iron uptake by Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic fungal pathogen for immunocompromised patients, and genes involved in siderophore metabolism have been identified as virulence factors. Recently, we identified the membrane transporters sit1 and sit2, which are putative virulence factors of A. fumigatus; sit1 and sit2 are homologous to yeast Sit1, and sit1 and sit2 gene expression was up-regulated after iron depletion. When expressed heterologously in Saccharomyces cerevisiae, sit1 and sit2 were localized to the plasma membrane; sit1 efficiently complemented ferrichrome (FC) and ferrioxamine B (FOB) uptake in yeast cells, whereas sit2 complemented only FC uptake. Deletion of sit1 resulted in a decrease in FOB and FC uptake, and deletion of sit2 resulted in a decrease in FC uptake in A. fumigatus It is of interest that a sit1 and sit2 double-deletion mutant resulted in a synergistic decrease in FC uptake activity. Both sit1 and sit2 were localized to the plasma membrane in A. fumigatus The expression levels of the sit1 and sit2 genes were dependent on hapX under low-but not high-iron conditions. Furthermore, mirB, and sidA gene expression was up-regulated and sreA expression down-regulated when sit1 and sit2 were deleted. Although sit1 and sit2 failed to affect mouse survival rate, these genes affected conidial killing activity. Taken together, our results suggest that sit1 and sit2 are siderophore transporters and putative virulence factors localized to the plasma membrane.

Severity of nonalcoholic fatty liver disease on sonography and risk of coronary heart disease.

PURPOSE: To evaluate the relationship between nonalcoholic fatty liver disease (NAFLD) on sonography (US) and the risk of coronary heart disease (CHD) as well as the predictive value of US-diagnosed NAFLD to determine intermediate/high CHD risk compared with as well as in combination on with NAFLD fibrosis score (NFS). METHODS: This retrospective study included 1,276 patients examined between November 2015 and August 2016. NAFLD was categorized as absent, mild, moderate, or severe based on liver-to-kidney echogenicity, visibility of intrahepatic vessel walls, and the diaphragm. The Framingham risk score (FRS) and NFS were used to predict CHD risk and hepatic fibrosis severity. Spearman correlation test, multivariate-adjusted logistic regression analysis, and receiver operating characteristic curves were used for statistical evaluation. RESULTS: FRS increased as NAFLD severity increased, and US-determined NAFLD severity and FRS were highly positively correlated (r = 0.683, p < 0.001). The odds ratios for intermediate/high CHD risk increased with increasing NAFLD severity. The predictive performance of US-determined NAFLD severity for determining intermediate/high CHD risk in NAFLD patients was 0.738. There was no significant difference between US-determined NAFLD severity and NFS in terms of identifying intermediate/high CHD risk (p = 0.88). However, the combination of US-determined NAFLD severity and NFS significantly improved the ability to distinguish intermediate/high CHD risk compared with that of US-determined NAFLD severity or NFS alone (p < 0.001 for both). CONCLUSIONS: US-determined NAFLD severity was well correlated with FRS and associated with the prevalence of intermediate/high CHD risk. The combination of US-determined NAFLD severity and NFS may be useful for predicting CHD risk. © 2017 Wiley Periodicals, Inc. J Clin Ultrasound 45:391-399, 2017.

Persimmon Fruit Powder May Substitute Indolbi, a Synthetic Growth Regulator, in Soybean Sprout Cultivation.

Soybean sprouts are a major food item in Korea. Various studies have been carried out to enhance their yield and nutritional values. The objective of the present study was to examine the influence of persimmon fruit powder and Indolbi, a synthetic plant growth regulator, on the yield and nutritional value of soybean sprouts. Seeds were soaked in tap water containing 0.5%, 1.0%, 2.5% and 5.0% (w/v) persimmon fruit powder and the samples were named as PT-1, PT-2, PT-3, and PT-4, respectively. The yield increment was almost doubled in PT-3 and PT-4 than in the Indolbi treated sprouts on basis of the control. Vitamin C, isoflavones, and total phenolic contents as well as antioxidant potentials (determined by 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radical scavenging assays) were also significantly (p < 0.05) higher in PT-3 compared to the Indolbi treatment and the control. However, total free amino acid and magnesium contents of Indolbi- applied sprouts were higher than in the fruit powder treatments. The overall results of the present study showed that persimmon fruit powder can be an option to enhance the yield and nutritional value of soybean sprouts since, due to potential health hazards, the use of synthetic chemicals like Indolbi is less preferred than the natural products.

Rck1 promotes pseudohyphal growth via the activation of Ubp3 phosphorylation in Saccharomyces cerevisiae.

Previously, we reported that Rck1 up-regulates Ras2 and pseudohyphal growth of Saccharomyces cerevisiae. Here, we further investigate the involvement of Rck1 in the activation of pseudohyphal growth. Rck1 activated phosphorylation of the deubiquitinase Ubp3 through a direct protein interaction between Rck1 and Ubp3. The N-terminal Bre5 binding region of Ubp3 physically interacted with Rck1, and Ubp3 and Rck1 co-precipitated. Overexpression of UBP3 using a high-copy plasmid resulted in the upregulation of Ras2, and deletion of UBP3 blocked the upregulation of Ras2 by RCK1 overexpression. Treatment with the proteasome inhibitor MG132 resulted in accumulation of Ras2, indicating that Rck1 is involved in Ras2 degradation in a proteasome-dependent manner. Furthermore, deletion of UBP3 blocked the upregulation of FLO11, a flocculin required for pseudohyphal and invasive growth induced by RCK1 overexpression in S. cerevisiae. Taken together, these results demonstrate that Rck1 promotes S. cerevisiae pseudohyphal growth via the activation of Ubp3 phosphorylation.

Physical interaction between Sit1 and Aft1 upregulates FOB uptake activity by inhibiting protein degradation of Sit1 in Saccharomyces cerevisiae.

Previously, we reported that Aft1 regulates Sit1 by modulating the ubiquitination of Sit1 in Saccharomyces cerevisiae. Here, we report the function of the physical interaction between Sit1 and Aft1 in ferrioxamine B (FOB) uptake. The interaction between Sit1 and Aft1 induced protein localization of Sit1 to the plasma membrane, and more Sit1 was detected in the plasma membrane when Sit1 and Aft1 were coexpressed compared with Sit1 expression alone. The MSN5-deletion mutant, which failed to translocate Aft1 to the cytosolic compartment, showed lower FOB uptake activity than the wild type. However, higher free iron uptake activity was detected in the MSN5-deletion mutant. Furthermore, the strain transformed with AFT1-1(up) plasmid, which failed to regulate Aft1 via iron concentration and accumulated Aft1 in the nucleus, showed lower FOB uptake activity. The Aft1 Y179F mutant, which contained a tyrosine residue that was changed to phenylalanine, failed to interact physically with Sit1 and showed more degradation of the Sit1 and, ultimately, lower FOB uptake activity. Additionally, we found that MG132 and PMSF, which are inhibitors of proteasomes and serine proteases, respectively, increased the Sit1 protein level. Taken together, these results suggest that the protein-protein interaction between Sit1 and Aft1 is an important factor in the FOB uptake activity of Sit1.

A novel protein, Pho92, has a conserved YTH domain and regulates phosphate metabolism by decreasing the mRNA stability of PHO4 in Saccharomyces cerevisiae.

The homologue of human YTHDF2, Ydr374c (Pho92), is the only protein that has a YTH (YT521-B homology) domain in Saccharomyces cerevisiae. Based on microarray analysis, genes involved in the phosphate signal transduction (PHO) pathway were up-regulated in the Δpho92 strain, as were genes regulated by Pho4, which is an important transcription factor in the PHO pathway. To identify the exact mechanism of Pho92 action with respect to phosphate metabolism, we investigated the effect of Pho92 on PHO4 expression. The half-life of PHO4 mRNA was increased in the Δpho92 strain; this phenotype was also observed in the deletion mutants UPF1 and POP2, which are components of the NMD (nonsense-mediated decay) pathway and the Pop2-Ccr4-Not deadenylase complex respectively. Pho92 interacts physically with Pop2 of the Pop2-Ccr4-Not deadenylase complex. Furthermore, Pho92 binding to the 3'-UTR of PHO4 was dependent on the phosphate concentration. Deletion of the PHO4 3'-UTR resulted in PHO4 mRNA resistance to Pho92-dependent degradation. The results of the present study indicate that Pho92 regulates Pho4 expression at the post-transcriptional level via the regulation of mRNA stability. Taken together, Pho92 participates in cellular phosphate metabolism, specifically via the regulation of PHO4 mRNA stability by binding to the 3'-UTR in a phosphate-dependent manner.

Identification of diverse modulators of central and peripheral circadian clocks by high-throughput chemical screening.

The circadian clock coordinates daily oscillations of essential physiological and behavioral processes. Conversely, aberrant clocks with damped amplitude and/or abnormal period have been associated with chronic diseases and aging. To search for small molecules that perturb or enhance circadian rhythms, we conducted a high-throughput screen of approximately 200,000 synthetic compounds using Per2lucSV reporter fibroblast cells and validated 11 independent classes of molecules with Bmal1:luciferase reporter cells as well as with suprachiasmatic nucleus and peripheral tissue explants. Four compounds were found to lengthen the period in both central and peripheral clocks, including three compounds that inhibited casein kinase Iε in vitro and a unique benzodiazepine derivative acting through a non-GABA(A) receptor target. In addition, two compounds acutely induced Per2lucSV reporter bioluminescence, delayed the rhythm, and increased intracellular cAMP levels, but caused rhythm damping. Importantly, five compounds shortened the period of peripheral clocks; among them, four compounds also enhanced the amplitude of central and/or peripheral reporter rhythms. Taken together, these studies highlight diverse activities of drug-like small molecules in manipulating the central and peripheral clocks. These small molecules constitute a toolbox for probing clock regulatory mechanisms and may provide putative lead compounds for treatment of clock-associated diseases.

Rck1 up-regulates pseudohyphal growth by activating the Ras2 and MAP kinase pathways independently in Saccharomyces cerevisiae.

Previously, we reported that Rck1 regulates Hog1 and Slt2 activities and affects MAP kinase activity in Saccharomyces cerevisiae. Recently, we found that Rck1 up-regulates phospho-Kss1 and phospho-Fus3. Kss1 has been known as a component in the pseudohyphal growth pathway, and we attempted to identify the function of Rck1 in pseudohyphal growth. Rck1 up-regulated Ras2 at the protein level, not the transcriptional level. Additionally, FLO11 transcription was up-regulated by RCK1 over-expression. RCK1 expression was up-regulated during growth on SLAD+1% butanol medium. On nitrogen starvation agar plates, RCK1 over-expression induced pseudohyphal growth of colonies, and cells over-expressing RCK1 showed a filamentous morphology when grown in SLAD medium. Furthermore, 1-butanol greatly induced filamentous growth when RCK1 was over-expressed. Moreover, invasive growth was activated in haploid cells when RCK1 was over-expressed. The growth defect of cells observed on 1-butanol medium was recovered when RCK1 was over-expressed. Interestingly, Ras2 and phospho-Kss1 were up-regulated by Rck1 independently. Together, these results suggest that Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 via independent pathways in S. cerevisiae.

The expression of PHO92 is regulated by Gcr1, and Pho92 is involved in glucose metabolism in Saccharomyces cerevisiae.

Ydr374c (Pho92) contains a YTH domain in its C-terminal region and is a human YTHDF2 homologue. Previously, we reported that Pho92 regulates phosphate metabolism by regulating PHO4 mRNA stability. In this study, we found that growth of the ∆pho92 strain on SG media was slower than that of the wild type and that PHO92 expression was up-regulated by non-fermentable carbon sources, such as ethanol and glycerol, but not by fermentable carbon sources. Furthermore, two conserved Gcr1-binding regions were identified in the upstream, untranslated region of PHO92. Gcr1 is an important factor involved in the coordinated regulation of glycolytic gene expression. Mutation of two Gcr1-binding sites of the PHO92 upstream region resulted in a growth defect on SD media. Finally, mutagenesis of the Gcr1-binding sites of the PHO92 upstream region and deletion of GCR1 resulted in up-regulation of PHO92, and this resulted from inhibition of PHO4 mRNA degradation. Based on these results, we suggest that Gcr1 regulates the expression of PHO92, and Pho92 is involved in glucose metabolism.