Exploring the dietary and therapeutic potential of licorice (Glycyrrhiza uralensis Fisch.) sprouts.

ETHNOPHARMACOLOGICAL RELEVANCE: This research substantiates the traditional use of Glycyrrhiza uralensis Fisch. for liver health, with scientific evidence of the non-toxic and lipid-lowering properties of licorice sprout extracts. The sprouts' rich mineral and amino acid content, along with their strong antioxidant activity, reinforce their value in traditional medicine. These findings bridge ancient herbal practices with modern science, highlighting licorice's potential in contemporary therapeutic applications. AIM OF THE STUDY: The study aimed to investigate the dietary and medicinal potential of G. uralensis sprouts by assessing their safety, nutritional content, and antioxidant properties using both plant and animal models. Specifically, the study sought to determine the effects of different sizes of licorice sprouts on lipid metabolism in human liver cancer cells and their overall impact on rat health indicators. MATERIALS AND METHODS: The study examined the effects of aqueous and organic extracts from G. uralensis sprouts of varying lengths on the cytotoxicity, lipid metabolism, and antioxidant activity in HepG2 cells, alongside in vivo impacts on Sprague-Dawley rats, using MTT, ICP, and HPLC. It aimed to assess the potential health benefits of licorice sprouts by analyzing their protective effects against oxidative stress and their nutritional content. RESULTS: Licorice sprout extracts from G. uralensis demonstrated no cytotoxicity in HepG2 cells, significantly reduced lipid levels, and enhanced antioxidant activities, with the longest sprouts (7 cm) showing higher mineral, sugar, and arginine content as well as increased glycyrrhizin and liquiritigenin. In vivo studies with Sprague-Dawley rats revealed weight gain and improved antioxidant enzyme activities in blood plasma and liver tissues after consuming the extracts, highlighting the sprouts' dietary and therapeutic potential. CONCLUSIONS: This study is the first to demonstrate that G. uralensis sprouts, particularly those 7 cm in length, have no cytotoxic effects, reduce lipids, and have high mineral and antioxidant contents, offering promising dietary and therapeutic benefits.

Vincristine Enhances the Efficacy of MEK Inhibitors in Preclinical Models of KRAS-mutant Colorectal Cancer.

Mutations in KRAS are found in more than 50% of tumors from patients with metastatic colorectal cancer (mCRC). However, direct targeting of most KRAS mutations is difficult; even the recently developed KRASG12C inhibitors failed to show significant benefit in patients with mCRC. Single agents targeting mitogen-activated protein kinase kinase (MEK), a downstream mediator of RAS, have also been ineffective in colorectal cancer. To identify drugs that can enhance the efficacy of MEK inhibitors, we performed unbiased high-throughput screening using colorectal cancer spheroids. We used trametinib as the anchor drug and examined combinations of trametinib with the NCI-approved Oncology Library version 5. The initial screen, and following focused validation screens, identified vincristine as being strongly synergistic with trametinib. In vitro, the combination strongly inhibited cell growth, reduced clonogenic survival, and enhanced apoptosis compared with monotherapies in multiple KRAS-mutant colorectal cancer cell lines. Furthermore, this combination significantly inhibited tumor growth, reduced cell proliferation, and increased apoptosis in multiple KRAS-mutant patient-derived xenograft mouse models. In vivo studies using drug doses that reflect clinically achievable doses demonstrated that the combination was well tolerated by mice. We further determined that the mechanism underlying the synergistic effect of the combination was due to enhanced intracellular accumulation of vincristine associated with MEK inhibition. The combination also significantly decreased p-mTOR levels in vitro, indicating that it inhibits both RAS-RAF-MEK and PI3K-AKT-mTOR survival pathways. Our data thus provide strong evidence that the combination of trametinib and vincristine represents a novel therapeutic option to be studied in clinical trials for patients with KRAS-mutant mCRC. SIGNIFICANCE: Our unbiased preclinical studies have identified vincristine as an effective combination partner for the MEK inhibitor trametinib and provide a novel therapeutic option to be studied in patients with KRAS-mutant colorectal cancer.

Combined inhibition of HMGCoA reductase and mitochondrial complex I induces tumor regression of BRAF inhibitor-resistant melanomas.

BACKGROUND: Primary and posttreatment resistance to BRAFV600 mutation-targeting inhibitors leads to disease relapse in a majority of melanoma patients. In many instances, this resistance is promoted by upregulation of mitochondrial oxidative phosphorylation (OxPhos) in melanoma cells. We recently showed that a novel electron transport chain (ETC) complex I inhibitor, IACS-010759 (IACS), abolished OxPhos and significantly inhibited tumor growth of high-OxPhos, BRAF inhibitor (BRAFi)-resistant human melanomas. However, the inhibition was not uniform across different high OxPhos melanomas, and combination with BRAFi did not improve efficacy. METHODS: We performed a high-throughput unbiased combinatorial drug screen of clinically relevant small molecules to identify the most potent combination agent with IACS for inhibiting the growth of high-OxPhos, BRAFi-resistant melanomas. We performed bioenergetics and carbon-13 metabolite tracing to delineate the metabolic basis of sensitization of melanomas to the combination treatment. We performed xenograft tumor growth studies and Reverse-Phase Protein Array (RPPA)-based functional proteomics analysis of tumors from mice fed with regular or high-fat diet to evaluate in vivo molecular basis of sensitization to the combination treatment. RESULTS: A combinatorial drug screen and subsequent validation studies identified Atorvastatin (STN), a hydroxymethylglutaryl-coenzyme A reductase inhibitor (HMGCRi), as the most potent treatment combination with IACS to inhibit in vitro cell growth and induce tumor regression or stasis of some BRAFi-resistant melanomas. Bioenergetics analysis revealed a dependence on fatty acid metabolism in melanomas that responded to the combination treatment. RPPA analysis and carbon-13 tracing analysis in these melanoma cells showed that IACS treatment decreased metabolic fuel utilization for fatty acid metabolism, but increased substrate availability for activation of the mevalonate pathway by HMGCR, creating a dependence on this pathway. Functional proteomic analysis showed that IACS treatment inhibited MAPK but activated AKT pathway. Combination treatment with STN counteracted AKT activation. CONCLUSIONS: STN and other clinically approved HMGCRi could be promising combinatorial agents for improving the efficacy of ETC inhibitors like IACS in BRAFi-resistant melanomas.

Quality Characteristics and Antioxidant Potential of Lemon (Citrus limon Burm. f.) Seed Oil Extracted by Different Methods.

Lemon (Citrus limon Burm. f.) is one of the most widely produced and consumed fruits in the world. The seeds of lemon are generally discarded as waste. The purpose of this study was to investigate the quality characteristics and antioxidant potential of lemon seed oil obtained by four extraction methods (roasted-pressing at 170°C, RP-170; roasted-pressing at 100°C, RP-100; cold-pressing, CP; and supercritical fluid, SF). No significant differences in the viscosity, density, and refractive index were observed in the oil obtained from different methods. In the case of Hunter's value, L (lightness) and b (yellowness) values of SF were higher than those of the others. The oil obtained by the CP method exhibited higher levels of Ca (252.17 mg/kg), Cu (2.38 mg/kg), K (225.98 mg/kg), and Mo (0.47 mg/kg) than that of other methods. The highest contents of total phenols (165.90 mg/mL) and flavonoids (21.69 mg/mL) were significantly high in oil obtained by the SF method. Oleic and linoleic acids consisted of principal fatty acids, which were significantly higher in oil obtained by RP-170. Higher amounts of volatile flavor compounds, such as γ-terpinene, sabinene, and limonene, were observed in CP compared to those observed for the other methods. This study elucidates the effects of different methods of oil extraction on the composition of lemon seed oil and highlights potential applications of these benefits in the food, cosmetic, pharmaceutical, and/or fragrance industries.

Therapeutically actionable signaling node to rescue AURKA driven loss of primary cilia in VHL-deficient cells.

Loss of primary cilia in cells deficient for the tumor suppressor von Hippel Lindau (VHL) arise from elevated Aurora Kinase A (AURKA) levels. VHL in its role as an E3 ubiquitin ligase targets AURKA for degradation and in the absence of VHL, high levels of AURKA result in destabilization of the primary cilium. We identified NVP-BEZ235, a dual PI3K/AKT and mTOR inhibitor, in an image-based high throughput screen, as a small molecule that restored primary cilia in VHL-deficient cells. We identified the ability of AKT to modulate AURKA expression at the transcript and protein level. Independent modulation of AKT and mTOR signaling decreased AURKA expression in cells confirming AURKA as a new signaling node downstream of the PI3K cascade. Corroborating these data, a genetic knockdown of AKT in cells deficient for VHL rescued the ability of these cells to ciliate. Finally, inhibition of AKT/mTOR using NVP-BEZ235 was efficacious in reducing tumor burden in a 786-0 xenograft model of renal cell carcinoma. These data highlight a previously unappreciated signaling node downstream of the AKT/mTOR pathway via AURKA that can be targeted in VHL-null cells to restore ciliogenesis.

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.

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.

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.

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.

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.

Suppression of IL-8 production in gastric epithelial cells by MUC1 mucin and peroxisome proliferator-associated receptor-γ.

MUC1 is a membrane-tethered mucin expressed on the apical surface of epithelial cells. Our previous report (Guang W, Ding H, Czinn SJ, Kim KC, Blanchard TG, Lillehoj EP. J Biol Chem 285: 20547-20557, 2010) demonstrated that expression of MUC1 in AGS gastric epithelial cells limits Helicobacter pylori infection and reduces bacterial-driven IL-8 production. In this study, we identified the peroxisome proliferator-associated receptor-γ (PPARγ) upstream of MUC1 in the anti-inflammatory pathway suppressing H. pylori- and phorbol 12-myristate 13-acetate (PMA)-stimulated IL-8 production. Treatment of AGS cells with H. pylori or PMA increased IL-8 levels in cell culture supernatants compared with cells treated with the respective vehicle controls. Prior small interfering (si)RNA-induced MUC1 silencing further increased H. pylori- and PMA-stimulated IL-8 levels compared with a negative control siRNA. MUC1-expressing AGS cells pretreated with the PPARγ agonist troglitazone (TGN) had reduced H. pylori- and PMA-stimulated IL-8 levels compared with cells treated with H. pylori or PMA alone. However, following MUC1 siRNA knockdown, no differences in IL-8 levels were seen between TGN/H. pylori and H. pylori-only cells or between TGN/PMA and PMA-only cells. Finally, TGN-treated AGS cells had increased Muc1 promoter activity, as measured using a Muc1-luciferase reporter gene, and greater MUC1 protein levels by Western blot analysis, compared with vehicle controls. These results support the hypothesis that PPARγ stimulates MUC1 expression by AGS cells, thereby attenuating H. pylori- and PMA-induced IL-8 production.

Prevention of lung injury by Muc1 mucin in a mouse model of repetitive Pseudomonas aeruginosa infection.

OBJECTIVE AND DESIGN: To determine whether repetitive airway Pseudomonas aeruginosa (Pa) infection results in lung inflammation and injury and, if so, whether these responses are affected by Muc1 mucin. Muc1 wild type (WT) and knockout (KO) mice were compared for body weights, lung inflammatory responses, and airspace enlargement using a chronic lung infection model system. MATERIALS: Mice were treated intranasally with Pa (10(7) CFU) on days 0, 4, 7 and 10. On day 14, body weights, inflammatory cell numbers in bronchoalveolar lavage fluid (BALF), and airspace enlargement were measured. Differences in inflammatory responses between groups were statistically analyzed by the Student's t test and ANOVA. RESULTS: Muc1 WT mice exhibited mild degrees of both inflammation and airspace enlargement following repetitive airway Pa infection. However, Muc1 KO mice exhibited significantly decreased body weights, greater macrophage numbers in the BALF, and increased airspace enlargement compared with Muc1 WT mice. CONCLUSIONS: This is the first report demonstrating that Muc1 deficiency can lead to lung injury during chronic Pa infection in mice. These results suggest that MUC1 may play a crucial role in the resolution of inflammation during chronic respiratory infections and that MUC1 dysfunction likely contributes to the pathogenesis of chronic inflammatory respiratory disease.

Membrane-tethered MUC1 mucin is phosphorylated by epidermal growth factor receptor in airway epithelial cells and associates with TLR5 to inhibit recruitment of MyD88.

MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of mucosal epithelial cells. Previous in vivo and in vitro studies established that MUC1 counterregulates airway inflammation by suppressing TLR signaling. In this article, we elucidate the mechanism by which MUC1 inhibits TLR5 signaling. Overexpression of MUC1 in HEK293 cells dramatically reduced Pseudomonas aeruginosa-stimulated IL-8 expression and decreased the activation of NF-κB and MAPK compared with cells not expressing MUC1. However, overexpression of MUC1 in HEK293 cells did not affect NF-κB or MAPK activation in response to TNF-α. Overexpression of MyD88 abrogated the ability of MUC1 to inhibit NF-κB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5/MyD88 compared with controls. The MUC1 cytoplasmic tail associated with TLR5 in all cells tested, including HEK293T cells, human lung adenocarcinoma cell line A549 cells, and human and mouse primary airway epithelial cells. Activation of epidermal growth factor receptor tyrosine kinase with TGF-α induced phosphorylation of the MUC1 cytoplasmic tail at the Y46EKV sequence and increased association of MUC1/TLR5. Finally, in vivo experiments demonstrated increased immunofluorescence colocalization of Muc1/TLR5 and Muc1/phosphotyrosine staining patterns in mouse airway epithelium and increased Muc1 tyrosine phosphorylation in mouse lung homogenates following P. aeruginosa infection. In conclusion, epidermal growth factor receptor tyrosine phosphorylates MUC1, leading to an increase in its association with TLR5, thereby competitively and reversibly inhibiting recruitment of MyD88 to TLR5 and downstream signaling events. This unique ability of MUC1 to control TLR5 signaling suggests its potential role in the pathogenesis of chronic inflammatory lung diseases.

PPARγ inhibits airway epithelial cell inflammatory response through a MUC1-dependent mechanism.

This study was conducted to examine the relationship between the peroxisome proliferator-associated receptor-γ (PPARγ) and MUC1 mucin, two anti-inflammatory molecules expressed in the airways. Treatment of A549 lung epithelial cells or primary mouse tracheal surface epithelial (MTSE) cells with phorbol 12-myristate 13-acetate (PMA) increased the levels of tumor necrosis factor (TNF)-α in cell culture media compared with cells treated with vehicle alone. Overexpression of MUC1 in A549 cells decreased PMA-stimulated TNF-α levels, whereas deficiency of Muc1 expression in MTSE cells from Muc1 null mice increased PMA-induced TNF-α levels. Treatment of A549 or MTSE cells with the PPARγ agonist troglitazone (TGN) blocked the ability of PMA to stimulate TNF-α levels. However, the effect of TGN required the presence of MUC1/Muc1, since no differences in TNF-α levels were seen between PMA and PMA plus TGN in MUC1/Muc1-deficient cells. Similarly, whereas TGN decreased interleukin-8 (IL-8) levels in culture media of MUC1-expressing A549 cells treated with Pseudomonas aeruginosa strain K (PAK), no differences in IL-8 levels were seen between PAK and PAK plus TGN in MUC1-nonexpressing cells. EMSA confirmed the presence of a PPARγ-binding element in the MUC1 gene promoter. Finally, TGN treatment of A549 cells increased MUC1 promoter activity measured using a MUC1-luciferase reporter gene, augmented MUC1 mRNA levels by quantitative RT-PCR, and enhanced MUC1 protein expression by Western blot analysis. These combined data are consistent with the hypothesis that PPARγ stimulates MUC1/Muc1 expression, thereby blocking PMA/PAK-induced TNF-α/IL-8 production by airway epithelial cells.

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.

Effect of guaifenesin on mucin production, rheology, and mucociliary transport in differentiated human airway epithelial cells.

Guaifenesin is widely used to alleviate symptoms of excessive mucus accumulation in the respiratory tract. However, its mechanism of action is poorly understood. The authors hypothesized that guaifenesin improves mucociliary clearance in humans by reducing mucin release, by decreasing mucus viscoelasticity, and by increasing mucociliary transport. To test these hypotheses, human differentiated airway epithelial cells, cultured at an air-liquid interface, were treated with clinically relevant concentrations of guaifenesin by addition to the basolateral medium. To evaluate the effect on mucin secretion, the authors used an anzyme-linked immunosorbent assay (ELISA) to measure the amounts of MUC5AC protein in apical surface fluid and cell lysates. To measure mucociliary transportability, additional cultures were treated for 1 or 6 hours with guaifenesin, and the movement of cell debris was measured from video data. Further, the authors measured mucus dynamic viscoelasticity using a micro cone and plate rheometer with nondestructive creep transformation. Guaifenesin suppressed mucin production in a dose-dependent manner at clinically relevant concentrations. The reduced mucin production was associated with increased mucociliary transport and decreased viscoelasticity of the mucus. Viability of the cultures was not significantly affected. These results suggest that guaifenesin could improve mucociliary clearance in humans by reducing the release and/or production of mucins, thereby altering mucus rheology.

TNF-α is a key regulator of MUC1, an anti-inflammatory molecule, during airway Pseudomonas aeruginosa infection.

Muc1 is a heterodimeric mucin that is expressed on the apical surface of airway epithelial cells as well as hematopoietic cells. Both in vivo and in vitro studies revealed that Muc1 suppresses inflammatory responses induced by Pseudomonas aeruginosa (PA). In this study, we sought to determine, using intact animals (C57BL/6 mice), whether the expression of Muc1 is important during airway PA infection, and how Muc1 levels are controlled during inflammation. Our results showed that: (1) Muc1 levels in the wild-type (WT) mice were initially low, but gradually increased after PA inhalation, reaching a peak on Day 2, remaining elevated until Day 4, and then gradually decreasing to basal levels on Day 7; (2) TNF receptor 1(-/-) mice failed to increase Muc1 levels after PA infection; (3) after PA inhalation, more inflammatory cells were present in the bronchoalveolar lavage fluid from either Muc1(-/-) or TNF receptor(-/-) mice compared with their WT control animals; (4) more apoptotic neutrophils were present in bronchoalveolar lavage fluid from WT mice compared with Muc1(-/-) mice. We conclude that Muc1(-/-) mice are more inflammatory than WT mice during airway PA infection as a result of both an increase in neutrophil influx and a decrease in neutrophil apoptosis. These results suggest that the up-regulation of Muc1 during airway PA infection might be crucial for suppressing excessive and prolonged inflammatory responses, and is induced mainly by TNF-α, the key proinflammatory mediator.

A chitosan hydrogel-based cancer drug delivery system exhibits synergistic antitumor effects by combining with a vaccinia viral vaccine.

Cancer treatment combining chemotherapy and immunotherapy has been vigorously exploited to further improve cancer therapeutic efficacy. This study investigated a new chemoimmunotherapy approach utilizing hydrogel as a local anti-cancer drug delivery system. Chitosan hydrogel containing doxorubicin (CH-DOX) and vaccinia virus vaccine expressing Sig/E7/LAMP-1 (Vac-Sig/E7/LAMP-1) were used as chemoimmunotherapeutic agents. It was found that intratumoral injection of CH-DOX effectively inhibited tumor growth itself and, in addition, exhibited a synergistic antitumor effect in combination with a vaccinia virus-based vaccine. This combination did not decrease but rather increased the number of tumor-specific CD8(+) T cells primed by vaccinia virus-mediated vaccination; the resulting antitumor effects were further improved up to 60 days as compared with monotherapy after tumor challenge, and the survival of tumor-bearing mice was dramatically prolonged. This study is a pioneer report that demonstrates the use of a biodegradable hydrogel system as an anti-cancer drug delivery system for successful chemoimmunotherapy. It is hoped that, this study can provide a foundation for a rational approach to improve antitumor efficacy of chemoimmunotherapy.

Physical and functional interaction of FgFtr1-FgFet1 and FgFtr2-FgFet2 is required for iron uptake in Fusarium graminearum.

FgFtr1 and FgFtr2 are putative iron permeases, and FgFet1 and FgFet2 are putative ferroxidases of Fusarium graminearum. They have high homologies with iron permease ScFtr1 and ferroxidase ScFet3 of Saccharomyces cerevisiae at the amino acid level. The genes encoding iron permease and ferroxidase were localized to the same chromosome in the manner of FgFtr1/FgFet1 and FgFtr2/FgFet2. The GFP (green fluorescent protein)-fused versions of FgFtr1 and FgFtr2 showed normal functions when compared with FgFtr1 and FgFtr2 in an S. cerevisiae system, and the cellular localizations of FgFtr1 and FgFtr2 in S. cerevisiae depended on the expression of their putative ferroxidase partners FgFet1 and FgFet2 respectively. Although FgFtr1 was found on the plasma membrane when FgFet1 and FgFtr1 were co-transformed in S. cerevisiae, most of the FgFtr1 was found in the endoplasmic reticulum compartment when co-expressed with FgFet2. Furthermore, FgFtr2 was found on the vacuolar membrane when FgFet2 was co-expressed. From the two-hybrid analysis, we confirmed the interaction of FgFtr1 and FgFet1, and the same result was found between FgFtr2 and FgFet2. Iron-uptake activity also depended on the existence of the respective partner. Finally, the FgFtr1 and FgFtr2 were found on the plasma and vacuolar membrane respectively, in F. graminearum. Taken together, these results strongly suggest that FgFtr1 and FgFtr2 from F. graminearum encode the iron permeases of the plasma membrane and vacuolar membrane respectively, and require their specific ferroxidases to carry out normal function. Furthermore, the present study suggests that the reductive iron-uptake system is conserved from yeast to filamentous fungi.

Chemotherapy enhances CD8(+) T cell-mediated antitumor immunity induced by vaccination with vaccinia virus.

The use of immunotherapy or chemotherapy alone is generally ineffective against well-established tumors. To overcome this intrinsic resistance against therapy for tumors, we have attempted to combine immunotherapy with chemotherapy. In this study, we tried to induce a rapid antitumor effect via chemoimmunotherapy using a vaccinia viral vaccine as an immunotherapeutic agent with anticancer agents including epigallocatechin-3-gallate (EGCG) and conventional anticancer drugs. Although a combination of vaccinia-mediated vaccination and chemotherapy led to a strong inhibition of tumor growth, monotherapy alone failed to completely cure tumors. In contrast, intravenous injection of cisplatin (CDDP) or cyclophosphamide (CTX) after vaccinia virus vaccination led to complete regression of the established tumors. Interestingly, anticancer drugs appear to augment the antitumor effect of the vaccinia virus-mediated immunotherapy. This effect is mainly associated with the enhanced tumor-specific CD8(+) T cell immune response induced by vaccinia virus, which was demonstrated by antibody depletion. However, anticancer drugs alone failed to induce a significant enhancement of the tumor-specific CD8(+) T cell immune response. Taken together, these results suggest that combining vaccinia virus-based immunotherapy with anticancer drugs is particularly effective against established tumors by increasing the tumor antigen-specific CD8(+) T cell immune response, which is primed by vaccinia virus-mediated vaccination.