Increase in mean diffusivity indicates the presence of interstitial brain edema. Mean diffusivity values increase as the grade of HE increases, suggesting that brain edema present in patients with HE may contribute to its pathogenesis.59 Mean diffusivity values decreased significantly and there was a corresponding improvement in neuropsychological test scores in patients with MHE after three weeks of lactulose therapy.59 MR imaging techniques therefore complement neuropsychological evaluation of MHE. 31 MRS, diffusion-weighted this website imaging, magnetization transfer imaging and diffusion tensor

imaging show abnormalities in cirrhotic patients with or without HE. (1b) By definition, patients with MHE have a normal neurological examination; however they may still be symptomatic. Symptoms relate to disturbances in sleep, memory, attention, concentration and other areas of cognition.60,61 Sleep disturbance is a classic sign of HE. On a sleep questionnaire, disturbance is seen in 47% of cirrhotics and 38% of patients with chronic renal failure compared to 4.5% of controls.60 Studies using HRQOL

questionnaires have confirmed a higher frequency of sleep disturbance in cirrhotic patients with MHE as well.3,14 However, sleep disturbance in cirrhosis is not associated with cognitive impairment; thus it may not truly be an MHE symptom. Unsatisfactory sleep is associated with higher scores for depression and anxiety, raising the possibility that the effects of chronic disease may underlie the pathogenesis of sleep disturbance. Disturbances in cirrhotics may also be related to abnormalities of circadian rhythm. Defective memory has also been shown to be a feature selleck chemicals of MHE. Weissenborn et al.61 have shown that patients with MHE have impaired short- and long-term memory. This impairment was predominantly related to deficits in attention and visual perception. Memory deficit of MHE seems to comprise short-term but not long-term memory impairment. This can be described as an encoding defect, in which memory recall (or retrieval) is intact.

Several cognitive statements (i.e. complaints), have predictive value for MHE, including impaired psychomotor performance selleck chemicals llc (‘I have difficulty doing handwork; I am not working at all’); impaired sleep or rest (‘I spend much of the day lying down in order to rest’); decreased attention (‘I am confused and start several actions at a time’); and poor memory (‘I forget a lot; for example, things that happened recently, where I put things, etc.’).14 It has been shown conclusively that cognitive functions improve with therapy for MHE.3,62–67 Such therapy may improve HRQOL of patients with MHE3,67 and delay the development of HE.68 Hence all patients with liver cirrhosis should be subjected to testing for MHE. Special attention should be given to those who have cognitive symptoms and high-risk groups such as active drivers, patients handling heavy machines or reporting decline in work performance.

Increase in mean diffusivity indicates the presence of interstitial brain edema. Mean diffusivity values increase as the grade of HE increases, suggesting that brain edema present in patients with HE may contribute to its pathogenesis.59 Mean diffusivity values decreased significantly and there was a corresponding improvement in neuropsychological test scores in patients with MHE after three weeks of lactulose therapy.59 MR imaging techniques therefore complement neuropsychological evaluation of MHE. 31 MRS, diffusion-weighted Selleck Compound Library imaging, magnetization transfer imaging and diffusion tensor

imaging show abnormalities in cirrhotic patients with or without HE. (1b) By definition, patients with MHE have a normal neurological examination; however they may still be symptomatic. Symptoms relate to disturbances in sleep, memory, attention, concentration and other areas of cognition.60,61 Sleep disturbance is a classic sign of HE. On a sleep questionnaire, disturbance is seen in 47% of cirrhotics and 38% of patients with chronic renal failure compared to 4.5% of controls.60 Studies using HRQOL

questionnaires have confirmed a higher frequency of sleep disturbance in cirrhotic patients with MHE as well.3,14 However, sleep disturbance in cirrhosis is not associated with cognitive impairment; thus it may not truly be an MHE symptom. Unsatisfactory sleep is associated with higher scores for depression and anxiety, raising the possibility that the effects of chronic disease may underlie the pathogenesis of sleep disturbance. Disturbances in cirrhotics may also be related to abnormalities of circadian rhythm. Defective memory has also been shown to be a feature Autophagy inhibitor of MHE. Weissenborn et al.61 have shown that patients with MHE have impaired short- and long-term memory. This impairment was predominantly related to deficits in attention and visual perception. Memory deficit of MHE seems to comprise short-term but not long-term memory impairment. This can be described as an encoding defect, in which memory recall (or retrieval) is intact.

Several cognitive statements (i.e. complaints), have predictive value for MHE, including impaired psychomotor performance click here (‘I have difficulty doing handwork; I am not working at all’); impaired sleep or rest (‘I spend much of the day lying down in order to rest’); decreased attention (‘I am confused and start several actions at a time’); and poor memory (‘I forget a lot; for example, things that happened recently, where I put things, etc.’).14 It has been shown conclusively that cognitive functions improve with therapy for MHE.3,62–67 Such therapy may improve HRQOL of patients with MHE3,67 and delay the development of HE.68 Hence all patients with liver cirrhosis should be subjected to testing for MHE. Special attention should be given to those who have cognitive symptoms and high-risk groups such as active drivers, patients handling heavy machines or reporting decline in work performance.

Increase in mean diffusivity indicates the presence of interstitial brain edema. Mean diffusivity values increase as the grade of HE increases, suggesting that brain edema present in patients with HE may contribute to its pathogenesis.59 Mean diffusivity values decreased significantly and there was a corresponding improvement in neuropsychological test scores in patients with MHE after three weeks of lactulose therapy.59 MR imaging techniques therefore complement neuropsychological evaluation of MHE. 31 MRS, diffusion-weighted click here imaging, magnetization transfer imaging and diffusion tensor

imaging show abnormalities in cirrhotic patients with or without HE. (1b) By definition, patients with MHE have a normal neurological examination; however they may still be symptomatic. Symptoms relate to disturbances in sleep, memory, attention, concentration and other areas of cognition.60,61 Sleep disturbance is a classic sign of HE. On a sleep questionnaire, disturbance is seen in 47% of cirrhotics and 38% of patients with chronic renal failure compared to 4.5% of controls.60 Studies using HRQOL

questionnaires have confirmed a higher frequency of sleep disturbance in cirrhotic patients with MHE as well.3,14 However, sleep disturbance in cirrhosis is not associated with cognitive impairment; thus it may not truly be an MHE symptom. Unsatisfactory sleep is associated with higher scores for depression and anxiety, raising the possibility that the effects of chronic disease may underlie the pathogenesis of sleep disturbance. Disturbances in cirrhotics may also be related to abnormalities of circadian rhythm. Defective memory has also been shown to be a feature EPZ015666 cell line of MHE. Weissenborn et al.61 have shown that patients with MHE have impaired short- and long-term memory. This impairment was predominantly related to deficits in attention and visual perception. Memory deficit of MHE seems to comprise short-term but not long-term memory impairment. This can be described as an encoding defect, in which memory recall (or retrieval) is intact.

Several cognitive statements (i.e. complaints), have predictive value for MHE, including impaired psychomotor performance see more (‘I have difficulty doing handwork; I am not working at all’); impaired sleep or rest (‘I spend much of the day lying down in order to rest’); decreased attention (‘I am confused and start several actions at a time’); and poor memory (‘I forget a lot; for example, things that happened recently, where I put things, etc.’).14 It has been shown conclusively that cognitive functions improve with therapy for MHE.3,62–67 Such therapy may improve HRQOL of patients with MHE3,67 and delay the development of HE.68 Hence all patients with liver cirrhosis should be subjected to testing for MHE. Special attention should be given to those who have cognitive symptoms and high-risk groups such as active drivers, patients handling heavy machines or reporting decline in work performance.

All animals received care in compliance with protocols approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School. Mice were gradually habituated to a Lieber-DeCarli liquid diet with 5% ethanol (vol/vol) over a period of 2 weeks, then maintained on the 5% diet for 4 weeks. Consumption was recorded

daily throughout and isocaloric amounts of a non–alcohol-containing diet (in which dextran-maltose replaced calories from ethanol) were dispensed to pair-fed animals. Weights were recorded weekly. Wild-type (WT) mice (C57/Bl6), Alb-Cre, and HIF-1flox/flox mice were purchased from Jackson Laboratories (Bar Harbor, ME). LSL-HIF1dPA mice were a

kind gift of William Kim (University of North Carolina, Chapel Hill, NC). The HIF1dPA allele was engineered by Kim et check details al.10 Briefly, a stop codon is flanked by loxP sites upstream of a HIF-1α transgene in which a proline-to-alanine substitution enables the transgene to escape recognition by proline hydroxylases and subsequent proteasomal degradation. Coexpression of the albumin-cre transgene excises the stop codon, and subsequently enables expression of the transgene in hepatocytes. LSL-HIF1dPA and HIF-1flox/flox mice were bred against Cre mice as described,10, 11 tagged by ear notching, and housed in separate

cages.10, 11 Prior to the conclusion of the study, some mice were randomly buy Nivolumab assigned to receive lipopolysaccharide (LPS) (Sigma) injection (500 μg/kg) or saline injection. Mice were sacrificed 18 hours after LPS injection. At the conclusion of the feeding, mice were weighed and euthanized. Livers were excised and weighed, and portions were snap-frozen in liquid nitrogen for protein and biochemical assays, preserved in 10% neutral-buffered formalin for histopathological analysis, or soaked in RNALater (Qiagen GmbH, Hilden, Germany) for RNA extraction. Blood was collected and serum was separated for see more biochemical analysis. Tail snips were collected for genotyping. Nuclear extracts were prepared via sucrose gradient centrifugation and two-step purification as described.17 Serum alanine aminotransferase (ALT) levels were determined using a commercially available reagent (Advanced Diagnostics Inc., Plainfield, NJ) as described.17 Liver triglycerides were quantified as described using a commercially available kit (Wako Chemicals USA Inc., Richmond, VA).17 Sections of formalin-fixed livers were stained with hematoxylin/eosin and analyzed via microscopy. Frozen sections were prepared from liver tissue frozen in OCT media and stained with Oil Red O. Photomicrographs were analyzed with Metamorph software.

All animals received care in compliance with protocols approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School. Mice were gradually habituated to a Lieber-DeCarli liquid diet with 5% ethanol (vol/vol) over a period of 2 weeks, then maintained on the 5% diet for 4 weeks. Consumption was recorded

daily throughout and isocaloric amounts of a non–alcohol-containing diet (in which dextran-maltose replaced calories from ethanol) were dispensed to pair-fed animals. Weights were recorded weekly. Wild-type (WT) mice (C57/Bl6), Alb-Cre, and HIF-1flox/flox mice were purchased from Jackson Laboratories (Bar Harbor, ME). LSL-HIF1dPA mice were a

kind gift of William Kim (University of North Carolina, Chapel Hill, NC). The HIF1dPA allele was engineered by Kim et ICG-001 price al.10 Briefly, a stop codon is flanked by loxP sites upstream of a HIF-1α transgene in which a proline-to-alanine substitution enables the transgene to escape recognition by proline hydroxylases and subsequent proteasomal degradation. Coexpression of the albumin-cre transgene excises the stop codon, and subsequently enables expression of the transgene in hepatocytes. LSL-HIF1dPA and HIF-1flox/flox mice were bred against Cre mice as described,10, 11 tagged by ear notching, and housed in separate

cages.10, 11 Prior to the conclusion of the study, some mice were randomly Dasatinib assigned to receive lipopolysaccharide (LPS) (Sigma) injection (500 μg/kg) or saline injection. Mice were sacrificed 18 hours after LPS injection. At the conclusion of the feeding, mice were weighed and euthanized. Livers were excised and weighed, and portions were snap-frozen in liquid nitrogen for protein and biochemical assays, preserved in 10% neutral-buffered formalin for histopathological analysis, or soaked in RNALater (Qiagen GmbH, Hilden, Germany) for RNA extraction. Blood was collected and serum was separated for this website biochemical analysis. Tail snips were collected for genotyping. Nuclear extracts were prepared via sucrose gradient centrifugation and two-step purification as described.17 Serum alanine aminotransferase (ALT) levels were determined using a commercially available reagent (Advanced Diagnostics Inc., Plainfield, NJ) as described.17 Liver triglycerides were quantified as described using a commercially available kit (Wako Chemicals USA Inc., Richmond, VA).17 Sections of formalin-fixed livers were stained with hematoxylin/eosin and analyzed via microscopy. Frozen sections were prepared from liver tissue frozen in OCT media and stained with Oil Red O. Photomicrographs were analyzed with Metamorph software.

, 2001b). Other suggested reasons why it might benefit subordinates to defer breeding include reduced foraging skills and associated energetic constraints, negative effects of breeding at the same time as dominants on the fitness of their own offspring, and costs to indirect components of their fitness if dominants are close relatives

(Young, 2009). Evidence of these effects has led to a debate over whether subordinate infertility should be interpreted as a consequence of constraints on subordinate Selleckchem R788 breeding imposed by dominants or of voluntary restraint by subordinates caused by the need to avoid attracting aggression from dominants or by high costs of breeding associated with reduced condition or inferior foraging skills (Saltzman, Digby et al., 2009; Young, 2009). However, the distinction between these arguments is not as clear as it may initially appear since subordinates may commonly show restraint because dominants constrain their reproductive

options (Young, 2009). For example, subordinates may respond to the presence of dominants by down-regulating their physiological systems because dominants are likely to evict them if they attempt to breed, so that the likely fitness benefits of competing to breed are low (a reproductive constraint). Evidence that other factors modify the frequency of breeding by subordinates

(such as condition or the absence of unrelated partners) does not necessarily argue for interpretations based on restraint, for effects of this Vorinostat datasheet kind would be expected under both scenarios. Perhaps the most realistic view is that subordinates commonly show restraint because dominants constrain their reproductive options (Young, 2009). Attempts see more by dominant females to prevent other females from breeding or to reduce their success in rearing offspring are sometimes regarded as examples of spite since they can occur at times when the benefits of reproductive suppression are not obvious or resources are abundant (Stockley & Bro-Jorgensen, 2011). However, although this is theoretically possible (Gardner & West, 2004), the fitness costs of attacks on subordinates and their offspring may often be low while simultaneous breeding by subordinates may often have long-term costs to dominants and their dependents (Clutton-Brock et al., 2010). Consequently, it is probably more realistic to regard attempts by dominants to suppress reproduction by subordinates as an example of selfish behaviour rather than spite. While infanticide by females has attracted less attention than infanticide by males, it is probably more widespread (Rödel et al., 2008) and frequently represents a threat for group-living females (Digby, 2000).

, 2001b). Other suggested reasons why it might benefit subordinates to defer breeding include reduced foraging skills and associated energetic constraints, negative effects of breeding at the same time as dominants on the fitness of their own offspring, and costs to indirect components of their fitness if dominants are close relatives

(Young, 2009). Evidence of these effects has led to a debate over whether subordinate infertility should be interpreted as a consequence of constraints on subordinate this website breeding imposed by dominants or of voluntary restraint by subordinates caused by the need to avoid attracting aggression from dominants or by high costs of breeding associated with reduced condition or inferior foraging skills (Saltzman, Digby et al., 2009; Young, 2009). However, the distinction between these arguments is not as clear as it may initially appear since subordinates may commonly show restraint because dominants constrain their reproductive

options (Young, 2009). For example, subordinates may respond to the presence of dominants by down-regulating their physiological systems because dominants are likely to evict them if they attempt to breed, so that the likely fitness benefits of competing to breed are low (a reproductive constraint). Evidence that other factors modify the frequency of breeding by subordinates

(such as condition or the absence of unrelated partners) does not necessarily argue for interpretations based on restraint, for effects of this AZD6244 cost kind would be expected under both scenarios. Perhaps the most realistic view is that subordinates commonly show restraint because dominants constrain their reproductive options (Young, 2009). Attempts find more by dominant females to prevent other females from breeding or to reduce their success in rearing offspring are sometimes regarded as examples of spite since they can occur at times when the benefits of reproductive suppression are not obvious or resources are abundant (Stockley & Bro-Jorgensen, 2011). However, although this is theoretically possible (Gardner & West, 2004), the fitness costs of attacks on subordinates and their offspring may often be low while simultaneous breeding by subordinates may often have long-term costs to dominants and their dependents (Clutton-Brock et al., 2010). Consequently, it is probably more realistic to regard attempts by dominants to suppress reproduction by subordinates as an example of selfish behaviour rather than spite. While infanticide by females has attracted less attention than infanticide by males, it is probably more widespread (Rödel et al., 2008) and frequently represents a threat for group-living females (Digby, 2000).

758) (Fig. 2C). Furthermore, the magnitude of enhancement of TAA-specific immune responses did not correlate significantly with the length of HCC recurrence-free survival (P = 0.267) (Fig. 2D). When univariate analysis of prognostic factors for HCC recurrence-free survival was performed, γ-glutamyltransferase (<30), AFP (<400), Okuda stage,1 and number

of TAA-specific T cells after RFA (≥50) were detected as factors that decrease HCC recurrence rate after RFA (Table 3). When multivariate learn more analysis including these three factors was performed, only the number of TAA-specific T cells after RFA (≥50) was found to be a factor that decreases HCC recurrence rate after RFA. To identify the factors that affect the number of TAA-specific T cells after RFA, we analyzed clinical parameters of patients

and the frequency of CD14+HLA-DR−/low selleck compound library MDSCs after HCC treatment. We could not find any clinical parameters correlated with the number of TAA-specific T cells after RFA. The frequency of CD14+HLA-DR−/low MDSCs after RFA showed various levels and depended on the patient (Fig. 3A,B). The frequency decreased significantly after RFA (P = 0.022) except in three patients (Fig. 3B) and correlated inversely with the number of TAA-specific T cells after RFA, but not with that of CMV-specific T cells (Fig. 3C). Next, we examined the naïve/effector/memory phenotype of increased TAA-specific T cells after RFA using a tetramer assay. The memory phenotype was investigated by the criterion of CD45RA/CCR7 expression.17 In tetramer analysis, the frequency of TAA-derived peptide-specific CD8+ T cells before RFA was 0.00%-0.03% of CD8+ cells (Fig. 4A). On the other hand, the frequency was increased after RFA in 10/12 (83.3%) patients, and the range was 0.00%-0.10%

of CD8+ cells. The frequency of CD45RA−/CCR7+ (central memory), CD45RA−/CCR7− (effector memory), and CD45RA+/CCR7− (effector) T cells in tetramer-positive cells depended on the patients, and the ratio of these cells changed after RFA (Fig. 4B). The frequency of tetramer-positive cells with CD45RA−/CCR7+ and CD45RA−/CCR7− in CD8+ cells see more was increased in 6/7 (85.7%) and 6/7 (85.7%) patients, respectively, whose samples were available for the assay before and after RFA. Interestingly, the tetramer-positive cells with CD45RA−/CCR7+ were newly induced after RFA in 5/7 (71.4%) patients. Although the number of TAA-specific T cells was a predictive factor of a decrease of HCC recurrence rate after RFA (as shown in Fig. 2A), more than 50% of the patients with a high number of TAA-specific T cells showed HCC recurrence for 25 months after treatment. To identify the relationship between TAA-specific T cell responses and HCC recurrence more precisely, we examined the kinetics of TAA-specific T cells in 16 patients whose PBMCs were available for analysis at 24 weeks after RFA.

758) (Fig. 2C). Furthermore, the magnitude of enhancement of TAA-specific immune responses did not correlate significantly with the length of HCC recurrence-free survival (P = 0.267) (Fig. 2D). When univariate analysis of prognostic factors for HCC recurrence-free survival was performed, γ-glutamyltransferase (<30), AFP (<400), Okuda stage,1 and number

of TAA-specific T cells after RFA (≥50) were detected as factors that decrease HCC recurrence rate after RFA (Table 3). When multivariate Copanlisib mw analysis including these three factors was performed, only the number of TAA-specific T cells after RFA (≥50) was found to be a factor that decreases HCC recurrence rate after RFA. To identify the factors that affect the number of TAA-specific T cells after RFA, we analyzed clinical parameters of patients

and the frequency of CD14+HLA-DR−/low Torin 1 MDSCs after HCC treatment. We could not find any clinical parameters correlated with the number of TAA-specific T cells after RFA. The frequency of CD14+HLA-DR−/low MDSCs after RFA showed various levels and depended on the patient (Fig. 3A,B). The frequency decreased significantly after RFA (P = 0.022) except in three patients (Fig. 3B) and correlated inversely with the number of TAA-specific T cells after RFA, but not with that of CMV-specific T cells (Fig. 3C). Next, we examined the naïve/effector/memory phenotype of increased TAA-specific T cells after RFA using a tetramer assay. The memory phenotype was investigated by the criterion of CD45RA/CCR7 expression.17 In tetramer analysis, the frequency of TAA-derived peptide-specific CD8+ T cells before RFA was 0.00%-0.03% of CD8+ cells (Fig. 4A). On the other hand, the frequency was increased after RFA in 10/12 (83.3%) patients, and the range was 0.00%-0.10%

of CD8+ cells. The frequency of CD45RA−/CCR7+ (central memory), CD45RA−/CCR7− (effector memory), and CD45RA+/CCR7− (effector) T cells in tetramer-positive cells depended on the patients, and the ratio of these cells changed after RFA (Fig. 4B). The frequency of tetramer-positive cells with CD45RA−/CCR7+ and CD45RA−/CCR7− in CD8+ cells check details was increased in 6/7 (85.7%) and 6/7 (85.7%) patients, respectively, whose samples were available for the assay before and after RFA. Interestingly, the tetramer-positive cells with CD45RA−/CCR7+ were newly induced after RFA in 5/7 (71.4%) patients. Although the number of TAA-specific T cells was a predictive factor of a decrease of HCC recurrence rate after RFA (as shown in Fig. 2A), more than 50% of the patients with a high number of TAA-specific T cells showed HCC recurrence for 25 months after treatment. To identify the relationship between TAA-specific T cell responses and HCC recurrence more precisely, we examined the kinetics of TAA-specific T cells in 16 patients whose PBMCs were available for analysis at 24 weeks after RFA.

Interestingly, FoxC1 is reported

to induce EMT. FoxC1 induces EMT through the inhibition of E-cadherin expression in mammary epithelial cells and promotes their migration and invasion. Additionally, FoxC1 overexpression is strongly correlated with poor survival in breast cancer patients.18, 19 Several recent studies also reported that FoxC1 increases the migration and invasion of breast cancer cells, and that FoxC1 overexpression predicts poor overall survival (OS) in patients with FG-4592 price breast cancer.20, 21 These studies indicate that FoxC1 might promote tumor metastasis and malignant progression by inducing EMT. To date, no studies have reported on the clinicopathologic significance of FoxC1 in HCC. In this study, we present the first evidence that FoxC1 promotes HCC invasion and metastasis by not only inducing selleck kinase inhibitor EMT, but also by up-regulating NEDD9 expression. FoxC1 overexpression predicts poor prognosis in HCC patients after curative resection. The molecular mechanism of these effects involves

the transactivation of Snai1 and NEDD9 expression by FoxC1 through direct binding to their promoters. BLI, bioluminescent imaging; ChIP, chromatin immunoprecipitation; CREB, cAMP response element-binding protein; EGF, epidermal growth factor; EMT, epithelial-mesenchymal transition; ERK, extracellular signal-related kinase; FoxC1, forkhead box C1; HBV, hepatitis B virus; HBx, hepatitis B virus x; HCC, hepatocellular carcinoma; IF, immunofluorescence; IHC, immunohistochemical; miRNAs, microRNAs; mRNA, messenger selleck chemicals RNA; NEDD9, neural precursor cell expressed, developmentally down-regulated 9; OS, overall survival; PCR, polymerase chain reaction; siRNA, short interfering RNA; TNM, tumor-node-metastasis; VEGF, vascular endothelial growth factor. Plasmids were constructed according to the standard procedures in our previous study.12 All of the primers are shown in Supporting Table 2. The Snai1 promoter construct (−1511/+140)snail was generated from human genomic DNA corresponding to the sequence from −1511 to +140 (relative to the transcriptional start site) of the 5′-flanking region of the human

Snai1 gene. This construct was generated with the forward and reverse primers incorporating KpnI and HindIII sites at the 5′- and 3′-ends, respectively. The polymerase chain reaction (PCR) product was cloned into the KpnI and HindIII sites of the pGL3-Basic vector (Promega, Madison, WI). The 5′-flanking deletion constructs of the FoxC1 promoter ([−922/+140]Snail, [−694/+140]Snail, and [−354/+140]Snail) were similarly generated with the (−1511/+140)Snail construct as a template. Other promoter constructs ([−2056/+121]NEDD9, [−1762/+121]NEDD9, [−1324/+121]NEDD9, [−1007/+121]NEDD9, [−478/+121]NEDD9, and pGL3-E-cadherin) were cloned in the same manner. FoxC1-binding sites in the Snai1 and NEDD9 promoters were mutated with a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA).