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The predominant expression of cancer stem cell marker ALDH1A3 in tumor infiltrative area is associated with shorter overall survival of human glioblastoma
Chao Gan1,2,
Daniela Pierscianek1,
Nicolai El Hindy1,3,
Yahya Ahmadipour1,
Kathy Keyvani4,
Ulrich Sure1 &
Yuan Zhu ORCID: orcid.org/0000-0002-5908-07681
ALDH1A3 is a cancer stem cell marker in neoplasms including glioblastoma (GBM). However, the comprehensive role of ALDH1A3 in GBM remains unclear. This study attempted to investigate the expression of ALDH1A3 in human GBM tissues and its association with clinical parameters.
Thirty primary GBM and 9 control were enrolled in this study. ALDH1A3 mRNA and protein expression levels were detected by RT2-PCR and western blot, respectively. Immunohistochemistry and immunofluorescence staining were performed to evaluate the regional and cellular expression manner of ALDH1A3. The association of ALDH1A3 expression with multiple clinical parameters was analyzed.
ALDH1A3 protein level, but not mRNA level, in a subgroup of GBM was significantly higher than that in the control group. ALDH1A3 immunoreactivity was detected heterogeneously in individual GBMs. Fifteen of 30 cases showed a positive of ALDH1A3 immunoreactivity which was predominantly observed in the tumor infiltrative area (TI). Double immunofluorescence staining revealed a co-localization of ALDH1A3 with GFAP in glial-shaped cells and in tumor cells. ALDH1A3 immunoreactivity was often merged with CD44, but not with CD68. Moreover, ALDH1A3 expression was positively associated with the tumor edema grade and inversely with overall survival (OS) (median OS: 16 months vs 10 months), but with neither MGMT promoter methylation status nor Ki67 index in GBM. An upregulation of ALDH1A3 was accompanied by a reduced expression of STAT3β and p-STAT3β.
Inter- and intra-tumoral heterogeneous expression of ALDH1A3 was exhibited in GBMs. A high immunoreactivity of ALDH1A3 in tumor infiltrative area was associated with shorter OS, especially in patients with MGMT promoter methylation. Our findings propose ALDH1A3 not only as a predictive biomarker but also as a potential target for personalized therapy of GBM.
Glioblastoma (GBM), the most common primary malignant brain tumor in adults, is genetically and histopathologically highly heterogeneous. A median survival period is 15 months despite the advanced treatment including surgical resection and chemoradiotherapy [1]. Increasing evidence suggests that cancer stem cells (CSCs) are crucial for tumorigenesis, therapeutic resistance and recurrence in GBM [2, 3]. Given that subventricular zone (SVZ) consists of enriched neural stem cells that possess the capacity to generate neurons and glia throughout adulthood [4, 5]. Gliomas are thus often presumed being initiated by neural stem cells in SVZ [6,7,8]. Indeed, expression of multiple CSC markers in GBM is negatively associated with overall survival in GBM patients [9, 10]. Therefore, targeting CSCs is considered as a promising therapeutic strategy.
Aldehyde dehydrogenases (ALDHs) are a group of enzymes consisting of 19 isoforms. Besides the metabolic functions [11, 12], high ALDH activity is considered as a hallmark of CSCs in various cancers [13]. Targeting ALDH inhibits the proliferation of GBM tumor cells and CSCs [14]. ALDH1A3 is the most upregulated between ALDH high and low subgroups of glioma cells among 19 isoform of ALDH family [15]. ALDH1A3 prominently emerges as a CSC marker to be targeted in multiple neoplasms [16,17,18]. Of note, ALDH1A3 is enriched in mesenchymal subtype (MES) of GBM patients, thereby being a sensitive and specific marker for MES GBM subtype [15]. ALDH1A3 is crucial for transition from proneural-CSCs to MES-CSC and is important for the maintenance of MES subtype [19]. Besides, ALDH1A3 plays also important roles in regulating self-renewal, differentiation and chemo/radio-resistance [20].
Most of the studies of ALDH1A3 expression in dataset were performed at transcriptional level by microarray in GBM. However, the comprehensive association of ALDH1A3 protein expression with clinical outcome remains elusive. The present study focused on the investigation of ALDH1A3 protein expression in a cohort of GBM patients with emphasis on its regional expression pattern and cellular localization and on its correlation with clinical parameters. We also explored the STAT3 and Akt/PTEN signaling cascades which might be involved in the regulation of ALDH1A3 expression and in its functions in GBM as studied by other entities. Through this study, we anticipate providing a broader perspective on this molecule as a prognostic biomarker as well as a potential therapeutic target for GBM.
Patient cohort, magnetic resonance imaging (MRI)-based edema grading and evaluation of the Karnofsky performance index (KPI)
Surgical specimens (n = 30) were collected from adult patients with primary GBM who were treated in the Department of Neurosurgery at our hospital. All enrolled patients were histopathologically diagnosed with primary GBM (WHO grade IV). Surgical specimens from patients who suffered temporal lobe epilepsy and underwent anterior temporal lobe resections without histopathological findings were used as control (n = 9).
Tumor edema appears as a region with increased T2 signal intensity outside the gadolinium-enhanced portion. Peritumoral edema is classified into four grades based on preoperative MRI scans [21]. Briefly, grade zero reflects no edema on preoperative scans; if edema is seen as less than, or approximately equal to, or greater than the tumor itself, the edema is graded as I, II, or III, respectively.
The preoperative KPI as one of the major prognostic indicators for GBM survival was used to assess all patients enrolled in the present study.
Evaluation of O6-methylguanine-methyltransferase (MGMT) promoter methylation and IDH1 mutation
For MGMT promoter methylation analysis, genomic DNA was isolated from paraffin sections of GBM. MGMT promoter methylation was analyzed by methylation-specific PCR as described previously [22].
IDH1-R132H, the most common glioma derived mutation, was determined immuno-histochemically in paraffin-embedded tumor specimens with a specific antibody as described previously [23].
TCGA database analysis of ALDH1A3 gene expression and OS dataset in GBM
The data of ALDH1A3 gene expression and its association with OS in 525 GBM cases and 10 normal control from The Cancer Genome Atlas (TCGA-GBM) were obtained from the GlioVis browser [24]. The gene expression profile was measured using the Affymetrix HT Human Genome U133a microarray platform. Kaplan–Meier survival curve was generated to show patient survival status between ALDH1A3 high and low group with optimal cutoff provided by GlioVis itself.
Real time-reverse transcription-polymerase chain reaction (RT2-PCR)
The extraction of total RNA (Qiagen, Hilden) and cDNA synthesis (Bio-Rad, Munich) were performed according to the manufacturer's instructions. The primer sequences and annealing temperatures are listed in Table 1. The following condition was used for real-time PCR: initial denaturation at 95 °C for 2 min, 35–45 cycles of amplification at 95 °C for 5 s and at annealing temperature for 25 s. Melting curve was obtained using the following settings: 95 °C for 1 min, and 55 °C for 1 min, and 55–95 °C with a heating increase rate of 0.5 °C every 10 s. Relative mRNA expression (fold change) was calculated according to the cycle threshold approach (2-ΔΔCT method), and normalized to the reference gene GAPDH as described [25].
Table 1 Primer sequences and annealing temperatures for real-time reverse-transcription PCR (RT2-PCR)
Immunohistochemistry staining and analysis
Immunohistochemistry was performed on formalin-fixed and paraffin-embedded (FFPE) GBM sections (n = 30). Briefly, after deparaffinization in gradient ethanol, heat-induced epitope retrieval and blocking the unspecific binding, sections were incubated with rabbit anti-ALDH1A3 primary antibody (1:250, Novus Biologicals) overnight at 4 °C. After the incubation with a HRP-conjugated secondary antibody (1:1000, Cell Signaling Technology), the sections were incubated with the substrate 3,3'-diaminobenzidine kit (Invitrogen) followed by hematoxylin counter staining. Negative control staining was done omitting primary antibody instead of a non-specific rabbit immunoglobulin fraction (DAKO).
The ALDH1A3 immunoreactivity in all stained sections was quantified according to previous description [26]. Briefly, the intensity of ALDH1A3 immunoreactivity was scored as 0–3: 0 = negative; 1 = weak; 2 = moderate; 3 = strong. The percentage of positive cells in microscopic images (magnification × 200) was counted using ImageJ, and four categories (0–3) were defined: category 0, < 1%; category 1, 1–5%; category 2, 5–10%; category 3, > 10%. The immunoreactive score (IRS) was calculated using the following formula: the score of the immuno-intensity × the score of positive percentage. According to IRS, the patients were classified to three groups: negative/low: IRS ≤ 2; medium: IRS > 2; and high: IRS ≥ 6.
Double immunofluorescence staining and imaging
For FFPE tissue sections, deparaffinization, heat-induced epitope retrieval, and blocking steps were performed as described previously [22]. Thereafter, the sections were incubated overnight at 4 °C in the primary antibody mixtures containing rabbit anti-ALDH1A3 (1:250) and a cell type specific marker antibody, i.e., mouse anti-GFAP (1:250, Sigma Aldrich) or mouse anti-CD68 (1:100, gift from Neuropathology in our hospital) or rat anti-CD44 (1:100, Invitrogen). The sections were incubated with the mixture of biotinylated goat anti-rabbit IgG and Texas red conjugated horse anti-mouse IgG at room temperature for 1 h followed by the substrate reaction with FITC-conjugated avidin. Counter staining was done with Hoechst-33,342. Images were acquired using Axio Imager M2 microscope (Zeiss) with the ApoTome.2 system for optical sections.
Total protein extraction and electrophoresis were performed as before [21]. The primary antibody reaction was done overnight at 4 °C with the following primary antibodies (excepting ALDH1A3 antibody, all from Cell Signaling Technology): anti-ALDH1A3 (1:1000, Novus Biologicals), anti-STAT3 (1:1000), anti-phospho-STAT3 (Tyr705) (p-STAT3) (1:2000), anti-GAPDH (1:1000), anti-phospho-Akt (Ser437) (p-Akt) (1:1000), anti-PTEN (1:1000). After the second antibody reaction, the blots were incubated with ECL solution (GE Healthcare) and the image was acquired by using ImageQuant LAS 500 (GE Healthcare, Freiburg).
The experimental data were presented as the mean ± standard deviation (SD). All statistical analysis were performed using the GraphPad Prism 5 and SPSS 23.0. Student t test with Welch's correction was used for data analysis between two groups; one way ANOVA followed by Bonferroni's multiple comparison test was applied for multi-group comparison. The survival curve was plotted using the Kaplan-Meier method and analyzed using log-rank test. A P value less than 0.05 was considered statistically significant.
Baseline characteristics of patient
The mean age of GBM patients at the first diagnosis was 60.1 ± 13.3 years. The ratio of male to female was 1: 1.1 (14 to16). Among 30 GBMs, 60% (18/30) patient had a gross total resection of tumor. Twenty-six patients (86.7%) showed KPI ≥ 70. Fourteen of 30 cases (46.7%) had a positive methylation status of MGMT promoter. A R132H point mutation of IDH1 was detected in 3 of 20 cases, whereas IDH mutation information was not assessed in the other 10 cases. A standard chemoradiotherapy [1] after surgical resection was applied to 63.3% (19/30) of patients. The mean of OS was 13.3 ± 9.9 months. Given that SVZ is suggested as one of the prognostic factors in GBM [6], GBM patients were grouped upon their tumor location, whether the tumor contacted SVZ (+) or not (SVZ−). In 20 of 30 (66.7%) cases GBM tumors were found at the SVZ+ location.
ALDH1A3 mRNA expression was downregulated in GBM: TCGA database and own data
Microarray-based TCGA database revealed a statistically reduced ALDH1A3 mRNA in GBM compared to control group (P < 0.05, Fig. 1a) and a negative correlation between the ALDH1A3 mRNA and the OS of GBM patients, i.e., higher ALDH1A3 mRNA expression and shorter OS with optimal cutoff of ALDH1A3 mRNA value at 4.22 (P < 0.01, Fig. 1b).
mRNA expression of ALDH1A3 in GBM. a TCGA database showed a lower expression of ALDH1A3 mRNA in GBM (n = 525) compared with the control (n = 10). b The higher expression of ALDH1A3 mRNA was associated with the shorter overall survival (OS) of GBM patients at optimal cutoff based on TCGA database. c mRNA expression of ALDH1A3 detected in the present study by real-time RT-PCR in primary GBM (n = 30) and control (n = 9) supported the data from TCGA database. d Subgroup analysis of mRNA expression of ALDH1A3 and other stem cell markers in subventricular zone (SVZ+) and in non-subventricular zone (SVZ−). Student's t test with Welch's correction was utilized between two groups in a, c, d (*P < 0.05)
To validate the finding from the TCGA datasets, RT-PCR was performed and found a significant downregulation of ALDH1A3 mRNA expression in our patient cohort (P < 0.05, Fig. 1c). To compare SVZ+ GBM with SVZ− GBM in the matter of CSCs characteristics, not only ALDH1A3 but also some other known CSC markers including Nestin, CD133, CD44, YKL40, OLIG2 and SOX2 were examined. Interestingly, the ALDH1A3 mRNA level was 2.59-fold higher in SVZ+ group compared to SVZ−. However, neither ALDH1A3 nor other detected CSC markers showed significant difference between SVZ+ and SVZ− groups (Fig. 1d).
Characterization of regional and cell type specific expression of ALDH1A3 in GBM
Immunohistochemistry (IHC) revealed a heterogeneous expression of ALDH1A3 in different GBM sections and in the different regions of individual cases. To characterize its expression pattern upon area of focus, we classified tumor regions into three: tumor center (TC), tumor infiltrative area (TI) and tumor distant area (TD) (Fig. 2a, lines). TI showed a gradually lower tumor cell density in comparison to TC, whereas TD exhibited normal cells as well as some scattered tumor cells. An apparent expression of ALDH1A3 was found in 50% of GBM specimens (15/30). Figure 2b and c were representative of a negative and a positive ALDH1A3 expression case, respectively. In the cases showing ALDH1A3 expression, ALDH1A3 immunoreactivity was dominantly detected in TI (Fig. 2c, d, g-j), whereas scattered ALDH1A3 positive cells were also found in both TC (Fig. 2e) and TD (Fig. 2f). For quantitative analysis of ALDH1A3 expression, IRS was determined upon area of focus in tumor and classified patient cases into three subgroups: negative/low (IRS ≤ 2), medium (IRS > 2) and high (IRS ≥ 6). According to IRS of 30 patient cases, 24, 3 and 3 cases in TC and 15, 4 and 11 in TI were subgrouped, respectively (Fig. 4a), indicating that TI had a significantly higher expression of ALDH1A3 than TC (P < 0,001).
Immunohistochemistry of ALDH1A3 in GBM. a H&E staining defined the different regions in GBM sections. TC: tumor center; TI: tumor infiltrative area; and TD: tumor distant area. b, c Immunostaining of ALDH1A3 in GBM sections revealed heterogeneous expression of ALDH1A3 in different GBM patient sections (n = 30). The representative photos show the absence of ALDH1A3 expression b and the positive expression of ALDH1A3 c, respectively, in two GBM cases. The positive ALDH1A3 expressing cells were mainly detected in TI, and much less in TC. d-f Images are representative of the expression of ALDH1A3 in the different regions of GBM tissue. g-k Expression of ALDH1A3 in typical tumor cells (arrows in g), in out-layer of glomeruloid tufts (arrows in h), in some vessels (arrows in i), in multi-nuclear cells (arrow in j) and in glial-shaped cells (arrowheads in j). ALDH1A3 immunoreactivity was not detected in the necrosis core (asterisk in k) and the pseudopalisade structure (arrows in k). l ALDH1A3 immunoreactivity was not detected in the non-tumoral control brain section. a-c, original magnification × 100; d-j, original magnification × 400; k-l, original magnification × 200
We observed ALDH1A3 expression in tumor cells (Fig. 2g, arrows), in outermost layer cells of some glomeruloid tufts (Fig. 2h, arrows), as well as in vessels (Fig. 2i, arrows). Some multiple-nucleus cells with dendritic-like structures (Fig. 2j, arrows) and small cells displaying glial-shaped morphology (Fig. 2j, arrowheads) also appeared ALDH1A3 positive. Of note, ALDH1A3 immunoreactivity was not detected in pseudopalisades (Fig. 2k, arrows) and necrosis centers (Fig. 2k, asterisk) in all investigated sections in this study. There was no apparent ALDH1A3 positive cells in control (Fig. 2l).
In order to identify ALDH1A3 expressing cells, immunofluorescence staining of ALDH1A3 in combination with cell type specific markers was performed. Some glial-shaped cells and tumor cells were found double positive for ALDH1A3 and GFAP (Fig. 3a, b, arrows, respectively). Interestingly, the immunoreactivity of ALDH1A3 and GFAP was merged also in the dendritic-processed cells (Fig. 3c, arrows) and in cells with multiple nuclei (Fig. 3d, arrows). Cells expressing ALDH1A3 are often found positive for a stem cell surface marker CD44 (Fig. 3f, arrows), but not for a macrophage marker CD68 (Fig. 3e, arrowheads).
Double staining of ALDH1A3 with different markers in GBM sections. a-d Double staining of ALDH1A3 with GFAP. In both TC and TI, the co-expression of ALDH1A3 (green) and GFAP (red) was detected in some glial-shaped cells (arrows in a), tumor cells (arrows in b) and multi-nucleated cells (arrows in c and d). e Double staining of ALDH1A3 with CD68. ALDH1A3 immunoreactivity (green) detected in the outer layer of glomeruloid tuft (arrows in e) was negative for the macrophage marker CD68 (red, arrowheads). f Double staining revealed a coexpression (arrows) of ALDH1A3 (red) with the stem cell marker CD44 (green). a-f, original magnification × 400
Correlation of ALDH1A3 expression with clinical parameters
According to IRS of ALDH1A3 in TI, the cohort of GBM patients was subgrouped as follows: negative/lower and medium/high expression of ALDH1A3 (Fig. 4a). The association of ALDH1A3 expression with clinical parameters in each subgroup was analyzed and summarized in Table 2. No statistical significance was found for the correlation of ALDH1A3 expression with all these analyzed parameters except OS and peritumoral edema. A higher expression of ALDH1A3 was significantly associated with a shorter median OS (P < 0.01, HR = 3.170, 95% CI: 1.328–7.566) (Fig. 4b). The median survival time of patients with medium/high ALDH1A3 expression was 10 months, while that of patients with negative/low ALDH1A3 group was 16 months. Furthermore, the higher expression of ALDH1A3 was also associated with a higher grade of peritumoral edema (P < 0.05, Fig. 4c). We further analyzed the association of MGMT promoter methylation status with OS, and no significant association was found in our patient cohort. The median survival in both MGMT promoter methylation positive (MGMT+, n = 14) and negative (MGMT−, n = 14) patients was 12 months. Next, we also studied association of OS with a combination of MGMT methylation status and ALDH1A3 expression. Of note, the median survival in the subgroup of MGMT+ with low ALDH1A3 IRS was found significantly longer than that in the subgroup of MGMT+ with higher ALDH1A3 IRS (17 months vs 7 months, respectively; P < 0.01). However, there was no significant difference in OS seen between low and higher ALDH1A3 expression in the subgroup of MGMT− patients (13.5 months vs 10.5 months).
Association of ALDH1A3 expression with clinical parameters. a Quantitative analysis of ALDH1A3 immunoreactivity in TC and in TI of GBMs. The immunoreactive score (IRS) was evaluated in TC and TI as described in the method (n = 30). Based on IRS, TI had a significantly higher ALDH1A3 expression than TC (P < 0.001). In according to TI, GBM with IRS ≤ 2 was grouped as negative/low (n = 15), whereas GBM with IRS > 2 was defined as high and medium group (n = 15). b The association of ALDH1A3 expression with the OS period of patients. A higher expression of ALDH1A3 was significantly associated with a shorter OS time. c The representative MRI images show the different grades of edema. Peritumoral edema grade II had a higher IRS of ALDH1A3 compared with edema grade I. Paired t test was performed to compare IRS between TI and TC in a (***P < 0.001). One way ANOVA followed by Bonferroni's multiple comparison test, comparing each column to all the other columns, was employed for multi-groups comparison in c (*P < 0.05)
Table 2 Analysis of the association of ALDH1A3 IRS with clinical parameters
In addition, Ki67 index, a proliferation parameter, was 16.3 and 17.3% in low and higher ALDH1A3 subgroup, respectively (Table 2), and appeared as not associated with ALDH1A3 expression.
Association of the expression of ALDH1A3 and signaling proteins in GBM
Figure 5a shows representative blots detecting ALDH1A3, STAT3 and p-STAT3, p-Akt, PTEN and GAPDH in GBM and in control. Western blot confirmed distinct expression level of ALDH1A3 in subgroups of GBMs (P < 0.01). As expected, an upregulation of ALDH1A3 was confirmed in the GBM cases with higher IRS (IRS > 2, lane 6–9), whereas a low protein level of ALDH1A3 was detected in the GBM cases with lower IRS (IRS ≤ 2, lane 3–5). Semi-quantitative analysis of the blots revealed a significant upregulation of p-STAT3α, p-STAT3β and p-Akt accompanied by a downregulation of PTEN in GBMs compared to control. Interestingly, the expression of STAT3β (P < 0.01) and p-STAT3β (P < 0.05), but not STAT3α and p-STAT3α, was inversely associated with the protein level of ALDH1A3 in two subgroups of GBM (Fig. 5b).
Detection of ALDH1A3 and the signaling proteins in GBM by western blot. a Representative blots detected the expression of STAT3 and p-STAT3, p-Akt, PTEN and GAPDH in the control (C, lane 1–2) and in GBM cases with lower immunoreactivity score (IRS ≤ 2, lane 3–5) and with higher immunoreactivity score (IRS > 2, lane 6–9) (See original images in Supplementary Fig. S1). b Semi-quantification of the blots. The integrated optical density (IOD) of the individual bands on blots was measured by ImageJ and the ratio of the target protein to the housekeeping protein GAPDH was calculated. The relative expression data were presented when the ratio of the control group was normalized as 1. One way ANOVA followed by Bonferroni's multiple comparison test was employed for multi-groups comparison in b. *P < 0.05, **P < 0.01 and ***P < 0.001: compared with the control (C); #P < 0.05 and ##P < 0.01, compared with the group "low" (IRS ≤ 2)
Increasing evidence indicates ALDH1A3 as an important molecule that influences a diverse range of biological processes in CSCs and in tumor cells, thereby being associated with the initiation, progression, and prognosis of various cancers including GBM [27]. The present study investigated the expression pattern of ALDH1A3 at both mRNA and protein levels in human GBM specimens with emphasis on its association with clinical parameters of the patients.
Analysis of the microarray based TCGA-GBM dataset revealed that the mean level of ALDH1A3 mRNA in the total cohort was lower than that in control. However, when considered in genomic subtypes of GBM [28, 29], its mRNA level appeared significantly higher in the MES than in the classical and proneural subtypes; even in the MES of GBM, the mean level of ALDH1A3 mRNA was comparable with that in control (Supplementary Fig. S2). On the other hand, the TCGA-GBM dataset showed an inverse association of ALDH1A3 mRNA expression with OS of GBM. These findings from the TCGA data encouraged us to further investigate implication of ALDH1A3 in GBM. In the present study, we demonstrated a significant downregulation of ALDH1A3 mRNA expression in our GBM cohort in comparison to control, consistent with the findings derived from the TCGA dataset. As the cellular, biological functions of a molecule are ultimately determined by its translational/post-translational levels, not by its transcriptional level, we next focused on ALDH1A3 expression at protein levels in individual GBM tissues of our cohort. As shown in Fig. 2l, the immunoreactivity of ALDH1A3 was not detected in the control (normal) brain tissue. Furthermore, by western blot we also demonstrated a 4.66-fold higher protein expression of ALDH1A3 in the subgroup of GBMs with IRS > 2 than that in the control group (Fig. 5b). These findings allow us to postulate that a higher mRNA level of ALDH1A3 does not necessarily refer to its higher protein level. Indeed, several potential mechanisms regulating the translational and post-translation of ALDH1A3 have been identified: the hypermethylation status of ALDH1A3 promoter predicts a low expression of ALDH1A3 protein accompanied by a better prognosis of GBM patients [30]; USP9X-mediated deubiquitinase plays an important role in ALDH1A3 protein stabilization [31]; temozolomide (TMZ) treatment at high concentrations does not alter ALDH1A3 mRNA levels, but protein levels through autophagy [32]. Thus, it is more reliable to analyze the association of clinical parameters with ALDH1A3 protein levels.
GBM tumor is composed of heterogeneous cell populations including a small population, glioma CSCs [2], which is related to therapeutic resistance of GBM [3, 33]. As the subventricular zone (SVZ) is often proposed to be the source of CSCs [6, 8], we evaluated mRNA expression levels of ALDH1A3 as well as other well-known CSC markers including Nestin, CD133, CD44, YKL40, OLIG2, SOX2 in GBM. Despite a 2.5-fold higher ALDH1A3 mRNA detected in the SVZ+ group, none of these investigated CSC markers showed a statistically significant alteration in mRNA levels (Fig. 1d). These results are consistent with previous reports [34, 35], the mechanism under which need to be further investigated.
The ALDH superfamily is the most important aldehyde metabolic enzyme family in human cells and has been linked to metabolism reprogramming in the initiation, metastasis, and recurrence of cancer [36]. Among the 19 members, the ALDH1A3 isoform has been served as the major source of total ALDH activity in GBM [15]. Thus, it is important to check not only ALDH1A3 mRNA levels but also its protein levels that is more relevant to its enzymatic activity. Our immunohistochemistry study revealed a distinct expression of ALDH1A3 in individual GBM patients and a high inter-tumoral heterogeneity. A clear positive immunoreactivity of ALDH1A3 was detected only in 15 of 30 (50%) GBMs. Among these positive cases, 4 and 11 cases showed medium (IRS > 2) and high (IRS ≥ 6) expression of ALDH1A3, respectively. ALDH1A3 positive cells were mostly located at the tumor infiltrative area, suggesting that ALDH1A3 may participate in tumor cell invasiveness and metastasis. In fact, knockdown of ALDH1A3 expression in vitro models also suppressed cancer cell invasion in different entities [37, 38]. We demonstrated that a higher ALDH1A3 IRS was significantly associated with a short OS. As supporting, the expression of ALDH1A3 was positively associated with the grade of peri-tumoral edema that is also a prognostic parameter for GBM patients [39, 40]. Regardless of treatment, MGMT promoter methylation is an independent and favorable prognostic factor in GBM [41]. MGMT encodes a DNA-repair protein that inhibits the effect of treatment through removing alkyl groups from guanine, a target site for alkylating chemotherapy agents such as TMZ. MGMT promoter methylation is associated with higher therapeutic effect of TMZ and longer OS in GBM patients [42]. However, controversial results are also observed in several studies [43, 44]. In the present study, MGMT promoter methylation status was not associated with OS in GBM. Interestingly, when combining MGMT and ALDH1A3 expression together, low expression of ALDH1A3 may sensitively predict a better prognosis than those with higher expression of ALDH1A3 in the subgroup of MGMT+ patients. Hence, evaluation of ALDH1A3 expression might be a powerful prognostic tool in combination with MGMT promoter methylation status. IDH1 mutation is another prognostic maker for GBM. In TCGA database ALDH1A3 mRNA expression is negatively associated with IDH1 mutation [15]. In our cohort only 3 of 30 patients were identified with IDH1 mutation, and nevertheless, we did analysis of the association of ALDH1A3 expression with IDH1 mutation status. As predicted, no association was found between the level of ALDH1A3 protein or mRNA with IDH1 mutation. Therefore, a larger patient cohort is needed to verify the association of ALDH1A3 protein with IDH1 mutation in the future. Finally, we also observed the expression of ALDH1A3 frequently in the outer layer of different stage of glomeruloid tufts and in some endothelial cells of tumor vessels. In line with the pro-angiogenic functions of ALDH1A3 in vitro [45, 46], our findings by tissue staining also implicate ALDH1A3 in microvascular proliferation and in neo-angiogenesis in a subgroup of GBM. These clinical associations highlight ALDH1A3 as a potential prognostic biomarker and as a therapeutic target preventing tumor invasion and angiogenesis.
The TI consists of various cell types including infiltrating tumor cells, CSCs, reactive astrocyte, microglia, oligodendrocytes, inflammatory and immune cells, endothelial cells, and stromal cells thereby creating a complex microenvironment feasible for tumor growth and invasion as well as survival and therapy-resistance [47, 48]. To identify a cell type expressing ALDH1A3, we performed double staining of ALDH1A3 with the following cell type specific markers: GFAP, CD68 and CD44. Among ALDH1A3 positive cells, there were some GFAP positive cells likely reactive astrocytes or tumor cells due to their distinct morphology. As the co-expression of ALDH1A3 with the stem cell marker CD44 was also found, we propose that ALDH1A3 positive tumor cells might possess the stem cell-like properties involved in tumor progression and therapy resistance. Tumor-associated macrophages (TAMs) are highlighted in GBM due to the considerable size of their population (30–50%) [49]. But, the present study did not observe cells co-expressing ALDH1A3 and CD68. The possible implication of ALDH1A3 in immunoreaction mediated by immune cells needs to be further studied.
Along with important roles of ALDH1A3 in cancer and cancer stem cells, its underlying regulatory mechanism has become of interest. Several non-coding microRNAs including miR-600hg [50], miR-7 [51], miR-187 and miR-125a/b [27] have been identified to recognize ALDH1A3 as a target gene and to suppress ALDH1A3 expression. Forkhead Box D1 (FOXD1) regulates the transcriptional activity of ALDH1A3 in glioma stem cells (GSCs) of MES subtype [52]. The hedgehog pathway can significantly increase the expression of ALDH1 in cancer stem cells of ovary, which can also be a possible mechanism in GBM [53]. In a recent study, ALDH1A3 is stabilized upon ubiquitin-specific protease 9X (USP9X) in the MES GSCs. In contrast, deletion of USP9X induces degradation of ALDH1A3, and a subsequent decrease in p-STAT3, whereas overexpression of ALDH1A3 can restore p-STAT3 expression in GSCs in which downregulation of ALDH1A3 was induced by USP9X depletion [31]. On the other hand, in NSCLC CSCs, activation of STAT3 pathway significantly increases ALDH1A3 expression while multiple inhibitors of STAT3 signaling can decreased ALDH1A3 expression [17]. Taken together, we assume that there might be a feedback loop between ALDH1A3 and STAT3 to promote tumor progression in cancers. Developing STAT3 signaling inhibitors has been a new strategy for anticancer therapy. However, so far none of them is satisfied in the clinic studies [54]. STAT3 has two mainly isoforms, STAT3α and STAT3β, of which the latter is a suppressive factor of STAT3α due to lacking the transactivation domain [55]. In the present study, we found that ALDH1A3 protein expression was inversely associated with the levels of total STAT3β and p-STAT3β but no association was seen between the expression of ALDH1A3 and STAT3α. The association and the underlying regulatory mechanism of ALDH1A3 and STAT3 in GBM need to be further elucidated by using gene techniques in the future. Akt/PTEN is an important signaling implicated in numerous malignant tumors including GBM. We demonstrated a significant increase in p-Akt levels concomitantly accompanied by a downregulation of PTEN in GBM. However, the level of PTEN/p-Akt did not appear to be associated with ALDH1A3 expression in GBMs, suggesting that PTEN/Akt may not be a direct downstream signaling cascade trigged by ALDH1A3.
The present study revealed a high intra- and inter-tumoral heterogeneity of ALDH1A3 expression manner in GBM. ALDH1A3 expression enriched in tumor infiltrative region highlights its crucial role in tumor invasiveness and progression. Moreover, we demonstrated an inverse association of ALDH1A3 expression with the prognosis of GBM, supporting ALDH1A3 as a prognostic marker and as a potential target for future GBM therapy.
All the data generated or analyzed during this study are included in this article.
ALDH1A3:
Aldehyde dehydrogenase 1 family, member A3
ALDHs:
Aldehyde dehydrogenases
CSCs:
Cancer stem cells
Formalin-fixed and paraffin-embedded
GAPDH:
Glyceraldehyde 3-phosphate dehydrogenase
GBM:
GFAP:
IDH1:
Isocitrate dehydrogenase 1
IRS:
Immunoreactive score
KPI:
Karnofsky performance index
Mesenchymal subtype
O6-methylguanine-methyltransferase
Overall survival
PTEN:
Phosphatase and tensin homolog
RT2-PCR:
Real time reverse transcriptase polymerase chain reaction
STAT3:
Signal transducer and activator of transcription 3
SVZ:
Subventricular zone
TAMs:
Tumor-associated macrophages
TC:
Tumor center
TCGA:
The cancer genome atlas
Tumor distant area
TI:
Tumor infiltrative area
TMZ:
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Hou C, Ishi Y, Motegi H, Okamoto M, Ou Y, Chen J, et al. Overexpression of CD44 is associated with a poor prognosis in grade II/III gliomas. J Neuro-Oncol. 2019;145(2):201–10.
Jackson B, Brocker C, Thompson DC, Black W, Vasiliou K, Nebert DW, et al. Update on the aldehyde dehydrogenase gene (ALDH) superfamily. Hum Genomics. 2011;5(4):283–303.
Marchitti SA, Brocker C, Stagos D, Vasiliou V. Non-P450 aldehyde oxidizing enzymes: the aldehyde dehydrogenase superfamily. Expert Opin Drug Metab Toxicol. 2008;4(6):697–720.
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Kuo YC, Wang LJ, Rajesh R. Targeting human brain cancer stem cells by curcumin-loaded nanoparticles grafted with anti-aldehyde dehydrogenase and sialic acid: Colocalization of ALDH and CD44. Mater Sci Eng C Mater Biol Appl. 2019;102:362–72.
Li G, Li Y, Liu X, Wang Z, Zhang C, Wu F, et al. ALDH1A3 induces mesenchymal differentiation and serves as a predictor for survival in glioblastoma. Cell Death Dis. 2018;9(12):1190.
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Durinikova E, Kozovska Z, Poturnajova M, Plava J, Cierna Z, Babelova A, et al. ALDH1A3 upregulation and spontaneous metastasis formation is associated with acquired chemoresistance in colorectal cancer cells. BMC Cancer. 2018;18(1):848.
Lambertz N, El Hindy N, Kreitschmann-Andermahr I, Stein KP, Dammann P, Oezkan N, et al. Downregulation of programmed cell death 10 is associated with tumor cell proliferation, hyperangiogenesis and peritumoral edema in human glioblastoma. BMC Cancer. 2015;15:759.
El Hindy N, Keyvani K, Pagenstecher A, Dammann P, Sandalcioglu IE, Sure U, et al. Implications of Dll4-notch signaling activation in primary glioblastoma multiforme. Neuro-Oncology. 2013;15(10):1366–78.
Ahmadipour Y, Jabbarli R, Gembruch O, Pierscianek D, Darkwah Oppong M, Dammann P, et al. Impact of Multifocality and molecular markers on survival of Glioblastoma. World Neurosurg. 2019;122:e461–e6.
GlioVis: TCGA_GBM dataset. http://gliovis.bioinfo.cnio.es/. Accessed 20 Feb 2020.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25(4):402–8.
Krajewska M, Krajewski S, Epstein JI, Shabaik A, Sauvageot J, Song K, et al. Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in prostate cancers. Am J Pathol. 1996;148(5):1567–76.
Duan JJ, Cai J, Guo YF, Bian XW, Yu SC. ALDH1A3, a metabolic target for cancer diagnosis and therapy. Int J Cancer. 2016;139(5):965–75.
Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD, et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 2010;17(1):98–110.
Wang Q, Hu B, Hu X, Kim H, Squatrito M, Scarpace L, et al. Tumor Evolution of Glioma-Intrinsic Gene Expression Subtypes Associates with Immunological Changes in the Microenvironment. Cancer Cell. 2017;32(1):42–56 e6.
Zhang W, Yan W, You G, Bao Z, Wang Y, Liu Y, et al. Genome-wide DNA methylation profiling identifies ALDH1A3 promoter methylation as a prognostic predictor in G-CIMP- primary glioblastoma. Cancer Lett. 2013;328(1):120–5.
Chen Z, Wang HW, Wang S, Fan L, Feng S, Cai X, et al. USP9X deubiquitinates ALDH1A3 and maintains mesenchymal identity in glioblastoma stem cells. J Clin Invest. 2019;129(5):2043–55.
Wu W, Schecker J, Wurstle S, Schneider F, Schonfelder M, Schlegel J. Aldehyde dehydrogenase 1A3 (ALDH1A3) is regulated by autophagy in human glioblastoma cells. Cancer Lett. 2018;417:112–23.
Kahlert UD, Joseph JV, Kruyt FAE. EMT- and MET-related processes in nonepithelial tumors: importance for disease progression, prognosis, and therapeutic opportunities. Mol Oncol. 2017;11(7):860–77.
Kappadakunnel M, Eskin A, Dong J, Nelson SF, Mischel PS, Liau LM, et al. Stem cell associated gene expression in glioblastoma multiforme: relationship to survival and the subventricular zone. J Neuro-Oncol. 2010;96(3):359–67.
Jungk C, Mock A, Exner J, Geisenberger C, Warta R, Capper D, et al. Spatial transcriptome analysis reveals notch pathway-associated prognostic markers in IDH1 wild-type glioblastoma involving the subventricular zone. BMC Med. 2016;14(1):170.
Vassalli G. Aldehyde dehydrogenases: not just markers, but functional regulators of stem cells. Stem Cells Int 2019;2019:3904645.
Feng H, Liu Y, Bian X, Zhou F, Liu Y. ALDH1A3 affects colon cancer in vitro proliferation and invasion depending on CXCR4 status. Br J Cancer. 2018;118(2):224–32.
Zhang W, Liu Y, Hu H, Huang H, Bao Z, Yang P, et al. ALDH1A3: a marker of Mesenchymal phenotype in Gliomas associated with cell invasion. PLoS One. 2015;10(11):e0142856.
Qiu XX, Wang CH, Lin ZX, You N, Wang XF, Chen YP, et al. Correlation of high delta-like ligand 4 expression with peritumoral brain edema and its prediction of poor prognosis in patients with primary high-grade gliomas. J Neurosurg. 2015;123(6):1578–85.
Wu CX, Lin GS, Lin ZX, Zhang JD, Liu SY, Zhou CF. Peritumoral edema shown by MRI predicts poor clinical outcome in glioblastoma. World J Surg Oncol. 2015;13:97.
Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352(10):997–1003.
Gilbert MR, Wang M, Aldape KD, Stupp R, Hegi ME, Jaeckle KA, et al. Dose-dense temozolomide for newly diagnosed glioblastoma: a randomized phase III clinical trial. J Clin Oncol. 2013;31(32):4085–91.
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Ohmura-Kakutani H, Akiyama K, Maishi N, Ohga N, Hida Y, Kawamoto T, et al. Identification of tumor endothelial cells with high aldehyde dehydrogenase activity and a highly angiogenic phenotype. PLoS One. 2014;9(12):e113910.
Hida K, Maishi N, Akiyama K, Ohmura-Kakutani H, Torii C, Ohga N, et al. Tumor endothelial cells with high aldehyde dehydrogenase activity show drug resistance. Cancer Sci. 2017;108(11):2195–203.
D'Alessio A, Proietti G, Sica G, Scicchitano BM. Pathological and Molecular Features of Glioblastoma and Its Peritumoral Tissue. Cancers (Basel). 2019;11(4).
Darmanis S, Sloan SA, Croote D, Mignardi M, Chernikova S, Samghababi P, et al. Single-cell RNA-Seq analysis of infiltrating neoplastic cells at the migrating front of human Glioblastoma. Cell Rep. 2017;21(5):1399–410.
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Yao Y, Li N. MIR600HG suppresses metastasis and enhances oxaliplatin chemosensitivity by targeting ALDH1A3 in colorectal cancer. Biosci Rep. 2020;40(4).
Pan M, Li M, You C, Zhao F, Guo M, Xu H, et al. Inhibition of breast cancer growth via miR-7 suppressing ALDH1A3 activity concomitant with decreasing breast cancer stem cell subpopulation. J Cell Physiol. 2020;235(2):1405–16.
Cheng P, Wang J, Waghmare I, Sartini S, Coviello V, Zhang Z, et al. FOXD1-ALDH1A3 signaling is a determinant for the self-renewal and Tumorigenicity of Mesenchymal Glioma stem cells. Cancer Res. 2016;76(24):7219–30.
Sneha S, Nagare RP, Sidhanth C, Krishnapriya S, Garg M, Ramachandran B, et al. The hedgehog pathway regulates cancer stem cells in serous adenocarcinoma of the ovary. Cell Oncol (Dordr). 2020. https://doi.org/10.1007/s13402-020-00504-w.
Wong ALA, Hirpara JL, Pervaiz S, Eu JQ, Sethi G, Goh BC. Do STAT3 inhibitors have potential in the future for cancer therapy? Expert Opin Investig Drugs. 2017;26(8):883–7.
Caldenhoven E, van Dijk TB, Solari R, Armstrong J, Raaijmakers JA, Lammers JW, et al. STAT3beta, a splice variant of transcription factor STAT3, is a dominant negative regulator of transcription. J Biol Chem. 1996;271(22):13221–7.
We thank Ms. Rita Haase and Ms. Michaela Hiber for their technical assistance. CG received a scholarship from the Medical Faculty, University of Duisburg-Essen. We also thank Dr. Su Na Kim for careful proofreading of the manuscript.
This study was supported financially by the IFORES-program at the Medical Faculty, University of Duisburg-Essen to Y.Z. The faculty paid the material necessary to elaborate the research work. Open access funding provided by Projekt DEAL.
Department of Neurosurgery and Spine Surgery, University hospital Essen, University of Duisburg-Essen, Hufelandstrasse 55, 45122, Essen, Germany
Chao Gan, Daniela Pierscianek, Nicolai El Hindy, Yahya Ahmadipour, Ulrich Sure & Yuan Zhu
Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Chao Gan
Present Address: Department of Spine- and Peripheral Nerve-Surgery, St. Christophorus 625 Hospital, Werne, Germany
Nicolai El Hindy
Institute of Neuropathology, University hospital Essen, University of Duisburg-Essen, Essen, Germany
Kathy Keyvani
Daniela Pierscianek
Yahya Ahmadipour
Ulrich Sure
Yuan Zhu
CG contributed to the main experiments, data collection and analysis and participated in the manuscript preparation; DP, YA and NEH: clinical data collection and analysis; KK: histopathological diagnosis of GBM, tissue sectioning and immunostaining; US and YZ: the study conception, design and manuscript preparation. All authors have read and approved the final manuscript.
Correspondence to Yuan Zhu.
This study was strictly performed in accordance with the Declaration of Helsinki and approved by the local ethics committee of the University Hospital Essen. Informed writing consent was obtained from all the patients before the sample collection.
Additional file 1: Supplementary Figure S1.
Original blots of western blotting. Supplementary Figure S2. ALDH1A3 mRNA expression in subtypes of GBM and normal control based on TCGA database. Among three subtypes of GBM, ALDH1A3 mRNA level in the classical and proneural subtypes of GBM was significantly lower than that in the control and in the mesenchymal GBM. Student t test with Welch's correction was used for data analysis between subgroups. **P < 0.01, ***P< 0.001, compared with control; ###P <0.001, compared with mesenchymal.
Gan, C., Pierscianek, D., El Hindy, N. et al. The predominant expression of cancer stem cell marker ALDH1A3 in tumor infiltrative area is associated with shorter overall survival of human glioblastoma. BMC Cancer 20, 672 (2020). https://doi.org/10.1186/s12885-020-07153-0
Primary glioblastoma
ALDH1A3
Cancer stem cell marker
Peritumoral edema


text:
Q: Are the following statements using pointer arithmetic involving out of bound aceess valid? char array[8] = "Raining";

I think all the below comments respective to the statements are true to my understanding.
char *p1 = array + 7;      --> points to '\0'

char *p2 = array + 8;      --> undefined behaviour

char *p3 = array + 9;      --> undefined behaviour

char *p4 = array + 10;     --> undefined behaviour

Is my understanding correct?

A: array has 8 elements in it due to the NULL-terminator.
char *p1 = array + 7; is well-defined.
char *p2 = array + 8; is actually defined. You're allowed to set a pointer to one-past the array. (Just don't dereference it.)
The other two are undefined. You're not allowed to set a pointer beyond one-past the array.

A: The first line points to a legal element inside the array and the second line is valid until dereferenced. The rest of your post is right.
char *p1 = array + 7;   \\-->points to '\0', within array

char *p2 = array + 8;   \\-->defined behaviour until dereferenced

The reason the past-the-end addressing is allowed is to make it easy to write a loop to go over an entire array, e.g., while (*d++ = *s++);.

The boundary markers for arrays describe a half-open range, starting with (and including) the first element, and ending with (but not including) the last element: [beginning,end).

A: In your case,
  char *p1 = array + 7;

and
  char *p2 = array + 8;

are valid, because, you are legally allowed to point to

*

*any object inside the array length

*one past the last array element until you dereference it.

OTOH,
  char *p3 = array + 9;

and
  char *p4 = array + 10;

are undefined.

Quoting C11, chapter §6.5.6, "Additive operators" (emphasis mine)
[same in C99, chapter §6.5.6/p8, for anybody interested]

When an expression that has integer type is added to or subtracted from a pointer, the
  result has the type of the pointer operand. If the pointer operand points to an element of
  an array object, and the array is large enough, the result points to an element offset from
  the original element such that the difference of the subscripts of the resulting and original
  array elements equals the integer expression. In other words, if the expression P points to
  the i-th element of an array object, the expressions (P)+N (equivalently, N+(P)) and
  (P)-N (where N has the value n) point to, respectively, the i+n-th and i−n-th elements of
  the array object, provided they exist. Moreover, if the expression P points to the last
  element of an array object, the expression (P)+1 points one past the last element of the
  array object, and if the expression Q points one past the last element of an array object,
  the expression (Q)-1 points to the last element of the array object. If both the pointer
  operand and the result point to elements of the same array object, or one past the last
  element of the array object, the evaluation shall not produce an overflow; otherwise, the
  behavior is undefined. [...]

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text:
The Wooden Maiden
By Douglas Van Dyke, Jr
It turns out that tales of a witch in the vale are true, as an unfortunate maiden named Evessa finds out. The witch forces her into the wilderness for the sake of magical experiments. A curse transforms her body and robs her freedom. She remains isolated and trapped, until the day a loner stumbles upon her predicament. Can this young man, outcast by choice, rekindle her lost hopes? More
Despite the common presence of magic in Dhea Loral, tales of a witch in the vale are treated as no more than a scary story to keep children in line. Myth becomes reality when a young maiden named Evessa finds herself face to face with a captivating woman. Bound by the witch's magical commands, Evessa is forced to march into the wilderness for the sake of magical experiments. The maiden's life and body are transformed by the witch's curse. Left isolated and trapped, she faces a future of despair. After many dark days, a whistling song heralds a new visitor. A loner that has forsaken civilized lands stumbles upon her exile. Can this man rekindle the hope she once thought lost?
This is a short story suitable for Young Adult readers, set in the fantasy realm of Dhea Loral. It is being offered free in many outlets on behalf of the author.
Download: epub mobi (Kindle) pdf more Online Reader
lrf pdb txt
Price: Free! USD
Category: Fiction » Fantasy » Short stories
Category: Fiction » Young adult or teen » Fairy Tales
Tags: fantasy magic witch teen young readers fairy tale free story curse transformation female lead
About Douglas Van Dyke, Jr
Douglas was born on Nov 28th, 1971 . He got to live many different places while growing up, courtesy of the assignments the US Army offered to his father. Too quiet and too shy for too long, there was always dreams of other worlds and places…and the desire to write about them.
He got into fantasy role-playing games in his mid-teens. The first such games played on a computer were offered by a Commodore 64. Often Douglas and his brother would create their own tabletop fantasy games and rules as well, all using very basic 6-sided die. Eventually, they also got into Dungeons and Dragons*. As MMORPGs (Massively Multiplayer Online Role Playing Games) appeared, he tried a hand at several of them and made several new friends across the internet. To this day he has friends whom he meets in tabletop role-playing games, as well as online adventures. Many of his characters evolved in games, and each developed their own personality.
Inheritance of a Sword and a Path is the second book he has written, though the first published. It starts The Earthrin Stones trilogy, set in the fantasy world of Dhea Loral…a world he created for tabletop role-playing. The Widow Brigade is set in the same world, but a different locale and characters. While The Earthrin Stones trilogy servers as a good overall introduction to Dhea Loral, The Widow Brigade is focused on the dwarf widows of Tok-Maurron and their struggles. Douglas continues to write novels and think up short stories, while pondering the changing world of print vs ebooks.
Douglas lives with his wife and two young autistic children in Minnesota. He works in health care, serving people's healthcare needs in imaging. When most people see him, he is wearing scrubs.
FUN FACTS:
Most influential authors: R.A. Salvatore, Ed Greenwood, Joel Rosenberg, Terry Brooks
Favorite meal: Sausage and pepperoni pizza, with a Pepsi
Favorite role-playing class: Rangers lead the way
Hobbies: Online fantasy games, collecting swords, local Pathfinder games (Paizo)
–Graduated high school at age 16, then faced resistance from the state of Wisconsin because they felt no one under 18 should hold a full-time job.
–Lived on an army base in Germany for 3 years.
–Most loved RPG character endured a long campaign in which he lost twelve horses out from under him and more magical weapons destroyed than he could count. The Dungeon Master finally let him earn an unbreakable sword and an intelligent, high-level monster mount. The sword melted in acid and the mount was accidentally killed by a party member.
–Wouldn't be surprised if he has Asperger's Syndrome, since the more he learns about it in his oldest the more it sounds like his own childhood. Douglas sees no point in trying to diagnose it since he's an adult.
–Won the middle school spelling bee, but didn't last long in the regional contest.
–Played the following characters in school plays: Blackbeard the Pirate, Squanto, Mr Smee (Capt Hook's First Mate), and…..R2 D2, whose lines were all beeps and whirring noises.
–Spent several years in Civil War reenactments, portraying the Red-Legged-Devils of the 14th Brooklyn, NYSM.
Learn more about Douglas Van Dyke, Jr
Fiction > Fantasy > General
Fiction > Fantasy > Epic
Fiction > Fantasy > Contemporary
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The Maxline 14 Piece Honeycomb Tool Set includes aluminum honeycomb handles (available in blue or black) with titanium nitride coated tips in a convenient storage case along with a set of Maxline stickers!
So I have purchased a few sets of high end hex drivers like Hudy, Arrowmax. I did not like the Hudy Hand tools. But did like the arrowmax brand. But then I saw these, so I purchased them and yeah.... Very nice tool set as it has every tool we need to work on R/c car and trucks, and what ever. These hand tools feel very good and are worth every penny they ask. If your building r/cs then you need a good set of hand tools, and with this set you can't go wrong.


text:
Home/News/India/Budget has taken care of all sectors, will create New India: Modi
Budget has taken care of all sectors, will create New India: Modi
By shameen| Published: 1st February 2019 7:16 pm IST
Photo: ANI
New Delhi: Prime Minister Narendra Modi said on Friday that the interim Budget will empower the poor, the middle class, farmers, labourers, and traders besides imparting speed to economy and infrastructure for creation of 'New India'.
In his reaction to the Budget presented earlier in the day by Finance Minister Piyush Goyal, Modi said it will provide direct benefit to 12 crore farmers, three crore middle class tax payers and about 40 crore workers in the informal sector.
"It will give power to the poor, empower the farmers and traders, increase dignity of labourers, fulfil dreams of the middle class, celebrate the honest tax payer, accelerate infrastructure development and give new strength to economy. It will increase the confidence of the country. It will give new hope to 130 crore people of the country to achieve goals of New India," Modi said.
He said the Budget is "all encompassing and all inclusive" and is "dedicated to welfare of all."
THe Prime Minister said the Budget covers all sectors such as manufacturing, infrastructure, MSME, housing, healthcare and will boost economy.
READ: No coercive action against Arnab Goswami till Jan 29 in TRP case: Cops
"The Budget has taken care of all. Our policies have made a positive change in the lives of everyone," he said.
Modi said the efforts of government have led to a "record" in the speed of poverty reduction. More and more people were getting out of poverty and entering the middle and the neo-middle class, he said. "This big section is making their dreams come true and are accelerating development of the country. In this scenario the aspirations of middle class get a boost, get support — the government has shown its commitment for that," he said.
The Prime Minister said that Ayushman Bharat has benefited 50 crore people, the Pradhan Mantri Jeevan Jyoti Yojana and Pradhan Mantri Suraksha Bima Yojana have benefited 21 crore poor, Swachh Bharat mission has benefited 9 crore families, Ujjawala scheme has given free gas connection to 1.5 crore families and 1.5 crore families have got permanent houses under the Pradhan Mantri Awas Yojana.
READ: Uighur women not 'emancipated' by forced abortions, sterilisations: Pompeo
He congratulated the middle class, considered a supporter of the BJP, for relief in income tax rates.
"It is the liberal nature, honesty and law-abiding nature of the upper middle class and middle class due to which taxes are paid and welfare measures are taken for the poor."
Modi said there had been a demand for long to raise the income tax exemption limit to Rs 5 lakh and his government has done so.
He said that efforts by previous governments had only benefited two-three per cent of farmers but the PM-Kisan programme of his government will benefit 12 crore farmers who have land up to 5 acres.
"In one way, this is the biggest scheme for farmers since Independence. Our government is taking one step after the other for welfare of farmers," he said, adding that creation of separate departments for cow development and fishries will help farmers increase their income.
[source_without_link]IANS[/source_without_link]
budget Infrastructure Narendra Modi


text:
Ola Timeline: The Tale of an Indian Consumer Startup Turning Into a Global Mobility Giant
Let's get on board Ola for a flashy flashback
By Aastha Singal
How often do we opt to cab it today? Pretty! Booking a cab on a few clicks seems like an everyday activity today for office goers and long-distance travellers. Had it been a few days ago, the case was not the same. Hailing taxis was among the last of options on the list of everyday commuters until the entry of an internet startup that was destined to change the way India travels.
This is Ola's, Startup Story!
3 December 2010 marked the historical date when India produced its own rival to the world's largest cab aggregator Uber in form of Ola. Founded by Bhavish Aggarwal and Ankit Bhati, the ride-hailing startup initiated operations in Mumbai before shifting base to Bangalore. 2011 brought along a wave of investments that breathed life into many enterprising start-ups.
Within 2 years of launch, Ola was successful in pocketing two cheques from big investors like Tiger Global and Matrix Partners. The startup was able to create a much-required hype around the ride-hailing business and bring the otherwise hesitant commuters to cab it! While India was opening up to taxis, auto remained to be a comparable economic source of travel.
Taking the aspect into consideration, Ola incorporated autos into its application in 2014. The step helped the startup elevate its status to a billion-dollar entity the next year. Ola became a Unicorn in 2015. The same year, it acquired its crosstown rival TaxiForSure for $200 million only to shut down its business the next year.
Gaining Market Leadership
While 2016 was relatively a laid back year for the consumer brand, not many would have anticipated such a striking comeback. Not only did the firm raise a staggering $1.1 billion funds led by led by Chinese Internet conglomerate Tencent and Japan-based SoftBank but also acquired the food tech startup Foodpanda.
Needless to say, 2018 was a bombastic year for the Indian unicorn. Apart from acquiring the public transport ticketing app, Ridlr, the firm indulged in financing a startup for the first time ever. Ola invested in Scooter rent startup Vogo and launched its international operations starting with Australia, followed by the UK and New Zealand.
Having established itself as one of the internet majors in the mobility space, Ola has been making efforts to gain market leadership against rival Uber, the most-valuable startup in the world through various efforts. Now that Uber has filed for an IPO, it would be interesting to see Ola's next act to overtake the ride-hailing giant.
Billion-dollar Funding Saga
Looking to take on Uber that has targeted India as a priority market outside the US, Ola is looking to raise over $1 billion in Series J round of funding. In a major boost, the start-up received around $92 million from Flipkart co-founder Sachin Bansal. This marks the largest investment by an individual in Ola to date.
Commenting on the investment, Sachin Bansal had stated, "Ola is one of India's most promising consumer businesses. On one hand, they have emerged as a global force in the mobility space and on the other, they continue to build deeper for various needs of a billion Indians through their platform, becoming a trusted household name today."
Earlier in January, Ola raised $74 million from its existing investor, a Hong Kong-and London-based hedge fund, Steadview Capital, through preference shares at a subscription price of $301 per preference share. The funding round is expected to take Ola's valuation to over $6 billion. Ola is currently operational in over 100 Indian cities and towns with a network of more than 10,00,000 vehicles.


The file utility shall conform to the Base Definitions volume of POSIX.1‐2008, Section 12.2, Utility Syntax Guidelines, except that the order of the −m, −d, and −M options shall be significant.
Apply any position-sensitive default system tests and context-sensitive default system tests to the file. This is the default if no −M or −m option is specified.
When a symbolic link is encountered, identify the file as a symbolic link. If −h is not specified and file is a symbolic link that refers to a nonexistent file, file shall identify the file as a symbolic link, as if −h had been specified.
Specify the name of a file containing position-sensitive tests that shall be applied to a file in order to classify it (see the EXTENDED DESCRIPTION). No position-sensitive default system tests nor context-sensitive default system tests shall be applied unless the −d option is also specified.
If the −m option is specified without specifying the −d option or the −M option, position-sensitive default system tests shall be applied after the position-sensitive tests specified by the −m option. If the −M option is specified with the −d option, the −m option, or both, or the −m option is specified with the −d option, the concatenation of the position-sensitive tests specified by these options shall be applied in the order specified by the appearance of these options. If a −M or −m file option-argument is −, the results are unspecified.
The standard input shall be used if a file operand is '−' and the implementation treats the '−' as meaning standard input. Otherwise, the standard input shall not be used.
The values for < type> are unspecified, except that in the POSIX locale, if file is identified as one of the types listed in the following table, < type> shall contain (but is not limited to) the corresponding string, unless the file is identified by a position-sensitive test specified by a −M or −m option. Each <space> shown in the strings shall be exactly one <space>.
This test is applied only if the −i option is specified.
This test is applied only if the −i option is not specified.
Position-sensitive default system tests and context-sensitive default system tests are not applied if the −M option is specified unless the −d option is also specified.
A file specified as an option-argument to the −m or −M options shall contain one position-sensitive test per line, which shall be applied to the file. If the test succeeds, the message field of the line shall be printed and no further tests shall be applied, with the exception that tests on immediately following lines beginning with a single '>' character shall be applied.
All type specifiers, except for s, can be followed by a mask specifier of the form & number. The mask value shall be AND'ed with the value of the input file before the comparison with the value field of the line is made. By default, the mask shall be interpreted as an unsigned decimal number. With a leading 0x or 0X, the mask shall be interpreted as an unsigned hexadecimal number; otherwise, with a leading 0, the mask shall be interpreted as an unsigned octal number.
The <backslash>-escape sequences as specified in the Base Definitions volume of POSIX.1‐2008, Table 5-1, Escape Sequences and Associated Actions ('\\', '\a', '\b', '\f', '\n', '\r', '\t', '\v'). In addition, the escape sequence '\ ' (the <backslash> character followed by a <space> character) shall be recognized to represent a <space> character. The results of using any other character, other than an octal digit, following the <backslash> are unspecified.
Octal sequences that can be used to represent characters with specific coded values. An octal sequence shall consist of a <backslash> followed by the longest sequence of one, two, or three octal-digit characters (01234567).
The message to be printed if the test succeeds. The message shall be interpreted using the notation for the printf formatting specification; see printf. If the value field was a string, then the value from the file shall be the argument for the printf formatting specification; otherwise, the value from the file shall be the argument.
The −f option was omitted because the same effect can (and should) be obtained using the xargs utility.
Historical versions of the file utility attempt to identify the following types of files: symbolic link, directory, character special, block special, socket, tar archive, cpio archive, SCCS archive, archive library, empty, compress output, pack output, binary data, C source, FORTRAN source, assembler source, nroff/troff/eqn/tbl source troff output, shell script, C shell script, English text, ASCII text, various executables, APL workspace, compiled terminfo entries, and CURSES screen images. Only those types that are reasonably well specified in POSIX or are directly related to POSIX utilities are listed in the table.
Historical systems have used a ``magic file'' named /etc/magic to help identify file types. Because it is generally useful for users and scripts to be able to identify special file types, the −m flag and a portable format for user-created magic files has been specified. No requirement is made that an implementation of file use this method of identifying files, only that users be permitted to add their own classifying tests.
In addition, three options have been added to historical practice. The −d flag has been added to permit users to cause their tests to follow any default system tests. The −i flag has been added to permit users to test portably for regular files in shell scripts. The −M flag has been added to permit users to ignore any default system tests.
The context-sensitive default system tests were always built into the file utility. These tests looked for language constructs in text files trying to identify shell scripts, C, FORTRAN, and other computer language source files, and even plain text files. With the addition of the −m and −M options the distinction between position-sensitive and context-sensitive default system tests became important because the order of testing is important. The context-sensitive system default tests should never be applied before any position-sensitive tests even if the −d option is specified before a −m option or −M option due to the high probability that the context-sensitive system default tests will incorrectly identify arbitrary text files as text files before position-sensitive tests specified by the −m or −M option would be applied to give a more accurate identification.
The historical −c option was omitted as not particularly useful to users or portable shell scripts. In addition, a reasonable implementation of the file utility would report any errors found each time the magic file is read.
The historical format of the magic file was the same as that specified by the Rationale in the ISO POSIX‐2:1993 standard for the offset, value, and message fields; however, it used less precise type fields than the format specified by the current normative text. The new type field values are a superset of the historical ones.


text:
Parroting squawkbox
And a dirty dozen more
Christopher Monckton
Dave Blount
Kathryn Marshall
Matt Barber
William Donohue
Daniel J. Mitchell is a wacky libertarian economist/blogger who serves as a senior fellow at the Cato Institute[1].
1 Foolishness
Foolishness[edit]
Mitchell is a serial global warming denialist, apparently believing it to just be an excuse for bigger government or a scam by scientists to take government grants.[2][3] He has gotten better about this over time but still says global warming may or may not be a serious concern, and that he doesn't trust those big government environmentalists to handle the issue. He has also endorsed myths surrounding DDT, primarily the myth that Rachel Carson killed millions by inspiring environmentalists to ban DDT in areas of the world where it could have been used to fight malaria, even though there were always exemptions to DDT bans when it was used to fight malaria.[4]
Mitchell has also tried to scare his audience from the idea of universal healthcare by making faulty claims about Britain's National Health Service. The most egregious example of this is when he wrote a column alleging that Josef Stalin would be proud of the NHS because they had allegedly starved 1,165 of their patients.[5] This is a false claim, since the vast majority of those numbers come from people who were malnourished when they died, which doesn't mean that they died from malnourishment. The distinction is an important one since many fatal conditions make it more difficult to retain nutrients, inevitably leading to malnourishment. Nevertheless, Mitchell tried to pass this off as a government attempt to kill patients. His main source for this information was the Daily Mail (whom he often cites in other columns).
For whatever reason Mitchell considers Paul Krugman his greatest enemy, probably because he's just one of the most famous Keynesians out there. Krugman has responded to Mitchell by calling him dishonest in his claims.[6]
Despite his fervent anti-government, anti-Keynesian tendencies Mitchell has, like most libertarians, stopped clock moments, having declared the War on Drugs a failure, along with the Iraq War. He is also largely indifferent to gay marriage.
Experts for hire: As you may have guessed by now, his PhD in economics is from George Mason University.
Townhall.com: Contributes often.
Wingnut welfare
His blog
↑ Cato's profile
↑ http://danieljmitchell.wordpress.com/2010/10/11/share-this-letter-with-everybody-you-know-who-has-been-seduced-by-the-global-warming-hype/
↑ http://danieljmitchell.wordpress.com/2009/12/20/the-perfect-christmas-gift-for-your-global-warming-friends/
↑ http://danieljmitchell.wordpress.com/2010/09/19/the-environmentalist-death-toll/
↑ http://danieljmitchell.wordpress.com/2013/06/29/josef-stalin-would-have-admired-the-u-k-s-government-run-healthcare-system/
↑ http://krugman.blogs.nytimes.com/2010/08/04/what-reagan-didnt-do/
Retrieved from "https://rationalwiki.org/w/index.php?title=Daniel_J._Mitchell&oldid=1994155"
Political pundits
Denialists
Internet kooks
Libertarian wingnuttery


Core Water Consulting has assisted in Montana Stream, Wetland, Floodplain & Other Water Bodies Development permitting, 310 Natural Streambed Alteration Permit with Conservation. Environmental cleanup and Phase 1 and 2 Environmental Site Assessments are primary avenues of expertise. Many of our projects are repeat customers as; some permits require years of monitoring to ensure impacts are negated. Proper planning and communication with clients and agencies can make projects seamless. Given perpetual reporting requirements, agencies become proponents for project completion knowing components of the applications are detailed and complete.
CWC has developed an excellent rapport with agencies through continual interactions. The Department of Natural Resources and Conservation (DNRC) Land and Water Right Divisions in Kalispell and the Department of Environmental Quality (DEQ) regularly receive applications on our projects.


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text:
Q: Can't send GET request to Django via Postman I perform request http://167.71.57.114/api/users/list-exercises
And receive error

User matching query does not exist.

How can I fix the error?
Found out that reason of the error is that request didn't have any data
But you can see that I am sendig the "user_id"

My code
class WorkoutUserProgramListViewAPI(GenericAPIView):
    permission_classes = (IsAuthenticated,)
    serializer_class = WorkoutUserProgramListSerializer

    def get(self, request):
        response = {}
        user = request.data.get("user_id") # user is None
        user = User.objects.get(pk=user) # Error User matching query does not exist.

A: Like @Klaus D said in the comments, you would rather put a user-id as a parameter. If you're sending user_id=29, I would add that as a parameter, which should change the URL to be something like
http://167.71.57.114/api/users/list-exercises?user_id=29

A: That error occurs when the objects.get method does not find any result.
If you use filter and verify that the result is not None you avoid that error in the case that the id passed by parameter does not belong to any user.
class WorkoutUserProgramListViewAPI(GenericAPIView):
    permission_classes = (IsAuthenticated,)
    serializer_class = WorkoutUserProgramListSerializer

    def get(self, request):
        response = {}
        user_id = request.data.get("user_id")
        user = User.objects.filter(pk=user_id).first()
        if user is not None:
            # code
        else:
            # code

Listen what Klaus D. tells you, too.


text:
Old Gold & Black • March 30, 2017 • https://wfuogb.com/3619/sports/final-four-set-after-wild-weekend/
Final Four set after wild weekend
Ethan Perellis
Another thrilling weekend of college basketball took place this past weekend.
Gonzaga, UNC, Oregon and South Carolina all punched their ticket to the Final Four in Glendale, Arizona this past weekend. The Sweet 16 and Elite 8 games ranged from huge blowouts to tight buzzer beaters.
At Madison Square Garden, the East Region had no shortage of drama. The four-seed Florida beat the eight-seed Wisconsin, 84-83, on a last second three-pointer by Chris Chiozza in overtime. Chiozza drove the length of the court in four seconds and hit a floating three-pointer to allow the Gators to escape.
The seven-seed South Carolina Gamecocks did not need any last second magic as they destroyed Baylor 70-50. In an all-SEC Elite Eight matchup, the Gamecocks rallied late to defeat the Gators, who were making poor offensive decisions in the final few minutes. Led by SEC Player of the Year Sindarius Thornwell, South Carolina is headed to the first Final Four in program history. The Cinderella team had not even won a game in the NCAA Tournament since 1973. South Carolina moves on to take on Gonzaga Saturday on CBS.
The 1-seed Gonzaga Bulldogs won the West Region to also advance to the Final Four for the first time in school history. In the Sweet 16, Gonzaga held-off the four-seed West Virginia Mountaineers that caught fire late.
With 25 points from Trevon Bluiett, the 11-seed Xavier squeaked-out a victory against two-seed Arizona, 73-71, to set-up a match-up with Gonzaga. Gonzaga marched into the Final Four with a commanding 83-59 win. The Bulldogs, coached by Mark Few, finally advance to the final weekend of the tournament, earning the respect the program deserves. Gonzaga has had at least 28 wins in each of the last four seasons and already 36 this season. The Bulldogs' remarkable consistency gets the benchmark victory needed to validate a top-tier college basketball program.
On the other side of the bracket in the Midwest Region, the one-seed Kansas Jayhawks ran past the four-seed Purdue 98-66 in a game that was never close in the second half. The three-seed Oregon beat the red-hot seven-seed Michigan, 69-68, with 20 points from Tyler Dorsey. Dorsey was forced to step-up with Pac-12 player of the year, Dillion Brooks, struggling mightily with his shot. Dorsey continued to be in-form against the Jayhawks. Oregon took down Kansas, 74-60, with 27 points from Dorsey and 17 points from Brooks. The Duck's Jordan Bell, who finished with 11 points, 13 rebounds and eight blocks, is being considered the tournaments breakout star.
The loss for the Jayhawks is another disappointment under Bill Self. Self is a mere 2-5 in the regional finals at Kansas, even though the Jayhawks have been a one-seed seven times in the last 14 years. Oregon enters their second Final Four all-time but first since 1939. The Ducks will play the University of North Carolina Tar Heels in the Final Four.
The one-seed Tarheels won the South Region, which featured multiple high-quality games from some of the sport's most storied programs. North Carolina scored at will against the four-seed Butler and won, 92-80. The two-seed Kentucky played 3-seed UCLA in a fast-paced game. The Wildcats outscored the Bruins, 86-75, in rematch from a December game won by UCLA. De'Aaron Fox had a career-high 39 points and outplayed Lonzo Ball to punch Kentucky's ticket into the Elite Eight. With a trip to Phoenix up for grabs, North Carolina and Kentucky found themselves in the best game of the entire NCAA Tournament. Freshman Kentucky guard Malik Monk hit a challenging three-pointer with 7.2 seconds left. With the game in the balance, UNC's Luke Maye hit a jumper with 0.3 seconds left to lift the Tar Heels over the Wildcats, 75-73. The Tar Heels enter their record 20th Final Four as the favorites for the national championship and look to win the tournament a year after losing at the buzzer to Villanova. North Carolina will play Oregon Saturday at 8:49 p.m. on CBS.
Even without many traditional college basketball powers, there are plenty of headlines heading into the Final Four. An all-Carolina game or all-west coast national championship remains possible. Regardless of the match-up, the final few games of March Madness rarely disappoint as the college basketball season comes to a close.


At 22, Saoirse Ronan has already built up a career to be envied. She's worked with the likes of Joe Wright, Wes Anderson, and Peter Jackson, and she's just snagged her second Oscar nomination for Brooklyn. Now for her next big-screen role, she'll be teaming up with another hot young talent. Ronan is set to star in Lady Bird, the solo directing debut of Greta Gerwig.
According to Deadline, who broke the news, Lady Bird follows a high school senior over her last year at home in Sacramento. Gerwig, whose writing credits also include Frances Ha and Mistress America, penned the script. The coming-of-age tale should be right in Gerwig's wheelhouse, especially given that Gerwig is herself a Sacramento native. And Ronan proved remarkable as a young woman coming into her own in Brooklyn, so she seems like an ideal choice to bring Gerwig's tale to life.
Powerhouse producer Scott Rudin is on board f0r Lady Bird, along with producers Eli Bush (The Grand Budapest Hotel) and Evelyn O'Neill. Lady Bird will shoot around Ronan's schedule, as she prepares to make her Broadway debut in The Crucible this spring. (That one is also a Rudin production.) Ronan has recently wrapped an adaptation of Anton Chekov's classic play The Seagull, which also stars Corey Stoll and Elisabeth Moss.
Gerwig was most recently seen in Mistress America. She hit the festival circuit last year with Maggie's Plan, which opens in theaters this May, and will do so again this year with Todd Solondz's Welcome to the Dollhouse sequel Wiener-Dog. She is now shooting Jackie, the Jackie Kennedy biopic which has Natalie Portman playing the former First Lady. Although Gerwig previously co-directed Nights and Weekends with Joe Swanberg, Lady Bird will mark her first time directing by herself.


text:
AUTHOR'S NOTE (BREAKING): McCAIN WILL ATTEND TONIGHT'S DEBATE
AUTHOR'S NOTE (REMINDER): THE KEY GRIP WILL BE LIVE-BLOGGING TONIGHT'S EVENT
To say of this election's fall finish that it hasn't unfolded the way John McCain and Steve Schmidt were planning is to engage in droll understatement. After a respectable (if also, for some of us, infuriating) Sarah Palin bounce from his own convention, McCain appeared to have all the initiative in the national dialogue--repositioning himself as the only candidate who could say he was for change and then actually deliver on it.
That message resonated with a still-credulous pool of undecided voters to such a degree that one of my colleagues had to stop wearing a button that said, "I'm Voting for CHANGE!" because people kept approaching her at gas pumps to ask which candidate she was intending to endorse with that sentiment. Everything seemed to be perfectly in place for the third incarnation of Lee Atwater: Set the other guy back on his heels with a convention that makes the discussion all about him instead of issues; grin smugly like the guy who's still holding his best pin-move in reserve; steal the other guy's big issue; attack, attack, attack.
Seems like a long time ago now, doesn't it.
So--what the hell happened? Well this author has been saying it almost from the very moment that it was unfolding in real time, but you'll have to forgive him if he takes this opportunity to say it now, again: The wheels started coming off exactly two weeks ago, when Sarah Palin's interview with Charlie Gibson aired on the same day that John McCain was savagely, pitilessly filleted by the undecided-friendly panelists of ABC's midmorning talk show, The View.
From there we had the Lehman / AIG / Merill meltdown the following Monday, during which Mr. McCain could be found standing on a stage in Jacksonville telling us in Bush-ian tones of befuddled helplessness that the fundamentals of our economy are strong. In the Lee Atwater playbook this is known as "stripping the bark off," by which one campaign wounds the other on likability grounds, then pins it with an unpopular position on a key issue. You show Mr. Dukakis riding in a tank, and then you run the Willie Horton ad. Not the other way around. If there's irony in this whole mess (and surely there is), it is that this time around the Republican nominee inflicted this exact, textbook maneuver--on himself.
After a week in which the Obama people hammered McCain mercilessly and deservedly for his track-record advocating for less government oversight, week-2 of the crisis began with Treasury Secretary Paulson pitching a three-page bailout bill with a price tag so whopping as to cripple the fiduciary discretion of all successive Presidents of this great land for the foreseeable future. The public outcry was immediate and bipartisan and left only Senator McCain with the narrow corridor for wiggling, since his patrons at the White House were (and are) just about the only people in the country who seem to want the bill.
Oh, and entirely by the way in case you missed it, the Treasury quietly let slip yesterday that they have no basis whatsoever for the $700 billion price-tag. "We didn't base it on a specific data-point," an un-named but high-ranking Treasury official said, "we mostly just needed a really big number."
...I'm sorry, let me just repeat that: The same Treasury Department that has been telling us all week that the economy will come totally unhinged if this bill isn't passed immediately, admitted yesterday afternoon that it doesn't actually have the faintest idea where it got the figure for how much this was going to cost. And if you can decide for yourself whether that's hilarious or horrifying, do me the favor of telling me too, please.
By Wednesday McCain had all but irretrievably lost Colorado (along with New Mexico and Iowa) and was receiving news that North Carolina and Missouri are both now tied. McCain's Senior Campaign Advisor and longest-serving confederate, Rick Davis, was accused--on the front page of the Wednesday New York Times--of having taken a retainer from Freddie Mac in the amount of $15,000 a month for over four years, right up to the very moment that the federal government seized control of the institution. Well after the McCain campaign had started running ads accusing Senator Obama of being in cahoots with that very same institution.
And then, a little after noon on Wednesday, the campaign could find itself signing at the front door for a small package from CBS, containing an advance copy of the final version of Katie Couric's two-part interview of Sarah Palin. The highlight of the first segment? Well, it could be that Governor Palin was unable to cite examples of McCain's track-record on encouraging government oversight. ("Yes, Katie, but he's also known as the maverick, takin' shots from his own party, and the other party, tryin' to get people to see what he's tryin' to accomplish.") The highlight of the second segment? How about Palin's inability to explain why Alaska's proximity to Russia qualifies her for foreign policy leadership. ("Alaska has a very narrow maritime border between a foreign country, Russia, and on our other side, the land-- boundary that we have with-- Canada. It-- it's funny that a comment like that was-- kind of made to-- cari-- I don't know, you know? Reporters.")
We all know what happened next.
In total exasperation, the McCain team pulled the grandaddy of all electoral stunts: what has increasingly been described even in mainstream media outlets as a "call for a time-out"--hoping that the drama of a campaign suspension would wipe all of these other stories off the media map, and maybe even scuttle a debate at which McCain would otherwise have little chance of escaping a discussion of his role in repealing the Glass/Steagal Act. And never mind the fact that the McCain team didn't actually suspend anything (its television ads have continued to run in full force, everywhere), or that his presence in Washington was understood to be useless by all but the most politically un-astute respondents in a hasty Survey-USA poll that found only 10% support for his actions.
It may well have been the biggest election gamble in modern history, and it hasn't worked. In an uncharacteristic fury, Dave Letterman invested over ten minutes of his Wednesday night program to assail the decision and its architect--including a cutaway that showed Mr. McCain not jetting-off to Washington, but rather being prepped by a makeup artist for an interview across town with the same Katie Couric who'd been planning to highlight her frightful encounter with the Senator's running-mate.
Then Mississippi Governor Haley Barbour went public with a call that the debate go forward as planned--together with all the major newspapers in the state. Meanwhile, when Mr. McCain finally arrived in Washington (over twenty-four hours after his suspension announcement), his appearance seemed to scuttle, rather than facilitate, a fragile consensus on the final language in the bill. Individuals loyal to McCain have spun this story in a variety of ways, but history will know better: McCain yesterday was the champion of nothing, the maverick standing up to nobody. Indeed he was considerably the more reckless and opportunistic of the two major candidates for President.
All of which leaves McCain with only today to salvage the situation. At the moment he is claiming (with a straight face) that the broad outlines of a compromise are in place (just as they were before he arrived), and that he may now therefore adjourn to Oxford. This is not an argument the public is likely to buy unless truly newsworthy progress on the bill ends up being made in the hours remaining for Friday business.
By taking this particular gamble, Mr. McCain has broken over his knee the first cardinal rule of gambling: always minimize your losses on the losing hands.
Instead, having been dealt a flop that consists of a two of clubs and a spoonful of ant poison, Mr. McCain has decided to bluff--by swallowing the ant poison right there at the table, and then betting the farm on the two of clubs. And none of the other players have anything to gain by folding: The general public doesn't want a Friday compromise on the bailout. The Republicans in the House of Representatives don't want a Friday compromise on the bailout. The Republicans in the Senate, who were for the compromise before Mr. McCain showed up to fan the partisan flames surrounding the issue, no longer want a Friday compromise on the bailout. Only Mssrs. McCain, Paulson, and Bush are still of the opinion that the sun will not rise tomorrow if this deal isn't done by the time Mr. Obama takes the stage in Oxford. And I know I've said it before, but my friends, that's just not the company Mr. McCain needs to be keeping at the moment.
My personal take is that a compromise bill that would give Mr. McCain enough cover to come to Mississippi as conquering hero is vanishingly unlikely, but beyond that relatively safe bet I haven't a clue what will actually happen instead. The GOP ticket can afford neither to show with its tail between its legs and no compromise on the bill, nor to allow Mr. Obama to have the initiative that would attach to yet another reversal. There can be little hope that Mr. McCain could conjure yet another "shakeup" of this election on such short notice, or that this as-yet hypothetical new shakeup would have any traction at all with an increasingly shakeup-fatigued electorate.
If there's a fourth option I haven't considered, then McCain supporters had better hope that he and Steve Schmidt are smarter than I am about what that option might be--and, more importantly, that they get started availing themselves of it, immediately.
I have to believe in my heart-of-hearts, that there are only 3 types of people that would vote for McCain/Palin at this point:
1. The woefully uninformed.
There are alot of them out there. The guy in the office next to me still plans to vote Republican. His take on the bailout for example? "Cut McCain some slack, it's a really complicated issue."
2. The woefully republican.
These folks will vote red regardless of the candidate. It simply doesn't matter who the candidate is.
3. Fearful for Israel.
McCain seems to be the bigger defender of Israel. I don't believe this, but the (elder) Jewish community has this perception.
This has been a great week for Democrats... hell, for Americans, with reagrd to the election. The bul$hit McCain has been spewing is finally coming out. There's no better disinfectant than sunshine, right?
I just hope it's enough. These two scare the living hell out of me.
isuyankee said...
I don't find it at all strange that Treasury doesn't know if they need $200 bln or $700 bln. I think they chose a big number simply so they don't have to keep coming back in front of Congress over and over again. Here's the deal as I see it....they need to start the auctions to find out how much they need to buy and what the pricing looks like. Again, this is not an expenditure, it's a purchase of assets. The price of the assets may go up or down and the success of the program from the standpoint of the national debt over time, will depend on whether Paulson and friends price the assets well. I understand the idea that we don't want to bail out Wall Street executives, but the government made a good deal on AIG (Bill Clinton made a point of this on the Daily Show) and has allowed other big players (Lehman for example) to fail. I know that people are angry over executive compensation but it will be taken care of in two ways. The FBI is already investigating Fannie, Freddie, AIG, and Lehman. Second, contact the Board of Directors of every company you own stock in and demand they eliminate golden parachutes, etc. There are good folks in the business world who will work without this stuff. They need to pass a bill and start cleaning up some of this mortgage paper and I think that the economy will slowly (very slowly) get better. Is McCain setting himself up here as the "maverick" who goes against Bush to fight for the taxpayer?


PARALIGN Roll Alignment - Preventive Maintenance Concepts, Inc.
PARALIGN 'learns' the ideal position of the rollers on a reference roller or plate. Simply by placing the system on the next roller surface, the actual position of the individual roller is compared with the ideal alignment and the deviation is shown immediately as a graphical and numerical information on the display. The data is transmitted wirelessly to a laptop. A side view of the machine is shown, with the deviations of every measured roller from its ideal position graphically and numerically displayed.