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The Efflux Transporter ABCG2 Maintains Prostate Stem Cells
Neha G. Sabnis, Austin Miller, Mark A. Titus and Wendy J. Huss
Neha G. Sabnis
1Department of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Buffalo, New York.
Austin Miller
2Department of Bioinformatics & Biostatistics, Roswell Park Cancer Institute, Buffalo, New York.
Mark A. Titus
3Department of Genitourinary Medical Oncology - Research, Division of Cancer Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas.
Wendy J. Huss
4Department of Urologic Oncology, Roswell Park Cancer Institute, Buffalo, New York.
For correspondence: Wendy.Huss@RoswellPark.org
DOI: 10.1158/1541-7786.MCR-16-0270-T Published February 2017
Prostate stem cells (PSC) are characterized by their intrinsic resistance to androgen deprivation therapy (ADT), possibly due to the lack of androgen receptor (AR) expression. PSCs resistance to ADT and PSC expansion in castration resistant prostate cancer (CRPC) has sparked great interest in using differentiation therapy as an adjuvant to ADT. Understanding the mechanisms, by which PSCs maintain their undifferentiated phenotype, thus has important implications in differentiation therapy. In the prostate, the ATP binding cassette sub-family G member 2 (ABCG2) transporters, which enrich for AR-positive, ADT-resistant PSCs, play an important role in regulating the intracellular androgen levels by effluxing androgens. We hypothesized that the ABCG2-mediated androgen efflux is responsible for maintaining PSCs in an undifferentiated state. Using the HPr-1-AR (nontumorigenic) and CWR-R1 (tumorigenic) prostate cell lines, it was demonstrated that inhibiting the ABCG2-mediated androgen efflux, with Ko143 (ABCG2 inhibitor), increased the nuclear AR expression due to elevated intracellular androgen levels. Increased nuclear translocation of AR is followed by increased expression of AR regulated genes, a delayed cell growth response, and increased luminal differentiation. Furthermore, Ko143 reduced tumor growth rates in mice implanted with ABCG2-expressing CWR-R1 cells. In addition, Ko143-treated mice had more differentiated tumors as evidenced by an increased percentage of CK8+/AR+ luminal cells and decreased percentage of ABCG2-expressing cells. Thus, inhibiting ABCG2-mediated androgen efflux forces the PSCs to undergo an AR-modulated differentiation to an ADT-sensitive luminal phenotype.
Implications: This study identifies the mechanism by which the prostate stem cell marker, ABCG2, plays a role in prostate stem cell maintenance and provides a rationale for targeting ABCG2 for differentiation therapy in prostate cancer. Mol Cancer Res; 15(2); 128–40. ©2016 AACR.
This article is featured in Highlights of This Issue, p. 115
The ATP binding cassette sub-family G member 2 (ABCG2) transporters belong to the ATP binding cassette (ABC) superfamily of transporters and use ATP hydrolysis to catalyze the transport of a wide range of substrates ranging from chemotherapeutic drugs, steroids, and xenobiotics from the intracellular to the extracellular milieu. The side population assay is a flow cytometric method that isolates stem cells based on the dye-efflux properties of the ABC transporters and ABCG2 is the molecular determinant of the stem cell–enriched side population phenotype in the hematopoietic, retinal, cardiac, limbal, and skeletal muscle system (1–5). The importance of ABCG2 to stem cell maintenance can be adjudged from the observations that ABCG2 expression is lost as stem cells undergo differentiation (1, 2, 6). In fact, overexpression of ABCG2 has been shown to promote stemness while the loss of ABCG2 expression has been shown to promote lineage commitment in the retinal, hematopoietic, and cardiac side population cells (1, 2, 7). While the importance of ABCG2 in stem cell maintenance has been shown, the precise mechanism by which ABCG2 prevents stem cells from undergoing differentiation is unknown.
An ABCG2-expressing side population is present in normal and cancerous prostate tissues and cell lines (8). ABCG2 expression is found in approximately 1% of the cells in the prostate basal compartment (9, 10). ABCG2-expressing cells are present in biopsies following androgen deprivation therapy (ADT)(9) and are upregulated upon androgen blockade in culture (9, 11). Although, there are reports of ABCG2-effluxing bicalutamide (12), there have been no studies that show enzalutamide efflux by ABCG2. Moreover, ABCG2-expressing side population in the prostate demonstrates multipotency and self-renewal properties, thus suggesting an enrichment of stem cells in this population (13). Transcriptome analysis of ABCG2-expressing prostate cells confirmed the expression of stem cell–associated genes such as CD133, Oct4, and nestin and the lack of androgen receptor (AR) expression which is typical of prostate stem cells (PSC; refs. 9, 10, 14). Although, ABCG2 transporters are suspected to play a role in maintaining the prostate stem cell phenotype, the mechanistic significance of ABCG2 to stem cell maintenance is largely unknown. Elucidating the mechanisms by which PSCs maintain their stemness will provide opportunities to target them in prostate cancer therapy. PSCs are implicated in the etiology of castration-resistant prostate cancer (CRPC) and are resistant to ADT (15). In fact, ABCG2-expressing prostate cells have been shown to not only survive ADT but are amplified in CRPC (9, 11), thus suggesting a role for the transporters in conferring ADT-resistance to PSCs and in the etiology of CRPC, in addition to stem cell maintenance.
Previous studies showed that mitoxantrone-resistant rat progenitor epithelial (Mx-RPE) cells, which have been selected for based on high ABCG2 expression, had elevated retention of radiolabeled DHT when treated with the ABCG2 inhibitors, novobiocin and fumitremorgin C, thus, suggesting that ABCG2 can regulate androgen levels (9). Several studies have demonstrated that the exogenous introduction of AR in prostate cells that either lack or have very low levels of endogenous AR such as PC3, HPr-1, and PSCs forces cells to undergo differentiation to a luminal phenotype (14, 16, 17). Thus, we hypothesized that ABCG2 plays a critical role in maintaining PSCs in an undifferentiated state via the efflux of androgens and inhibiting the ABCG2-mediated androgen efflux would force the ABCG2-expressing PSCs to undergo an AR-modulated differentiation to a luminal phenotype. In the current study, we investigate the effects of pharmacologic inhibition of ABCG2 on cellular differentiation in the ABCG2-expressing HPr-1-AR (nontumorigenic prostate epithelial cell line) and the CWR-R1 (tumorigenic CRPC cell line derived from CWR22 xenografts) cells by sphere-formation assays and immunoblots. In vivo differentiation is assessed by treating nude mice implanted with ABCG2-expressing CWR-R1 cells with either placebo or Ko143. Inducing luminal differentiation in PSCs and sensitizing them to ADT is the basis of differentiation therapy. Our findings provide a rationale for targeting ABCG2 for differentiation therapy in prostate cancer.
Cell culture and treatments
The HPr-1-AR cells were obtained from Dr. Dominic Smiraglia and cultured in keratinocyte-serum-free media (Thermo Fisher Scientific) supplemented with 1× penicillin-streptomycin. CWR-R1 cells were obtained from Dr. James Mohler and were cultured in Improved MEM supplemented with 2% FBS (FBS), 1× penicillin–streptomycin, 0.2× insulin-transferrin-selenium (ITS; Life Technologies), 0.9 mg/L linoleic acid (Sigma-Aldrich), and 100 μg/mL EGF (Thermo Fisher Scientific). Our preliminary data shows that prolonged charcoal stripping leads to the downregulation of ABCG2 membrane expression in mitoxantrone-resistant CWR-R1 cells (data not shown). Thus, the ABCG2-expressing CWR-R1 cells were cultured in FBS-supplemented media. All cells were maintained in an atmosphere of 5% CO2 at 37°C. All experiments were done before cells reached passage 65. Treatments were administered in the appropriate media such that DHT (Sigma) was used at a concentration of 0.1 nmol/L for the HPr-1-AR cells and 1 nmol/L for the CWR-R1 cells. Ko143 (Axon chemicals) and enzalutamide (Selleck Chemicals) were administered at a concentration of 1 μmol/L each.
Magnetic isolation of ABCG2+ cells
Cells were grown to 75% confluency and the HumanSep FITC Selection Kit (StemCell Technologies) was used in conjunction with the anti-BCRP antibody, clone 5D3, FITC-conjugated (Millipore) to isolate the ABCG2-expressing cells as per the manufacturer's protocol.
Immunofluorescence microscopy
Cells were seeded onto coverslips, fixed with 10% formalin, and permeabilized with 0.1% Triton X. Cells were blocked with 1% BSA and immunostained with the anti-AR antibody (1:200, Millipore). The goat anti-rabbit antibody, Alexa Fluor 594 conjugate (1:1,000, Invitrogen) was used as a secondary antibody. Coverslips were mounted onto slides with Vectashield mounting medium containing DAPI (Vector Laboratories) for analysis under an immunofluorescent microscope.
Intracellular androgens from cells treated with [3H]DHT (Perkin Elmer) were extracted using methyl tert butyl ether (Sigma-Aldrich) and reconstituted in a mixture of 9:1 chloroform:acetone. The reconstituted mixture along with steroid standards (Steraloids, Inc) were loaded onto 20 cm × 20 cm silica-coated TLC plates (Millipore). The 9:1 chloroform:acetone mixture was used as the mobile phase to separate the steroids. The standards were visualized on the TLC plate by spraying the plate with p-Anisaldehyde stain. Zones corresponding to the reference steroid standards were scraped into vials containing the BetaMax Scintillation Cocktail (MP Biomedicals) and tritium levels were measured using a liquid scintillation counter.
RIPA buffer (Invitrogen) was used to prepare cell lysates and protein concentration was measured by the BCA Protein Assay Kit (Thermo Scientific). Cell lysates (20 μg) were loaded in the wells of Novex NuPAGE 4%–12% Bis-Tris Gels (Thermo Scientific). Electrophoresed proteins from gels were transferred onto polyvinylidene difluoride membranes. Membranes were blocked and stained with anti-AR (1:250, Millipore), anti-Sox2 (1 μg/mL, Millipore), p21 (1:200, C-19; Santa Cruz Biotechnology), PSA (1:500, Dako), Cytokeratin-8 (1:500, Covance), and β-actin (1:10,000, BioVision Inc.) antibodies and counterstained with the appropriate horseradish peroxidase–conjugated secondary antibodies (GE Healthcare). Signal from the secondary antibodies was detected using an enhanced chemiluminescence detection system (Pierce).
Sphere-formation assay
Cells were plated at a density of 2,000 cells per well or 4,000 cells per well (after xenograft tumor digestion) in 24-well ultralow attachment plates (Sigma-Aldrich) coated with Matrigel (Thermo Fisher Scientific). Cells in media were suspended in Matrigel at a ratio of 2:3 (cells in media volume: Matrigel volume) and this suspension was plated onto the edges of the wells of the plates and allowed to solidify at 37°C for 45 minutes. Upon solidification, treatments were administered and the number and sizes of spheres were quantitated 10–14 days later. Sphere area was measured using the Basic SPOT imaging software. The CWR-R1 spheres were maintained in improved MEM, original growth media, whereas the HPr-1-AR spheres were cultured in prostate epithelial cell growth medium (Lonza).
RNA isolation and quantitative real-time PCR
RNA was isolated using the RNeasy Mini Kit (Qiagen) and cDNA was synthesized using the SuperScript III Reverse Transcriptase Kit (Invitrogen) and oligodT primers. Gene expression was determined using SYBR Green PCR mix (Bio-Rad). Primer sequences are enlisted in Supplementary Table S1. qRT-PCR was performed on the Applied Biosystems 7300 machine. The following conditions were used to carry out the RT-PCR reactions: 300 seconds at 95°C; 50 cycles of 10 seconds at 95°C; 20 seconds at 60°C and 20 seconds at 72°C.
In vivo tumor xenograft studies
Twenty-six male nude mice (Harlan Sprague Dawley Inc.) aged 4–5 weeks were castrated and implanted with a testosterone silastic tubing to normalize the serum testosterone levels to 4 ng/mL. Mice were subcutaneously injected with 1 × 106 ABCG2-expressing CWR-R1 cells suspended in Matrigel (1:1 ratio). Seven days postinoculation; mice were subdivided into two groups of 13 mice each. The first group of mice received placebo while the second group of mice received 15 mg/kg of Ko143 by oral gavage. Treatments were administered three times a week and tumors were measured at the time of drug delivery. Tumor volume was measured by digital calipers and calculated using the formula L1×(L2)2×0.523. Once tumors reached 1,500 mm3 volume, mice were euthanized and tumors were excised for further analysis.
Three mice from each group with multiple tumors originating from the same injection site or whose tumors failed to reach a volume of 250 mm3 on day 45 of cell inoculation were excluded from the study. Linear Mixed Model analysis was used to compute differences in the tumor growth rates between control and Ko143-treated animals. Data obtained from in vitro experiments was analyzed either by one-way or two-way ANOVA tests. All data analyses were generated using SAS/STAT software, Version 9.4.
IHC was done on one section of the paraffin-embedded tumors. Tissue specimens were immunostained with primary antibodies: anti-Cytokeratin-8 (1:1000, Covance), anti-BCRP antibody, clone BXP-21 (1:500, Millipore) and anti-AR antibody (1:200, Millipore). Biotinylated horse anti-mouse IgG antibody (1:1000, Dako) and biotinylated goat anti-rabbit (1:1,000, Abcam) were used as the secondary antibodies as appropriate. Vectastain Elite ABC HRP kit (Vector Laboratories) was used to visualize the immunostained tissue sections. Staining was performed on Dako Omni. Images were scanned using the Aperio scanner.
Inhibiting ABCG2-mediated androgen efflux increases nuclear AR expression
On the basis of previous studies showing the increased retention of radiolabeled DHT in Mx-RPE cells upon incubation with Novobiocin and Fumitremorgin C (9), we propose that inhibiting ABCG2 in the presence of DHT would increase the nuclear AR expression due to increased intracellular retention of androgens. Studies were conducted in the HPr-1-AR and the CWR-R1 cell lines. The HPr-1-AR and CWR-R1 cell lines were chosen due to the presence of functional ABCG2 in a subpopulation and an androgen-responsive phenotype. In addition, the HPr-1-AR and the CWR-R1 cells offer comparison between nontumorigenic and tumorigenic cells respectively. Optimal DHT concentrations for treatment were determined by assessing growth curves of the unsorted HPr-1-AR and CWR-R1 cells in response to DHT (Supplementary Fig. S1A and B) and TLC analysis of the intracellular androgens extracted from unsorted HPr-1-AR and CWR-R1 cells treated with increasing concentrations of [3H]DHT (Supplementary Fig. S1C and D). TLC analysis was performed to determine whether DHT is metabolized by hydroxysteroid dehydrogenase or aldo-keto reductase expressed in prostate cells. On the basis of these studies, a concentration of 0.1 nmol/L DHT was used for the HPr-1-AR cells (Supplementary Fig. S1A and C) and 1 nmol/L DHT was used for the CWR-R1 cells (Supplementary Fig. S1B and D) in all further experiments unless otherwise mentioned as these were the lowest concentrations of DHT at which there was both, a retardation in the growth response as well as an observable change in the amount of intracellular androgens. Using DHT concentrations greater than the ones used would overwhelm the ABCG2 transporters and lower concentrations would be undetectable.
To determine the effects of inhibiting the ABCG2-mediated androgen efflux on the expression and cellular localization of AR, ABCG2-expressing HPr-1-AR and CWR-R1 cells were treated with the vehicle control, DHT, Ko143, or a combination of DHT and Ko143 and subjected to immunofluorescent staining. ABCG2-negative cells treated with DHT served as the positive control. In our study, ABCG2 was only pharmacologically inhibited as approximately 1% of the cell population express ABCG2, thus genetic silencing of ABCG2 expression in such a small population is technically challenging due to low cell yield and genetic silencing of ABCG2 expression in a mixed population of cells would lead to undetectable changes.
Our results indicate that in both, the HPr-1-AR and the CWR-R1 cells, a combination of DHT and Ko143, significantly increased the nuclear AR expression; the nuclear AR signal was comparable with the ABCG2-negative cells treated with DHT (Fig. 1). The vehicle control and Ko143-treated ABCG2-expressing HPr-1-AR cells showed the presence of cytoplasmic AR due to the transfected AR vector (Fig. 1A). Upon addition of DHT, cytoplasmic AR translocated to the nucleus; however, the nuclear AR signal was very weak (Fig. 1A and C). The control and Ko143-treated ABCG2-expressing CWR-R1 cells showed very weak AR signal, which is in agreement with previous studies that have demonstrated undetectable AR expression in PSCs (10, 14). The addition of DHT to the ABCG2-expressing CWR-R1 cells slightly increased the nuclear AR expression compared with the control and Ko143-treated cells; however, a combination of DHT and Ko143, led to a significant increase in the nuclear AR expression compared with DHT alone (Fig. 1B and D). To confirm that the increased nuclear AR expression upon treatment with a combination of DHT and Ko143 is due to increased intracellular androgen levels, the efflux of androgens by ABCG2 and the consequent increase in the intracellular androgen levels upon inhibiting the ABCG2 transporters was confirmed in the steroidogenic, HPr-1-AR, and CWR-R1 cells and the nonsteroidogenic Hek293 cells stably transfected with pcDNA3.1ABCG2 vector (Supplementary Fig. S2). Thus, our results indicate that inhibiting the ABCG2-mediated androgen efflux increases the nuclear translocation of AR.
Inhibiting the ABCG2-mediated androgen efflux leads to the nuclear translocation of AR. ABCG2-expressing (ABCG2+ve) HPr-1-AR (A) and CWR-R1 cells (B) obtained by magnetic sorting were treated with either vehicle control, 1 μmol/L Ko143, DHT, or DHT+ 1 μmol/L Ko143 for 24 hours. ABCG2-negative (ABCG2−ve) cells treated with DHT were used as the positive control. Nuclear AR translocation was determined by IF staining. C and D, The quantitative representation of IF data in the HPr-1-AR and CWR-R1 cells, respectively. The mean red intensity in the nucleus of individual cells from 11–15 different fields was computed using ImageJ. Experiments were done three times and statistical analysis was done using the one-way ANOVA test. (*, P < 0.05; **, P < 0.01).
AR nuclear translocation, with ABCG2 inhibition, upregulates expression of AR target genes
To determine whether elevated nuclear AR expression leads to increased AR transcriptional activity, the expression of AR target genes was quantitated by qRT-PCR. To determine the change in the expression of AR target genes upon inhibiting the ABCG2-mediated androgen efflux, ABCG2-expressing HPr-1-AR and CWR-R-1 cells were incubated with either the vehicle control, DHT, Ko143 or DHT in combination with Ko143 for 24 hours. The AR antagonist, enzalutamide, was added to evaluate the role of AR in the upregulation of AR target genes. Expression of AR target genes was analyzed by qRT-PCR. Different genes were quantitated in the HPr-1-AR (MME, GHRL1, AQP3, and TMEM) and the CWR-R1 cells (TIPARP, MME, FKBP5, and GADD45B) as AR targets different sets of genes in the two cell lines (18, 19). qRT-PCR analysis revealed that both cell lines treated with a combination of DHT and Ko143 showed a significant increase in the expression of AR target genes compared with cells treated with the vehicle control and DHT (Fig. 2) and that enzalutamide antagonized the ABCG2-mediated upregulation of AR target genes (Fig. 2). Although the AR target gene expression showed a maximal increase upon treatment with a combination of DHT and Ko143, we observed an upregulation of AR target genes upon treatment with DHT alone (Fig. 2); this could be due to the incomplete efflux of DHT by ABCG2 due to saturation of the transporters with DHT. In addition, we did not observe any change/downregulation in the AR target gene expression upon treatment with enzalutamide (Fig. 2), possibly due to the low levels of nuclear AR in the ABCG2-expressing cells (Fig. 1). Our results confirm that inhibiting the ABCG2-mediated androgen efflux leads to an upregulation of AR target genes and suppression of AR target gene upregulation by enzalutamide confirms the direct role of AR in upregulating the AR target genes when ABCG2-mediated androgen efflux is inhibited.
Inhibiting the ABCG2-mediated androgen efflux increases the expression of AR target genes. ABCG2-expressing HPr-1-AR (A–D) and CWR-R1 (E–H) cells obtained by magnetic sorting were treated with either vehicle control, DHT, DHT+ 1 μmol/L Ko143 or 1 μmol/L Ko143 for 24 hours. Enzalutamide at 1 μmol/L concentration was added to confirm the role of AR. m-RNA was extracted and reverse transcribed to cDNA. The expression of AR target genes in both the cell lines was evaluated by qRT- PCR. All experiments were done three times in triplicates and statistical analysis was done using the two-way ANOVA test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Inhibiting the ABCG2-mediated androgen efflux leads to a delayed cell growth response and cell differentiation mediated by AR
Exogenous introduction of AR in cells that lack endogenous AR leads to a delayed cell growth response and cell differentiation (14, 16, 17). Thus, we propose that increased nuclear AR expression in the HPr-1-AR and the CWR-R1 cells, upon inhibiting the ABCG2-mediated androgen efflux, would lead to a delayed cell growth response due to increased luminal differentiation of cells. Cell growth response was assessed in the ABCG2-expressing HPr-1-AR and CWR-R1 cells treated with increasing concentrations of DHT with/without Ko143, by the sphere-forming assay, which serves as a direct indicator of cell growth response of a single sphere-forming cell in a 3D setting. In addition, the sphere-forming assay can also serve as an indicator of the relative number of stem and differentiated cells in tumor samples. While the proliferative stem cells possess the ability to multiply and form spheres, the differentiated cells are unable to grow as spheres. The sphere-forming efficiency was computed by measuring the sphere sizes and assessing sphere number. Sphere number serves as a direct indicator of the proliferative potential of cells capable of forming spheres, whereas a change in sphere size indicates the eventual loss/gain of proliferative potential thus leading to smaller/larger spheres, respectively. We observed that in both, the HPr-1-AR and the CWR-R1 cells, inhibiting the ABCG2-mediated androgen efflux, significantly impaired the sphere formation efficiency as evidenced by a decrease in sphere size and sphere number (Fig. 3A, B, E, and F)). Enzalutamide rescued the impaired sphere formation efficiency of the ABCG2-expressing HPr-1-AR and CWR-R1 cells, thus confirming that the delayed cell growth response upon inhibiting the ABCG2-mediated androgen efflux is modulated by AR (Fig. 3C, D, G, H).
Inhibiting the androgen efflux by ABCG2 leads to an AR-modulated delayed cell growth response and cell differentiation to a luminal phenotype. ABCG2-expressing HPr-1-AR (A–D) and CWR-R1 cells (E–H) were plated onto 24-well ultralow adhesion plates coated with Matrigel at a density of 2,000 cells per well for sphere-forming assay. Cells were treated either with different combinations of DHT, Ko143, and enzalutamide. Sphere sizes were measured (A, C, E, G) and spheres were counted between 10 and 14 days (B, D, F, H). Experiments were done three times and spheres from 4–6 wells were counted each time. Statistical analysis was done using the two-way ANOVA test (**, P <0.01; ***, P < 0.001; ****, P < 0.0001). I and J, Cell differentiation was assessed by subjecting the cell lysates obtained from DHT-, Ko143-, and enzalutamide-treated cells to immunoblot analysis and determining the expression levels of luminal markers.
Previous studies have shown that the exogenous introduction of AR in PSCs drive differentiation to a luminal phenotype (16). To investigate whether inhibiting the ABCG2-mediated androgen efflux leads to an AR-modulated cell differentiation, ABCG2-expressing cells, treated with different combinations of DHT, Ko143, and enzalutamide for 5 days were analyzed for the expression of differentiation and stem cell markers. We observed that ABCG2-expressing cells treated with the vehicle control lacked the expression of prostate luminal markers. However, DHT in combination with Ko143, increased the expression levels of the prostate luminal markers, CK8 and CK18, in both the cell lines (Fig. 3I and J). In the ABCG2-expressing HPr-1-AR cells, treatment with a combination of DHT and Ko143 led to an upregulation of PSA and the downregulation of Sox2 (stem cell marker; Fig. 3I), whereas in the ABCG2-expressing CWR-R1 cells, treatment with a combination of DHT and Ko143 led to an increase in AR and phosphorylated p21 (cell-cycle arrest marker) expression (Fig. 3J). Different markers were analyzed in both the cell lines as (i) we could not detect phosphorylated p21 in the ABCG2-expressing HPr-1-AR cells and Sox2 in the ABCG2-expressing CWR-R1 cells (data not shown) (ii) In the CWR-R1 cells, PSA expression is not altered by DHT (19) and (iii) HPr-1-AR cells have a stably transfected AR vector, such that AR is cytosolic in the absence of androgens and nuclear upon treatment with androgens. As the analysis was done on whole-cell lysates, the intracellular AR expression in the ABCG2-expressing HPr-1AR cells would remain unchanged. Of note, the CWR-R1 cells are known to have both, mutated AR as well as AR variants (Supplementary Fig. S3B; ref. 20). However, we did not observe AR variants in the ABCG2-expressing cells upon treatment with DHT and Ko143 (Fig. 3J). The reason for this observation could be that AR variants are generated in response to ADT; however, PSCs are intrinsically resistant to ADT (21, 22).
Interestingly, high levels of CK8 and CK18 were also observed in the Ko143-treated ABCG2-expressing CWR-R1 cells (Fig. 3J). However, the Ko143-treated CWR-R1 cells showed the absence of phosphorylated p21 (p-p21) expression (Fig. 3J). The ABCG2-expressing CWR-R1 cells were cultured in FBS-supplemented media because growth in charcoal-stripped FBS-supplemented media leads to the downregulation of ABCG2-expressing CWR-R1 cells. The HPr-1-AR cells are routinely cultured in a serum-free media, thus obviating the need for charcoal-stripped serum. Serum contains a wide range of steroids such as testosterone, progesterone, and cholesterol. Furthermore, prostate cells can synthesize DHT from the steroids through the intact, cholesterol, and the backdoor pathway (23). Pump inhibition with Ko143 could have trapped the steroidal androgens from the serum in the cells thus eliciting a weak AR response. Thus, inhibiting ABCG2, albeit in the absence of exogenous DHT, was sufficient to induce a very weak AR expression and consequently upregulate CK8 and CK18 but not p-p21 (Fig. 3J). The immunoblot results suggest that steroidal androgens from serum can partially induce the expression of some luminal differentiation markers but not induce cell growth arrest. These results are in accordance with the observations from the sphere-forming assay where Ko143-treated ABCG2-expressing CWR-R1 cells showed no change in the sphere formation efficiency compared with cells treated with the vehicle control (Fig. 3E and F). The role of AR in the forced differentiation of ABCG2-expressing cells was confirmed by enzalutamide (Fig. 3I and J). Thus, our results suggest that inhibiting the ABCG2-mediated androgen efflux forces prostate cells to undergo AR-regulated luminal differentiation.
Inhibiting ABCG2 leads to delayed tumor progression and increased overall survival in vivo
On the basis of our in vitro observations, we proposed that inhibiting ABCG2 in vivo would slow tumor progression and increase the overall survival in mice. To determine whether Ko143 slows tumor progression, tumor volume was monitored in testosterone-supplemented nude mice subcutaneously implanted with ABCG2-expressing CWR-R1 cells treated with either placebo or 15 mg/kg Ko143. Survival was computed by the time it took for tumors to reach a volume of 1,500 mm3. Our results show that mice treated with Ko143 showed significantly slower tumor growth rates. Linear mixed model analysis that took the individual tumor volume trajectories into account revealed statistically significant differences between the two cohorts (P < 0.01; Fig. 4A–C).
Inhibiting the ABCG2 transporters by Ko143 slows tumor progression and increases the overall survival in vivo. Tumor trajectories of individual mice in the control (n = 10; A) and Ko143-treated group (n = 10; B), respectively. The black arrows represent day 0, when the treatments were started. C, The average tumor growth rate of mice in the control and Ko143-treated group. Statistical analysis was done using the Linear mixed modeling. D, The overall survival of mice in both the groups as computed by the time taken for the tumors to reach a volume of 1,500 mm3. Difference in the slopes of placebo and Ko143-treated animals was computed using the log-rank test.
In addition to slowing down tumor progression, mice treated with Ko143 demonstrated a trend for increased survival. The median survival in the control group was 56 days as opposed to 70 days in the treated cohort. We observed a strong tendency of Ko143 to increase the overall survival; however, the log-rank analysis revealed that the differences were not statistically significant (Fig. 4D).
Ko143 disrupts the sphere-forming ability of cells isolated from tumor xenografts
Results from our in vivo data show that Ko143 slows the tumor growth rate. Slower tumor growth rates are a direct indicator of impaired cellular proliferation. To confirm the results of our in vivo observation that Ko143 leads to a delayed cell response, we isolated cells from the tumors of mice by protease digestion and subjected the isolated cells to sphere-formation assay. Sphere formation assay revealed that cells isolated from tumors of Ko143-treated group formed significantly smaller and fewer spheres (Fig. 5). On the basis of our sphere formation data, we can infer that the tumors of mice treated with Ko143 had a significantly higher percentage of differentiated cells, which are unable to grow into spheres, whereas tumors of mice in the placebo group had comparatively greater number of stem cells.
Cells isolated from tumors of mice in the treated group showed impaired sphere formation. Cells were isolated from tumors of mice in the control (n = 3) and Ko143 group (n = 3) by protease digestion and plated in 24-well ultralow attachment plates coated with Matrigel at a density of 4,000 cells per well. On day 14, spheres were imaged under 20× magnification (A), sphere sizes were measured by the basic SPOT software (B), and spheres were counted (C). The Student t test was used for statistical analysis (**, P < 0.01; ****, P < 0.0001).
Mice treated with Ko143 have a higher percentage of luminal cells and a lower percentage of ABCG2-expressing cells in tumors
On the basis of our observations from in vitro experiments, showing that the inhibition of the ABCG2-mediated androgen efflux forces prostate stem cells to differentiate to a luminal phenotype, we hypothesized that Ko143-treated mice would have more differentiated tumors compared with mice from the control group. Luminal differentiation was assessed by immunostaining the paraffin-embedded tumor sections for CK8 and AR. We observed that tumors from the Ko143-treated group had a higher percentage of CK8-expressing cells (∼86.4% in the Ko143 group vs. ∼63.75% in the control group) and AR-expressing cells (∼74% in the Ko143 group vs. ∼57% in the control group) compared with the control group (Fig. 6A–D). Also, we observed a general overlap between CK8- and AR-expressing cells. In addition, tumor sections from the Ko143 cohort showed a 2.5-fold reduction in the number of ABCG2-expressing cells compared with tumors from the control group. In the control group, ∼87.5% of the tumors contained at least one foci (areas with ≥1 ABCG2-expressing cell) of ABCG2-expressing cells as opposed to only 55.5% of the tumors from the treated cohort (Fig. 6E and F). Our results thus suggest that the Ko143-treated group had more differentiated tumors compared with the control group as evidenced by an increased percentage of luminal and decreased percentage of ABCG2-expressing cells. The reduction in tumor growth rate and increased survival in the treated group could be attributed to increased differentiation.
IHC analyses of the paraffin-embedded tumor sections. Tumors from control (n = 8) and Ko143 (n = 9) treated mice were dissected upon autopsy and a part of the tumors were subjected to paraffin embedding. The paraffin-embedded sections were immunostained with AR (A and B), CK8 (C and D), and ABCG2 (E and F) and imaged under 40× magnification. Analysis was done in one section of the tumor from each animal.
Elucidating the mechanisms by which stem cells maintain their undifferentiated phenotype is important to improve the efficacy of cancer therapy due to their role in the etiology of cancer and their intrinsic resistance to conventional therapies. Although, ABCG2 is suspected to play an important role in stem cell maintenance, the precise mechanism is unknown. Our study dissects the mechanism by which ABCG2 protects PSCs from undergoing differentiation. Using the HPr-1-AR and CWR-R1 cell lines, we have shown that ABCG2 maintains PSCs in an undifferentiated state by effluxing androgens. Inhibiting the ABCG2-mediated androgen efflux leads to an AR-modulated luminal differentiation and a delayed cell growth response (Fig. 7). Our observations are consistent with previous reports, which show that inducing AR expression in PSCs leads to a delayed cell growth response and luminal differentiation rather than cell proliferation (14, 16). In accordance with our in vitro observations, we observed that Ko143 treatment reduced the tumor growth rate, increased survival, and led to more differentiated tumors in mice transplanted with ABCG2-expressing CWR-R1 cells. Most of the studies on ABCG2 and its relevance to stem cells have shown that ABCG2 maintains the stem cell phenotype either by effluxing cytotoxic substrates or by acting as a downstream effector of stem cell–specific pathways such as the Wnt, Notch, and Hedgehog pathways (2, 24, 25). This is the first study to show that ABCG2 plays a role in stem cell maintenance by effluxing differentiation-inducing substrates. In addition, our study provides a plausible reason for the lack of AR expression in PSCs. Previous studies show that PSCs have low AR levels due to the methylation of the AR promoter CpG islands (14). However, we detected AR transcripts in ABCG2-expressing HPr-1-AR and CWR-R1 cells (data not shown). Previous reports have confirmed the presence of AR transcripts and shown that AR is unstable and degraded in the absence of androgens in PSCs (26, 27). Our study suggests that androgen efflux by ABCG2 may facilitate AR degradation, whereas inhibiting the ABCG2-mediated androgen efflux may lead to stabilization and the nuclear translocation of AR in the PSCs.
Schematic representation of our proposed model. ADT targets only the AR+ve cancer cells, whereas a small population of AR−ve prostate cancer stem cells survives and over time proliferates to give rise to CRPC. Combining differentiation therapy with ADT would in theory target the AR−ve stem cell as well as the AR+ve cancer cell population. In our model, we show that the ABCG2 transporters efflux androgens. Inhibiting this ABCG2-mediated androgen efflux would induce the nuclear translocation of AR and consequently force the prostate cells to differentiate to an ADT-sensitive luminal phenotype. Subjecting the tumor to ADT after the introduction of Ko143 would then target both, the pre-existing cancer cells as well as the newly differentiated prostate cancer stem cells.
The important role played by prostate cancer stem cells in the etiology of CRPC have sparked a lot of interest in differentiation therapy as an adjuvant to ADT as reviewed in ref. 28. However, initial attempts at applying differentiation therapy in prostate cancer management failed to yield significant results (29, 30). The failure of differentiation therapy could be attributed to the focus on bulk tumor response, not the prostate cancer stem cell population. In addition, most of the studies evaluated differentiation therapy in the LnCaP cell line, which is primarily comprised of luminal-differentiated cells (31, 32). In contrast, our study was conducted in the ABCG2-enriched undifferentiated subpopulation derived from both normal and tumorigenic prostate cell lines. Future studies will test the direct role of ABCG2 in delaying the progression to CRPC in mouse models such as such as the CWR22 and LuCaP35 xenografts. These models can progress from an androgen-dependent to an androgen-independent phenotype, thus mimicking CRPC disease progression in human.
As we could successfully induce luminal differentiation in the HPr-1-AR and CWR-R1 cell lines and CWR-R1 xenografts, our approach could be applied in the clinical setting, in both hormone naïve as well as castration recurrent scenario. Our proposed model relies on restored AR activity to induce differentiation, while this seems counterintuitive to the current standard of care, which focuses on inhibiting AR activity, we intend to highlight the dual role of AR, in inducing differentiation and growth retardation in PSCs and inducing proliferation in AR-expressing cancer cells. Ko143 is proposed to restore AR activity and induce luminal differentiation only in the small population of ABCG2-expressing PSCs, while the bulk of the ABCG2-negative cancer cells will not be affected. In addition, we demonstrated that inhibiting the ABCG2-mediated androgen efflux in the CWR-R1 cells increases only the full-length AR expression (which subsequently causes luminal differentiation) despite the presence of AR variants in the ABCG2-negative cell population (Supplementary Fig. S3). Blocking AR activity after Ko143 administration is proposed to target the newly differentiated stem cells as well as the pre-existing AR-expressing cancer cells (Fig. 7). The presence of androgens is important for this approach. Hence, in the hormone-naïve setting, Ko143 could be administered during intermittent ADT, when the androgen sensitivity is restored, to delay the progression to CRPC or prior to enzalutamide in the CRPC setting, when androgens are synthesized by intratumoral steroidogenesis to improve survival. However, further investigation is needed to fully understand the significance of targeting ABCG2 for differentiation therapy. Although ABCG2 expression enriches for PSCs, it is likely that a subpopulation of stem cells lack ABCG2 expression, thus it is important to evaluate the percentage of ABCG2-negative stem cells in the prostate and assess their contribution to the etiology of CRPC. In addition, it would be interesting to study the expression profile of ABCG2 as prostate cancer progresses from hormone naïve to CRPC and the effects of ADT and AR antagonists on ABCG2 expression.
The initial discovery of ABCG2 and its role in multidrug resistance sparked a lot of interest in the ABC transporters. However, the subsequent lack of clinical benefit and neurotoxicity associated with ABCG2 inhibitors led to a lot of disappointment (33). Despite the lack of clinical benefit associated with first- and second-generation ABCG2 inhibitors, Ko143 has shown immense potential in preclinical studies. In addition to being the most specific ABCG2 inhibitor to date, it is also well tolerated in mice at extremely high dosages (34) and for a long period of time, as confirmed by our study. Most of the studies conducted on clinically targeting ABCG2 have focused on targeting drug resistance by administering ABCG2 inhibitors in combination with chemotherapeutic drugs to increase the bioavailability of drugs (35–37). However, combination therapy is associated with increased toxicity as ABCG2 is expressed in organs that influence drug excretion. We propose a novel use of Ko143 in differentiation therapy, which entails administering Ko143 as a single agent in the presence of endogenous androgens, thus potentially minimizing the toxicity associated with combination therapy. In conclusion, we provide a strategy to induce luminal differentiation in the PSCs by targeting the ABCG2 transporters and sensitize them to ADT.
Conception and design: N.G. Sabnis, M.A. Titus, W.J. Huss
Development of methodology: N.G. Sabnis, M.A. Titus, W.J. Huss
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N.G. Sabnis, W.J. Huss
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N.G. Sabnis, A. Miller, M.A. Titus, W.J. Huss
Writing, review, and/or revision of the manuscript: N.G. Sabnis, A. Miller, M.A. Titus, W.J. Huss
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): N.G. Sabnis
Study supervision: N.G. Sabnis, W.J. Huss
This study was supported by the NIHRO1CA095367 (to W.J. Huss), Pharmacology & Therapeutics Departmental Funding (to W.J. Huss), Mark Diamond Research Fund (to N.G. Sabnis) and the NCI Cancer Center Support Grant (P30CA016056) to RPCI supported Mouse Tumor Model Resource and Laboratory Animal Resources.
We thank Drs. Barbara Foster, Michael Moser, James Mohler, Kalyan Gangavarapu, Mugdha Samant, and Vineet Dhiman for insightful discussions about the in vivo experiments, sphere formation assays, and cell line. We thank Ellen Karasik and Bryan Gillard for their technical assistance with the in vivo experiments. We thank the following cores at RPCI supported by NCI Cancer Center Support Grant: Laboratory Animal Resources (LAR) Bioinformatics and Biostatistics and the Mouse Tumor Model Resource (MTMR) Core.
Revision received October 10, 2016.
Accepted October 19, 2016.
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You are going to email the following The Efflux Transporter ABCG2 Maintains Prostate Stem Cells
Mol Cancer Res February 1 2017 (15) (2) 128-140; DOI: 10.1158/1541-7786.MCR-16-0270-T


text:
from django.core import mail
from django.core.urlresolvers import resolve
from django.template.loader import render_to_string
from django.test import TestCase
from django.utils.html import escape

from blog.views import (about, about_site, category, contact, index, show_post)
from .factories import *

class HomePageTest(TestCase):

    def test_root_url_resolves_to_correct_view(self):
        found = resolve('/')
        self.assertEqual(found.func, index)

    def test_uses_index_template(self):
        response = self.client.get('/')
        self.assertTemplateUsed(response, 'blog/index.html')

    def test_home_page_returns_correct_html(self):
        response = self.client.get('/')
        expected_html = render_to_string('blog/index.html')
        self.assertEqual(response.content.decode(), expected_html)

    def test_home_page_displays_post_title(self):
        published = PublishedPostFactory.create()

        response = self.client.get('/')

        self.assertContains(response, "Published Post")

    def test_home_page_displays_only_published_items(self):
        unpublished = UnPublishedPostFactory.create()
        published = PublishedPostFactory.create()

        response = self.client.get('/')

        self.assertContains(response, "Published Post")
        self.assertNotContains(response, "My Unpublished Post")

class PaginationTest(HomePageTest):
    """Pagination Tests for the Index Page"""

    def test_page_number_not_an_integer_redirects_to_home_page(self):
        published = PublishedPostFactory.create()

        response = self.client.get('/?page=index')

        self.assertEqual(response.status_code, 200)
        self.assertContains(response, "Published Post")

    def test_page_number_out_of_range_redirects_to_last_page(self):
        PostFactory.reset_sequence()
        posts = PostFactory.create_batch(10, published=True, content='content')

        response = self.client.get('/?page=999')

        self.assertEqual(response.status_code, 200)
        self.assertContains(response, posts[0].title)
        self.assertNotContains(response, posts[9].title)

    def test_index_page_displays_next_page_link(self):
        PostFactory.reset_sequence()
        posts = PostFactory.create_batch(
            10, published=True, content='some content')

        response = self.client.get('/')

        self.assertContains(response, 'Older Posts')

    def test_next_page_link_not_displayed_if_not_enough_posts(self):
        published = PublishedPostFactory.create()

        response = self.client.get('/')

        self.assertNotContains(response, 'Older Posts')

    def test_previous_page_link_shown_if_enough_posts(self):
        PostFactory.reset_sequence()
        posts = PostFactory.create_batch(
            10, published=True, content='some content')

        response = self.client.get('/?page=2')

        self.assertContains(response, 'Newer Posts')

class CategoryTest(TestCase):

    def test_category_view_resolves_to_correct_view(self):
        cat = resolve('/category/kevin/')
        self.assertEqual(cat.func, category)

    def test_uses_category_template(self):
        response = self.client.get('/category/abcde/')
        self.assertTemplateUsed(response, 'blog/category.html')

    def test_category_view_returns_correct_html(self):
        response = self.client.get('/category/abcde/')
        expected_html = render_to_string('blog/category.html')
        self.assertEqual(response.content.decode(), expected_html)

    def test_category_view_displays_only_published_items(self):
        test_category = CategoryFactory(name='test category')
        unpublished = UnPublishedPostFactory.create(category=test_category)
        published = PublishedPostFactory.create(category=test_category)

        response = self.client.get('/category/%s/' % test_category.slug)

        self.assertContains(response, "Published Post")
        self.assertNotContains(response, "My Unpublished Post")

    def test_invalid_category_slug(self):
        response = self.client.get('/category/does-not-exist/')

        self.assertContains(response, 'Error')
        self.assertContains(response, 'The specified category does not exist!')

    def test_empty_category(self):
        test_category = CategoryFactory(name='test category')

        response = self.client.get(
            '/category/{slug}/'.format(slug=test_category.slug))

        self.assertContains(
            response, 'Nothing has been posted in this category yet.')

class CategoryPaginationTest(CategoryTest):
    """Pagination Tests for the Category View"""

    def test_page_number_not_an_integer_redirects_to_category_view(self):
        cat = CategoryFactory()
        published = PublishedPostFactory.create(category=cat)

        response = self.client.get(
            '/category/{slug}/?page=ab'.format(slug=cat.slug))

        self.assertEqual(response.status_code, 200)
        self.assertContains(response, "Published Post")

    def test_page_number_out_of_range_redirects_to_last_page(self):
        cat = CategoryFactory()
        PostFactory.reset_sequence()
        posts = PostFactory.create_batch(
            10, published=True, content='content', category=cat)

        response = self.client.get(
            '/category/{slug}/?page=999'.format(slug=cat.slug))

        self.assertEqual(response.status_code, 200)
        self.assertContains(response, posts[0].title)
        self.assertNotContains(response, posts[9].title)

    def test_category_view_displays_next_page_link(self):
        cat = CategoryFactory(name='test')
        PostFactory.reset_sequence()
        posts = PostFactory.create_batch(
            10, published=True, content='some content', category=cat)

        response = self.client.get(
            '/category/{slug}/'.format(slug=cat.slug))

        self.assertContains(response, 'Older Posts')

    def test_next_page_link_not_displayed_if_not_enough_posts(self):
        cat = CategoryFactory()
        published = PublishedPostFactory.create(category=cat)

        response = self.client.get('/category/{slug}/'.format(slug=cat.slug))

        self.assertNotContains(response, 'Older Posts')

    def test_previous_page_link_shown_if_enough_posts(self):
        cat = CategoryFactory()
        PostFactory.reset_sequence()
        posts = PostFactory.create_batch(
            10, published=True, content='some content', category=cat)

        response = self.client.get(
            '/category/{slug}/?page=2'.format(slug=cat.slug))

        self.assertContains(response, 'Newer Posts')

class ContactViewTest(TestCase):
    """Tests the Contact View"""

    def post_invalid_input(self):
        return self.client.post(
            '/contact/',
            data={
            'name': '',
            'email': 'me@gmail.com',
            'message': 'The name is Bond, James Bond.'
            }
        )

    def post_valid_input(self):
        return self.client.post(
            '/contact/',
            data={
            'name': 'Blog Fan',
            'email': 'myemail@email.com',
            'message': 'I am your biggest fan',
                }
            )

    def test_contact_view_resolves_to_correct_view(self):
        cat = resolve('/contact/')
        self.assertEqual(cat.func, contact)

    def test_uses_contact_template(self):
        response = self.client.get('/contact/')
        self.assertTemplateUsed(response, 'blog/contact.html')

    def test_contact_form_has_csrf_token(self):
        response = self.client.get('/contact/')

        self.assertIn('csrfmiddlewaretoken', response.content.decode())

    def test_mail_sent_on_successful_POST_request(self):
        self.post_valid_input()

        self.assertEqual(len(mail.outbox), 1)
        self.assertIn(
            '[kevgathuku] New Message', mail.outbox[0].subject)

    def test_correct_reponse_on_successful_POST_request(self):
        resp = self.post_valid_input()

        self.assertJSONEqual(resp.content.decode(), {'success': '1'})

    def test_correct_response_on_invalid_POST_rquest(self):
        resp = self.post_invalid_input()

        self.assertEqual(resp.status_code, 403)
        self.assertEqual(
            resp.reason_phrase, 'Make sure all fields are entered and valid.')

    def test_raises_403_on_invalid_headers(self):
        resp = self.client.post(
            '/contact/',
            data={
            'name': 'Blog Fan',
            'email': 'myemail\n@email.com',
            'message': 'I am your biggest fan',
                }
            )

        self.assertEqual(resp.status_code, 403)
        self.assertEqual(
            resp.reason_phrase, 'Invalid header found.')

class ShowPostTest(TestCase):
    """Test for individual post view"""

    def test_post_url_resolves_to_correct_view(self):
        found = resolve('/post/new/')
        self.assertEqual(found.func, show_post)

    def test_uses_post_template(self):
        published = PublishedPostFactory.create()

        response = self.client.get('/post/{}/'.format(published.slug))
        self.assertTemplateUsed(response, 'blog/post.html')

    def test_show_post_displays_post_content(self):
        published = PublishedPostFactory.create()

        response = self.client.get('/post/{}/'.format(published.slug))

        self.assertContains(response, "Published Post")

    def test_resolves_only_published_posts(self):
        unpublished = UnPublishedPostFactory.create()
        published = PublishedPostFactory.create()

        response = self.client.get('/post/{}/'.format(published.slug))
        bad_response = self.client.get('/post/{}/'.format(unpublished.slug))

        self.assertEqual(response.status_code, 200)
        self.assertEqual(bad_response.status_code, 404)

class AboutMeTest(TestCase):
    """Test for About me view"""

    def test_about_url_resolves_to_correct_view(self):
        found = resolve('/about/')
        self.assertEqual(found.func, about)

    def test_uses_correct_template(self):
        response = self.client.get('/about/')
        self.assertTemplateUsed(response, 'blog/about.html')

    def test_about_me_page_returns_correct_html(self):
        response = self.client.get('/about/')
        expected_html = render_to_string('blog/about.html')
        self.assertEqual(response.content.decode(), expected_html)

class AboutSiteTest(TestCase):
    """Test for About Site view"""

    def test_about_site_url_resolves_to_correct_view(self):
        found = resolve('/about-this-site/')
        self.assertEqual(found.func, about_site)

    def test_uses_correct_template(self):
        response = self.client.get('/about-this-site/')
        self.assertTemplateUsed(response, 'blog/about-this-site.html')

    def test_about_site_page_returns_correct_html(self):
        response = self.client.get('/about-this-site/')
        expected_html = render_to_string('blog/about-this-site.html')
        self.assertEqual(response.content.decode(), expected_html)

redpajama_set_name: RedPajamaGithub


text:
CycT1 protein was undetectable in freshly isolated monocytes and
CycT1 protein was undetectable in freshly isolated monocytes and induced in monocyte-differentiated macrophages, while the expression
of CDK9 remained constant. Transient expression of CycT1 in undifferentiated monocytes could not rescue Tat transactivation, Smad inhibitor suggesting that CycT1 is not the only limiting factor of HIV-1 infection in monocytes. Furthermore, monocyte differentiation into macrophages appeared to enhance the phosphorylation of CDK9, which correlated with significantly increased HIV-1 infection in macrophages. Our results provide new insights into HIV-1 infection and regulation in primary monocytes and viral pathogenesis."
"Intracellular clearance of toxic protein aggregates represents a promising therapeutic approach to treat protein-misfolding diseases such as Parkinson's and Huntington's diseases. Intracelluarly expressed
antibody fragments or intrabodies can be used to bind specific intracellular targets. Addition of a non-traditional secretion signal sequence enables the intrabody to first bind its target inside the cell and then shuttle the bound target through learn more the cell membrane, secreting it from the cell. We intracellularly expressed two different single chain antibody (scFv) fragments targeting either monomeric or oligomeric alpha-synuclein (a-syn), in a mammalian cell model that overexpresses a-syn. Two versions of each intrabody were studied, one with and one without the Levetiracetam non-traditional secretion signal. The scFv targeting monomeric a-syn provided little or no reduction in toxicity induced by overexpression of a-syn, however binding and secretion of oligomeric a-syn totally reduced toxicity. Non-traditional
intrabody secretion therefore represents an effective method to target and clear a variety of harmful intracellular species. (C) 2009 Elsevier Ireland Ltd. All rights reserved."
"The bacillus Calmette-Guerin ( BCG) strain of Mycobacterium bovis is used in many parts of the world as a vaccine against Mycobacterium tuberculosis. Some epidemiological evidence has suggested that BCG immunization may have unpredicted effects on resistance to other pathogens. We show here in a mouse model that BCG immunization followed by antibiotic treatment to clear the host of the pathogen rendered three strains of mice partially resistant to infection with vaccinia virus (VV) but not to lymphocytic choriomeningitis virus (LCMV). VV-challenged BCG-immune mice developed a striking splenomegaly and elevated CD4 and CD8 T-cell responses by 6 days postinfection (p.i.). However, resistance to VV infection could be seen as early as 1 to 2 days p.i. and was lost after antibody depletion of CD4 T-cell populations. BCG-but not LCMV-immune memory phenotype CD4 T cells preferentially produced gamma interferon (IFN-gamma) in vivo after VV challenge.

redpajama_set_name: RedPajamaCommonCrawl


Postpone income until 2017 and accelerate deductions into 2016 to lower your 2016 tax bill. This strategy may enable you to claim larger deductions, credits, and other tax breaks for 2016 that are phased out over varying levels of adjusted gross income (AGI). These include child tax credits, higher education tax credits, and deductions for student loan interest. Postponing income also is desirable for those taxpayers who anticipate being in a lower tax bracket next year due to changed financial circumstances. Note, however, that in some cases, it may pay to actually accelerate income into 2016. For example, this may be the case where a person's marginal tax rate is much lower this year than it will be next year or where lower income in 2017 will result in a higher 2017 tax credit for an individual who plans to purchase health insurance on a health exchange and is eligible for a premium assistance credit.
Take required minimum distributions (RMDs) from your IRA or 401(k) plan (or other employer-sponsored retirement plan). RMDs from IRAs must begin by April 1 of the year following the year you reach age 70-½. That start date also applies to company plans, but non-5% company owners who continue working may defer RMDs until April 1 following the year they retire. Failure to take a required withdrawal can result in a penalty of 50% of the amount of the RMD not withdrawn. Although RMDs must begin no later than April 1 following the year in which the IRA owner attains age 70-½, the first distribution calendar year is the year in which the IRA owner attains age 70-½. Thus, if you turn age 70-½ in 2016, you can delay the first required distribution to 2017, but if you do, you will have to take a double distribution in 2017—the amount required for 2016 plus the amount required for 2017. Think twice before delaying 2016 distributions to 2017, as bunching income into 2017 might push you into a higher tax bracket or have a detrimental impact on various income tax deductions that are reduced at higher income levels. However, it could be beneficial to take both distributions in 2017 if you will be in a substantially lower bracket that year.


text:
NFL Players Now Required to Stand for Anthem
Rob Carr, Getty Images
Concluding months of drama and debate within the NFL community, NFL commissioner Roger Goodell has issued an official statement that states that players who do not stand for the national anthem will be fined by the NFL. This is not to say that football players are not permitted to protest; in Goodell's official statement, players who do not wish to stand for the national anthem are permitted to wait within their respective locker room until the anthem is fully finished.
This is the first time in NFL history that the NFL operation manual permits players to not be present for the United States' national anthem. The removal of the requirement to stay on-field goes hand in hand with the addition of the rule prohibiting kneeling during the anthem, unless the player who kneels wishes to risk a fine. Teams will have their own specific rules on how a player is punished if they do not follow these new regulations.
In 2016, Colin Kaepernick of the 49ers was the first to kneel during the national anthem in protest against the oppression of minorities, though most visibly by police. This received great backlash from many who perceived the protest as unpatriotic and called for the silent protests to be forcibly ceased. After nearly two years of negotiation, NFL team owners have voted unanimously to pass this new policy; however, this unanimous vote does not include teams who abstained.
LOS ANGELES, CA - DECEMBER 31: Several members of the San Francisco 49ers kneel during the anthem prior to the football game against Los Angeles Rams at Los Angeles Memorial Coliseum on December 31, 2017 in Los Angeles, California. (Photo by Kevork Djansezian, Getty Images)
Among those who abstained was Jed York, the CEO of the 49ers. While unable to comment on other teams who abstained from this vote, Mr. York was able to say that he wanted the 49ers to focus on "the progress aspect, not focus on the protests." In addition, the 49ers will also close concession sales during the national anthem, insisting that it is disrespectful to profit during the anthem.
However, not everyone is happy with this. The NFL Players Association was kept in the dark while team owners were having talks, and have stated that the new policy will be reviewed for inconsistencies with the union's "collective bargaining agreement." The NFL Players Association said that the new policy contradicts information they had been given about the League's ideals. What parts of this new policy will be challenged, if any, is yet to be seen.


Diagnos Inc. (TSXV:ADK), Canada's 29th largest Application software company by market cap, has dropped 0.50c (2.8%) from its last trading session of January 31 to close at 17.50c. The volume was 0.5 times the average trading volume of 655,981 shares. In the past week the shares have sunk 5.4%, the biggest weekly fall since October 18, 2016.


text:
Home Slider PSD2: Opening Up a New World of Opportunity
PSD2: Opening Up a New World of Opportunity
by internationalbanker April 19, 2018
By Matt Williamson, Global Head, Payments and Cash Management, Finastra
The full impact of Open Banking and PSD2 (The Second Payment Services Directive), which came into effect on January 13 of this year, is yet to be seen. There has been much talk about delivering greater choice to customers by exposing incumbent banks to competition from a new range of accredited service providers—but with only four out of nine banks in the United Kingdom so far complying with the legislation, there's still some way to go.
Of course, even once fully up and running, customers will still need to be convinced of the value of sharing their data with new providers. But in the same way that digital has disrupted the way products and services are bought and consumed across industries from retail to travel, we can be certain that banking and payments, too, are on the cusp of a dramatic transformation.
How can banks prepare?
It's becoming clear that while some banks are taking a proactive approach and looking to seize the opportunities presented by PSD2, others are being more cautious and focusing primarily on the compliance aspects. Adopting a "wait and see" approach to assess if anything really changes is a risky and short-sighted strategy. While it will, of course, take time for Open Banking to fully take off, it would be wrong to expect things to simply continue with business as usual over the long-term.
Banks must move beyond mere compliance and view PSD2 as an opportunity to ensure their relevance in the ecosystem. They should be looking at new, innovative ways to monetize products and services that arise as a result. They have to cultivate partnerships when appropriate so that they can continue to "own the customer relationship", while allowing customers to consume third-party products and services through their existing banking interfaces or online apps. In truth, the consumer has no interest in "who" does "what", as long as it's intuitive, seamless and secure. It is here that banks will need to do everything they can to support and reassure consumers, and ensure they are protected from "potential exploitation", as appropriate.
For example, it is predicted that the uptake of new direct payment offerings from a range of new payment service providers (PSPs) will result in a 37-percent decline in card transactions. It is also anticipated that some 20 percent of online transactions will be made by mobile devices in 2018—this will cause an onslaught of data requests into banks. Peak traffic and payment requests will potentially be while consumers are on the train to or from work, or even just past midnight on Sunday mornings (as seen in Sweden with Swish) as opposed to the traditional timings and methods. Old ways of working will effectively be "turned on their heads"—and banks need to build in the agility to adjust quickly to changing customer needs.
The banking community also needs to invert its current concern that exposing its core to third-party providers to share data will leave it at a disadvantage. In fact, these banks could also act as third-party providers themselves, benefitting from access to another bank's or provider's data.
Banks that are able to harness technologies such as artificial intelligence (AI) and predictive learning tools to deliver more personalized and tailored service to customers based on the wealth of data they share will be best positioned to succeed.
Impact across all business lines
As Open Banking takes off, the lines will start to blur—with the expectations of corporate customers rising just as fast as their retail counterparts'. The implications of PSD2 and Open Banking are far-reaching across all areas of business. The possibilities for change are almost limitless. Some examples include:
Predictive FX (foreign exchange) services and FX bulk-buying—with developments in IoT (Internet of things) and AI fueling real-time trades/spreads and catering to both manual and automated trading.
Order management that allows complete initiation services (both from small/medium-sized enterprises and corporates) as well as transformation from any format and straight-through processing.
Cash and liquidity services that include balance reporting (for the AISP, or Account Information Service Provider), sweeping/pooling services, forecasting and account reconciliation.
Information-messaging services that include requests for pay, addressing and aliases and remittance information.
Collaboration and open APIs
It is in the interests of both the incumbent banks and the new entrants and payment service providers to collaborate. For the PSP, it's about gaining access to a bank's established customer base; while for the bank, it's about delivering greater choice and flexibility to customers. It's also about having the agility to capitalize on new, possibly unforeseen opportunities and ensuring they are viewed as innovators.
Previously, banks developed everything in-house or heavily customized procured services to make them proprietary. The focus has now changed to how, working together, banks and fintechs can add value and drive change.
The requirement to be agile is not a new concept, but it's all too frequently perceived as a destination rather than a continuous journey. Growing API (application programming interface) acceptance and adoption by the banking community over the past 18 months are helping to accelerate this. And the effective use and development of API libraries will be essential in enabling all parties to tap into new revenue streams swiftly and adeptly. But a paradigm shift in culture is paramount to ensuring continual momentum.
Many of today's incumbent banks are still operating in business silos with little crossover across business lines. Along with developing APIs that allow them to connect with third parties in the outside world, they should also look at applying the same approach to bursting through these silos and creating fully integrated back offices. In doing this, they will realize their own internal value-added, which in turn can be delivered back to their client bases in the form of better service and tailor-made products.
Time to take action
We cannot underestimate the challenges banks face today in adapting to Open Banking and PSD2. The fact that, at this stage, Finastra still has some banks asking for a one-size-fits-all "PSD2 solution" demonstrates that many in the industry don't yet fully realize the far-reaching impacts of the regulation across all areas of their businesses.
Of course, timelines have been tight. The European Commission published its FAQ (frequently asked questions) on PSD2 only the day before the January 13 launch. And certain aspects of compliance, including strong customer authentication and standards for secure communication, won't now come into effect until September 2019.
But while PSD2 may be off to a slow start, and we still have some time before the full impact of the regulation will be realized, banks must adopt a mindset that allows them to make the most of the "new order", whatever that may be. A lack of initial public understanding didn't prevent innovators such as Jeff Bezos or Elon Musk from articulating and achieving their visions. The same will happen in financial services. Don't risk being left behind because the traditional ways are comfortable.
European CommissionFinastraFinTechMatt WilliamsonOpen BankingPSD2Technology
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Both taste of honey and top girls are often referred to as feminist texts. Do happy days also belong to this category? This is has for many days been a matter of debate following the recent years human exploration. In an attempt to answer this various scholars have tried to come up with various hypotheses in order to shade some light on this.
A taste of honey has also been referred to as a feminist texts because its used proverbially as it the case that taste by taste empties the honey jar. This literally means that a small drain on limited resources will finish it eventually. Like feminist texts ,this maneuvering from where the women were not even considered as existing, since some text shows that even women were not counted during censures, to a period when they can hold the presidency. Thus even without enough space to put their grievances, one can be able to manipulate the available, even if they are scarce in order to come up with whatever he or she desires.
Top girls expression as also been used metaphorically to mean beautiful ladies who can attract all sorts of men. Thus they have eyes which bore a purity of a sheen mirror which despite their reflective brilliance, they shore through their spirit and resolve. This means that a single lady of this nature can attract a lot of men who cant resist the temptation of being left behind. Like the feminists text, the top girls can be classified as in this group together with a taste of honey. The issue that remains is whether happy days also belong to this category.
As from the above explanations, its evident that feminists texts can be some form of struggle to entangle oneself or a group of disabled individuals in order to achieve a recognition beyond expectation of the many, As pertains to happy days that can bring about happiness, then there are various day that lie to this category like the Christmas day, birthday, valentines day, public holiday and many others (The New York Times, 2006).
Starting with Valentines Day as a day of happiness, it can lie to a category of feminist texts since it tries to bring into celebrations that many people view to be obvious known as love. It is a day that is being celebrated every 14th day of February. It has gained lots of fame in the entire world. One thing that makes this day look like a feminist text is that it was stated by on catholic saint known as valentine who decided to do away with the monk business and decided to give himself a chance of entertaining a wife. It did not occur into his mind that he brought such changes that were later come to be celebrated everywhere until a day was set aside for these celebrations. It started from an individual and later transformed into a global affair (Simckins, 1988).
There is also another day that has gained recognition among Christians known as the Christmas day. Its an annual event that is being celebrated world over by Christians in remembrance of the birth of Jesus Christ. It is an event that started from a small town in Nazareth and later spread to all parts of the world. This can also be equivalent to feminist texts in that it originated from a small region and later spread to other parts of the world. In most countries in the world, charismas is the most less active day in all daily activities like business and commerce. All the institutional premises are closed and almost all the activities of the industries are paralyzed during that day (Nora, 2009).
Another happy day that belongs to this category is the birthdays. These are the days that people commemorate the period in which they were born. The history of birthdays can be traced back to the period before Christianity in which the pagans believed in the existence of evil sprit that pervaded human being during birth. In order to protect a person from the evil sprits, people used to surround him and make merry. A lot of noises were being made during this period in order to scare away the evil spirits. But nowadays they have turned out to gain a lot of popularity. However in the past gusts did not bring gifts. But nowadays they have gained a lot importance in that almost every individual can be able to carry it out and in the function flowers have become popular as gifts. About hundred years ago birthday cards were introduced in England that is now popularly being used. This can be classified to the category of feminist texts in that it started as small event for particular people who never had even world recognition and becoming a world event that in a matter of time is likely to become an important event as it has been seen in Washington where there a birthday set a side for the commemoration of the birth of Washington DC.
There are other days of happiness that are being celebrated world over. These are the national public holidays. In most cases these are those days that are set aside by each country in order to celebrate either the time they gained independence or some other internationally recognized days like that of the international labor organization day. These days have a long history in which states started learning from other sates that had held such events (Brian, 2006).
As it been discussed, happy days are of the same category as a taste of honey and top girls also as feminists' texts. In the world over various scholarly arguments have been made and has shown that in essence happy days are of various categories ranging from those that are celebrated after people lost their lives like wars of independence or those which, commemorate the genocide like that of Rwanda and there are some which try to make some disastrous events to comfort the living like the world aids day.
Like feminist texts, most events that occur globally have also been attributed to the same like the growth of towns. Amid all the fanfare in these towns, the main attraction is something for more mundane, the post offices, banks, and other infrastructural facilities. Like in every year, thousands of people flock in Bethlehem for post marks, a town in which Jesus was born ,which are often stamped with special cachets like an image of the three wise men.
In terms of public holidays, it ensured that all individuals have leisure time at the same time. But they do not typically force individuals to consume more leisure in any given year. In some places like Bavaria there are 17 public holidays but workers still have 348 other days each year in which they could vary their working time to compensate for the public holiday days. So happy days are like feminist texts like a taste of honey and top girls.


text:
The tale of Glasgow's romantic link with St Valentine - and how he ended up in city's southside
One of the city's most fascinating treasures can be found in a little box - sealed with a Glasgow Kiss.
Rebecca ParkerAudience Editor
The bones of St Valentine's forearm lie in this little wooden box (Image: PA)
Maybe Glasgow isn't the first place you think of when you hear the phrase 'city of love' - but we've certainly earned the accolade thanks to a wee box hidden in the heart of the southside.
Inside the Church of Blessed John Duns Scotus in the Gorbals lies the bones of the Saint Valentine himself, in a small wooden casket.
The bones of the saint's forearm were "rediscovered" in 1999, having been kept in a cardboard box, on top of a wardrobe, for six years.
Today he's earned a far more amorous spot. When lockdown eases, you can visit and look upon the 'Corpus Valentini Martyris'.
Church of Blessed John Duns Scotus
You might be wondering, why Glasgow? And specifically, why the Gorbals?
St Valentine's remains were brought to Glasgow by Franciscan monks who were impressed by the faith of locals. His bones were originally taken to St Francis' Church in 1868 where they were contained before being moved to the former St Luke's Church on Ballater Street in 1993.
It's also become common for men to propose to their girlfriend's on Valentine's Day in the church beside the container and the statue of St Valentine.
According to Reverend Antony Collins, Blessed St John Duns Scotus Church carries the forearm of St Valentine, with the rest of him scattered across cities such as Rome, Dublin and Birmingham.
So, when you're thinking of some post-lockdown date ideas, why not take a trip to see the good old romantic saint himself? While we can't guarantee it to set the mood, it will definitely score you points if you're keen to impress your date...


Gel-ee Grippers are more dense and compact than traditional stress relievers. They retain their shap..
Milk Cow, Holstein, 2-tone Cow, Udder, Key Fob, Split Ring, Chain Link, Stress Reliever, Tension Red..
Tow truck. Complies with CPSIA. 4\" W x 1 3/4\" H x 1 5/8\" D ..
Pill - round. Complies with CPSIA. 3\" Diameter x 1 1/2\" H ..


Create fun placeholders that your guests will love with these Meal Markers Thanksgiving Treats. Simple to personalize and decorate, these easy Thanksgiving desserts double as seat assignments for your friends and family. Made of Candy Melts candy and fondant, these Thanksgiving treats are fine sitting out at room temperature. Decorate the edges of these meal markers with festive sprinkles to add a harvest touch to your candy plaques.
Reserve 1 Candy Melts disk for each plaque to make easel. Mold candy plaques 1/4 in. thick in pan cavities. Refrigerate until firm; unmold. Brush melted candy on edges and dip in sprinkles. Refrigerate until firm.
Make pilgrims. Tint melted white candy with a little orange. Mold candy faces in Candy Melting Plate cavities. Using fondant, make clothes and hair; roll out 1/8 in. thick.
For collars, cut a 1/4 x 1 1/2 in. white strip; cut out a notch in center and attach to face with melted candy.
For man's hat, tint fondant black, blue, and yellow (combine Lemon Yellow and Golden Yellow for yellow shown). Cut a black strip 1 x 5/8 in., tapering to 1/2 in. wide at top. Attach to face with candy.
For hatband, cut a 1/8 in. blue strip; trim to fit and attach to hat with damp brush.
For brim, cut a 1/4 x 1 1/4 in. black strip. Attach to face with damp brush.
For buckle, hand shape a yellow log into a square; attach with damp brush.
For top of woman's bonnet, cut a 1 3/8 x 5/8 in. white strip, tapering ends to 1 1/8 in. wide toward bottom; attach to face with melted candy.
For woman's brim, cut a 1/4 x 1 1/2 in. white strip; attach with melted candy.
For hair, cut small pieces of yellow fondant; using scissors, cut slits for fringe; attach to bonnet with damp brush. Attach to plaque with melted candy.
For name, tint orange candy with red candy color. Pipe names and facial features using melted candy in cut parchment bag. Make easels using reserved Candy Melts wafers. Cut wafers in half; cut one side flat to create a right angle. Attach 2 easels to back of each plaque with melted candy; let set.


text:
// The MIT License (MIT)
//
// Copyright (c) 2021 Henk-Jan Lebbink
//
// Permission is hereby granted, free of charge, to any person obtaining a copy
// of this software and associated documentation files (the "Software"), to deal
// in the Software without restriction, including without limitation the rights
// to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
// copies of the Software, and to permit persons to whom the Software is
// furnished to do so, subject to the following conditions:
//
// The above copyright notice and this permission notice shall be included in all
// copies or substantial portions of the Software.
//
// THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
// IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
// FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE
// AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
// LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
// OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE
// SOFTWARE.

namespace IntrinsicsDude.QuickInfo
{
    using System.ComponentModel.Composition;
    using IntrinsicsDude.SyntaxHighlighting;
    using IntrinsicsDude.Tools;
    using Microsoft.VisualStudio.Language.Intellisense;
    using Microsoft.VisualStudio.Text;
    using Microsoft.VisualStudio.Text.Tagging;
    using Microsoft.VisualStudio.Utilities;

    /// <summary>
    /// Factory for quick info sources
    /// </summary>
    [Export(typeof(IQuickInfoSourceProvider))]
    [ContentType(IntrinsicsDudePackage.IntrinsicsDudeContentType)]
    [Name("Intrinsics QuickInfo Source Provider")]
    [Order(After = "default")] // order after the default quickinfo provider such the default tooltips can be removed
    internal sealed class IntrinsicsQuickInfoSourceProvider : IQuickInfoSourceProvider
    {
        [Import]
        private readonly IBufferTagAggregatorFactoryService _aggregatorFactory = null;

        public IQuickInfoSource TryCreateQuickInfoSource(ITextBuffer buffer)
        {
            IntrinsicsQuickInfoSource sc()
            {
                ITagAggregator<IntrinsicTokenTag> aggregator = IntrinsicsDudeToolsStatic.GetAggregator(buffer, this._aggregatorFactory);
                return new IntrinsicsQuickInfoSource(buffer, aggregator);
            }

            return buffer.Properties.GetOrCreateSingletonProperty(sc);
        }
    }
}


text:
IMS - Integrated Marketing Systems
A Challenge to Build Back Better
Investments in infrastructure
A historic disaster recovery program will invest $1 billion to help communities identify and develop new industries through regional growth clusters. With the goal of spurring economic recovery during the COVID-19 pandemic and building resilient regions, the U.S. Department of Commerce's Economic Development Administration opened the Build Back Better Regional Challenge in July 2021.
In late October, the Economic Development Administration announced that regions in all 50 states had submitted 529 applications for Phase 1 funding. On December 13, the Department of Commerce announced the 60 finalists that will each receive $500,000 technical assistance grants in the first phase of the program.
"These projects will help revitalize local economies and tackle our biggest challenges related to climate change, manufacturing, supply chains and more," said Alejandra Castillo, Assistant Secretary of Commerce for Economic Development, in a news release.
Those 60 coalitions will be the finalists for awards in the second phase, where 20-30 groups will each receive $25 million to $75 million (and up to $100 million!) to implement their projects. Many of the initiatives developed in Phase 1 have the potential to transform a community.
The Build Back Better program has selected 60 finalists around the US for Phase 2 funding. (Source: EDA)
The City of Tucson and the Southern Arizona Coalition for Climate Adaptation and Resilience was awarded Phase 1 funding for a "cluster to drive sustainable agriculture, preserve water resources and develop clean energy." One component of the plan calls for "expansion of infrastructure projects that promote the sustainable use of limited resources and the enablement of new climate adaptive technologies." As an example of this, the coalition cited the transformation of Tucson's landfill site into the Los Reales Sustainability Campus. A story in the Arizona Daily Star described how the city is "reimagining" its 1,200-acre landfill to include areas for economic development, recreation, more recycling, and even a tree nursery.
In California, the SoCal Tribal Accelerated Clean Energy Economy, led by the Pala Band of Mission Indians, was selected in Phase 1 for its Energy and Resilience proposal to establish a SCTACEE facility, develop a solar panel manufacturing plant, and establish tribally owned clean energy facilities. The group's goal to demonstrate wireless charging for electric vehicles would "advance this new zero-emissions transportation technology while serving tribal clean energy, resilience, and economic development needs."
In Maine, where the forest products sector represents $8 billion in annual economic benefits, a coalition was selected in Phase 1 for a project titled "Accelerating the Northern Forest Bioeconomy: Through Innovation, Commercialization, Workforce and Community Redevelopment." The plan calls for redeveloping industrial sites such as closed mills, supporting innovations for the use of biomaterials, and the development of a new Green Engineering and Materials Laboratory.
According to the coalition's proposal: "UMaine will develop a Factory of the Future facility (45k sf) to usher in a new era of AI-enabled large-scale bio-based additive manufacturing. GEM will house AI-enabled arrays of large-scale, 3D printers and sensors to manufacture and test new products from wood-derived cellulose and other advanced biomaterials, collaborate with industry, and train the next generation manufacturing workforce."
Like those three examples, many of the successful Phase 1 coalitions have proposed new facilities and infrastructure as part of their plans, and IMS expects that architectural, engineering and consulting services will be needed in various stages of these ambitious projects. (IMS covers current RFPs on a daily basis and tracks future projects through Advance Notices for its A/E/C clients.)
A wide range of infrastructure projects are being considered for Build Back Better funding. (Source: American Public Power Association)
Prior to the Phase 1 submittals, IMS covered several RFPs for professional planning and management services for the Build Back Better Regional Challenge.
* The Northeast Ohio Areawide Coordinating Agency released an RFP in November 2021 to support the Build Back Better Regional Challenge by developing conceptual plans to guide the initial planning and development of coalition projects, as well as for a public participation plan (IMS 598885).
* The West Virginia Department of Economic Development released an RFP in December 2021 for technical, consulting, and grant writing services for the US EDA ARPA Build Back Better Regional Challenge grant and other potential grants, as well as program and project development, strategic planning, and project management for procedures for funding for efforts to increase economic development opportunities (IMS 602715).
More of these RFPs may be issued as Coalitions develop their plans for Phase 2. In fact, a few days after being selected in Phase 1, the Piedmont Triad Regional Council in North Carolina released an RFQ for a consultant to provide environmental, preliminary engineering, and cost estimating services to support its next submission for the SITE Next-Gen: A Piedmont Triad Cluster Strategy (IMS 604094).
The Piedmont Triad Regional Council's plan has seven components, ranging from projects to support a National Hub for Regenerative Medicine Manufacturing to the next generation of furniture design and manufacturing. "These projects are highly feasible to complete by September 2027, and transformative to move the Triad beyond a long history of manufacturing strength to a new era of Next-Generation Manufacturing innovation focused on high-growth industries, high-paying jobs, resiliency, and equity," the Council said. "By accelerating advances in equitable innovation and bringing new products to market, the Triad will bolster its resilience and address generational racial and geographic disparities."
Key utilities, like water treatment, are among the myriad types of projects needed across the country. (Source: Ivan Bandura)
The Build Back Better Regional Challenge is the largest component of the Commerce Department's "Investing in America's Communities" program, which includes the Good Jobs Challenge ($500 million), Economic Adjustment Assistance Challenge ($500 million), Indigenous Communities Challenge ($100 million), Travel, Tourism, and Outdoor Recreation Grants ($750 million), and Statewide Planning, Research, and Networks Grants ($90 million).
This level of funding is not business as usual. "The EDA investment is the largest economic development initiative from the Department of Commerce in decades," the Department said.
IMS is tracking several federal funding programs, such as the massive investments in water and sewer infrastructure, as the country recovers from the pandemic. This initiative, however, has a range of development projects and new facilities. For communities with big ideas, the Build Back Better Regional Challenge is an opportunity to find an area of competitive advantage, jumpstart an economy, and position a region for decades to come.
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