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Nancy Butler, Above All Else, Success in Life and Business
For commercial real estate investors, a real estate investment trust (REIT) can be the right "match." A look at the basic features of REITs and the advantages and disadvantages to investors can help investors make an appropriate investment decision.
Wikipedia Definition
A REIT is a company that owns, and in most cases, operates income-producing real estate. REITs own many types of commercial real estate, ranging from office and apartment buildings to warehouses, hospitals, shopping centers, hotels and even timberlands. Some REITs also engage in financing real estate.
REITs can provide the opportunity for both small and large investors to acquire ownership in real estate ventures, and in some cases operate commercial properties.
Traded vs. Non-Traded REIT
Many REITs are registered with the SEC but are not publicly traded. These are known as non-traded REITs (also known as non-exchange traded REITs). Others are registered with the SEC and are publicly traded on a stock exchange. These are known as publicly traded REITs. This is one of the most important distinctions between these two kinds of REITs.
REITs are required to:
• Have at least 100 shareholders, no five of whom can hold more than 50% of shares between them
• Maintain dividend payout ratios of at least 90%
• Have at least 75% of gross income derived from real estate.
• Have at least 75% of a REIT's assets invested in real estate, cash or U.S. Treasuries
This can make REITs a favorite for income-seeking investors.
REIT History
REITs have existed for more than 50 years in the U.S. Congress granted legal authority to form REITs in 1960 as an amendment to the Cigar Excise Tax Extension of 1960. That year The National Association of Real Estate Investment Funds, a professional group for the promotion of REITs is founded. The following year it changed its name to the National Association of Real Estate Investment Trusts (NAREIT).
In 1965 the first REIT was listed on the N.Y. Stock Exchange (NYSE).
International REITs
In 1969 the first European REIT legislation (the Fiscal Investment Institution Regime [fiscale beleggingsinstelling:FBI]) is passed in The Netherlands.
ââ¡Ã'¬Ãƒ¢Ã¢â€š¬Ã'¹Since their development in Europe, REITs have become available in many countries outside the United States on every continent on Earth.
Three Main Kinds of REITs in the U.S.
1. Equity REITs invest in and own properties, and are responsible for the equity or value of their real estate assets. Revenues come principally from leasing space. They may sell property holdings.
2. Mortgage REITs, invest primarily in mortgages and other debt products related to real estate. They receive income from the payments borrowers make toward the mortgages the REIT owns.
3. Hybrid REITs invest in both properties and mortgages.
Individuals can invest in REITs either by purchasing shares directly on an open exchange or by investing in a mutual fund that specializes in public real estate. Some REITs are SEC-registered and public, but not listed on an exchange; others are private.
Some REITs will invest specifically in one area of real estate – shopping malls, for example – or in one specific region, state or country. Others are more diversified.
What are the benefits and risks of REITs?
Non-traded REITs, may provide diversification to an investment portfolio through investments in many types of commercial real estate properties. Additional benefits can include:
• Since Real Estate investments historically have had a low correlation to stocks and bonds, it may reduce volatility by providing asset class diversification for investors. Of course this is true for Real Estate investing in general. The additional advantage of a REIT over an individual Real Estate purchase is that for the same amount invested in a single piece of commercial Real Estate, you may able to own "a piece" of multiple properties which may help to better diversify your portfolio and therefore reduce risk.
• REITs may provide access to global real estate investments that own properties in the U.S. and abroad.
• REITs can offer investors the ability to invest in real estate without the responsibility of managing properties.
• Some REITs may offer higher dividend yields than some other investments.
Disadvantages and Risks
An investment in a non-traded REIT is subject to significant risk. Some of the risks and disadvantages include:
• Principal risk. Investments in non-traded REITs are not guaranteed
• Lack of Liquidity: Non-traded REITs are illiquid investments. They generally cannot be sold readily on the open market
• Distributions may be derived from the proceeds of the offering, from borrowings or from the sale of assets.
• Potential risk of reduced or no distributions to shareholders
• Value Transparency: It can be difficult to determine the value of a share of a non-traded REIT which can reduce the value of the shares.
Special Tax Considerations
Most REITS pay out 100% of their taxable income to their shareholders and can deduct these dividends and avoid most or all tax liabilities. The holders of a REIT are responsible for paying taxes on any dividends and capital gains they receive in connection with their investment in the REIT. Dividends paid by REITs generally are treated as ordinary income and are not entitled to the reduced tax rates on other types of corporate dividends.
Important note: Some states impose restrictive suitability standards for purchasers of Non-Traded REITs, such as requiring qualification and concentration limits using liquid net worth.
Nancy Butler, CFP, CDFA, CLTC is founder of Above All Else, Success in Life and Business, Waterford, Conn.


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Provide Automated Feedback to Customer Regarding their URL recategorization Request
Several customers have taken issue with our URL recategorization page:https://secure2.sophos.com/en-us/threat-center/reassessment-request.aspx and the fact that they receive no feedback regarding their requests. They would like to be notified when the request has been processed and information regarding if it was approved or denied. Other vendors do provide this feature.
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The fusion gene AML1-ETO is the product of t(8;21)(q22;q22), one of the most common chromosomal translocations associated with acute myeloid leukemia. To investigate the impact of AML1-ETO on hematopoiesis, tetracycline-inducible AML1-ETO-expressing cell lines were generated using myeloid cells. AML1-ETO is tightly and strongly induced upon tetracycline withdrawal. The proliferation of AML1-ETO+ cells was markedly reduced, and most of the cells eventually underwent apoptosis. RNase protection assays revealed that the amount of Bcl-2 mRNA was decreased after AML1-ETO induction. Enforced expression of Bcl-2 was able to significantly delay, but not completely overcome, AML1-ETO-induced apoptosis. Prior to the onset of apoptosis, we also studied the ability of AML1-ETO to modulate differentiation. AML1-ETO expression altered granulocytic differentiation of U937T-A/E cells. More significantly, this change of differentiation was associated with the down-regulation of CCAAT/enhancer binding protein α (C/EBPα), a key regulator of granulocytic differentiation. These observations suggest a dichotomy in the functions of AML1-ETO: (i) reduction of granulocytic differentiation correlated with decreased expression ofC/EBPα and (ii) growth arrest leading to apoptosis with decreased expression of CDK4, c-myc, andBcl-2. We predict that the preleukemic AML1-ETO+ cells must overcome AML1-ETO-induced growth arrest and apoptosis prior to fulfilling their leukemogenic potential.
Acute myeloid leukemia (AML) is a common hematopoietic malignancy characterized by the abnormal proliferation and differentiation of myeloid progenitor cells. The fusion gene AML1-ETO is the product of the t(8;21)(q22;q22) chromosomal translocation, which is associated with approximately 40% of cases with the M2 subtype of AML (8, 10, 30, 44). The chimeric protein (AML1-ETO) contains the N terminus of the AML1 (RUNX1, CBFα, PEBP2αB) transcription factor and nearly the full-length ETO (MTG8) (39, 40). The AML1 moiety of the AML1-ETO fusion protein has the Drosophila melanogasterprotein runt homology domain, which is required for DNA binding and interaction with its heterodimerization partner, CBFβ, but which lacks the transcriptional activation domain of AML1 (35, 46, 61, 68, 70). AML1 is a common target for chromosomal translocations and is involved in several other hematopoietic malignancies, such as the TEL-AML1 fusion observed in t(12;21), which is involved in approximately 25% of childhood pre-B-cell acute lymphoblastic leukemias (14, 56, 59);AML1-MTG16 from t(16;21) with AML1 fused toETO-related gene MTG16 (11); andAML1-EVI1 from t(3;21) in lymphoblast crises evolving from chronic myelogenous leukemia and in therapy-related AML (37, 42, 43). These translocations point to a fundamental role of AML1 in hematopoiesis. Furthermore, analysis of AML1knockout mice demonstrates that AML1 is a crucial factor for definitive hematopoiesis (48, 67). The ETO moiety of AML1-ETO is able to recruit the nuclear receptor corepressor (N-CoR)–mammalian Sin3 (mSin3)–histone deacetylase-1 (HDAC1) complex (13, 32, 65). ETO recruitment of the N-CoR–mSin3–HDAC1 complex leads to lower levels of histone acetylation and less-accessible chromatin. Inhibition of HDAC has been shown to decrease ETO-mediated repression, inducing partial differentiation of AML1-ETO-positive Kasumi-1 cells (66). Understanding how AML1 and its fusion proteins are involved in hematopoiesis and leukemogenesis will provide valuable insight into cell differentiation and cancer development.
Various attempts to develop a mouse model with AML1-ETO have so far failed to generate AML in adult mice. AML1-ETO heterozygous knockin mice died at the embryonic stage and displayed a block in definitive hematopoiesis (47, 75). Inducible AML1-ETO-expressing transgenic mice were generated with a tetracycline-inducible system (55). Unlike the knockin model mice, these mice are viable but fail to develop any malignancies despite strong expression of AML1-ETO in the bone marrow upon withdrawal of tetracycline. More recently, a promising murine model that mimics the t(8;21) chromosomal translocation through Cre/loxP-mediated recombination has been generated (5). However, hematopoietic studies have yet to reveal any malignancies. These results suggested that AML1-ETO expression at a particular stage of hematopoietic cell development may be critical for its leukemogenic potential and that AML1-ETO-associated leukemogenesis may also require a secondary event or "hit" for AML1-ETO-positive cells to adopt leukemogenic behavior, including block of differentiation, dysregulation of proliferation, and/or reduction of apoptotic potential. Various cell lines have been established to study AML1 and AML1-ETO proteins. AML1 has been shown to promote cell cycle progression of myeloid progenitor cell line 32Dcl3 and to transform NIH 3T3 cells (25, 62). Ectopic expression of AML1-ETO inhibits granulocytic differentiation of 32Dcl3 and L-G myeloid cell lines, monocytic differentiation of U937 cells, and erythroid differentiation of K562 and TF-1 cells (1, 13, 22, 24, 26, 71). In addition, it has been reported that AML1-ETO expression decreases proliferation by up to threefold in interleukin-3-dependent L-G cells and induces cell-cycle arrest in 32Dcl3 and Ba/F3 cell lines (22, 29). Furthermore, ectopic expression of AML1-ETO in NIH 3T3 cells has induced cell transformation (9).
Here, we report the generation of inducible AML1-ETO-expressing cell line U937T-A/E using human myeloid cells to investigate the effect of AML1-ETO on hematopoiesis. Unlike most previously reported stable cell lines that continuously express AML1-ETO (1, 24, 58), these cell lines present the advantage of not having been directly selected for AML1-ETO expression during their establishment. As such, these cell lines have not been preselected with particular features such as reduced apoptosic potential or enhanced proliferation prior to the analysis of AML1-ETO expression. In U937T-A/E cells, AML1-ETO is tightly regulated and strongly induced only upon tetracycline withdrawal at both mRNA and protein levels. Upon induction, the proliferation of AML1-ETO+ cells was severely reduced as the cells arrest in G1phase of the cell cycle. Eventually, most of these cells underwent apoptosis and exhibited decreased Bcl-2 expression. Enforced expression of Bcl-2 using retrovirus infection was able to significantly delay AML1-ETO-induced apoptosis, while the growth arrest remained. Furthermore, we also studied the effect of AML1-ETO on U937T cell differentiation. Induced expression of AML1-ETO in U937T-A/E cells significantly decreased the expression of CCAAT/enhancer binding protein α (C/EBPα) and altered the neutrophilic differentiation of U937T cells. These observations suggest a dichotomy of AML1-ETO properties in that the protein is able not only to block cell differentiation but also to induce cell cycle arrest, which eventually leads to apoptosis. We predict that the preleukemic AML1-ETO+ cell needs to overcome the growth arrest and/or the apoptotic potential of AML1-ETO prior to fulfilling its leukemogenic potential.
Cell cultureMyeloid U937 and AML1-ETO-expressing Kasumi-1 cells were cultured in RPMI 1640 medium (GIBCO/BRL, Rockville, Md.) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, Utah) and 2 mM l-glutamine (GIBCO/BRL). Phoenix amphotropic cells (21) were maintained in Dulbecco's modified Eagle's medium (GIBCO/BRL) supplemented with 10% FBS, 2 mM l-glutamine, and 5 μM β-mercaptoethanol (Fisher Scientific, Fair Lawn, N.J.). All cells were maintained in a 37°C incubator with 5% CO2. U937T cells are U937 cells stably transfected with a tet-VP16 fusion gene under the control of a tetracycline-inducible promoter; the cells were kindly provided by Gerald Grossveld (3, 15). U937T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mMl-glutamine, 1 μg of tetracycline (Sigma, St. Louis, Mo.)/ml, and 0.5 μg of puromycin (Sigma)/ml.
Plasmid construction.The construction of pUHD-AML1/ETO was described previously (55). The cDNAs for Bcl-2(GenBank accession no. GI4557356) and for CDK4 (GenBank accession no. GI6753379) were subcloned into the EcoRI site of the MSCV-internal ribosome entry signal (IRES)-enhanced green fluorescent protein (EGFP) coding sequence (16) to form murine stem cell virus (MSCV)-Bcl-2-IRES-EGFP and MSCV-CDK4-IRES-EGFP (17). The cDNA for c-myc (GenBank accession no. GI34820) was subcloned into HpaI of MSCV-IRES-EGFP to form MSCV-cMyc-IRES-EGFP. The BamHI and XhoI cDNA fragment for C/EBPα (GenBank accession no. GI4757971) was subcloned into BglII and XhoI of MSCV-IRES-EGFP to form MSCV-C/EBPα-IRES-EGFP.
Establishment of U937T-A/E stable transformants.U937T cells were washed in RPMI 1640 medium without FBS and resuspended at 2 × 107 cells/ml in 500 μl of RPMI 1640 medium without FBS. Fifty micrograms of linearized pUHD-AML1/ETO (55) and 1 μg of linearized pGK-Neo in 45 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) were mixed with the cells, and the mixture was transferred to electroporation cuvettes with a 0.4-cm gap (Bio-Rad, Hercules, Calif.). Electroporations were performed using a Gene-Pulser II (Bio-Rad) at 300 V and 960 μF. The samples were then transferred to complete RPMI 1640 medium containing 1 μg of tetracycline and 0.5 μg of puromycin/ml and incubated at 37°C in 5% CO2. Twenty-four hours later, G418 (GIBCO/BRL) was added at a concentration of 1 mg/ml. Positive polyclonal populations (pools) were identified based on Southern blot hybridization and on the induction of AML1-ETO expression following tetracycline withdrawal and were named U937T-A/E. Individual U937T-A/E clones were eventually isolated by limiting dilution and assayed for AML1-ETO inducibility. U937T-A/E cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM l-glutamine, and 1 μg of tetracycline, 0.5 μg of puromycin, and 1 mg of G418/ml.
Retrovirus infection of cell lines.For the production of retrovirus, Phoenix amphotropic cells were transfected with MSCV–Bcl-2–IRES–EGFP, MSCV-CDK4-IRES-EGFP, MSCV-cMyc-IRES-EGFP, MSCV-C/EBPα-IRES-EGFP, or MSCV-IRES-EGFP vector by calcium phosphate precipitation (21). Retrovirus supernatants were harvested 48 h after transfection, filtered through 0.45-μm-pore-size filters, and stored at −80°C. Filtered supernatant was used to infect U937T and U937T-A/E cells in the presence of 8 μg of Polybrene (Sigma)/ml. The infected cells were then centrifuged at 700 ×g for 1 h at room temperature and incubated at 37°C in 5% CO2. The medium was changed 24 h after virus infection. Cells infected with the various MSCV-IRES-EGFP constructs were identified on the basis of green fluorescence.
Northern blot analysis.Total RNA was isolated from the U937T-A/E cell lines by extraction with guanidine isothiocyanate and centrifugation through cesium chloride, and Northern blot analysis was performed as described previously (55, 76). Blots were exposed to MR X-ray films (Kodak, Rochester, N.Y.).
RPA.The RNA samples were hybridized with the hAPO-2c (BD Pharmingen, La Jolla, Calif.) set of probes against the main members of the Bcl-2 family. The RNase protection assay (RPA) was performed using the RiboQuant RPA starter package according to the manufacturer's instructions (BD Pharmingen). The level of modulation of Bcl-2 was determined by using a Storm phosphorimager (Molecular Dynamics, Sunnyvale, Calif.) and Image software (Scion Corporation, Frederick, Md.).
Transient transfection assays.Electroporation was performed with a Bio-Rad Gene-Pulser II (230 V, 975 μF). U937T cells were transfected with 25 μg of total DNA, including 13 μg of pXP1 (GenBank accession no. GI3929277) or 20 μg of pBcl2-luc (equimolarity) or 20 μg of MDR-1-luc (63) or 1.4 μg of pCMV5 (empty expression vector; GenBank accession no. GI7542546) or 2 or 4 μg of pCMV5-AML1-ETO and 100 ng of pRL-Null (GenBank accession no. GI7638455) to normalize transfection efficiency. Alternatively, U937T and U937T-A/E cells were induced for 12 h prior to transfection. Transfected cells were harvested 24 h after electroporation, and luciferase activity was measured using the dual-luciferase reporter assay system (Promega).
Western blot analysis.Cells were washed in phosphate-buffered saline (PBS) and resuspended in Laemmli buffer (1% sodium dodecyl sulfate [SDS], 0.1275 M Tris-HCl [pH 6.8], 1% β-mercaptoethanol). The protein concentration of the supernatant was assessed using the Bio-Rad protein assay according to the manufacturer's instructions. Following SDS-polyacrylamide gel electrophoresis (PAGE), proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) using a Mini-Trans-Blot cell (Bio-Rad). The blot was then blocked in 5% dry milk in Tris-buffered saline with 0.1% Tween 20. The blot was then incubated with a 1:1,000 dilution of rabbit anti-ETO antibody (a gift from Scott Hiebert), a 1:1,000 dilution of rabbit anti-human Bcl-2 antibody (a gift from John Reed), and a 1:5,000 dilution of mouse anti-human β-actin (AC15) (Sigma). The blot was then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, N.J.). The blot was developed using a chemiluminescent substrate (Renaissance; NEN, Boston, Mass.) and exposed to MR X-ray film (Kodak). In all Western blot analyses, β-actin or Coomassie staining of the SDS-PAGE gel after transfer was used as a loading control.
Analysis of cell proliferation, differentiation, morphology, and cell cycle.For the induction of tetracycline-controlled AML1-ETO, the cells were washed three times in 50 ml of PBS and seeded at 105 cells/ml in the maintenance medium in the presence of the desired tetracycline concentration. The viability of the cells was assessed daily by trypan blue exclusion. The cells were induced to differentiate by adding 65 nM phorbol 12-myristate 13-acetate (TPA) (Sigma) to the culture medium 24 h after tetracycline withdrawal. Morphology was determined by Wright-Giemsa staining of air-dried cytospin preparations. Cell cycle distribution was evaluated by flow cytometry analysis of propidium iodide-stained nuclei (41). A minimum of 104 cells were analyzed using a FACSCalibur (BD Immunocytometry, San Jose, Calif.). The cell cycle distribution was assessed using ModFit LT software (Verity Software House, Topsham, Maine)
Detection of apoptosis.Apoptotic cells were identified by double staining with acridine orange (100 μg/ml; Sigma) and ethidium bromide (100 μg/ml; Sigma) (36). The level of apoptosis was also assessed by flow cytometry of cells stained with annexin V-phycoerythrin (PE) and 7-aminoactinomycin D (7-AAD; BD Pharmingen). The cells (105) were washed twice in PBS and resuspended in annexin V binding buffer (0.01 M HEPES [pH 7.4], 0.14 M NaCl, 2.5 mM CaCl2) and then double stained with annexin V-PE and 7-AAD. A minimum of 104cells were analyzed.
Differentiation marker analysis.For each flow cytometry analysis, 106 cells were washed twice in washing buffer (PBS, 0.1% [wt/vol]NaN3, 1% FBS) and resuspended in 100 μl of washing buffer with 2 μl of anti-human CD11b-PE (clone ICRF44), CD18-fluorescein isothiocyanate (FITC) (clone 6.7), or immunoglobulin G1(κ)–PE-FITC (clone MOPC-21; BD Pharmingen) as an isotype control. Incubation was performed at room temperature for 30 min. A minimum of 104 cells were analyzed by flow cytometry.
Establishment of myeloid cell lines with inducible expression of AML1-ETO.To study the effect of AML1-ETO on the growth, survival, and differentiation of myeloid cells, we made inducible AML1-ETO-expressing U937T cell lines. The AML1-ETO cDNA was inserted into tetracycline-responsive expression vector pUHD10–3 to form pUHD-AML1/ETO (55). pUHD-AML1/ETO was introduced into the U937T cells, which contain stably transfected pUHD-tTA (a tetracycline-responsive transcription activator [tTA], whose expression is under the control of tetracycline) to establish the inducible cell lines (3). In principle, in the presence of tetracycline, the expression of tTA and AML1-ETO should be extremely low; in the absence of tetracycline, tTA activates its own promoter to produce more tTA and also activates AML1-ETO expression. Forty-eight pools of U937T cells stably transfected with pUHD-AML1/ETO were confirmed by Southern blot hybridization. Cells were cultured in the presence or absence of tetracycline to test the inducibility ofAML1-ETO expression. Northern blot hybridization using RNA harvested from these cultures demonstrated that only two pools (pools 18 and 48) of U937T-A/E cells were inducible for AML1-ETO expression upon withdrawal of tetracycline (Fig.1A). All the other pools did not showAML1-ETO expression in either the presence or absence of tetracycline (data not shown). Pools 18 and 48 showed very tight control of AML1-ETO expression. Furthermore, the inducible up-regulation of AML1-ETO was also demonstrated at the protein level in Western blot analysis (Fig. 1B).
Inducible expression of AML1-ETO reduces proliferation and affects the viability of U937T-A/E cells.We first tested the effect of AML1-ETO expression on cell proliferation. U937T-A/E pool 18 and 48 cells and their parental cell line, U937T, were cultured in the presence of tetracycline (no induction of AML-ETO expression) and in the absence of tetracycline (induction of AML1-ETO expression).AML1-ETO transcripts were clearly detectable 24 h after the induction and reached a maximum at 48 h postinduction (Fig.2A). The growth curve showed a significant reduction of cell growth when AML1-ETO was expressed. This delay was initiated 2 days after induction and continued, with the proliferation being substantially reduced compared to that for uninduced cells (Fig. 2B and C). The proliferation of the parental U937T cells was the same in the presence and the absence of tetracycline, indicating that the effect was due solely to the expression of AML1-ETO. Besides the reduction of the proliferative potential of the U937T-A/E pool 18 and 48 cells, a clear reduction of the viability of AML1-ETO-expressing cells was observed 3 days after induction. To determine how the expression of AML1-ETO affected the cell cycle, we analyzed by flow cytometry the DNA content of propidium iodide-stained nuclei from induced cells using pool 48 cells and clones U937T-A/E 48–5 and 48–9 (no significant differences between the different populations were detected; data from pool 48 are represented in Fig. 3). After 57 h of AML1-ETO induction, the proportion of cells in S phase decreased from 45 to 22% and the percentage of cells in G0/G1 phase increased from 45 to 69% (Fig. 3), indicating the blockage of the G1-to-S phase transition. At this time point, no cells with a sub-G1 DNA content were detected. However, by 81 h, 13% of cells displayed a sub-G1 DNA content, and the proportion rose to 33% after 100 h. Nicoletti et al. (41) have shown that a cell with hypoploid content is an indication of the cell becoming apoptotic. As shown in Fig. 4, dual acridine orange and ethidium bromide staining of the AML1-ETO-induced U937T-A/E pool 48 cells showed an accumulation of cells with chromatin condensation and fragmentation, which confirmed that AML1-ETO-induced cell death was caused by apoptosis. Furthermore, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assays to detect double-strand DNA breaks also demonstrated apoptotic events in AML1-ETO-expressing cells (data not shown) (12).
AML1-ETO-induced apoptosis is associated with the decrease of Bcl-2 expression.To further analyze the molecular components involved in the induction of apoptosis in U937T-A/E cells, we examined the RNA expression pattern of the members of the Bcl-2 family by RPA (Fig.5A). Three days after withdrawal of tetracycline to induce AML1-ETO expression, the amounts ofBcl-2 transcripts were significantly reduced in both U937T-A/E pool 18 and 48 cells. Densitometry analysis showed a 10-fold difference in Bcl-2 RNA levels between AML1-ETO-induced cells and uninduced cells, compared to the difference for housekeeping gene transcripts. Some of the other members of antiapoptosis or proapoptosis genes of the Bcl-2 gene family also showed different levels of expression, but the difference was not as dramatic as that for the Bcl-2 gene. Furthermore, a reduction of Bcl-2 at the protein level was also observed by Western blotting (Fig.5B).
Bcl-2 is an important antiapoptosis factor (7, 54). Bcl-2 expression has also been reported to affect cell cycle progression (4, 33, 49). Therefore, to determine whether the suppression of Bcl-2 expression significantly contributed to the alteration of cell proliferation and apoptosis in induced U937T-A/E cells, Bcl-2 was ectopically expressed in U937T and U937T-A/E cells using an MSCV-Bcl2-IRES-EGFP retrovirus construct. Viral particles prepared from Phoenix amphotropic cells were used to infect U937T and U937T-A/E cells. EGFP expressed through the IRES was used to monitor ectopic Bcl-2-expressing cells. Following retrovirus infection, the proportion of EGFP-positive cells infected with control virus (MSCV-IRES-EGFP) and Bcl-2-expressing virus (MSCV–Bcl-2–IRES–EGFP) remained stable at approximately 20% for more than 1 month in both U937T and U937T-A/E pool 48 cells in the presence of tetracycline as determined by flow cytometry (data not shown). Upon induction of AML1-ETO expression, the proportion of viable EGFP+ U937T-A/E cells remained steady at 22% for 2 days before progressively increasing to 47% 5 days after induction in U937T-A/E cells infected with MSCV–Bcl-2–IRES–EGFP, whereas the proportion of viable EGFP+ cells remained constant in U937T cells and U937T-A/E cells infected with vector control MSCV-IRES-EGFP (Fig. 6A). Although the proportion of EGFP+Bcl-2+ cells increased to up to one-half of the viable U937T-A/E cells, the overexpression of Bcl-2 was unable to restore the proliferation of those cells and to suppress apoptosis, as all cells eventually underwent apoptosis. However, the increase of Bcl-2-positive cells in the culture indicated that Bcl-2 expression was able to delay apoptosis. To better understand the effect of Bcl-2, the cells infected with MSCV–Bcl-2–IRES–EGFP were sorted by flow cytometry on the basis of the EGFP expression. Later, the cell cycle distributions of both EGFP+ and EGFP− cells were analyzed upon AML1-ETO induction. As seen previously in the mix population, the ectopic expression of Bcl-2 dramatically reduced apoptosis over a period of 96 h (Fig. 6C). However, the ectopic expression of Bcl-2 was unable to prevent the AML1-ETO+ cells from arresting in G0/G1 phase (Fig. 6B). We also confirmed that the level of ectopic Bcl-2 expression was essentially unchanged for the length of experiments, whereas AML1-ETO was significantly up-regulated by 72 h in the absence of tetracycline (Fig. 6D).
The effect of AML1-ETO on Bcl-2 expression has been studied previously. Klampfler et al. reported the transactivation of the Bcl-2promoter by AML1-ETO via an AML1 binding consensus sequence (23). Two cell lines derived from t(8;21) leukemia patient cells showed higher expression of Bcl-2 (23, 24). However, studies using 29 (57) and 17 (2) primary t(8;21) leukemia patient samples indicated that Bcl-2 expression was generally down-regulated compared to that for nonleukemic or non-t(8;21) AML samples. Therefore, we performed studies to determine whether the down-regulation of Bcl-2 was the direct effect of AML1-ETO on a 3.7-kb Bcl-2 promoter in U937T and U937T-A/E cells using transient transfection assays (Fig.7A and B). The data indicated that AML1-ETO down-regulation of Bcl-2expression in this particular cell line was not a direct effect of AML1-ETO on the 3.7-kb Bcl-2 gene upstream regulatory element. To ensure the accuracy of the data presented, as the current results did not agree with previous studies (23), we assessed whether AML1-ETO was also able to repress the expression of the (−137 to +30) multidrug resistance 1 (MDR-1) promoter as previously reported (31). The data confirmed that AML1-ETO represses transcription of the MDR-1 promoter in U937T (Fig.7C).
AML1-ETO alters myeloid differentiation.The effect of AML1-ETO on U937T cell differentiation was also studied with these inducible AML1-ETO-expressing cells. Parental U937T cells and U937T-A/E cells were treated with TPA. Interestingly the differentiation patterns of U937 and U937T cells were different, as U937 cells treated with TPA rapidly assumed (24 h) a macrophage-like morphology (data not shown) whereas U937T and uninduced U937T-A/E pool 48 cells slowly displayed "doughnut-shaped" nuclei characteristic of granulocytes upon TPA treatment (Fig. 8A). In contrast, the surviving fraction of induced U937T-A/E cells treated with TPA did not exhibit doughnut-shaped nuclei. Nonetheless, the cells displayed some signs of maturation, with cytoplasm larger than that of untreated samples, which displayed lymphoblast-like morphologies. As in previous studies, the effects of AML1-ETO on differentiation were further analyzed by flow cytometry (13, 28), which revealed a clear increase of the cell surface differentiation markers CD11b and CD18 upon TPA treatment (Fig. 8B; from CD11blow and CD18low to CD11bhigh and CD18high). The increase in the CD11b and CD18 cell surface markers precedes the occurrence of the doughnut-shaped nuclei mentioned previously. However, the induction of AML1-ETO significantly reduced the differentiation of U937T-A/E cells. These cells lost their original lymphoblast-like morphology without displaying granulocytic morphology (Fig. 8A) and exhibited a block of differentiation at an early stage (Fig. 8B; CD11blow and CD18low).
C/EBPα is down-regulated upon AML1-ETO expression.The alteration of granulocytic differentiation suggested that AML1-ETO expression might affect certain critical factors involved in myeloid lineage commitment. Since C/EBPα and C/EBPɛ are important transcription factors for granulocytic cell differentiation, we hypothesized that down-regulation of their expression may be involved in the block of granulocytic differentiation (53, 72-74, 78). Therefore, Northern blot analysis was used to study the expression of C/EBPα andC/EBPɛ. C/EBPɛ expression is barely detectable in uninduced U937T-A/E cells. Upon induction, no obvious change of C/EBPɛ was detected (data not shown). C/EBPα was expressed at high levels in U937 and U937T parental cells and uninduced U937T-A/E cells (Fig. 9A). Upon induction of AML1-ETO expression, C/EBPα was considerably down-regulated (Fig. 9A). In a time course analysis, the decrease ofC/EBPα transcripts showed a tight correlation with the induced expression of AML1-ETO (Fig. 9B). Furthermore, the expression of two cell cycle-related genes, cdk4 and c-myc, were also down-regulated (Fig. 9B). The effects of TPA treatment on the level of C/EBPα were also assayed (Fig. 9C). Treatment with TPA did not alter the down-regulation of C/EBPα by AML1-ETO.
To determine whether the down-regulation of C/EBPα was a central part of the inhibition of differentiation by AML1-ETO,C/EBPα was ectopically expressed in U937T and U937T-A/E using an MSCV-C/EBPα-IRES-EGFP retrovirus construct. Following retrovirus infection, the proportion of EGFP-positive cells decreased rapidly from 30 to 10% within 5 days after the infection (data not shown). The reduction is the result of a reduced proliferation caused by the spontaneous differentiation of both U937T and uninduced U937T-A/E due to C/EBPα expression as determined by CD11b staining (Fig. 10). Interestingly, the induction of AML1-ETO halted the differentiation induced by ectopic C/EBPα as determined by CD11b staining (Fig. 10). The addition of TPA in conjunction with the ectopic expression of C/EBPα was only able to cause a small increase of CD11b-positive cells. Even in the presence of TPA, the ectopic expression of C/EBPα was unable to antagonize the effect of AML1-ETO.
To investigate the role of AML1-ETO in leukemogenesis, we examined the biological properties of the AML1-ETO fusion protein by inducible expression in U937T cells. The most interesting finding from this study is that AML1-ETO plays antagonistic roles regarding cancer development. AML1-ETO expression favors leukemogenesis by blocking cell differentiation associated with the decrease of C/EBPα protein level and opposes leukemogenesis by blocking cell proliferation, leading to apoptosis.
AML1-ETO blocks granulocytic differentiation and down-regulates C/EBPα expression.The treatment of U937T and uninduced U937T-A/E cells with TPA leads to progressive granulocytic maturation associated with an increased expression of cell surface markers CD11b and CD18. This differentiation was significantly reduced in AML1-ETO+ cells. This altered differentiation potential is correlated with the down-regulation of C/EBPα upon induction of AML1-ETO (Fig. 9). C/EBPα is the founding member of the C/EBP transcription factor family, regulating many genes involved in myeloid lineage commitment and differentiation (6, 19, 20, 27, 45, 52, 60, 69, 77; A. Khanna-Gupta, T. Zibello, C. Simkevich, A. G. Rosmarin, and N. Berliner, Abstr. Third Int. Meet. Mol. Aspects Myeloid Stem Cell Dev., abstr. 10, 1999). C/EBPα is up-regulated during granulocytic differentiation, and overexpression of C/EBPα in U937 cells leads to granulocytic differentiation (53). Furthermore, C/EBPα-deficient mice lack mature granulocytes, indicating a critical role in granulocytic differentiation (78). Interestingly, C/EBPα is consistently expressed at much lower level in AML M2 subtype patients with t(8;21) translocation than in patients with other subtypes of AML (50). Moreover, dominant-negative mutations of C/EBPα leading to a failure to induce granulocytic differentiation have been detected in 16% of AML M2 patients without t(8;21) translocation, emphasizing the important contribution of C/EBPα to AML leukemogenesis (51).
Although U937 cells constitutively express C/EBPα, U937T and uninduced U937T-A/E cells express markedly higher levels ofC/EBPα mRNA than the parental U937 cells. This difference may be reflected in the higher propensity for granulocytic differentiation of the U937T cells.
The ectopic expression of C/EBPα causes a rapid up-regulation of the CD11b cell surface marker in both U937T and uninduced U937T-A/E cells. However, the induction of AML1-ETO prevents the CD11b up-regulation induced by C/EBPα in U937T-A/E cells, suggesting that the down-regulation of C/EBPα probably contributes to the inhibition of differentiation but is not enough to fully explain this inhibition. In contrast, ectopic expression of C/EBPα in Kasumi-1 cells leads to granulocytic differentiation (50). One explanation could be that the cell cycle arrest caused by the induction of AML1-ETO blocks the differentiation despite the presence of C/EBPα, in contrast to what is found for Kasumi-1 cells. Alternatively, Westendorf et al. have previously demonstrated that AML1-ETO physically interacts with C/EBPα, abrogates C/EBPα-dependent transcription, and ultimately blocks granulocytic differentiation (71). C/EBPα activates its own promoter through an upstream stimulatory factor (USF) binding site (63). It has been shown that AML1-ETO decreases C/EBPα promoter activity also via a USF binding site (50). Therefore, it is possible that AML1-ETO prevents the autoactivation of the C/EBPα promoter by USF through protein-protein interaction with C/EBPα
Shimizu et al. (58) reported recently that AML1-ETO up-regulates C/EBPɛ expression without identifying an AML1 binding site in the C/EBPɛ promoter, suggesting the existence of additional steps between the expression of AML1-ETO and the up-regulation of C/EBPɛ. Interestingly, the expression of C/EBPɛ was also reported in cell lines derived from AML M2 patients but not in other hematopoietic cell lines such as U937. In the present studies, we did not observe any significant modulation of C/EBPɛ expression. The difference in C/EBPɛ expression between AML M2-derived cell lines and the present model could be reconciled by the presence or absence of a yet to be identified factor that would cooperate with AML1-ETO to induce the expression of C/EBPɛ.
AML1-ETO blocks cell proliferation by inducing cell arrest in G0/G1.The induction of AML1-ETO in U937T-A/E cells causes a progressive cell cycle arrest in G0/G1 phase. This growth arrest coincides with the down-regulation of c-myc and CDK4 (Fig. 9B). Lou et al. have recently reported that the tamoxifen-inducible fusion protein KRAB-AML1-ER prolongs G1 phase (29), possibly through the down-regulation of CDK4. Furthermore, Hermeking et al. have also identified CDK4 as a transcriptional target of c-myc (18). These authors suggest that the ability of c-myc to promote cell cycle reentry is in part due to its ability to directly induce the transcription ofCDK4. We assume that the down-regulation of c-myc causes the down-regulation of CDK4, thereby contributing to blocking the cell cycle. Upon induction of AML1-ETO, the expression pattern ofCDK4 closely matches that of c-myc expression, as transcription culminates 12 h after induction before decreasing to its lowest level by 48 h (highest level of AML1-ETO; Fig. 9B). The overexpression of CDK4 or c-myc in U937T-A/E cells using the MSCV-CDK4-IRES-EGFP or MSCV-cMyc-IRES-EGFP retrovirus construct was not able to prevent the growth arrest and apoptosis caused by the conditional expression of AML1-ETO in U937T-A/E cells (data not shown). Interestingly, the down-regulation of C/EBPα may actually antagonize growth arrest, as C/EBPα has been reported in hepatocytes to increase the level of p21/WAF-1, thereby causing growth arrest (64). Therefore, we hypothesize that AML1-ETO deregulates the expression of a yet to be identified factor that may be responsible for the down-regulation of both c-myc and CDK4. However, c-myc and CDK4 may not be the only targets of this putative factor to provoke cell cycle arrest of AML1-ETO+ cells. If such a factor were identified, we predict that the separation of its function with those of AML1-ETO would potentially unlock the growth arrest and contribute significantly to the leukemogenic process. Another interesting question raised by these results is whether DNA binding is required for AML1-ETO to induce cell cycle arrest or whether the growth arrest occurs through protein-protein interaction possibly mediated through the ETO moiety as reported recently by Melnick et al. (34).
AML1-ETO induces apoptosis and reduces Bcl-2 level.Eventually, the induction of AML1-ETO results in the onset of apoptosis in U937T-A/E cells. We also report the down-regulation of Bcl-2 at the RNA and protein levels following induction of AML1-ETO, suggesting that down-regulation of Bcl-2 contributed to the reduced survival of U937T-A/E cells. However, ectopic expression of Bcl-2 delays apoptosis without preventing AML1-ETO-induced G1/G0 arrest. Moreover, the inability of Bcl-2, which acts essentially downstream of the cell cycle, to prevent cell cycle arrest despite delaying apoptosis suggests that Bcl-2 is not a direct target of AML1-ETO (Fig. 6). Therefore, the AML1-ETO+ cells may initiate apoptosis through their inability to overcome cell cycle arrest.
Two groups have reported that t(8;21)-bearing de novo leukemia cells express, in general, lower levels of Bcl-2 protein than normal bone marrow, other non-t(8;21) AML samples, and Kasumi-1 and SKNO-1 cells (2, 23, 24, 57). Klampfer et al. report that AML1-ETO is able to transactivate the Bcl-2 promoter via a consensus AML1 binding sequence (23). As our results indicate that the induction of AML1-ETO results in the down-regulation ofBcl-2, we reexamined the effects of transient expression or induction of AML1-ETO in the specific context of U937T and U937T-A/E cells using the same Bcl-2 promoter fragment (Fig. 7) (23, 38). AML1-ETO does not transactivate theBcl-2 promoter in U937T cells, indicating a cell type-specific effect of AML1-ETO on the regulation of theBcl-2 promoter. Furthermore, the delay in the onset of apoptosis also reinforces the idea that Bcl-2 is not a target of AML1-ETO in U937T-A/E cells.
In summary, our studies with inducible cells indicate that expression of AML1-ETO blocks cell differentiation and proliferation and consequently induces apoptosis. The reduced granulocytic maturation in AML1-ETO+ cells correlates with the down-regulation of C/EBPα. Modulation ofC/EBPα is critical for the commitment to the granulocytic lineage (53, 78). The low level of C/EBPα reported in t(8;21) patients also suggests that the reduction of C/EBPα is central for blocking AML cells at the M2 differentiation stage (50). In view of the effects of AML1-ETO on the regulation of C/EBPα function, further studies are required to clarify whether overexpression of C/EBPα in t(8;21) patients is enough to induce the maturation of the leukemic cells and improve their clinical outcome.
In addition to blocking cell differentiation, AML1-ETO also blocks proliferation by inducing cell cycle arrest. This finding is further supported by the data from AML1-ETO knockin mice and the tetracycline-inducible mouse model. In previous studies, the effect of AML1-ETO on the cell cycle or apoptosis in the bone marrow of tetracycline-inducible AML1-ETO mice could not be determined. However, the new perspective offered by our findings now supports the negative effect of AML1-ETO on cell proliferation and survival observed in primary bone marrow and CFU–granulocyte-macrophage (GM) assays. First, AML1-ETO knockin mice died in midgestation without the development of definitive hematopoietic cells, raising the possibility of AML1-ETO expression blocking the proliferation of hematopoietic precursor cells (47, 75). Second, the infection of adult murine bone marrow with MSCV-AML1-ETOneo leads to a lower number of colonies than with the retrovirus vector control (75). Last, AML1-ETO-inducible transgenic mice, although viable, do not develop leukemia, but induction of AML1-ETO in the bone marrow of those mice exerts a detrimental effect on CFU GM number and size as well as blocking cell differentiation (55). The homeostasis in vivo and the high concentration of growth factors used in in vitro CFU assays may contribute to a decrease of AML1-ETO effects compared to those reported in U937T-A/E cells. Both Yergeau et al. and Okuda et al. followed the same experimental procedure for the CFU assay, including the use of fetal liver cells from the same developmental stage, for assessing the outcome of AML1-ETO expression. Nonetheless, the different origins of cells used to establish the AML1-ETO knockin mice could be another source of discrepancies between both models. While Okuda et al. reports the presence of dysplastic hematopoietic colonies with high replating efficiency, Yergeau et al. did not detect any dysplastic replating colonies (47, 75). In fact, no colonies were detected in the fetal liver culture. Additional mutations could reconcile the observed differences.
If growth arrest and apoptosis are general features associated with the expression of AML1-ETO, we predict that the putative link that unites them to AML1-ETO must also be severed prior to the occurrence of the t(8;21) translocation to allow the survival and expansion of the preleukemic cells. Inhibition of proliferation or apoptosis would not be favorable to the propagation of cells harboring the t(8;21) translocation. Therefore, we favor the hypothesis that other oncogenic events promoting proliferation or cell survival are necessary for the development of t(8;21) AML.
We thank John Reed, Stephen Nimer, Nancy Speck, Beatrice Muller, and Alan Friedman for valuable discussion and Gerald Grosveld, Scott Hiebert, Warren Pear, Gary Nolan, Jonathan Licht, Stanley Korsmeyer, Philip Koeffler, Kimiko Shimizu, Scott Kogan, and John Schuetz for DNA constructs, cell lines, and antibodies. We thank Ernest Beutler and Kenneth Ritchie for editing the manuscript.
This work was supported by National Institutes of Health grant CA72009 and American Cancer Society grant LBC-99438. D.E.Z. is a Leukemia and Lymphoma Society Scholar. S.A.B. was supported by a fellowship from the Lady Tata Memorial Trust. The Stein Endowment Fund partially supported the Departmental Molecular Biology Service Laboratory for DNA Sequencing and Oligonucleotide Synthesis.
- Received 20 November 2000.
- Returned for modification 8 January 2001.
- Accepted 7 May 2001.
- Copyright © 2001 American Society for Microbiology
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"TerraCycle offers a range of free programs that are funded by conscientious companies, as well as recycling solutions available for purchase for almost every form of waste." -terracycle.com
It's 2018. The technology exists to launch a Tesla Roadster into outer space, but here we are on Earth still figuring out how to best address the vast array of materials that are difficult to recycle. So what's to do with the all the materials with a potential for recycling that end up in our landfills?
Meet TerraCycle.
This is the company taking the materials that no one else wants to deal with and actually recycling them. If it's difficult to recycle, TerraCycle wants it. They have creatively harnessed the potential of these otherwise landfill-destined items through tailored, industry-sponsored recycling programs. These programs are now more accessible than ever and have in a short time taken out some pretty massive shovel-loads out of a mountain of trash.
With a current offering of 40 free recycling programs, TerraCycle has created the ultimate incentive by offering a virtually zero cost way of recycling common, hard to recycle everyday items. These programs are available to anyone who wants to participate. The fact that these programs are free to the public is a testament to not only TerraCycle's effort towards making recycling accessible and hassle-free to anyone, but also the effort from the companys which these products originate from to connect with the public and encourage participation. The sense of shared responsibility from both industry and end-user to adopt responsible material handling practices has proved to be an effective way to address the issue.
How It Works
Collect & box the items you plan to recycle with TerraCycle
Print a free shipping label from your TerraCycle account
That's it.
TerraCycle® & Clif Bar®
Over 2015, sales for granola/energy bars came close to 550 million units. Imagining the literal mountain of trash that follows a continued rise in sales of this market is just one of many areas that TerraCycle has focused on addressing over the years. In an effort to chip away at this issue, TerraCycle teamed-up with Clif Bar to offer a free program specific to the collection and recycling of energy bar wrappers. Upon collection, the used wrappers are cleaned and melted down to create new recycled products. As a result of this collaboration and the efforts of participants, TerraCycle has recycled 46,655,309 wrappers to date. This is just one example of many measurable impacts that TerraCycle has made through collaborative industry-sponsored recycling programs.
Taking steps towards responsible recycling practices should be on the minds of everyone. As creators of packaging, it's not only our responsibility to take part in these practices, but we must also help propagate and support programs such as those offered by TerraCycle. Stay up to date with new recycling program offerings from TerraCycle in the future.


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Doctor who is passionate about acupuncture The blog is for information purpose only
Welcome to my blog
My profile
Acupuncture practice contact for appointments and addresses
Kensington: book online or call at 02030110355 or email at firstname.lastname@example.com
Address: Anamaya 1 Adam and Eve Mews, Kensington, London W8 6UG
2 min walk from High Street Kensington underground station
Chelsea: book online or call 02033623366
Address: Triyoga Chelsea 372 King's road, SW3 5UZ
20 min walk from Sloan Square tube station
My background: I became a qualified medical doctor in Western medicine in China and was well trained in Western medicine together with Chinese medicine in the best Medical University in Beijing, China. Particularly I was trained with Ji-sheng Han famous professor and neuroscientist in China and with Dr Zheren Xuan--famous orthopedics expert and founder of soft tissue surgery in China. Furthermore I had training in dermatology and oral and maxilofacial surgery in China. Also I had training in fertility and had research experiences in uterine smooth muscles and blood vessels in China and the UK. I am dedicated to treat patients with acupuncture and am recognised as one of the world leading acupuncture specialists.
I obtained a PhD degree in the University of Leeds in the UK.
I had post doctoral training and worked as a senior researcher in St George's hospital, London, UK.
I practiced acupuncture at Harley street after finished post doctoral training.
I had frequently presented my research findings in the top international conferences in the field.
I have many publications including ebooks and articles.
I have many year clinical experiences. Over the years of practicing in London, I have developed unique effective treatment approaches for cosmetic acupuncture, acne, pain relief including vulvodynia, bladder pain, pelvic pain, chronic prostatitis, neck pain, headache, migraine, shoulder pain, back pain, stress relief, anxiety, fatigue, fertility, hot flushes, nerve pain, insomnia to achieve best treatment results.
My devotion and skills are highly praised by my patients.
My eBooks
Fertility and acupuncture10 Reasons why you should try acupuncture for
Guest blog articles and Reviews
Wednesday, 31 October 2018
Suffer from muscle pain? Acupuncture can help
There are four types of trigger points
An active trigger point is an area of extreme tenderness that usually lies within the skeletal muscle and which is associated with a local or regional pain.
A latent trigger point is a dormant (inactive) area that has the potential to act like a trigger point. It may cause muscle weakness or restriction of movement.
A secondary trigger point is a highly irritable spot in a muscle that can become active due to a trigger point and muscular overload in another muscle.
A satellite myofascial point is a highly irritable spot in a muscle that becomes inactive because the muscle is in the region of another trigger pain.
Acupuncture can reduce myofascial pain effectively by releasing muscle tension and removing trigger points in the muscles.
Acupuncture helps acne
Acne is a chronic inflammation in the hair follicles and sebaceous glands. The most affected areas are the face and chest and back, because ...
Can acupuncture help patients with stroke? Stroke is a serious condition which blood supply to the brain is cut off. It is the second most...
Hemiplegia which is severer than hemiparesis is paralysis of one half of the body on the same side. Brain damage is one of the causes. The p...
Thigh is the area between the hip and the knee. There is only single bone in the thigh called fumar, but there are a few groups of muscles i...


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Over the last 12 years I have worked in the field of marketing helping businesses small and large to build their brands and develop a stronger commitment to their customer base.
Developing a brand strategy is important for all companies, because it helps the ideal customers to relate to a product, and to help them become brand cheerleaders who inspire others to become loyal to your company too.
Brand development is partly built on your corporate mission, is based on how well you know your typical customer and mixed with proving your authenticity and organic love for what you do, sell, or buy. These three components can be lethal when infused into all advertising, events, and customer interactions you make.
In 2001 my team of ambassadors helped to build an awareness of South Beach Beverage Company and their new Pepsi connection. My team sampled to more than 450,000 people in just 4 months in the Northeast region of the US, and were awarded with a trip to Hawaii for our outstanding reach.
In 2004 my work as brand ambassador for Jelly Belly Jellybeans helped to introduce new flavors to the public and increased sales in the region by more than 11% during the duration of the tour.
In 2006 and 2007 I managed a national promotional tour for Pepsi called the "SoBe Luv Bus Tour". During the two years, my assistant and I drove a 36 ft school bus across more than a dozen states executing grass-roots events and promotions. On our best selling day we sold more than 33 cases of SoBe per hour at a single retail outlet. Although that in itself was a top accomplishment, I am most proud of our daily reporting work as ambassadors. The customer feedback reporting we did helped to inspire SoBe/Pepsi to make their drinks more heathy for their adoring fans by removing the HFCS from all their drinks.
From 2008 to 2010 I managed the marketing efforts of a technical apparel company, Corazzo Designs, where I implemented new brand promotions, strategies, and regular customer communications through a highly successful email program. The company had a stronghold on the national scooter riding community, but sought to build on the overlap of vintage motorcycle riding enthusiasts. Through the development of regular email communications, a branded ad campaign, several event promotions and a new brand image and website, Corazzo was able to attract new customers and generate additional sales and website traffic.
In 2010 and 2011 I developed the marketing processes, strategies, and branding for a newly formed children's book publisher in Portland, OR. The combination of developing a brand image, message driven advertising and promotional campaigns, as well as targeted content for potential customers in new industries, helped to increase brand awareness overall. Our involvement in event promotions and my publicity efforts helped sell more than 7,000 books in the fist year and gained the company national recognition and distribution with Ingram, a major distribution center for publishing houses.


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Although the Ivy League schools review the Common Application or Coalition Application essay, they also require supplemental essay responses. These help you to convey in greater detail how the specific school and program of study are a good fit for you and how you can contribute to the collegiate environment. The additional essay questions are geared to help these elite schools glean a better understanding of you as a potential student. To respond well, think about your future goals and how attending Cornell will help you achieve them!
It is important to familiarize yourself with the specific character of the school before sitting down to write your essays. You can begin by visiting the school website. Whenever possible, a campus visit is also helpful to get a feel for the school and gain a sense of how it supports your interests. Take a campus tour and speak with current students. Cornell's curriculum focuses on the collaborative nature of liberal arts education and fundamental knowledge. In addition, its practical approach to education is intentionally designed to impact societal and world problems. As you respond to each prompt, think about your personal objectives, the mission of the school, and why Cornell is the best place for you.
Cornell boasts 14 undergraduate colleges and schools with over 80 majors. Through the broad scope of majors and the individual course of study options, it prides itself on being "a place where any person can find instruction in any study." It fosters creative collaborations with a bottom-up approach. If you are unsure of which major is right for you, the Courses of Study catalog provides degree requirements for each college.
Get a free consultation:
Click here to schedule a call to find out how our admissions experts
can help YOU get accepted to Cornell!
The Cornell Writing Supplement is based on the undergraduate college(s) or school(s) to which you are applying. Each essay response should be a maximum of 650 words, which is up from the 500-word limit in 2018. These questions are fairly straightforward and the content is somewhat similar between colleges/schools. Your goal is to do your research and convincingly explain why your selected school and what you plan to study at Cornell is the best fit for you and vice versa.
Note, if you are utilizing the Primary/Alternate admission option, you must complete an essay for both colleges/schools that correspond to your primary and alternate selections.
Cornell University writing supplement essay prompts (by college/school)
College of Agriculture and Life Sciences
Why are you drawn to studying the major you have selected? Please discuss how your interests and related experiences have influenced your choice. Specifically, how will an education from the College of Agriculture and Life Sciences (CALS) and Cornell University help you achieve your academic goals?
College of Architecture, Art, and Planning
What is your "thing"? What energizes you or engages you so deeply that you lose track of time? Everyone has different passions, obsessions, quirks, and inspirations. What are yours?
College of Arts and Sciences
Students in Arts and Sciences embrace the opportunity to delve into multifaceted academic interests, embodying in 21st century terms Ezra Cornell's "any person…any study" founding vision. Tell us about the areas of study you are excited to explore, and specifically why you wish to pursue them in our College.
College of Engineering
Tell us about your interest in engineering or what you hope to achieve with a degree in engineering. Describe what appeals to you about Cornell Engineering and how it specifically relates to your engineering interest or aspirations.
School of Hotel Administration (SHA)
How have your interests and experiences influenced your decision to apply to the School of Hotel Administration? How does this decision relate to your future plans and aspirations?
College of Human Ecology
How has your decision to apply to the College of Human Ecology been influenced by your related experiences? How will your choice of major impact your goals and plans for the future?
School of Industrial and Labor Relations
Tell us about your intellectual interests, how they sprung from your course, service, work or life experiences, and what makes them exciting to you. Describe how ILR is the right school for you to pursue these interests.
College of Business
Charles H. Dyson School of Applied Economics and Management: The Dyson School is unique by design. What motivates you to apply to Dyson and where do you plan to go from here?
Your responses should enthusiastically discuss your past and present with an emphasis on your future. Consider the overall Cornell experience. What sparked your interest in a particular field? Then go on to identify why the program at Cornell is the best fit and how your chosen area of study will prepare your future plans. These essay prompts ask you to discuss specific examples from your life experience (academic and otherwise) that support your interest in a specific school/college at Cornell. In addition, they ask you to look toward life after college and how your educational experience at Cornell might support your goals. Keep in mind; you also need to reflect on how you can enrich the collegiate environment at the school. These questions allow you to focus on what excites you about certain subjects and how studying at Cornell makes sense for you. Consider academics, campus atmosphere, location in Ithaca, and your long-term objectives. This is your opportunity to convey your passion for Cornell!
Final thoughts on applying to Cornell
Cornell has a highly competitive applicant pool. It received 49,114 undergraduate applications for the class of 2023. Only 5,330 or 10.85% were offered admission and 83% of the students admitted were in the top 10% of their high school class with average SAT scores of 720 in evidenced-based reading and writing, 760 in math, and an average ACT score of 33.5. The best way to distinguish yourself from your peers is through your essays.
Applying to an Ivy League school can seem like a daunting process. It is reassuring to remember that these supplemental essays are a chance for you to share your personal stories and real-life experiences. Pay attention to deadlines and word limits as you craft each response to represent your unique perspective. Start early to allow time for reflection and revision. Your goal is to demonstrate that Cornell is the right school for you and that you are the right student for Cornell.
If you're applying to Cornell University, you already know you're up against tight competition. Don't be overwhelmed. Get the guidance of an experienced admissions specialist who will help you stand out from the highly competitive applicant pool so you can apply with confidence, and get accepted! Click here to get started!
***Disclaimer: Information is subject to change. Please check with individual programs to verify the essay questions, instructions and deadlines.***Marie Todd has been involved in college admissions for over twenty years. Marie has both counseled applicants to top colleges and evaluated 5000+ applications for the University of Michigan's College of Literature, Science and the Arts; College of Engineering; School of Kinesiology; School of Nursing; and Taubman College of Architecture. Want Marie to help you get accepted? Click here to get in touch with Marie Todd.
Related Resources:
• 5 Fatal Flaws to Avoid in Your College Application Essays, a free guide
• Stand Out or Fit In? 4 Application Strategies to Help You Do Both
• Tips For Answering Common Application Essay Prompts 2019-2020


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Your employees spend 8+ hours a day at work, so the air quality should be one of your topmost concerns. Happy and content coworkers will make the difference between a running and walking pace of work.
Temperature Regulation
Working in a sticky heat creates an uncomfortable and unpleasant work environment. Keeping your employees comfortable is one of the key components to making sure your business doesn't take any unnecessary setbacks. Keep the temperature a few degrees above room temperature to remove the need for bundling up. Some establishments have poorly built ventilation systems and distribute air unevenly throughout the building,
Dusty Atmosphere
Can you literally see the air? Do particles get trapped in the sunlight beaming out of the windows? Having a good air flow might have the opposite effect on everyone in the building if all it's doing is moving dust around the office. Be sure to frequently clean the office.r. Your air will never be good quality if it's constantly clogged up with dust. Dust can trap allergens and further spread illness throughout the building.
Combat Constant Illness
A poor quality air system could essentially trap all of the bad germs in the still air around the office, or worse, spread it to other areas around the building. Having air purifiers that use ultraviolet light installed into your vents can destroy any germs that pass through them; this will significantly decrease the possibility of spreading germs throughout the building. If installing a purifier is not an option, consider having the filters and air ducts cleaned. Sometimes blockages of mold or dust can be the main issue in having poor quality air.
Good quality air is definitely worth having and is very important to the longevity of a business. Don't let you or your coworkers suffer because of a problem that can be fixed by a quick call to your local air conditioner specialist. The AC repair St.Cloud team at All Temp want to help keep your location comfortable and breathable. Contact us at (407)857-7800 to schedule an appointment.


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Take a look at this seasons goals and key players for 2019 sports
September 28, 2019
Fall sports are in full swing and this year's coaches have big aspirations for their players. This season's varsity sports that students can support at events are football, dance, cheer, volleyball, soccer, cross country, and swimming. This year's coaches shared who their key players are, their goals for the team, their overall moto, their outlook on this season. Read their responses below.
Ryan Knowles
Key players
Senior Christian Niemela
Senior Brandon Casey
Senior Jaxon Pettit
Junior Tag Benefield
Senior Connor Hutchings
Goals for this season
Win the IEL, win a playoff game, and win state.
Team moto
"We are Sandpoint"
In a second year of rebuilding some long standing traditions, the team has worked hard this off season to put themselves in a position to compete. We return 5 all league players including our QB, Jaxon Pettit. Niemela provides a viable deep threat option. The RB's have depth with Braden Kappen and Gerrit Cox. They will be a great 1-2 punch. Our offensive line is blue collar and works. They will double as our DL lead by U of Montana commit Brandon Casey. Our linebackers have depth and experience. Tag Benefield returns as the IEL Defensive Player of the Year and has not disappointed with 38 tackles in the first two games. Tyler Lehman is a 2 time All IEL LB that is now contributing on offense as well. The secondary can run and is intelligent. Elijah Larson is in position to have a very good year as a lockdown corner with Jake Ryan holding down the Strong Safety position. The kicking duties will be handled by Dylan Peterson and look for All-IEL return specialist Niemela to do work returning kicks.
Laurie Buck
Key team members
All our team are key to us but we really want to give special attention to our Seniors and officers.
Sydney Clary
April Radford
Gracie Reichart
Josie Topp
Erica Etzwiler
Goals for this season
To see our girls improve in all areas of their dance from flexibility to turns and jumps.
Team moto
Everyone is a part of our Positive team and we work together as 1.
These girls have all trained in dance and so we anticipate a higher level of skills in all their performances and competitions.
Jessica Lester
Key team members
My captains, Cori Lester and Mandy Brown, both exemplify what I'm looking for in an athlete. They both strive for personal improvement while working hard to make the team better.
Goals for this season
One goal for this season is to increase the skill and difficulty of the routines while getting the crowd involved in the games! I would also like to see the team earn an academic award.
Team Motto
"Practice Hard"
My outlook is they will get out as much as they put in. This year is all about trying new things, setting goals and hitting them. From the small stuff to the big stuff, seeing how hard work pays off and remembering that the cheer team is all about uplifting others. The mark has been set high and this team has a great chance of surpassing it. Go dogs! We are all super excited for Football season and so far it has been very fun. We would love to see record student attendance at the home games this season. So all students and community come on out for some football!
Karen Alsager
Key players
Senior Jenny Slaveck
Senior Katie Korn
Junior Gabby Hicks
(the entire team and everyone on the roster are key players as well)
Goals for this season
Goals for this season are to be competitive in every match vs. 5A schools, to improve daily in order to win the District Tournament, and to have a completely positive season all around.
Team Motto
"Do it right, do it hard, or do it again!"
I'm loving the 2019 volleyball varsity team! They work hard, they are good teammates to one another, and they support one another. All 3 of our teams and coaches are doing a great job!
Tanner French
Key Players
Team captains:
Junior Zander Moore
Junior Chris Koch
Senior Teigen Edmundson
All three play important positions and are valuable leaders to our team.
Goals for the Season
With any Sandpoint high school team, the ultimate goal is bringing home a 1st place trophy. With this year's group, that is still the main goal, but I believe winning a game at state will be a good test for the boys to progress to.
Team Motto
I don't necessarily have one because I aim to have players within the program possess a large set of attributes that defines our mission to not only be a top student-athlete, but also be a servant to the community.
I am happy to be at the helm of this outstanding program, but it will take slow and steady steps to regain the dynasty that once was here in Sandpoint. I have a motivated staff that were part of those historic years, and we are positive in reattaining that pedigree in years to come.
Conor Baranski
Key players
So far we've had a number of players step up to become key parts of our success. The seniors, as a whole, have been excellent in leading by example with their work rate, and also their positivity.
Our seniors are:
Emi Lynch
Kiley Webster
Hannah Eddy
Shelby Mohler
Holly Kassa
Goals for this season
My goal for this program is always to have us playing high quality soccer as soon as possible, to be united as a team, and to steadily improve as soccer players and teammates throughout the season.
Team moto
This year, I'm focusing on our core values that the seniors and I created. Those are Commitment, Unity, Excellence, and Character.
If, as a team, we can come together to be united and work hard for ourselves and each other every day, there's no reason we won't be challenging for a state title.
Matt Brass
Key Runners
Junior Nikolai Braedt
Junior Jett Lucas
Senior Gabe Christman
Senior Seth Graham
Senior Brady Nelsen
Junior Keegan Nelson
Sophomore Trey Clark
Junior Tyler McNamee
Goals for this season
The goal for this season is to win 7th straight District I/II Regional Championship in both JV and Varsity. Another goal is to content for a top 4 place at State Championships.
Team Motto
"Rise as One"
We feel very strong about this years team. Their first races have been faster than any team in school history, and there is the drive and depth to be very successful. We have to battle one of the most competitive 4A environments in State history, but feel up to the challenge.
Angie Brass
Key Runners
Our captains, Bionce Vincent and Paige Davidson, are seniors. Our Varsity Women's Team is: Bionce Vincent, Paige Davidson, Megan Oulman (soph.), Ara Clark (fr.), Camille Neuder (jr.), Mackenzie Suhy-Gregoire (soph.), Quinn Hooper (jr.), Maddie Morgan (sr.).
Goals for this season
A goal each season is Regional Champion Teams for Men's and Women's Teams – both Varsity and JV. We will be going for our 7th consecutive Regional Title for Women's Varsity. Another goal each year is improvement and strengthening our pack running, as this is how teams improve, develop depth and grow!
Team motto
"Rise as One". (Women's Team also collectively utilizes a mantra of "For Each Other!")
Each season is filled with opportunity. The Women's Team is starting to gain depth after losing some big senior classes the last 2 years, looking to be Regional Champions and looking to be competitive at the state level.
Greg Jackson
Key team members
Everyone. Great motivated team, men's and women's. Captains Kate Bokowy (jr.), Mikayla Schoening (sr.), and Hayden Norling (jr.) have really been a huge part of that motivation.
Goals for this season
Goals for this season include personal improvement, district champions, and top 3 at state.
Team Motto
"Show up"
"Work smart"
"Work hard"
"Be consistent"
"Have fun"
Competitiveness throughout against 4A and 5A. We hope to be the standard all teams in Districts 1 & 2 are trying to catch.


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How Do Auto Insurance Company Ratings Work?
Auto insurance company ratings work a bit differently from rankings, which some often confuse. The ratings generally refer to the financial stability of the company. The ranking is how well they perform specific functions, like customer satisfaction.
Take a moment to enter your ZIP code into the FREE toolbox for great car insurance rates fast and easy!
Auto insurance company ratings are a direct result of the financial reports they provide. The three main auto insurance ratings companies include, A.M. Best, Standard and Poor's and JD Power. They each evaluate the way auto insurance companies meet or exceed their financial obligations.
They all differ in their methodology. A.M. Best, for example, uses a grade system based on the financial strength, credit rating, and debt rating. J.D. Power uses stars.
Regardless of how they arrive at their conclusion, they are highly respected and useful resources. Consumers consistently look to these rating companies. It helps them to decide if they want to do business with these auto insurance providers. They trust their reports and consider them to be honest and without bias.
An auto insurance company with exceptional ratings is bound to deliver results. If they are responsible and have a solid foundation, they can provide great benefits. This is one of the most important steps to research.
Different Factors Surrounding Auto Insurance Ratings
To determine how well an auto insurance company rates, there must be certain factors. These factors are indicators and the range varies greatly. However, the majority are quite measurable. They include, price, policy offerings, billing, and customer service.
When an auto insurance company submits these four factors to an evaluation, it is clear from the statistical data that some perform better than others do. It is also very easy to determine where the gap between companies that receive high scores lies between those that do not.
The car insurance providers take all of this into consideration. They look for ways to improve each year. If the issues are largely due to poor customer service, the changes can sometimes take effect in a short while.
However, if the problems are centered on price or policy offerings, it can take some time before improvement. This is because many times they must start from scratch.
A company that is attempting to find new and inventive ways to implement car insurance policies faces a challenge. They must also maintain a profit.
Finding the Best Auto Insurance Companies
The rating of an auto insurance company is a great place to start. You can definitely find detailed information. However, there are other ways to find the best company, besides ratings. You need personal experiences to factor into your equation.
Loyal customers are not easy to find in today's competitive auto insurance market, but they do exist. These are the testimonies that you need to review. For decades, Consumer Affairs has been the advocate for the American people. It is a free resource dedicated to providing consumers with information.
If you really want to find out how well your car insurance company performs, this is the best place to look. You can read actual complaints from real customers. If you do not see any or very few for your auto insurance provider, you can be assured that they are doing a good job. Their ratings are probably high. Their finances are strong due to the number of satisfied customers.
Remember, customers who have been treated unfairly are seldom quiet. They will look for any avenue available to expose the company at fault. This site is trusted. It has proven to be a wonderful outlet for anyone seeking justice. This is why so many people rely on the contents.
The Policy and Coverage is Different for Every State
The policy ratings and coverage are different for every state. As you review the information, remember some customers will expect certain levels of service. In addition, demographically speaking, it is not unusual for some regions to have higher expectations than others do.
For instance, if you reside in a state where there are over 500 licensed car insurance companies, the odds of you receiving personal service may be very low. Alternatively, a smaller state with only 60 licensed car insurance companies may have that opportunity.
Remember this while you review the ratings for each auto insurance provider you see. The complaints may be lower because of the expectations. Take your time to see if you can understand the main reason each customer felt unappreciated.
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Auto Insurance Companies Ratings for Customers
Customer feedback and financial reports provide the rating for the auto insurance industry. But what about the customers? How do the car insurance agencies see you? Everyone knows there are similar factors involved, like age and driving history. That is only one part. Each car insurance company also relies on a base price. They then introduce personal data into their calculations.
The person responsible for supplying the base price is called the actuary. The definition of an insurance actuary, as described in the Perdue University Department of Mathematics, is a professional analyzer of financial risks and consequences.
An actuary utilize mathematics, financial theory, and statistics to determine future events.
During the analysis, they create complex mathematic equations. They use them to arrive several numbers. Each number represents a specific risk group.
This is how car insurance companies develop their base rates. The role of an actuary is simple, but not really. Their knowledge is crucial to the auto insurance industry. They are responsible for analyzing the losses through the data they receive. They review geographical locations, variations of losses due to age groups and of course, the make, and models of vehicles.
Now you understand how base rates are calculated. It should be easy to see how car insurance companies calculate your individual rates. By using the information provided by the actuary, they need only ask a few questions about your age, type of vehicle and location.
Because data differs from state to state, so do the rates. Historically speaking, a young driver residing in New Jersey is more likely to pay high rates. The loss in this zip code may be higher than other states. It all depends on your demographic and the history associated with the amount of loss.
The Risk Groups
In the auto insurance industry, there are really only two risk groups, high and low. While some feel there is a medium risk group, the reality is car insurance is absolute. Auto insurance agencies seldom deal in anything other than black and white; there is no grey.
Low risk groups are the most valuable to car insurance providers. They fall into this category due to age, geographical location, and type of vehicle. Other factors include the driving history and number of claims filed.
A low risk driver is between the age of 30 and 50. They have a safe vehicle that does not appeal to thieves and only drive occasionally. They seldom use their car for work, choosing to commute by train or bus. They also carpool. They do not put more than 10,000 to 12,000 miles on the vehicle in a 12-month period.
Low risk drivers do not have at-fault accidents, which cause their car insurance premiums to increase. Instead, if they are involved in a collision, it is the fault of another driver and they pay for the repairs. They live in low-crime communities and their vehicles are parked in a garage overnight. They do not speed or receive traffic violations.
A high-risk driver is someone under the age of 30 or over the age of 70. Inexperience is the downfall for an under 30 driver. Slow reflexes for a senior over 70 places them in a high-risk group.
In addition, high-risk drivers use their vehicles to travel back and forth to work, more than 35 miles each day. They park their cars on the street and sometimes live in areas where crime is an issue. This makes their vehicles susceptible to theft and vandalism.
High-risk drivers often speed excessively and have multiple traffic violations. They may have had their license suspended or revoked. Their driving history is extensive, but not in a positive way.
Improve Your Insurance Ratings by Making Changes
To improve your ratings you will need to make some immediate changes. One of the quickest is to reduce the number of points on your license. This can be done by contacting your local Motor Vehicle Commission.
If you reside in New York, ask about the Point and Insurance Reduction Program. If you qualify and are successful in passing the course, you can have up to four points removed from your license and driving record. You may also receive a 10% discount off your car insurance premium.
Virtually every state has some type of driver improvement program. Start now and see how fast things can turn around. If you have a teen driver in the home, they are your high-risk driver because of age. Today, there are multiple programs dedicated for 16-19 year-old drivers. Make sure they enroll and you should see your rates decrease sharply.
Other changes may include where you park your vehicle. If you can afford to rent an overnight garage, do this immediately. Street parking is a way to keep your rates high. Install more anti-theft devices if you reside in a high crime area. You may not be able to change locations, but you can insure the safety of your vehicle.
Click here and enter your ZIP code into the FREE toolbox for a personal quote on great car insurance today!