Journal: Pharmaceuticals

Article Title: Chemical Composition, Biological Activity, and Potential Uses of Oregano ( Origanum vulgare L.) and Oregano Essential Oil

doi: 10.3390/ph18020267

Figure Lengend Snippet: Antimicrobial activity of O. vulgare L.

Article Snippet: Supercritical Fluid Extraction (SFE), leaf and flower extracts , Wild oregano ( O. vulgare ) purchased from Alfred Galke GmbH (Samtgemeinde Bad Grund, Germany) , B. subtilis S. aureus —MRSA , - , , [ ] .

Techniques: Activity Assay, Inhibition, Extraction

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: Leukemic progenitor assays replicate patterns of patient response to DRD2 antagonist TDZ (A) Leukemic blast counts were monitored before and after treatment with TDZ as a monotherapy in 11 relapsed or refractory AML patients (NCT02096289). Percentage change in blasts in the peripheral blood on day 5 versus day 1 is reported after treatment with TDZ. Percentage change in BM blast content is reported for trial patient 2T and 9T in the absence of circulating blast values. Partial response and progressive disease patterns are indicated as “response” and “no response” and are illustrated as gray versus black silhouettes, respectively. (B) Candidate trial patient samples from either response group were interrogated for progenitor content at baseline (day 1) and after clinical exposure to TDZ (day 5) using limiting dilution analysis (LDA). Leukemic progenitor frequency was estimated by LDA analysis and normalized to day 1. Baseline progenitor frequency of 1 in 75,000 cells was considered the progenitor frequency for trial patient 3T at day 1 since an absolute frequency was not achieved with the analysis of 75,000 cells for this patient. Dashed lines represent 95% confidence interval. Raw colony counts are shown in Figure S1 D. (C) Trial patient samples obtained at baseline were exposed to TDZ (“+TDZ”) versus DMSO control (“−TDZ”) for 24 h, followed by analysis of progenitor cell function in CFU assays. Data are normalized to DMSO control. Before normalization, the average DMSO control values were 79 and 2 colonies for trial patients 1T and 8T (non-responders) and 61, 28, 56, 2, 11, 28, and 14 colonies for trial patients 2T, 4T, 6T, 7T, 9T, 10T, and 11T, respectively (responders). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (D) Correlation between percentage change in leukemic blast levels versus percentage change in progenitor capacity (demonstrated in C). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (E) Schematic illustrating in vivo AML xenografts were treated with TDZ (22.5 mg/kg “+”) or 30% captisol (vehicle control “−”) in vivo , followed by analysis of leukemic chimerism levels (F), gene expression analysis (G), and progenitor CFU assays (H). (F) Leukemic chimerism levels (hCD45 + CD33 + ) after in vivo treatment with TDZ relative to vehicle control (“−“). Symbols represent individual recipient mice. ∗p = 0.05 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. (G) Gene set enrichment analysis (GSEA) plot of a gene set representing cellular pathways associated with AML (Kyoto Encyclopedia of Genes and Genomes [KEGG]; ), applied to transcription profiles from TDZ-treated versus vehicle control-treated AML xenografts derived from AMLs 1, 3, and 4. (H) Human AML grafts were recovered from mouse BM and evaluated in progenitor CFU assays. Symbols represent individual CFU wells, plated using cells recovered from a minimum of 2 individual mice per condition. Colony-forming capacity for AML 4 was not detectable with up to 150,000 human cells assayed. ∗∗∗p ≤ 0.0001 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. Data are summarized as means ± SEMs. See also and and .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Control, Cell Function Assay, In Vivo, Gene Expression, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: DRD2 expression profiles reliably predict functional response to DRD antagonism (A) DRD2 expression patterns within leukemic CD34 + cells. Dotted line represents FMO control (left). Comparison of DRD2 protein levels in CD34 + cells of AML patient versus healthy donor samples (right). Healthy donor samples consist of cord blood (n = 3), adult mobilized peripheral blood (n = 3), and adult non-mobilized peripheral blood (n = 5). Blue versus red shading indicates the threshold of normal versus aberrant DRD2 levels. ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test). (B) DRD2 protein expression within CD34 + subset of low versus intermediate-/high-risk AML patients based on ELN criteria. Dots represent individual AML patients. ∗∗p = 0.006 (Mann-Whitney U test). (C) Mononuclear cells (MNCs) isolated from healthy donors and AML patients were treated with TDZ or DMSO (vehicle control, “−”) for 24 h and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n = 3–10 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). Source data can be found in . (D) Proliferative capacity of leukemic versus healthy progenitor units was compared after in vitro exposure to TDZ for 24 h. Cell number output per colony was evaluated by custom scripts as a measure of proliferation. (E) Representative FACS plots demonstrate gating strategy to purify DRD2 + vs DRD2 − human AML cells (left) and human leukemic chimerism in mice transplanted with 1 million DRD2 + or DRD2 − human AML cells. (F) Western blot of DRD2, activated CREB (p-CREB at Ser-133), and histone H3 (loading control) in DRD2 + versus DRD2 − sorted fractions illustrated in (E). (G) Representative whole-well CFU images after treatment with dopamine (DA) at physiological levels (10 nM) versus DMSO control (-DA). (H) Progenitor cell activity was quantified in n = 6 distinct AML patients after treatment with physiological levels of DA (10–100 nM) relative to DMSO control. n = 2–3 CFU wells per AML sample. ∗p = 0.03 (unpaired t test). (I) Circulating DA levels in healthy individuals (n = 8 healthy adult peripheral blood (PB) and 11 cord blood (CB) samples, as hollow circles and squares, respectively) versus n = 11 AML patients (black circles). ∗p = 0.04 (unpaired t test). Data are summarized as means ± SEMs relative to vehicle control. See also Figure S3 and .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Expressing, Functional Assay, Control, Comparison, MANN-WHITNEY, Isolation, In Vitro, Western Blot, Activity Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: cAMP elevation is associated with leukemic progenitor suppression (A) Trial patients (NCT02096289) were exposed to TDZ in vitro , followed by analysis of cAMP level changes. Trial patients with abundant cell numbers available were prioritized for this analysis, including patients 1T and 3T from non-responders, and patients 7T, 10T, and 11T for responders. n = 3–6 technical replicates per condition. ∗p ≤ 0.05 (unpaired t test). (B) cAMP levels in response to DRD1 agonist (SKF 38393) relative to DMSO control. n ≥ 4 replicates across OCI-AML3 and NB4 cell lines. ∗∗p = 0.008 (Mann-Whitney U test). Progenitor response was evaluated after treatment with DRD1 agonist (SKF 38393) relative to DMSO control. n = 2–3 CFU replicates per AML sample (n = 5 AML samples total). (C) cAMP levels in response to anti-DRD1 antibody alone or in combination with DRD1 antagonist (SCH 23390) in AML cell lines OCI-AML3 and NB4. n = 2–4 replicates per condition. (D) Western blot of activated CREB (p-CREB at Ser-133) after exposure to anti-DRD1 antibody in OCI-AML3 cell line (top). Western blot of activated CREB (p-CREB at Ser-133) exposure to TDZ in OCI-AML3 and NB4 cell lines (bottom). (E) MNCs isolated from healthy donors and AML patients were treated with anti-DRD1 antibody or immunoglobulin G (IgG) control (“−“) for 30 min, and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n =3–7 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). (F) Cytospin preparations of AML cells from patient 2 after exposure to TDZ or vehicle control (DMSO). Yellow arrowheads indicate evidence of hematopoietic maturation (increased cell size, reduced nuclear:cytoplasmic ratio, increased cytoplasmic vacuolization). (G) FACS plot showing expression of granulocytic cell marker (CD15) after in vitro exposure to TDZ or DMSO control (“-TDZ“) in representative DRD2 lo and DRD2 + AML samples. CD15 frequencies were quantified for AMLs 1, 6, and 7 (n = 2 technical replicates per AML sample in each condition). ∗∗p = 0.002 (Mann-Whitney U test). (H) AML patient cells were treated with TDZ or DMSO for 24 h and evaluated in progenitor CFU assays, followed by analysis of re-plating capacity. ∗∗p = 0.004 (unpaired t test). (I) cAMP levels in response to TDZ relative to DMSO control. DRD2 + AML includes AML 1, 6, OCI-AML3, and NB4. DRD2 − AML and healthy controls include AML 12 and 3 CB samples, respectively. n ≥ 3 replicates per condition. ∗∗∗p = 0.007 (unpaired t test). (J) cAMP levels in response to forskolin (FSK) relative to DMSO control. n = 6 replicates per condition, across 1 AML cell line and n = 2 healthy donor cells. ∗∗∗p ≤ 0.0001 (unpaired t test). Data are summarized as means ± SEMs. See also Figure S4 .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: In Vitro, Control, MANN-WHITNEY, Western Blot, Isolation, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: TDZ + displays superior potency and reduced toxicity relative to TDZ (A) Chiral separation of TDZ using supercritical fluid chromatography. Chromatograms show the first and second peaks, indicating the (−) enantiomer “TDZ − ” and (+) enantiomer “TDZ + ,” respectively. Purified enantiomers were evaluated for effects on cAMP levels (B), and in progenitor CFU assays (C and D). (B) cAMP levels were evaluated after in vitro treatment with TDZ and its two enantiomers in AML cell lines (NB4 and OCI-AML3) and primary patient cells (AMLs 2, 9, and 27). Symbols represent individual CFU wells. ∗p ≤ 0.05 and ∗∗p ≤ 0.01 (unpaired t test). (C) AML patient cells were exposed to TDZ and its 2 enantiomers for 24 h in a dose-response assay in vitro , and subsequently evaluated in progenitor CFU assays. Bar graphs summarize half-maximal inhibitory concentration (IC 50 ) in progenitor CFU assays performed with AML patient cells. ∗∗p ≤ 0.01 and ∗∗∗p ≤ 0.001 (paired t test). (D) Comparison of TDZ and TDZ + IC 50 for individual AML patients in CFU assays (represented in C). ∗∗p = 0.004 (paired t test). (E) A 30-min monitoring of QTc level changes after intravenous injection of TDZ and TDZ + in a guinea pig assay (n = 5 animals per cohort). QTc increases over 5% were considered indicators of safety risks. No group averages were statistically different from baseline values (repeated-measures ANOVAs). (F) DRD2 transcript (Gene: 1813) was analyzed from TGCA (tumor and normal tissue) and GTEx (normal tissue) RNA-sequencing projects. Data points represent normalized gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) for DRD2 from individual cancer patients or healthy donors. ∗∗∗p ≤ 0.001 and ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test), ∗∗p = 0.01 (Kolmogorov-Smirnov test). Data are summarized as means ± SEMs. See also Figure S5 .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Supercritical Fluid Chromatography, Purification, In Vitro, Concentration Assay, Comparison, Injection, RNA Sequencing, Gene Expression, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet:

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Recombinant, Binding Assay, Purification, Microarray, Software, Imaging