Pathophysiological Response to SARS-CoV-2 Infection Detected by Infrared Spectroscopy Enables Rapid and Robust Saliva Screening for COVID-19.

Fourier transform infrared (FTIR) spectroscopy provides a (bio)chemical snapshot of the sample, and was recently used in proof-of-concept cohort studies for COVID-19 saliva screening. However, the biological basis of the proposed technology has not been established. To investigate underlying pathophysiology, we conducted controlled infection experiments on Vero E6 cells in vitro and K18-hACE2 mice in vivo.

Discovery and optimization of 2-phenyloxazole derivatives as diacylglycerol acyltransferase-1 inhibitors.

In a discovery effort to find safe and effective DGAT-1 inhibitors, we have identified 2-phenyloxazole 4-carboxamide 1 as a conformationally constrained analog of a hydrazide hit, which was previously identified from high-throughput screening. Further optimization of this series has led to chemically more stable 2-phenyloxazole-based DGAT-1 inhibitor 25 with improved solubility, cell-based activity, and pharmacokinetic properties. Compound 25 also demonstrated in vivo efficacy in a diet-induced obesity (DIO) rat model.

Cell-based screening approach for antitumor drug leads which exploits sensitivity differences between normal and cancer cells: identification of two novel cell-cycle inhibitors.

A cell-based in vitro screening approach for identification of antitumor drug leads that exploits the differential sensitivity between normal and cancer cells was developed. It is a three-step, high-throughput screen for antiproliferative and/or cytotoxic activity measured by a 7 day MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromidel assay using small panels of proliferating primary human cells and established cancer cell lines. Proof-of-concept experiments successfully identified 11 known cancer drugs randomly mixed with 5000 test compounds.

Quantification of telomerase activity by direct scintillation counting.

An improved telomerase assay was developed that allows direct quantification of the enzyme activity by scintillation counting of the labeled telomerase product. The assay measures the incorporation of 32P-dGTP into telomeric repeats synthesized at the 3' end of a biotinylated primer. Telomerase reaction product is separated from the reaction mix by streptavidin-coated magnetic beads and counted.

Myeloid differentiation and retinoblastoma phosphorylation changes in HL-60 cells induced by retinoic acid receptor- and retinoid X receptor-selective retinoic acid analogs.

The ability of subtypes of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) singly and in combination to elicit myeloid differentiation, G1/0-specific growth arrest, and retinoblastoma (RB) tumor suppressor protein dephosphorylation was determined in the human myeloblastic leukemia cell line HL-60 using subtype-selective retinoic acid (RA) analogs. RA analogs that selectively bind only to RARs (Am580 and/or TTNPB) or to RXRs (Ro 25-6603, SR11237, and/or SR11234) did not elicit the above-mentioned three cellular responses. In contrast, simultaneous treatment with both an RAR-selective ligand (Am580 or TTNPB) and an RXR-selective ligand (Ro 25-6603, SR11237, or SR11234) induced all three cellular processes.

Conformationally defined 6-s-trans-retinoic acid analogs. 2. Selective agonists for nuclear receptor binding and transcriptional activity.

We recently demonstrated in animal models that a new conformationally defined RA isomer (Vaezi et al. J. Med.

Binding of 9-cis-retinoic acid and all-trans-retinoic acid to retinoic acid receptors alpha, beta, and gamma. Retinoic acid receptor gamma binds all-trans-retinoic acid preferentially over 9-cis-retinoic acid.

Both 9-cis-retinoic acid (RA) and all-trans-RA (t-RA) compete for [3H]9-cis-RA binding to RA receptors (RAR alpha, beta, and gamma) in nucleosol fractions from transiently transfected COS-1 cells with IC50 values of approximately 12 and 5 nM, respectively. Curiously, 9-cis-RA competes for [3H]t-RA binding to mouse RAR alpha, beta, and gamma with IC50 values of 31, 8, and 60 nM, respectively, while t-RA itself does not exhibit such differential competition (IC50 values for RARs, 5 nM). A similar pattern is observed with human retinoic acid receptors (RARs).

Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.

Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs.

Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids.

The binding of endogenous retinoids and stereoisomers of retinoic acid (RA) to the retinoid nuclear receptors, RA receptor (RARs) and retinoid X receptors (RXRs), was characterized using nucleosol preparations from transiently transfected COS-1 cells. Among several stereoisomers of RA tested, including 7-cis-, 9-cis-, 11-cis-, 13-cis-, and all-trans-RA, only 9-cis-RA effectively competes with 9-cis-[3H]RA binding to the RXRs. Additionally, the endogenous retinoid trans-didehydro-RA (t-ddRA) does not interact with RXRs, whereas the 9-cis form of ddRA competes effectively.

9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha.

Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction.

The effect of culture conditions and toxicity on the Ames Salmonella/microsome agar incorporation mutagenicity assay.

Various methods were used to reconstruct an environment similar to a chemically bactericidal or bacteriostatic effect in the Ames mutagenicity assay in order to determine whether a false-positive result could occur. Neither population reduction, sonication, nor increased histidine produced an increase of spurious revertant colonies.

Use of rat/hamster S-9 mixture in the Ames mutagenicity assay.

Based on the findings of Nagao et al [1978] that phenacetin is negative in the standard Ames test with Aroclor induced rat S-9 and positive with hamster S-9, the test was performed with a mixture of rat/hamster S-9. Phenacetin was mutagenic with the mixture. The activity of the mixture was compared to the rat S-9 alone with low concentrations of 2-aminoanthracene (a strong promutagen for Salmonella typhimurium TA 1535, TA 100, TA 1537, and TA 98), nitrosopiperidine (a weak promutagen), and 1,2 epoxybutane (a weak, direct-acting mutagen).

An analysis of weak mutagens in the Ames assay.

Nitrosopiperidine, sodium nitrite and 1,2 epoxybutane were tested in the Ames agar incorporation assay in an attempt to establish exact criteria for detecting the activity of these weak mutagens. As regards minimum concentrations it was determined that at 500 microgram per plate a statistical analysis and several concentrations of the activating system are required in order to manifest an indication of the mutagenicity of 1,2 epoxybutane. The mutagenicity of sodium nitrite was detectable at 80 microgram/plate while nitrosopiperidine produced a 2.

Chromosomal effects of carcinogens and non-carcinogens on WI-38 after short term exposures with and without metabolic activation.

The human diploid fibroblast culture, WI-38 was analyzed for chromosomal damage after 24 h exposures to benzo(a)pyrene (BP), 3-methylcholanthrene (MCA), n-methyl-n'-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide (4NQO), pyrene and caffeine. A low concentration of 4NQO (0.15 micron) and MNNG (1.

The effect of methylxanthines on chromosomes of human lyphocytes in culture.

The effect of caffeine (I,3,7-trimethylxanthine), theophylline (I,3-dimethylxanthine), theobromine (3,7-dimethylxanthine), paraxanthine (I,7-dimethylxanthine) I-methylxanthine, 3-methylxanthine, and 7-methylxanthine added at the 48th h on the chromosomes of human lymphocytes in 72-h cultures has been investigated. Caffeine and the dimethylxanthines cause breakage at 750 mug/ml, with caffeine the most, and paraxanthine the least clastogenic. I-Methylxanthine and dimethylxanthines with a methyl group in the I-position are the most effective in depressing mitotic indices.