Total Synthesis of Thiamyxins A-C and Thiamyxin E, a Potent Class of RNA-Virus-Inhibiting (Cyclo)depsipeptides.

We present the first total synthesis of the thiamyxins A-C and the now fully characterized thiamyxin E, an interesting class of thiazole- and thiazoline-rich depsipeptides with diverse antiviral activity. The synthesis features a parallel closing of two methyl thiazoline units, with low epimerization of the very labile adjacent stereocenter. It also includes the three-step synthesis of an uncommon hydroxy acid and the oxidation-free elimination of a phenylselenide to form a dehydroalanine moiety. The exploitation of the acid-labile stereocenter at the isoleucine moiety and the reopening of the macrolactones gave access to the four thiamyxins with good yields and diastereomeric purities from a single precursor. The modular total synthesis allows further testing of the biological activity and gives opportunities to explore the pharmacophore and antiviral target through derivatization.

Bioprinting on Live Tissue for Investigating Cancer Cell Dynamics.

A challenge in cancer research is the lack of physiologically responsive in vitro models that enable tracking of cancer cells in tissue-like environments. A model that enables real-time investigation of cancer cell migration, fate, and function during angiogenesis does not exist. Current models, such as 2D or 3D in vitro culturing, can contain multiple cell types, but they do not incorporate the complexity of intact microvascular networks. The objective of this study was to establish a tumor microvasculature model by demonstrating the feasibility of bioprinting cancer cells onto excised mouse tissue. Inkjet-printed DiI+ breast cancer cells on mesometrium tissues from C57Bl/6 mice demonstrated cancer cells' motility and proliferation through time-lapse imaging. Colocalization of DAPI+ nuclei confirmed that DiI+ cancer cells remained intact postprinting. Printed DiI+ 4T1 cells also remained viable after printing on Day 0 and after culture on Day 5. Time-lapse imaging over 5 days enabled tracking of cell migration and proliferation. The number of cells and cell area were significantly increased over time. After culture, cancer cell clusters were colocalized with angiogenic microvessels. The number of vascular islands, defined as disconnected endothelial cell segments, was increased for tissues with bioprinted cancer cells, which suggests that the early stages of angiogenesis were influenced by the presence of cancer cells. Bioprinting cathepsin L knockdown 4T1 cancer cells on wild-type tissues or nontarget 4T1 cells on NG2 knockout tissues served to validate the use of the model for probing tumor cell versus microenvironment changes. These results establish the potential for bioprinting cancer cells onto live mouse tissues to investigate cancer microvascular dynamics within a physiologically relevant microenvironment. Impact statement To keep advancing the cancer biology field, tissue engineering has been focusing on developing in vitro tumor biomimetic models that more closely resemble the native microenvironment. We introduce a novel methodology of bioprinting exogenous cancer cells onto mouse tissue that contains multiple cells and systems within native physiology to investigate cancer cell migration and interactions with nearby microvascular networks. This study corroborates the manipulation of different exogenous cells and host microenvironments that impact cancer cell dynamics in a physiologically relevant tissue. Overall, it is a new approach for delineating the effects of the microenvironment on cancer cells and vice versa.

Comparative ecotoxicity of potential biofuels to water flea (Daphnia magna), zebrafish (Danio rerio) and Chinese hamster (Cricetulus griseus) V79 cells.

The ecotoxicity of two biofuel candidates (1­octanol and 2­butanone) was investigated by an integrative test strategy using three bioassays: the acute immobilisation test with water flea (D. magna), the fish embryo acute toxicity test with zebrafish (Danio rerio) and the in vitro micronucleus assay with Chinese hamster (Cricetulus griseus) V79 cells. The median effective concentration (EC50) values were 14.9±0.66mgL-1 for 1­octanol, and 2152.1±44.6mgL-1 for 2­butanone in the D. magna test. Both 1­octanol and 2­butanone caused teratogenic and lethal effects on zebrafish embryos, while exposure to 1­octanol significantly induced these effects at concentrations ≥2.0mgL-1. These results indicate that 1­octanol exert much higher ecotoxicity than 2­butanone to D. magna and zebrafish embryos. Moreover, both 1­octanol and 2­butanone did not cause significant genotoxic effects, while their metabolites significantly induced micronuclei in V79 cells. The present study proposed an integrative test approach to evaluate the potential ecotoxicity of biofuels using simple, quick and inexpensive bioassays.

Efficacy, pharmacokinetics, and metabolism of tetrahydroquinoline inhibitors of Plasmodium falciparum protein farnesyltransferase.

New antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparum PFT and are effective at killing P. falciparum in vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei-infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Zn-binding N-methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N-methyl-imidazole from dealkylation.

Protein farnesyltransferase inhibitors exhibit potent antimalarial activity.

New therapeutics to combat malaria are desperately needed. Here we show that the enzyme protein farnesyltransferase (PFT) from the malaria parasite Plasmodium falciparum (P. falciparum) is an ideal drug target. PFT inhibitors (PFTIs) are well tolerated in man, but are highly cytotoxic to P. falciparum. Because of their anticancer properties, PFTIs comprise a highly developed class of compounds. PFTIs are ideal for the rapid development of antimalarials, allowing "piggy-backing" on previously garnered information. Low nanomolar concentrations of tetrahydroquinoline (THQ)-based PFTIs inhibit P. falciparum PFT and are cytotoxic to cultured parasites. Biochemical studies suggest inhibition of parasite PFT as the mode of THQ cytotoxicity. Studies with malaria-infected mice show that THQ PFTIs dramatically reduce parasitemia and lead to parasite eradication in the majority of animals. These studies validate P. falciparum PFT as a target for the development of antimalarials and describe a potent new class of THQ PFTIs with antimalaria activity.

Resistance to a protein farnesyltransferase inhibitor in Plasmodium falciparum.

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. Inhibition of protein farnesyltransferase (PFT) is lethal to the pathogenic protozoa Plasmodium falciparum. Parasites were isolated that were resistant to BMS-388891, a tetrahydroquinoline (THQ) PFT inhibitor. Resistance was associated with a 12-fold decrease in drug susceptibility. Genotypic analysis revealed a single point mutation in the beta subunit in resistant parasites. The resultant tyrosine 837 to cysteine alteration in the beta subunit corresponded to the binding site for the THQ and peptide substrate. Biochemical analysis of Y837C-PFT demonstrated a 13-fold increase in BMS-388891 concentration necessary for inhibiting 50% of the enzyme activity. These data are consistent with PFT as the target of BMS-388891 in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents for effective therapy.

Preoperative intravenous midazolam: benefits beyond anxiolysis.

STUDY OBJECTIVE: To evaluate the effect of midazolam on the global perioperative experience, including patient satisfaction, postoperative nausea and vomiting, postoperative pain, and perioperative anxiety and amnesia. DESIGN: Prospective, randomized, placebo-controlled study. SETTING: Ambulatory surgical center affiliated with a tertiary-care hospital. PATIENTS: 88 ASA physical status I, II, and III patients scheduled for outpatient surgery. INTERVENTIONS: Patients were randomized into two groups to receive either 0.04 mg/kg of midazolam or placebo intravenously (IV) 20 minutes preoperatively. MEASUREMENTS: Perioperative measurements included blood pressure, heart rate, and oxygen saturation and the patient's level of anxiety; type of anesthetic administered; the anesthesiologist's guess at the treatment arm; perioperative dosages of fentanyl, morphine, and ondansetron; recovery room length of stay; frequency of nausea and vomiting, and level of postoperative pain in the 24 hours after surgery; the patient's overall satisfaction with the anesthetic, and whether the patient would recommend the premedication to a friend. MAIN RESULTS: Patient demographics, type of surgery/anesthesia, vital signs, case duration, recovery duration, and postoperative pain were all similar between the midazolam and placebo groups. As expected, IV midazolam was an effective anxiolytic. There was no evidence of retrograde amnesia. Fewer patients in the midazolam group suffered from postoperative nausea than did those in the placebo group (25%vs. 50%;p = 0.03), despite receiving similar perioperative antiemetic and opioid administration. Similarly, fewer patients in the midazolam group experienced postoperative vomiting than placebo group patients (8%vs. 21%), although this difference did not reach statistical significance. Only 42% of patients in the placebo group would recommend their premedication to a friend, compared with 85% of patients in the midazolam group (p < 0.001). CONCLUSIONS: In addition to the known anxiolytic effects of midazolam, midazolam premedication is an effective way to reduce the frequency of postoperative nausea, and perhaps vomiting, and increase patient satisfaction.