Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ∼0.9 in as little as six-weeks with a mean firing rate of ∼13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of blood-brain barrier breakdown by using human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ~0.9 in as little as six-weeks with a mean firing rate of ~13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of sporadic Alzheimer's disease by mimicking blood-brain barrier breakdown using a human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

Amyloid beta acts synergistically as a pro-inflammatory cytokine.

The amyloid beta (Aß) peptide is believed to play a central role in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. However, the natural, evolutionarily selected functions of Aß are incompletely understood. Here, we report that nanomolar concentrations of Aß act synergistically with known cytokines to promote pro-inflammatory activation in primary human astrocytes (a cell type increasingly implicated in brain aging and AD). Using transcriptomics (RNA-seq), we show that Aß can directly substitute for the complement component C1q in a cytokine cocktail previously shown to induce astrocyte immune activation. Furthermore, we show that astrocytes synergistically activated by Aß have a transcriptional signature similar to neurotoxic "A1" astrocytes known to accumulate with age and in AD. Interestingly, we find that this biological action of Aß at low concentrations is distinct from the transcriptome changes induced by the high/supraphysiological doses of Aß often used in in vitro studies. Collectively, our results suggest an important, cytokine-like function for Aß and a novel mechanism by which it may directly contribute to the neuroinflammation associated with brain aging and AD.

Application of Mycobacterium smegmatis as a surrogate to evaluate drug leads against Mycobacterium tuberculosis.

Discovery of new anti-tuberculosis (TB) drugs is a time-consuming process due to the slow-growing nature of Mycobacterium tuberculosis (Mtb). A requirement of biosafety level 3 (BSL-3) facility for performing research associated with Mtb is another limitation for the development of TB drug discovery. In our screening of BSL-1 Mycobacterium spp. against a battery of TB drugs, M. smegmatis (ATCC607) exhibits good agreement with its drug susceptibility against the TB drugs under a low-nutrient culture medium (0.5% Tween 80 in Middlebrook 7H9 broth). M. smegmatis (ATCC607) enters its dormant form in 14 days under a nutrient-deficient condition (a PBS buffer), and shows resistance to a majority of TB drugs, but shows susceptibility to amikacin, capreomycin, ethambutol, and rifampicin (with high concentrations) whose activities against non-replicating (or dormant) Mtb were previously validated.

Semisynthesis of an Anticancer DPAGT1 Inhibitor from a Muraymycin Biosynthetic Intermediate.

We have explored a method to convert a muraymycin biosynthetic intermediate 3 to an anticancer drug lead 2 for in vivo and thorough preclinical studies. Cu(OAc)2 forms a stable complex with the amide 4 and prevents electrophilic reactions at the 2-((3-aminopropyl)amino)acetamide moiety. Under the present conditions, the desired 5″-primary amine was selectively protected with (Boc)2O to yield 6. The intermediate 6 was converted to 2 in two steps with 90% yield.

An antimycobacterial pleuromutilin analogue effective against dormant bacilli.

Pleuromutilin is a promising pharmacophore to design new antibacterial agents for Gram-positive bacteria. However, there are limited studies on the development of pleuromutilin analogues that inhibit growth of Mycobacterium tuberculosis (Mtb). In screening of our library of pleuromutilin derivatives, UT-800 (1) was identified to kill replicating- and non-replicating Mtb with the MIC values of 0.83 and 1.20 µg/mL, respectively. UT-800 also kills intracellular Mtb faster than rifampicin at 2× MIC concentrations. Pharmacokinetic studies indicate that 1 has an oral bioavailability with an average F-value of 27.6%. Pleuromutilin may have the potential to be developed into an orally administered anti-TB drug.

Novel FR-900493 Analogues That Inhibit the Outgrowth of Clostridium difficile Spores.

The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 µM concentration.

A New Combination of a Pleuromutilin Derivative and Doxycycline for Treatment of Multidrug-Resistant Acinetobacter baumannii.

Multidrug-resistant (MDR) Acinetobacter baumannii is one of the most difficult Gram-negative bacteria to treat and eradicate. In a cell-based screening of pleuromutilin derivatives against a drug sensitive A. baumannii strain, new molecules (2-4) exhibit bacteriostatic activity with 3.13 µg/mL concentration and 1 shows bactericidal activity with an MBC of 6.25 µg/mL. The pleuromutilin derivative 1 displays strong synergistic effects with doxycycline in a wide range of concentrations. A 35/1 ratio of 1 and doxycycline (1-Dox 35/1) kills drug susceptible A. baumannii with the MBC of 2.0 µg/mL and an MDR A. baumannii with the MBC of 3.13 µg/mL. In vitro anti-Acinetobacter activity of 1-Dox 35/1 is superior to that of clinical drugs such as tobramycin, tigecycline, and colistin. The efficacy of 1-Dox 35/1 is evaluated in a mouse septicemia model; treatment of the infected C57BL/6 mice with 1-Dox 35/1 protects from lethal infection of A. baumannii with an ED50 value of <2.0 mg/kg.