Shear Forces during Blast, Not Abrupt Changes in Pressure Alone, Generate Calcium Activity in Human Brain Cells

Blast-Induced Traumatic Brain Injury (bTBI) describes a spectrum of injuries caused by an explosive force that results in changes in brain function. The mechanism responsible for primary bTBI following a blast shockwave remains unknown. We have developed a pneumatic device that delivers shockwaves, similar to those known to induce bTBI, within a chamber optimal for fluorescence microscopy. Abrupt changes in pressure can be created with and without the presence of shear forces at the surface of cells. In primary cultures of human central nervous system cells, the cellular calcium response to shockwaves alone was negligible. Even when the applied pressure reached 15 atm, there was no damage or excitation, unless concomitant shear forces, peaking between 0.3 to 0.7 Pa, were present at the cell surface. The probability of cellular injury in response to a shockwave was low and cell survival was unaffected 20 hours after shockwave exposure

Streamlining bioactive molecular discovery through integration and automation

The discovery of bioactive small molecules is generally driven via iterative design–make–purify–test cycles. Automation is routinely harnessed at individual stages of these cycles to increase the productivity of drug discovery. Here, we describe recent progress to automate and integrate two or more adjacent stages within discovery workflows. Examples of such technologies include microfluidics, liquid-handling robotics and affinity-selection mass spectrometry. The value of integrated technologies is illustrated in the context of specific case studies in which modulators of targets, such as protein kinases, nuclear hormone receptors and protein–protein interactions, were discovered. We note that to maximize impact on the productivity of discovery, each of the integrated stages would need to have both high and matched throughput. We also consider the longer-term goal of realizing the fully autonomous discovery of bioactive small molecules through the integration and automation of all stages of discovery

Discovery of a thiamin-utilizing α-keto acid decarboxylase ribozyme: Implications for RNA’s role in primordial metabolism

Vitamins are hypothesized to be relics of an RNA World, and likely participants in an RNA-mediated primordial metabolism. If catalytic RNAs could harness vitamin cofactors to aid their function, in a manner similar to enzymes, it would enable ribozymes to catalyze a much larger set of chemical reactions. The cofactor thiamin diphosphate, a derivative of vitamin B1 (thiamin), is used by enzymes to catalyze difficult metabolic reactions, including decarboxylation of stable α-keto acids such as pyruvate. Here I report a ribozyme that uses free thiamin to decarboxylate a pyruvate-based suicide substrate (LnkPB). Thiamin conjugated to biotin was used to isolate catalytic individuals from a pool of random sequence RNAs attached to LnkPB. Analysis of a stable guanosine adduct obtained via digestion of an RNA sequence (clone dc4) showed the expected decarboxylation product. Discovery of a prototypic thiamin-utilizing ribozyme has implications for RNA\u27s role in orchestrating early metabolic cycles

The Formal Cross-Coupling of Amines and Carboxylic Acids to Form sp3–sp2 Carbon–Carbon Bonds

Amines and carboxylic acids are abundant building blocks for synthesis that classically are united to form an amide bond. To access new pockets of chemical space we are interested in the development of complementary amine–acid coupling reactions. In particular, the formation of carbon–carbon bonds by formal deamination and decarboxylation would be an impactful addition to the synthesis toolbox. Here we report a formal cross coupling of alkyl amines and aryl carboxylic acids to form C(sp3)–C(sp2) bonds following pre-activation of the amine–acid building blocks as a pyridinium salt and N-acyl-glutarimide respectively. Under nickel-catalyzed reductive cross-coupling conditions, a diversity of simple and complex substrates are united in good to excellent yield. High-throughput experimentation was essential to the development of the reaction, and to the discovery of performance-enhancing additives such as cyclic imides, RuCl3 and GaCl3. Preliminary mechanistic investigations suggest that RuCl3 supports the decarbonylation event, increasing reaction selectivity. Numerous amine or acid containing pharmaceuticals are successfully diversified under the optimized conditions

Pressurized intraperitoneal aerosol chemotherapy, a new surgical technique for the treatment of unresectable peritoneal carcinomatosis

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications

TRPV1 activation results in disruption of the blood–brain barrier in the rat

1. We have examined the role of TRPV1 activation in disrupting the blood–brain barrier by measuring the permeability of single pial venular capillaries in anaesthetized rats. 2. Capsaicin application to the brain surface resulted in increased permeability, maximal 2.1±0.12 × 10(−6) cm s(−1) (mean±s.e.m.) with log EC(50) −4.5±0.10. Substance P methyl ester gave a similar response (maximal 2.0±0.07, n=6, log EC(50) −4.8±0.07), but the selective NK(2) agonist, β-Ala(8)-NKA(4–10) peptide, had no effect. Although CGRP decreased the permeability of venules (log EC(50) 10.3±0.11), its receptor antagonist CGRP(8–37) had no effect on the response to capsaicin. 3. The TRPV1 antagonist capsazepine (1 mM) reduced the response to capsaicin (100 μM), from 1.78±0.15 to 0.63±0.10 (n=4). The NK(1) receptor antagonists GR205171 (100 μM) and SDZ NKT 376 (1 mM) also reduced the response to capsaicin (from 1.75±0.14 to 0.46±0.08; n=6, and from 1.85±0.13 to 0.48±0.05; n=5, respectively), indicating that capsaicin acts via TRPV1 in series with NK(1). 4. Starch microspheres were used to produce transient focal ischaemia. Permeability was increased on reperfusion to a greater extent and more rapidly in vessels with diameter greater than 40 μm than those less than 15 μm. Capsazepine given intraperitoneally during ischaemia reduced the permeability increase in small venules from 5.9±0.3 to 2.4±0.1, and from 11.4±0.8 to 5.1±0.9 in large venules. 5. In conclusion, the TRPV1 receptor is active in the brain microvasculature and has its permeability-increasing effect via substance P. It also plays a role in the immediate blood–brain barrier disruption following ischaemia–reperfusion