Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer

Chimeric antigen receptor T (CAR‐T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR‐T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody‐based switch. Herein, we apply this approach to Her2‐expressing breast cancers by engineering switch molecules through site‐specific incorporation of FITC or grafting of a peptide neo‐epitope (PNE) into the anti‐Her2 antibody trastuzumab (clone 4D5). We demonstrate that both switch formats can be readily optimized to redirect CAR‐T cells (specific for the corresponding FITC or PNE) to Her2‐expressing tumor cells, and afford dose‐titratable activation of CAR‐T cells ex vivo and complete clearance of the tumor in rodent xenograft models. This strategy may facilitate the application of immunotherapy to solid tumors by affording comparable efficacy with improved safety owing to switch‐based control of the CAR‐T response.     

Manufacturing Man‐Made Magnetosomes: High‐Throughput In Situ Synthesis of Biomimetic Magnetite Loaded Nanovesicles

A new synthetic method for the production of artificial magnetosomes, i.e., lipid‐coated vesicles containing magnetic nanoparticles, is demonstrated. Magnetosomes have considerable potential in biomedical and other nanotechnological applications but current production methods rely upon magnetotactic bacteria which limits the range of sizes and shapes that can be generated as well as the obtainable yield. Here, electrohydrodynamic atomization is utilized to form nanoscale liposomes of tunable size followed by electroporation to transport iron into the nanoliposome core resulting in magnetite crystallization. Using a combination of electron and fluorescence microscopy, dynamic light scattering, Raman spectroscopy, and magnetic susceptibility measurements, it is shown that single crystals of single‐phase magnetite can be precipitated within each liposome, forming a near‐monodisperse population of magnetic nanoparticles. For the specific conditions used in this study the mean particle size is 58 nm (±8 nm) but the system offers a high degree of flexibility in terms of both the size and composition of the final product.

     

Four Classes of Structurally Unusual Peptides from Two Marine-Derived Fungi: Structures and Bioactivities

The structures and biological properties of peptides produced by two genera of marine-derived fungi, an atypical Acremonium sp., and a Metarrhizium sp., were explored. The Acremonium strain was isolated from a marine sponge and has previously been shown by our group to produce peptides from the efrapeptin and RHM families. The isolation and structural elucidation of the new linear pentadecapeptides efrapeptins Eα (1) and H (2) and N-methylated octapeptides RHM3 (3) and RHM4 (4) were carried out through a combination of 1D and 2D NMR techniques and tandem MS. Additional known efrapeptins E, F, and G and the known syctalidamides A and B were also isolated. The absolute configurations of 1-4 are proposed to be the same as the original compound families. The marine sponge-derived Metarrhizium sp. was shown to produce destruxin cyclic depsipeptides including A, B, B2, desmethyl B, E chlorohydrin, and E2 chlorohydrin. Efrapeptins Eα (1), F, and G each displayed IC₅₀s of 1.3 nM against H125 cells, and destruxin E2 chlorohydrin displayed an IC₅₀ of 160 nM against HCT-116 cells. An initial therapeutic assessment suggested a continuous (168 h) exposure of at least 2 ng/mL, or a daily (24 h) exposure of at least 300 ng/mL for H125 cells treated with efrapeptin G, and a continuous (168 h) exposure of at least 190 ng/mL for HCT-116 cells treated with destruxin E2 chlorohydrin, will cause 90% tumor cell death in vitro.     

Functionalization of bicyclic 2-pyridones targeting pilus biogenesis in uropathogenic Escherichia coli

Substituted bicyclic 2-pyridones, termed pilicides, prevent pilus assembly in uropathogenic Escherichia coli. Based on the bioactive methyl ester protected 2-pyridone 4, further functionalization at C-6 has yielded a set of new compounds, which have been evaluated for their ability to inhibit pilus formation in uropathogenic E. coli. The key intermediate in the synthesis was formylated 2-pyridone 5, which could be obtained via a Vilsmeier reaction. This versatile intermediate was converted into secondary and tertiary amines via reductive amination and could also be converted to other interesting functionalities using simple chemical transformations.     

Thermodynamics of carbon monoxide adsorption on polycrystalline titania studied by static adsorption microcalorimetry

The adsorption of CO on polycrystalline TiO2 was investigated by static adsorption microcalorimetry. The initial differential heat of adsorption (qdiff,0) of CO on polycrystalline titania is 40 kJ/mol, and the standard adsorption entropy (Deltas0) is -104 J mol(-1) K(-1). These results are consistent with those derived from temperature-programmed desorption and FTIR results in the literature. The good reproducibility of the isotherms and the stable qdiff indicate that the lattice oxygen and hydroxyl groups on titania surface are basically not reactive to adsorbed CO.     

Polymer-induced synthesis of stable gold and silver nanoparticles and subsequent ligand exchange in water

A simple, inexpensive, single-step synthesis of gold and silver nanoparticles using poly(allylamine) (PAAm) as a reducing and stabilizing agent is reported. The synthetic process was carried out in aqueous solution, making the method versatile and environmentally friendly. The synthesized polymer-stabilized nanoparticles are stable in water without particle aggregation at room temperature for at least a month. We demonstrate successful ligand exchange on the polymer-stabilized gold nanoparticles (AuNPs) with a variety of omega-functionalized acid-, alcohol-, amine-, and biotin-terminated alkylthiols. The methodologies, including ligand exchange, also are applicable for the generation of finely dispersed silver nanoparticles. The synthesized gold and silver nanoparticles are characterized by UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The different ligand-stabilized AuNPs are also analyzed by Fourier transform infrared (FTIR) spectroscopy.     

Kibdelones: novel anticancer polyketides from a rare Australian actinomycete

The kibdelones are a novel family of bioactive heterocyclic polyketides produced by a rare soil actinomycete, Kibdelosporangium sp. (MST-108465). Complete relative stereostructures were assigned to kibdelones A-C (1-3), kibdelone B rhamnoside (5), 13-oxokibdelone A (7), and 25-methoxy-24-oxokibdelone C (8) on the basis of detailed spectroscopic analysis and chemical interconversion, as well as mechanistic and biosynthetic considerations. Under mild conditions, kibdelones B (2) and C (3) undergo a facile equilibration to kibdelones A-C (1-3), while kibdelone B rhamnoside (5) equilibrates to a mixture of kibdelone A-C rhamnosides (4-6). A plausible mechanism for this equilibration is proposed and involves air oxidation, quinone/hydroquinone redox transformations, and a choreographed sequence of keto/enol tautomerizations that aromatize ring C via a quinone methide intermediate. Kibdelones exhibit potent and selective cytotoxicity against a panel of human tumor cell lines and display significant antibacterial and nematocidal activity.     

Modulation of bacterial quorum sensing with synthetic ligands: systematic evaluation of N-acylated homoserine lactones in multiple species and new insights into their mechanisms of action

Bacteria use a language of low molecular weight ligands to assess their population densities in a process called quorum sensing. This chemical signaling process plays a pivotal role both in the pathogenesis of infectious disease and in beneficial symbioses. There is intense interest in the development of synthetic ligands that can intercept quorum-sensing signals and attenuate these divergent outcomes. Both broad-spectrum and species-selective modulators of quorum sensing hold significant value as small-molecule tools for fundamental studies of this complex cell-cell signaling process and for future biomedical and environmental applications. Here, we report the design and synthesis of focused collections of non-native N-acylated homoserine lactones and the systematic evaluation of these approximately 90 ligands across three Gram-negative bacterial species: the pathogens Agrobacterium tumefaciens and Pseudomonas aeruginosa; the model symbiont Vibrio fischeri. This study is the first to report and compare the activities of a set of ligands across multiple species and has revealed some of the most potent synthetic modulators of quorum sensing to date. Moreover, several of these ligands exhibit agonistic or antagonistic activity in all three species, while other ligands are only active in one or two species. Analysis of the screening data revealed that at least a subset of these ligands modulate quorum sensing via a partial agonism mechanism. We also demonstrate that selected ligands can either inhibit or promote the production of elastase B, a key virulence factor in wild-type P. aeruginosa, depending on their concentrations. Overall, this work provides broad insights into the molecular features required for small-molecule inhibition or activation of quorum sensing in Gram-negative bacteria. In addition, this study has supplied an expansive set of chemical tools for the further investigation of quorum-sensing pathways and responses.     

The duplicate method of uncertainty estimation: are eight targets enough?

This paper presents methods for calculating confidence intervals for estimates of sampling uncertainty (s(samp)) and analytical uncertainty (s(anal)) using the chi-squared distribution. These uncertainty estimates are derived from application of the duplicate method, which recommends a minimum of eight duplicate samples. The methods are applied to two case studies--moisture in butter and nitrate in lettuce. Use of the recommended minimum of eight duplicate samples is justified for both case studies as the confidence intervals calculated using greater than eight duplicates did not show any appreciable reduction in width. It is considered that eight duplicates provide estimates of uncertainty that are both acceptably accurate and cost effective.