Biomimetic droplets for artificial engagement of living cell surface receptors: the specific case of the T-cell

Liquid colloids, in the form of droplets grafted with specific biomolecules, are emerging as potential biomimetic systems. Here we show for the first time the possibility of forming hybrid conjugates between an advanced living cell model, the T-cell of the Jurkat cell line, and a specifically grafted droplet. Using T-cells expressing a fluorescent chimeric protein associated with the TCR/CD3 complex and fluorescent ligand-grafted droplets, we demonstrate formation of an interfacial contact concentrated in linking molecules, the morphology and dynamics of which strongly depend on the targeted receptor. The sequence of events ranges from the initial concentration of molecules following an unbound molecule gradient to active actin-driven spreading and fragmentation of the contact, ending with droplet internalization. We observed synchronized colocalization of receptors and ligands driven by cell dynamics and closely mirrored by the droplet interface. Using intracellular calcium probe Fura-2, we also showed that the cell/droplet interaction can trigger the T-cell signaling cascade. By examining molecular dynamics using FRAP measurements, we observed a nearly frozen cell droplet joining interface. Taken together, our results point to liquid colloids as promising new tools both for probing cell surface interactions and receptor dynamics and for manipulating biological cell functions.     

Total synthesis and structural elucidation of ent-micropyrone and (+)-ascosalipyrone

The total synthesis of 6-[(1S,3S)-1,3-dimethyl-2-oxopentyl]-4-hydroxy-3,5-dimethyl-2H-pyran-2-one (22), the enantiomer of the natural product micropyrone (1), was achieved in 9 linear steps (10% overall yield), from Evans auxiliary (R)-12 with key coupling of the dianion of dione 17 and aldehyde 11. Formation of the pyrone ring and subsequent oxidation at C7 was achieved without epimerization of the sensitive position α to both the pyrone ring and the carbonyl. The same sequence using the alternate dione 24 achieved the total synthesis of 6-[(1S,3S)-1,3-dimethyl-2-oxopentyl]-4-hydroxy-3-methyl-2H-pyran-2-one (28), the (+)-enantiomer of the natural product, ascosalipyrone (2). In both cases diastereomeric aldehydes 11 and 16 were taken through the synthetic sequence to give two possible diastereomers of the natural products. Comparison of the (1)H and (13)C NMR data for the synthetic isomers with that reported for the natural products determined their relative stereochemistry. Comparison of the optical rotation obtained for 22 established it to be the enantiomer of micropyrone.     

Size-sorted anionic iron oxide nanomagnets as colloidal mediators for magnetic hyperthermia

Iron oxide colloidal nanomagnets generate heat when subjected to an alternating magnetic field. Their heating power, governed by the mechanisms of magnetic energy dissipation for single-domain particles (Brown and Néel relaxations), is highly sensitive to the crystal size, the material, and the solvent properties. This study was designed to distinguish between the contributions of Néel and Brownian mechanisms to heat generation. Anionic nanocrystals of maghemite and cobalt ferrite, differing by their magnetic anisotropy, were chemically synthesized and dispersed in an aqueous suspension by electrostatic stabilization. The particles were size-sorted by successive electrostatic phase separation steps. Parameters governing the efficiency of nanomagnets as heat mediators were varied independently; these comprised the particle size (from 5 to 16.5 nm), the solvent viscosity, magnetic anisotropy, and the magnetic field frequency and amplitude. The measured specific loss powers (SLPs) were in quantitative agreement with the results of a predictive model taking into account both Néel and Brown loss processes and the whole particle size distribution. By varying the carrier fluid viscosity, we found that Brownian friction within the carrier fluid was the main contributor to the heating power of cobalt ferrite particles. In contrast, Néel internal rotation of the magnetic moment accounted for most of the loss power of maghemite particles. Specific loss powers were varied by 3 orders of magnitude with increasing maghemite crystal size (from 4 to 1650 W/g at 700 kHz and 24.8 kA/m). This comprehensive parametric study provides the groundwork for the use of anionic colloidal nanocrystals to generate magnetically induced hyperthermia in various media, including complex systems and biological materials.     

Synthesis, characterization and potential biomedical applications of N-succinimidyl ester functionalized, polypyrrole-coated polystyrene latex particles

N-Succinimidyl ester functionalized polypyrrole-coated polystyrene latex particles (PS_E-PPyNSE) were prepared by the in situ copolymerization of pyrrole and the active ester-functionalized pyrrole (pyrrole-NSE) in the presence of polystyrene latex particles. Polystyrene microspheres were prepared by emulsion polymerization (PS_E) leading to particles having a diameter of 450 nm. These PS_E particles were precoated with poly(N-vinylpyrrolidone) prior to the in situ copolymerization of pyrrole and pyrrole-NSE. The initial comonomer concentration fractions were 25/75, 50/50 and 75/25 for pyrrole and pyrrole-NSE, respectively. The PPy-coated PS_E particles were characterized in terms of morphology, particle size, electrophoretic mobility and chemical composition. The study of morphology by means of scanning electron microscopy showed roughening of the underlying PS_E particles owing to the addition of PPyNSE, the overlayer thickness of which was estimated to be around 7 nm. Moreover, loading PPyNSE overlayers resulted in a shift of the electrophoretic mobility from –5.31 m cm/V s to a very small but positive value (0.082–0.112 m cm/V s). X-ray photoelectron spectroscopy and IR spectroscopy permitted the detection of pyrrole-NSE repeat units at the surface indicating that pyrrole and pyrrole-NSE did indeed copolymerize. The PS_E-PPyNSE particles were further evaluated as bioadsorbents of human serum albumin used as a test protein. For this study, PS_E-PPyNSE₅₀ particles, synthesized from a comonomer feed ratio of 50/50 in pyrrole/pyrrole-NSE, were used and were shown to attach efficiently human serum albumin macromolecules with a maximum amount of 0.2 mg m^–2.

Epothilone Analogues with Benzimidazole and Quinoline Side Chains: Chemical Synthesis,Antiproliferative Activity,and Interactions with Tubulin

A series of epothilone B and D analogues bearing isomeric quinoline or functionalized benzimidazole side chains has been prepared by chemical synthesis in a highly convergent manner. All analogues have been found to interact with the tubulin/microtubule system and to inhibit human cancer cell proliferation in vitro, albeit with different potencies (IC₅₀ values between 1 and 150 nM ). The affinity of quinoline‐based epothilone B and D analogues for stabilized microtubules clearly depends on the position of the N‐atom in the quinoline system, while the induction of tubulin polymerization in vitro appears to be less sensitive to N‐positioning. The potent inhibition of human cancer cell growth by epothilone analogues bearing functionalized benzimidazole side chains suggests that these systems might be conjugated with tumor‐targeting moieties to form tumor‐targeted prodrugs.     

Synthetic and structural studies of donor-functionalized alkoxy derivatives of gallium

The synthesis of a range of alkyl/chloro-gallium alkoxide and amido/alkoxide compounds was achieved via a series of protonolysis and alcoholysis steps. The initial reaction involved the synthesis of [Me(Cl)Ga{N(SiMe(3))(2)}](2) (1) via methyl group transfer from the reaction of GaCl(3) with two equivalents of LiN(SiMe(3))(2). Reaction of 1 with varying amounts of ROH resulted in the formation of [Me(Cl)Ga(OR)](2) (2, R = CH(2)CH(2)OMe; 3, CH(CH(3))CH(2)NMe(2)), [Me(Cl)Ga{N(SiMe(3))(2)}(μ(2)-OR)Ga(Cl)Me] (4, R = CH(2)CH(2)NMe(2)), or [MeGa(OR)(2)] (5, R = CH(CH(3))CH(2)NMe(2)). Compound 4 represents an intermediate in the formation of dimeric complexes, of the type [Me(Cl)Ga(OR)](2), when formed from compound [Me(Cl)Ga{N(SiMe(3))(2)}](2). A methylgallium amido/alkoxide complex [MeGa{N(SiMe(3))(2)}(OCH(2)CH(2)OMe)](2) (6) was isolated when 2 was further reacted with LiN(SiMe(3))(2). In addition, reaction of 2 with HO(t)Bu resulted in a simple alcohol/alkoxide exchange and formation of [Me(Cl)Ga(O(t)Bu)](2) (7). In contrast to the formation of 1, the in situ reaction of GaCl(3) with one equivalent of LiN(SiMe(3))(2) yielded [Cl(2)Ga{N(SiMe(3))(2)}](2) in low yield, where no methyl group transfer has occurred. Reaction of alcohol with [Cl(2)Ga{N(SiMe(3))(2)}](2) was then found to yield [Cl(2)Ga(OR)](2) (8, R = CH(2)CH(2)NMe(2)), and further reaction of 8 with LiN(SiMe(3))(2) yielded the gallium amido alkoxide complex, [ClGa{N(SiMe(3))(2)}(OR)](2) (9, R = CH(2)CH(2)NMe(2)), similar to 6. The structures of compounds 4, 5, 7, and 8 have been determined by single-crystal X-ray diffraction.     

Identification of a lacosamide binding protein using an affinity bait and chemical reporter strategy: 14-3-3 ζ

We have advanced a useful strategy to elucidate binding partners of ligands (drugs) with modest binding affinity. Key to this strategy is attaching to the ligand an affinity bait (AB) and a chemical reporter (CR) group, where the AB irreversibly attaches the ligand to the receptor upon binding and the CR group is employed for receptor detection and isolation. We have tested this AB&CR strategy using lacosamide ((R)-1), a low-molecular-weight antiepileptic drug. We demonstrate that using a (R)-lacosamide AB&CR agent ((R)-2) 14-3-3 ζ in rodent brain soluble lysates is preferentially adducted, adduction is stereospecific with respect to the AB&CR agent, and adduction depends upon the presence of endogenous levels of the small molecule metabolite xanthine. Substitution of lacosamide AB agent ((R)-5) for (R)-2 led to the identification of the 14-3-3 ζ adduction site (K120) by mass spectrometry. Competition experiments using increasing amounts of (R)-1 in the presence of (R)-2 demonstrated that (R)-1 binds at or near the (R)-2 modification site on 14-3-3 ζ. Structure-activity studies of xanthine derivatives provided information concerning the likely binding interaction between this metabolite and recombinant 14-3-3 ζ. Documentation of the 14-3-3 ζ-xanthine interaction was obtained with isothermal calorimetry using xanthine and the xanthine analogue 1,7-dimethylxanthine.     

Generation, characterisation, and applications of atomic and molecular alignment and orientation

The gas phase is generally defined as a state of matter in which atoms or molecules are in constant, rapid, random Brownian motion. However, a range of techniques exist for preparing distributions of gas phase atoms and molecules whose motion is far from random, and whose orientation in space is well defined. In this Perspective, we will explore the nature of atomic and molecular alignment and orientation, the various techniques by which samples of spatially oriented species may be prepared and characterised, and some of the ways in which oriented molecules are being exploited to further our knowledge of molecular structure and dynamics.     

Discovery of Chemicals to Either Clear or Indicate Amyloid Aggregates by Targeting Memory‐Impairing Anti‐Parallel Aβ Dimers

Amyloid‐β (Aβ) oligomers are implicated in Alzheimer disease (AD). However, their unstable nature and heterogeneous state disrupts elucidation of their explicit role in AD progression, impeding the development of tools targeting soluble Aβ oligomers. Herein parallel and anti‐parallel variants of Aβ(1–40) dimers were designed and synthesized, and their pathogenic properties in AD models characterized. Anti‐parallel dimers induced cognitive impairments with increased amyloidogenesis and cytotoxicity, and this dimer was then used in a screening platform. Through screening, two FDA‐approved drugs, Oxytetracycline and Sunitinib, were identified to dissociate Aβ oligomers and plaques to monomers in 5XFAD transgenic mice. In addition, fluorescent Astrophloxine was shown to detect aggregated Aβ in brain tissue and cerebrospinal fluid samples of AD mice. This screening platform provides a stable and homogeneous environment for observing Aβ interactions with dimer‐specific molecules.