Correlative single-cell hard X-ray computed tomography and X-ray fluorescence imaging.

X-ray computed tomography (XCT) and X-ray fluorescence (XRF) imaging are two non-invasive imaging techniques to study cellular structures and chemical element distributions, respectively. However, correlative X-ray computed tomography and fluorescence imaging for the same cell have yet to be routinely realized due to challenges in sample preparation and X-ray radiation damage. Here we report an integrated experimental and computational workflow for achieving correlative multi-modality X-ray imaging of a single cell. The method consists of the preparation of radiation-resistant single-cell samples using live-cell imaging-assisted chemical fixation and freeze-drying procedures, targeting and labeling cells for correlative XCT and XRF measurement, and computational reconstruction of the correlative and multi-modality images. With XCT, cellular structures including the overall structure and intracellular organelles are visualized, while XRF imaging reveals the distribution of multiple chemical elements within the same cell. Our correlative method demonstrates the feasibility and broad applicability of using X-rays to understand cellular structures and the roles of chemical elements and related proteins in signaling and other biological processes.

Coarse-Grained Simulations for the Characterization and Optimization of Hybrid Protein-DNA Nanostructures.

We present here the combination of experimental and computational modeling tools for the design and characterization of protein-DNA hybrid nanostructures. Our work incorporates several features in the design of these nanostructures: (1) modeling of the protein-DNA linker identity and length; (2) optimizing the design of protein-DNA cages to account for mechanical stresses; (3) probing the incorporation efficiency of protein-DNA conjugates into DNA nanostructures. The modeling tools were experimentally validated using structural characterization methods like cryo-TEM and AFM. Our method can be used for fitting low-resolution electron density maps when structural insights cannot be deciphered from experiments, as well as enable in-silico validation of nanostructured systems before their experimental realization. These tools will facilitate the design of complex hybrid protein-DNA nanostructures that seamlessly integrate the two different biomolecules.

Structural and Functional Analysis of Disease-Linked p97 ATPase Mutant Complexes.

IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97R155H with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97R155H mutant all show up configurations in ADP- or ATPγS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97R155H ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97R155H.

Conserved L464 in p97 D1-D2 linker is critical for p97 cofactor regulated ATPase activity.

p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1-D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4-Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97L464P and revealed the conformational change of the D1-D2 linker, resulting in a movement of the helix-turn-helix motif (543-569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ∼50 Šaway.

A library of aminoglycoside-derived lipopolymer nanoparticles for delivery of small molecules and nucleic acids.

Simultaneous delivery of small molecules and nucleic acids using a single vehicle can lead to novel combination treatments and multifunctional carriers for a variety of diseases. In this study, we report a novel library of aminoglycoside-derived lipopolymers nanoparticles (LPNs) for the simultaneous delivery of different molecular cargoes including nucleic acids and small-molecules. The LPN library was screened for transgene expression efficacy following delivery of plasmid DNA, and lead LPNs that showed high transgene expression efficacies were characterized using hydrodynamic size, zeta potential, 1H NMR and FT-IR spectroscopy, and transmission electron microscopy. LPNs demonstrated significantly higher efficacies for transgene expression than 25 kDa polyethyleneamine (PEI) and lipofectamine, including in presence of serum. Self-assembly of these cationic lipopolymers into nanoparticles also facilitated the delivery of small molecule drugs (e.g. doxorubicin) to cancer cells. LPNs were also employed for the simultaneous delivery of the small-molecule histone deacetylase (HDAC) inhibitor AR-42 together with plasmid DNA to cancer cells as a combination treatment approach for enhancing transgene expression. Taken together, our results indicate that aminoglycoside-derived LPNs are attractive vehicles for simultaneous delivery of imaging agents or chemotherapeutic drugs together with nucleic acids for different applications in medicine and biotechnology.

Hypoelectronic isomeric diiridaboranes [(Cp*Ir)2B6H6]: the "Rule-Breakers"(Cp* = η(5)-C5Me5).

In an effort to synthesize supraicosahedral iridaboranes, pyrolysis of [Cp*IrCl2]2 with excess [BH3·] was carried out, and this synthesis afforded the isomeric iridaborane [(Cp*Ir)2B6H6] clusters 1 and 2. The geometry of 1 was determined to be dodecahedral, i.e., similar to that of [B8H8](2-), whereas 2 was found to exhibit a cluster shape that can be derived from a nine-vertex tricapped trigonal prism by removing one of the capped vertices. The calculation of a large HOMO-LUMO gap further rationalized the isocloso structures for these isomers.