The Combination of Vascular Endothelial Growth Factor A (VEGF-A) and Fibroblast Growth Factor 1 (FGF1) Modified mRNA Improves Wound Healing in Diabetic Mice: An Ex Vivo and In Vivo Investigation.

BACKGROUND: Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1). METHODS: An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination. RESULTS: The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth. CONCLUSION: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.

An enhanced biophysical screening strategy to investigate the affinity of ASOs for their target RNA.

The recent and rapid increase in the discovery of new RNA therapeutics has created the perfect terrain to explore an increasing number of novel targets. In particular, antisense oligonucleotides (ASOs) have long held the promise of an accelerated and effective drug design compared to other RNA-based therapeutics. Although ASOs in silico design has advanced distinctively in the past years, especially thanks to the several predictive frameworks for RNA folding, it is somehow limited by the wide approximation of calculating sequence affinity based on RNA-RNA/DNA sequences. None of the ASO modifications are taken into consideration, losing hybridization information particularly fundamental to ASOs that elicit their function through RNase H1-mediated mechanisms. Here we present an inexpensive and enhanced biophysical screening strategy to investigate the affinity of ASOs for their target RNA using several biophysical techniques such as high throughput differential scanning fluorimetry (DSF), circular dichroism (CD), isothermal calorimetry (ITC), surface plasmon resonance (SPR) and small-angle X-ray scattering (SAXS).

Multiomics analysis of naturally efficacious lipid nanoparticle coronas reveals high-density lipoprotein is necessary for their function.

In terms of lipid nanoparticle (LNP) engineering, the relationship between particle composition, delivery efficacy, and the composition of the biocoronas that form around LNPs, is poorly understood. To explore this we analyze naturally efficacious biocorona compositions using an unbiased screening workflow. First, LNPs are complexed with plasma samples, from individual lean or obese male rats, and then functionally evaluated in vitro. Then, a fast, automated, and miniaturized method retrieves the LNPs with intact biocoronas, and multiomics analysis of the LNP-corona complexes reveals the particle corona content arising from each individual plasma sample. We find that the most efficacious LNP-corona complexes were enriched with high-density lipoprotein (HDL) and, compared to the commonly used corona-biomarker Apolipoprotein E, corona HDL content was a superior predictor of in-vivo activity. Using technically challenging and clinically relevant lipid nanoparticles, these methods reveal a previously unreported role for HDL as a source of ApoE and, form a framework for improving LNP therapeutic efficacy by controlling corona composition.

Solution and Membrane Interaction Dynamics of Mycobacterium tuberculosis Fatty Acyl-CoA Synthetase FadD13.

The very-long-chain fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis activates fatty acids for further use in mycobacterial lipid metabolism. FadD13 is a peripheral membrane protein, with both soluble and membrane-bound populations in vivo. The protein displays a distinct positively charged surface patch, suggested to be involved in membrane association. In this paper, we combine structural analysis with liposome co-flotation assays and membrane association modeling to gain a more comprehensive understanding of the mechanisms behind membrane association. We show that FadD13 has affinity for negatively charged lipids, such as cardiolipin. Addition of a fatty acid substrate to the liposomes increases the apparent affinity of FadD13, consistent with our previous hypothesis that FadD13 can utilize the membrane to harbor its very-long-chain fatty acyl substrates. In addition, we unambiguously show that FadD13 adopts a dimeric arrangement in solution. The dimer interface partly buries the positive surface patch, seemingly inconsistent with membrane binding. Notably, when cross-linking the dimer, it lost its ability to bind and co-migrate with liposomes. To better understand the dynamics of association, we utilized two mutant variants of FadD13, one in which the positively charged patch was altered to become more negative and one more hydrophobic. Both variants were predominantly monomeric in solution. The hydrophobic variant maintained the ability to bind to the membrane, whereas the negative variant did not. Taken together, our data indicate that FadD13 exists in a dynamic equilibrium between the dimer and monomer, where the monomeric state can adhere to the membrane via the positively charged surface patch.

Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles.

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP's plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens.

Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5-7. Here we describe a group of RNR proteins in Mollicutes-including Mycoplasma pathogens-that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR-some of which are developing resistance to antibiotics-are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.

Structure and Cooperativity of the Cytosolic Domain of the CorA Mg2+ Channel from Escherichia coli.

Structures of the Mg2+ bound (closed) and apo (open) states of CorA suggests that channel gating is accomplished by rigid-body motions between symmetric and asymmetric assemblies of the cytosolic portions of the five subunits in response to ligand (Mg2+) binding/unbinding at interfacial sites. Here, we structurally and biochemically characterize the isolated cytosolic domain from Escherichia coli CorA. The data reveal an Mg2+-ligand binding site located in a novel position between each of the five subunits and two Mg2+ ions trapped inside the pore. Soaking experiments show that cobalt hexammine outcompetes Mg2+ at the pore site closest to the membrane. This represents the first structural information of how an analog of hexa-hydrated Mg2+ (and competitive inhibitor of CorA) associates to the CorA pore. Biochemical data on the isolated cytoplasmic domain and full-length protein suggests that gating of the CorA channel is governed cooperatively.

Synchrotron-based Nickel Mössbauer Spectroscopy.

We used a novel experimental setup to conduct the first synchrotron-based (61)Ni Mössbauer spectroscopy measurements in the energy domain on Ni coordination complexes and metalloproteins. A representative set of samples was chosen to demonstrate the potential of this approach. (61)NiCr2O4 was examined as a case with strong Zeeman splittings. Simulations of the spectra yielded an internal magnetic field of 44.6 T, consistent with previous work by the traditional (61)Ni Mössbauer approach with a radioactive source. A linear Ni amido complex, (61)Ni{N(SiMe3)Dipp}2, where Dipp = C6H3-2,6-(i)Pr2, was chosen as a sample with an "extreme" geometry and large quadrupole splitting. Finally, to demonstrate the feasibility of metalloprotein studies using synchrotron-based (61)Ni Mössbauer spectroscopy, we examined the spectra of (61)Ni-substituted rubredoxin in reduced and oxidized forms, along with [Et4N]2[(61)Ni(SPh)4] as a model compound. For each of the above samples, a reasonable spectrum could be obtained in ∼1 d. Given that there is still room for considerable improvement in experimental sensitivity, synchrotron-based (61)Ni Mössbauer spectroscopy appears to be a promising alternative to measurements with radioactive sources.

The solution configurations of inactive and activated DntR have implications for the sliding dimer mechanism of LysR transcription factors.

LysR Type Transcriptional Regulators (LTTRs) regulate basic metabolic pathways or virulence gene expression in prokaryotes. Evidence suggests that the activation of LTTRs involves a conformational change from an inactive compact apo- configuration that represses transcription to an active, expanded holo- form that promotes it. However, no LTTR has yet been observed to adopt both configurations. Here, we report the results of structural studies of various forms of the LTTR DntR. Crystal structures of apo-DntR and of a partially autoinducing mutant H169T-DntR suggest that active and inactive DntR maintain a compact homotetrameric configuration. However, Small Angle X-ray Scattering (SAXS) studies on solutions of apo-, H169T- and inducer-bound holo-DntR indicate a different behaviour, suggesting that while apo-DntR maintains a compact configuration in solution both H169T- and holo-DntR adopt an expanded conformation. Models of the SAXS-obtained solution conformations of apo- and holo-DntR homotetramers in complex with promoter-operator region DNA are consistent with previous observations of a shifting of LTTR DNA binding sites upon activation and a consequent relaxation in the bend of the promoter-operator region DNA. Our results thus provide clear evidence at the molecular level which strongly supports the 'sliding dimer' hypothesis concerning LTTR activation mechanisms.

Sound velocities of compressed Fe3C from simultaneous synchrotron X-ray diffraction and nuclear resonant scattering measurements.

The applications of nuclear resonant scattering in laser-heated diamond anvil cells have provided an important probe for the magnetic and vibrational properties of (57)Fe-bearing materials under high pressure and high temperature. Synchrotron X-ray diffraction is one of the most powerful tools for studying phase stability and equation of state over a wide range of pressure and temperature conditions. Recently an experimental capability has been developed for simultaneous nuclear resonant scattering and X-ray diffraction measurements using synchrotron radiation. Here the application of this method to determine the sound velocities of compressed Fe(3)C is shown. The X-ray diffraction measurements allow detection of microscale impurities, phase transitions and chemical reactions upon compression or heating. They also provide information on sample pressure, grain size distribution and unit cell volume. By combining the Debye velocity extracted from the nuclear resonant inelastic X-ray scattering measurements and the structure, density and elasticity data from the X-ray diffraction measurements simultaneously obtained, more accurate sound velocity data can be derived. Our results on few-crystal and powder samples indicate strong anisotropy in the sound velocities of Fe(3)C under ambient conditions.

Electronic and magnetic structures of the postperovskite-type Fe2O3 and implications for planetary magnetic records and deep interiors.

Recent studies have shown that high pressure (P) induces the metallization of the Fe(2+)-O bonding, the destruction of magnetic ordering in Fe, and the high-spin (HS) to low-spin (LS) transition of Fe in silicate and oxide phases at the deep planetary interiors. Hematite (Fe(2)O(3)) is an important magnetic carrier mineral for deciphering planetary magnetism and a proxy for Fe in the planetary interiors. Here, we present synchrotron Mössbauer spectroscopy and X-ray diffraction combined with ab initio calculations for Fe(2)O(3) revealing the destruction of magnetic ordering at the hematite --> Rh(2)O(3)-II type (RhII) transition at 70 GPa and 300 K, and then the revival of magnetic ordering at the RhII --> postperovskite (PPv) transition after laser heating at 73 GPa. At the latter transition, at least half of Fe(3+) ions transform from LS to HS and Fe(2)O(3) changes from a semiconductor to a metal. This result demonstrates that some magnetic carrier minerals may experience a complex sequence of magnetic ordering changes during impact rather than a monotonic demagnetization. Also local Fe enrichment at Earth's core-mantle boundary will lead to changes in the electronic structure and spin state of Fe in silicate PPv. If the ultra-low-velocity zones are composed of Fe-enriched silicate PPv and/or the basaltic materials are accumulated at the lowermost mantle, high electrical conductivity of these regions will play an important role for the electromagnetic coupling between the mantle and the core.

Phonon density of states of metallic Sn at high pressure.

Determination of the lattice dynamics of Sn at high pressure has represented a major experimental challenge and eluded previous attempts. Here we report the first successful measurement of the phonon density of states of Sn at high pressure to 64 GPa using nuclear resonant inelastic x-ray scattering. We also present density functional theory calculations that are in excellent agreement with the measured data. The results of this combined experimental and theoretical study establish reliable phonon density of states of Sn at high pressure. It makes possible an accurate description of its thermodynamic properties that are of great importance and interest in high pressure research.