Delivery of siRNAs Against Selective Ion Channels and Transporter Genes Using Hyaluronic Acid-coupled Carbonate Apatite Nanoparticles Synergistically Inhibits Growth and Survival of Breast Cancer Cells.

Introduction: Dysregulated calcium homeostasis and consequentially aberrant Ca2+ signalling could enhance survival, proliferation and metastasis in various cancers. Despite rapid development in exploring the ion channel functions in relation to cancer, most of the mechanisms accounting for the impact of ion channel modulators have yet to be fully clarified. Although harnessing small interfering RNA (siRNA) to specifically silence gene expression has the potential to be a pivotal approach, its success in therapeutic intervention is dependent on an efficient delivery system. Nanoparticles have the capacity to strongly bind siRNAs. They remain in the circulation and eventually deliver the siRNA payload to the target organ. Afterward, they interact with the cell surface and enter the cell via endocytosis. Finally, they help escape the endo-lysosomal degradation system prior to unload the siRNAs into cytosol. Carbonate apatite (CA) nanocrystals primarily is composed of Ca2+, carbonate and phosphate. CA possesses both anion and cation binding domains to target negatively charged siRNA molecules. Methods: Hybrid CA was synthesized by complexing CA NPs with a hydrophilic polysaccharide - hyaluronic acid (HA). The average diameter of the composite particles was determined using Zetasizer and FE-SEM and their zeta potential values were also measured. Results and Discussion: The stronger binding affinity and cellular uptake of a fluorescent siRNA were observed for HA-CA NPs as compared to plain CA NPs. Hybrid CA was electrostatically bound individually and combined with three different siRNAs to silence expression of calcium ion channel and transporter genes, TRPC6, TRPM8 and SLC41A1 in a human breast cancer cell line (MCF-7) and evaluate their potential for treating breast cancer. Hybrid NPs carrying TRPC6, TRPM8 and SLC41A1 siRNAs could significantly enhance cytotoxicity both in vitro and in vivo. The resultant composite CA influenced biodistribution of the delivered siRNA, facilitating reduced off target distribution and enhanced breast tumor targetability.

A Homochiral Porous Organic Cage-Polymer Membrane for Enantioselective Resolution.

Membrane-based enantioselective separation is a promising method for chiral resolution due to its low cost and high efficiency. However, scalable fabrication of chiral separation membranes displaying both high enantioselectivity and high flux of enantiomers is still a challenge. Here, the authors report the preparation of homochiral porous organic cage (Covalent cage 3 (CC3)-R)-based enantioselective thin-film-composite membranes using polyamide (PA) as the matrix, where fully organic and solvent-processable cage crystals have good compatibility with the polymer scaffold. The hierarchical CC3-R channels consist of chiral selective windows and inner cavities, leading to favorable chiral resolution and permeation of enantiomers; the CC3-R/PA composite membranes display an enantiomeric excess of 95.2% for R-(+)-limonene over S-(-)-limonene and a high flux of 99.9 mg h-1 m-2. This work sheds light on the use of homochiral porous organic cages for preparing enantioselective membranes and demonstrates a new route for the development of next-generation chiral separation membranes.

Optical photothermal infrared spectroscopy for nanochemical analysis of pharmaceutical dry powder aerosols.

The aim of this research was to chemically analyse the distribution of drugs and excipients in pharmaceutical dry powder inhalation (DPI) aerosol particles of various sizes in solid state. The conventional wet assay of the chemical composition of particles after collection in a cascade impactor lacks the capability to differentiate spatially resolved morphology and chemical composition of particles in complex DPI formulations. In this proof-of-concept study, we aim to demonstrate the feasibility of using optical photothermal infrared spectroscopy (O-PTIR) to characterize micro- to nano-scale chemical composition of size-segregated particles of pharmaceutical DPI formulations. These formulations were prepared by spray drying a solution or a suspension comprising an inhaled corticosteroid fluticasone propionate, a long-acting ß2-agonist salmeterol xinafoate, and excipient lactose. The active ingredients fluticasone propionate and salmeterol xinafoate are widely used for the treatment of asthma and chronic obstructive pulmonary disease. Spatially resolved O-PTIR spectra acquired from the particles collected from stages 1-7 of a Next Generation Impactor (NGI) for both formulations confirmed the presence of peaks related to fluticasone propionate (1746 cm-1, 1702 cm-1, 1661 cm-1 and 1612 cm-1), salmeterol xinafoate (1582 cm-1), and lactose (1080 cm-1). There was no significant difference in the drug to lactose peak ratio among various size fractions of particles spray dried from solution indicating a homogeneity in drug and lactose content in the aerosol formulation. In contrast, the suspension-spray dried formulation showed the drug content increased while the lactose content decreased in the particles collected down the NGI from stage 1 to stage 7, indicating heterogeneity in the ratio of drug-excipient distribution. The qualitative chemical compositions from O-PTIR were comparable to conventional wet chemical assays of various size fractions, indicating the suitability of O-PTIR to serve as a valuable analytical platform for screening the physicochemical properties of DPIs in solid state.

Scope and challenges of nanoparticle-based mRNA delivery in cancer treatment.

Messenger RNA (mRNA) recently emerged as an appealing alternative to treat and prevent diseases ranging from cancer and Alzheimer's disease to COVID-19 with significant clinical outputs. The in vitro-transcribed mRNA has been engineered to mimic the structure of natural mRNA for vaccination, cancer immunotherapy and protein replacement therapy. In past decades, significant progress has been noticed in unveiling the molecular pathways of mRNA, controlling its translatability and stability, and its evolutionary defense mechanism. However, numerous unsolved structural, biological, and technical difficulties hamper the successful implementation of systemic delivery of mRNA for safer human consumption. Advances in designing and manufacturing mRNA and selecting innovative delivery vehicles are mandatory to address the unresolved issues and achieve the full potential of mRNA drugs. Despite the substantial efforts made to improve the intracellular delivery of mRNA drugs, challenges associated with diverse applications in different routes still exist. This study examines the current progress of mRNA therapeutics and advancements in designing biomaterials and delivery strategies, the existing translational challenges of clinical tractability and the prospects of overcoming any challenges related to mRNA.

Fe/Mg-Modified Carbonate Apatite with Uniform Particle Size and Unique Transport Protein-Related Protein Corona Efficiently Delivers Doxorubicin into Breast Cancer Cells.

Breast cancer is the abnormal, uncontrollable proliferation of cells in the breast. Conventional treatment modalities like chemotherapy induce deteriorating side effects on healthy cells. Non-viral inorganic nanoparticles (NPs) confer exclusive characteristics, such as, stability, controllable shape and size, facile surface modification, and unique magnetic and optical properties which make them attractive drug carriers. Among them, carbonate apatite (CA) particles are pH-responsive in nature, enabling rapid intracellular drug release, but are typically heterogeneous with the tendency to self-aggregate. Here, we modified the nano-carrier by partially substituting Ca2+ with Mg2+ and Fe3+ into a basic lattice structure of CA, forming Fe/Mg-carbonate apatite (Fe/Mg-CA) NPs with the ability to mitigate self-aggregation, form unique protein corona in the presence of serum and efficiently deliver doxorubicin (DOX), an anti-cancer drug into breast cancer cells. Two formulations of Fe/Mg-CA NPs were generated by adding different concentrations of Fe3+ and Mg2+ along with a fixed amount of Ca2+ in bicarbonate buffered DMEM (Dulbecco's Modified Eagle's Medium), followed by 30 min incubation at 37 °C. Particles were characterized by turbidity analysis, z-average diameter and zeta potential measurement, optical microscopy, field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray (EDX), flame atomic absorption spectroscopy (FAAS), pH dissolution, drug binding, cellular uptake, thiazolyl blue tetrazolium bromide (MTT) assay, stability analysis, and protein corona study by LCMS (Liquid chromatography-mass spectrometry). Both formulations of Fe/Mg-CA displayed mostly uniform nano-sized particles with less tendency to aggregate. The EDX and FAAS elemental analysis confirmed the weight (%) of Ca, Fe and Mg, along with their Ca/P ratio in the particles. A constant drug binding efficiency was noticed with 5 µM to 10 µM of initial DOX concentration. A pH dissolution study of Fe/Mg-CA NPs revealed the quick release of DOX in acidic pH. Enhancement of cytotoxicity for the chemotherapy drug was greater for Fe/Mg-CA NPs as compared to CA NPs, which could be explained by an increase in cellular internalization as a result of the small z-average diameter of the former. The protein corona study by LCMS demonstrated that Fe/Mg-CA NPs exhibited the highest affinity towards transport proteins without binding with opsonins. Biodistribution study was performed to study the effect of DOX-loaded Fe/Mg-CA NPs on the tissue distribution of DOX in Balb/c 4T1 tumor-bearing mice. Both formulations of Fe/Mg-CA NPs have significantly increased the accumulation of DOX in tumors. Interestingly, high Fe/Mg-CA NPs exhibited less off-target distribution compared to low Fe/Mg-CA NPs. Furthermore, the blood plasma analysis revealed prolonged blood circulation half-life of DOX-loaded low and high Fe/Mg-CA NPs compared to free DOX solution. Modifying CA NPs with Fe3+ and Mg2+, thereby, led to the generation of nano-sized particles with less tendency to aggregate, enhancing the drug binding efficiency, cellular uptake, and cytotoxicity without hampering drug release in acidic pH, while improving the circulation half-life and tumor accumulation of DOX. Therefore, Fe/Mg-CA which predominantly forms a transport protein-related protein corona could be a proficient carrier for therapeutic delivery in breast cancer.

Homochiral MOF-Polymer Mixed Matrix Membranes for Efficient Separation of Chiral Molecules.

Homochiral metal-organic framework (MOF) membranes have been recently reported for chiral separations. However, only a few high-quality homochiral polycrystalline MOF membranes have been fabricated due to the difficulty in crystallization of a chiral MOF layer without defects on porous substrates. Alternatively, mixed matrix membranes (MMMs), which combine potential advantages of MOFs and polymers, have been widely demonstrated for gas separation and water purification. Here we report novel homochiral MOF-polymer MMMs for efficient chiral separation. Homochirality was successfully incorporated into achiral MIL-53-NH2 nanocrystals by post-synthetic modification with amino acids, such as l-histidine (l-His) and l-glutamic acid (l-Glu). The MIL-53-NH-l-His and MIL-53-NH-l-Glu nanocrystals were then embedded into polyethersulfone (PES) matrix to form homochiral MMMs, which exhibited excellent enantioselectivity for racemic 1-phenylethanol with the highest enantiomeric excess value up to 100 %. This work, as an example, demonstrates the feasibility of fabricating diverse large-scale homochiral MOF-based MMMs for chiral separation.

Estrogen depletion and drug treatment alter the microstructure of type I collagen in bone.

The impact of estrogen depletion and drug treatment on type I collagen fibril nanomorphology and collagen fibril packing (microstructure) was evaluated by atomic force microscopy (AFM) using an ovariectomized (OVX) rabbit model of estrogen deficiency induced bone loss. Nine month-old New Zealand white female rabbits were treated as follows: sham-operated (Sham; n = 11), OVX + vehicle (OVX + Veh; n = 12), OVX + alendronate (ALN, 600 µg/kg/wk., s.c.; n = 12), and OVX + cathepsin-K inhibitor L-235 (CatKI, 10 mg/kg, daily, p.o.; n = 13) in prevention mode for 27 weeks. Samples from the cortical femur and trabecular lumbar vertebrae were polished, demineralized, and imaged using AFM. Auto-correlation of image patches was used to generate a vector field for each image that mathematically approximated the collagen fibril alignment. This vector field was used to compute an information-theoretic entropy that was employed as a quantitative fibril alignment parameter (FAP) to allow image-to-image and sample-to-sample comparison. For all samples, no change was observed in the average FAP values; however significant differences in the distribution of FAP values were observed. In particular, OVX + Veh lumbar vertebrae samples contained a tail of lower FAP values representing regions of greater fibril alignment. OVX + ALN treatment resulted in a FAP distribution with a tail indicating greater alignment for cortical femur and less alignment for trabecular lumbar vertebrae. OVX + CatKI treatment gave a distribution of FAP values with a tail indicating less alignment for cortical femur and no change for trabecular lumbar vertebrae. Fibril alignment was also evaluated by considering when a fibril was part of discrete bundles or sheets (classified as parallel) or not (classified as oblique). For this analysis, the percentage of parallel fibrils in cortical femur for the OVX group was 17% lower than the Sham group. OVX + ALN treatment partially prevented the proportion of parallel fibrils from decreasing and OVX + CatKI treatment completely prevented a change. In trabecular lumbar vertebrae, there was no difference in the percentage of parallel fibrils between Sham and any of the other treatment groups.

Generation 3 PAMAM dendrimer TAMRA conjugates containing precise dye/dendrimer ratios.

The synthesis, isolation, and characterization of generation 3 poly(amidoamine) (G3 PAMAM) dendrimer containing precise ratios of 5-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dye (n = 1-3) per polymer particle are reported. Stochastic conjugation of TAMRA dye to the dendrimer was followed by separation into precise dye-polymer ratios using rp-HPLC. The isolated materials were characterized by rp-UPLC, MALDI-TOF-MS, and 1H NMR spectroscopy, UV-vis, and fluorescence spectroscopies.

High-resolution NMR characterization of low abundance oligomers of amyloid-ß without purification.

Alzheimer's disease is characterized by the misfolding and self-assembly of the amyloidogenic protein amyloid-ß (Aß). The aggregation of Aß leads to diverse oligomeric states, each of which may be potential targets for intervention. Obtaining insight into Aß oligomers at the atomic level has been a major challenge to most techniques. Here, we use magic angle spinning recoupling (1)H-(1)H NMR experiments to overcome many of these limitations. Using (1)H-(1)H dipolar couplings as a NMR spectral filter to remove both high and low molecular weight species, we provide atomic-level characterization of a non-fibrillar aggregation product of the Aß1-40 peptide using non-frozen samples without isotopic labeling. Importantly, this spectral filter allows the detection of the specific oligomer signal without a separate purification procedure. In comparison to other solid-state NMR techniques, the experiment is extraordinarily selective and sensitive. A resolved 2D spectra could be acquired of a small population of oligomers (6 micrograms, 7% of the total) amongst a much larger population of monomers and fibers (93% of the total). By coupling real-time (1)H-(1)H NMR experiments with other biophysical measurements, we show that a stable, primarily disordered Aß1-40 oligomer 5-15 nm in diameter can form and coexist in parallel with the well-known cross-ß-sheet fibrils.

Crystallinity and compositional changes in carbonated apatites: Evidence from 31P solid-state NMR, Raman, and AFM analysis.

Solid-state (magic-angle spinning) NMR spectroscopy is a useful tool for obtaining structural information on bone organic and mineral components and synthetic model minerals at the atomic-level. Raman and 31P NMR spectral parameters were investigated in a series of synthetic B-type carbonated apatites (CAps). Inverse 31P NMR linewidth and inverse Raman PO43- ν1 bandwidth were both correlated with powder XRD c-axis crystallinity over the 0.3-10.3 wt% CO32- range investigated. Comparison with bone powder crystallinities showed agreement with values predicted by NMR and Raman calibration curves. Carbonate content was divided into two domains by the 31P NMR chemical shift frequency and the Raman phosphate ν1 band position. These parameters remain stable except for an abrupt transition at 6.5 wt% carbonate, a composition which corresponds to an average of one carbonate per unit cell. This near-binary distribution of spectroscopic properties was also found in AFM-measured particle sizes and Ca/P molar ratios by elemental analysis. We propose that this transition differentiates between two charge-balancing ion-loss mechanisms as measured by Ca/P ratios. These results define a criterion for spectroscopic characterization of B-type carbonate substitution in apatitic minerals.

Quantitative analysis of generation and branch defects in G5 poly(amidoamine) dendrimer.

Although methods have been developed to synthesize and isolate generation 5 (G5) PAMAM dendrimers containing precise numbers of ligands per polymer particle, the presence of skeletal and generational defects in this material can substantially hamper the process. Here we provide a quantitative analysis of G5 PAMAM dendrimer defects via high performance liquid chromatography, potentiometric titration, mass spectrometry, size exclusion chromatography, and nuclear magnetic resonance. We identified, isolated, and characterized the major structural defects of G5 dendrimer, trailing generations, and dimer, trimer, and tetramer species. We determine that the G5 material present in the as-received mixture contains 93 arms on average. We have developed two model systems capable of generating the experimentally observed mass range and polydispersity at defect rates of 8-15%.

Type I collagen self-assembly: the roles of substrate and concentration.

Collagen molecules, self-assembled into macroscopic hierarchical tissue networks, are the main organic building block of many biological tissues. A particularly common and important form of this self-assembly consists of type I collagen fibrils, which exhibit a nanoscopic signature, D-periodic gap/overlap spacing, with a distribution of values centered at approximately 67 nm. In order to better understand the relationship between type I collagen self-assembly and D-spacing distribution, we investigated surface-mediated collagen self-assembly as a function of substrate and incubation concentration. Collagen fibril assembly on phlogopite and muscovite mica as well as fibrillar gel coextrusion in glass capillary tubes all exhibited D-spacing distributions similar to those commonly observed in biological tissues. The observation of D-spacing distribution by self-assembly of type I collagen alone is significant as it eliminates the necessity to invoke other preassembly or postassembly hypotheses, such as variation in the content of collagen types, enzymatic cross-linking, or other post-translational modifications, as mechanistic origins of D-spacing distribution. The D-spacing distribution on phlogopite mica is independent of type I collagen concentration, but on muscovite mica D-spacing distributions showed increased negative skewness at 20 µg/mL and higher concentrations. Tilted D-spacing angles were found to correlate with decreased D-spacing measurements, an effect that can be removed with a tilt angle correction, resulting in no concentration dependence of D-spacing distribution on muscovite mica. We then demonstrated that tilted D-spacing is uncommon in biological tissues and it does not explain previous observations of low D-spacing values in ovariectomized dermis and bone.

Variation in type I collagen fibril nanomorphology: the significance and origin.

Although the axial D-periodic spacing is a well-recognized nanomorphological feature of type I collagen fibrils, the existence of a distribution of values has been largely overlooked since its discovery seven decades ago. Studies based on single fibril measurements occasionally noted variation in D-spacing values, but accredited it with no biological significance. Recent quantitative characterizations supported that a 10-nm collagen D-spacing distribution is intrinsic to collagen fibrils in various tissues as well as in vitro self-assembly of reconstituted collagen. In addition, the distribution is altered in Osteogenesis Imperfecta and long-term estrogen deprivation. Bone collagen is organized into lamellar sheets of bundles at the micro-scale, and D-spacings within a bundle of a lamella are mostly identical, whereas variations among different bundles contribute to the full-scale distribution. This seems to be a very general phenomenon for the protein as the same type of D-spacing/bundle organization is observed for dermal and tendon collagen. More research investigation of collagen nanomorphology in connection to bone biology is required to fully understand these new observations. Here we review the data demonstrating the existence of a D-spacing distribution, the impact of disease on the distribution and possible explanations for the origin of D-spacing variations based on various collagen fibrillogenesis models.

Dendrimer-based multivalent vancomycin nanoplatform for targeting the drug-resistant bacterial surface.

Vancomycin represents the preferred ligand for bacteria-targeting nanosystems. However, it is inefficient for emerging vancomycin-resistant species because of its poor affinity to the reprogrammed cell wall structure. This study demonstrates the use of a multivalent strategy as an effective way for overcoming such an affinity limitation in bacteria targeting. We designed a series of fifth generation (G5) poly(amidoamine) (PAMAM) dendrimers tethered with vancomycin at the C-terminus at different valencies. We performed surface plasmon resonance (SPR) studies to determine their binding avidity to two cell wall models, each made with either a vancomycin-susceptible (D)-Ala-(D)-Ala or vancomycin-resistant (D)-Ala-(D)-Lac cell wall precursor. These conjugates showed remarkable enhancement in avidity in the cell wall models tested, including the vancomycin-resistant model, which had an increase in avidity of four to five orders of magnitude greater than free vancomycin. The tight adsorption of the conjugate to the model surface corresponded with its ability to bind vancomycin-susceptible Staphylococcus aureus bacterial cells in vitro as imaged by confocal fluorescent microscopy. This vancomycin platform was then used to fabricate the surface of iron oxide nanoparticles by coating them with the dendrimer conjugates, and the resulting dendrimer-covered magnetic nanoparticles were demonstrated to rapidly sequester bacterial cells. In summary, this article investigates the biophysical basis of the tight, multivalent association of dendrimer-based vancomycin conjugates to the bacterial cell wall, and proposes a potential new use of this nanoplatform in targeting Gram-positive bacteria.

Estrogen depletion results in nanoscale morphology changes in dermal collagen.

Tissue cryo-sectioning combined with atomic force microscopy imaging reveals that the nanoscale morphology of dermal collagen fibrils, quantified using the metric of D-periodic spacing, changes under the condition of estrogen depletion. Specifically, a new subpopulation of fibrils with D-spacings in the region between 56 and 59 nm is present 2 years following ovariectomy in ovine dermal samples. In addition, the overall width of the distribution, both values above and below the mean, was found to be increased. The change in width due to an increase in lower values of D-spacings was previously reported for ovine bone; however, this report demonstrates that the effect is also present in non-mineralized collagen fibrils. A nonparametric Kolmogorov-Smirnov test of the cumulative density function indicates a statistical difference in the sham and OVX D-spacing distributions (P<0.01).

Acetonitrile shortage: use of isopropanol as an alternative elution system for ultra/high performance liquid chromatography.

Acetonitrile is a choice of solvent for almost all chromatographic separations. In recent years, researchers around the globe have faced an acetonitrile shortage that affected routine analytical operations. Researchers have tried to counter this shortage by applying many innovative solutions, including using ultra performance liquid chromatography (UPLC) columns that are shorter and smaller in diameter than traditional high performance liquid chromatography (HPLC) columns, thus significantly decreasing the volume of eluent required. Although utilizing UPLC in place of HPLC can alleviate the solvent demand to some extent, acetonitrile is generally thought of as the solvent of choice due to its versatility. In the following communication, we describe an alternative eluent system that uses isopropanol in place of acetonitrile as an organic modifier for routine chromatographic separations. We report here the development of an isopropanol based UPLC protocol for G5 PAMAM dendrimer based conjugates that was transferred to semi-preparative applications.

Effect of osteogenesis imperfecta mutations on free energy of collagen model peptides: a molecular dynamics simulation.

We have carried out stochastic boundary molecular dynamics simulations to estimate free energy changes for substitutions of Gly with Val, Arg and Trp residues in a collagen-like peptide. The relative free energy change differences of mutants containing a Val, an Arg and a Trp relative to the wild type are 5.7, 8.1 and 9.5 kcal/mol, respectively. The corresponding free energy change differences of mutants containing two mutated residues are on average 7.6, 10.5 and 14.7 kcal/mol, respectively. We show that the free energy change differences are correlated with the severity of OI from statistical analysis and mechanical properties of the individual tropocollagen molecules. This simulation result indicates an atomistic-level mechanistic understanding of the effect of OI mutations in terms of stability of the mutants relative to the wild type, which could eventually provide a new strategy for diagnosis and treatment of the disease.

Polyvalent saccharide-functionalized generation 3 poly(amidoamine) dendrimer-methotrexate conjugate as a potential anticancer agent.

A saccharide-terminated generation 3 (G3) polyamidoamine (PAMAM) dendrimer was synthesized as a drug carrier. Utilizing this dendritic platform, we have successfully synthesized polyvalent conjugates (G3-MTX) containing the drug methotrexate (MTX). Surface Plasmon Resonance (SPR) results showed that G3-MTX presented three orders of magnitude enhancement in binding avidity to folate-binding protein (FBP) as compared to the free folic acid (FA). Flow cytometric and confocal microscopic analysis showed that conjugate (G3-MTX-FI) containing imaging agent fluorescein-5(6)-carboxamidohexanoic acid (FI) was internalized into folate receptor (FR)-expressing KB cells in dose-dependent and receptor-mediated fashion. The G3-MTX induced a dose-dependent cytotoxicity in the KB cells. Therefore, the polyvalent G3-MTX may have potential as an anticancer nanodevice for the specific targeting and killing of FR-expressing tumor cells.

Dual-wavelength linear regression phase unwrapping in three-dimensional microscopic images of cancer cells.

We present a study of the three-dimensional structure of cancer cells using dual-wavelength phase-imaging digital holographic microscopy. Phase imaging of objects with optical height variation greater than the wavelength of light is ambiguous and causes phase wrapping. By comparing two phase images recorded at different wavelengths, the images can be accurately unwrapped. The unwrapping method is computationally fast and straightforward, and it can process complex topologies. Additionally, the limitations on the total optical height are significantly relaxed. This new methodology is widely applicable to other phase-imaging techniques as well as in applications beyond optical microscopy.

Free energy simulation to investigate the effect of amino acid sequence environment on the severity of osteogenesis imperfecta by glycine mutations in collagen.

Molecular dynamics simulations were carried out to calculate the free energy change difference of two collagen-like peptide models for Gly --> Ser mutations causing two different osteogenesis imperfecta phenotypes. These simulations were performed to investigate the impact of local amino acid sequence environment adjacent to a mutation site on the stability of the collagen. The average free energy differences for a Gly --> Ser mutant relative to a wild type are 3.4 kcal/mol and 8.2 kcal/mol for a nonlethal site and a lethal site, respectively. The free energy change differences of mutant containing two Ser residues relative to the wild type at the nonlethal and lethal mutation sites are 4.6 and 9.8 kcal/mol, respectively. Although electrostatic interactions stabilize mutants containing one or two Ser residues at both mutation sites, van der Waals interactions are of sufficient magnitude to cause a net destabilization. The presence of Gln and Arg near the mutation site, which contain large and polar side chains, provide more destabilization than amino acids containing small and nonpolar side chains.