Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells.

The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation

Observability Gramian for Bayesian Inference in Nonlinear Systems With Its Industrial Application

In this letter, we present a novel (empirical) observability Gramian for nonlinear stochastic systems in the light of Bayesian inference. First, we define our observability Gramian, which we refer to as the estimability Gramian, based on the relation to the so-called Bayesian Fisher Information Matrix for initial state estimation. Then, we study the fundamental properties of an empirical version of the estimability Gramian. The practical usefulness of the proposed framework is examined through its application to a parameter and initial state estimation in a natural gas engine cylinder

GROUND REACTION FORCE ESTIMATION USING MUSCULOSKELETAL SIMULATION

There are many situations in sports when external forces such as ground reaction force are unobtainable. Musculoskeletal kinetics including joint moments can be calculated when all external force information including ground reaction force are provided. In this study, we developed a simulation method that can predict the center of pressure on the foot and the ground reaction force from kinematics data utilizing artificial muscles between the foot and ground. The artificial muscles on the foot were activated when the distance between a foot node and a ground node is smaller than a pre-definded value. In this study we demonstrated the predictions of ground reaction force and center of pressure on the foot during normal walking. The results were validated against the measured values from a force plate

Shape Effect in the Design of Nanowire Coated Microparticles as Transepithelial Drug Delivery Devices

While the oral drug delivery route has traditionally been the most popular among patients, it is estimated that 90 % of therapeutic compounds possess oral bioavailability limitations. Thus, the development of novel drug carriers for more effective oral delivery of therapeutics is an important goal. Composite particles made by growing nanoscopic silicon wires from the surface of narrowly dispersed, microsized silica beads were previously shown to be able to: (a) adhere well onto the epithelium by interdigitating their nanowires with the apical microvilli; and (b) increase the permeability of Caco-2 cell monolayers with respect to small organic molecules in direct proportion to their concentration. A comparison between the effects of spherical and planar particle morphologies on the permeability of the epithelial cell layer in vitro and in vivo presented the subject of this study. Owing to their larger surface area, the planar particles exhibited a higher drug loading efficiency than their spherical counterparts, while simultaneously increasing the transepithelial permeation of a moderately sized model drug, insulin. The insulin elution profile for planar nanowire-coated particles displayed a continual increase in the cumulative amount of the released drug, approaching a constant release rate for 1 – 4 h period of the elution time. An immunohistochemical study confirmed the ability of planar silica particles coated with nanowires to loosen the tight junction of the epithelial cells to a greater extent than the spherical particles did, thus enabling a more facile transport of the drug across the epithelium. Transepithelial permeability tests conducted for model drugs ranging in size from 0.4 to 150 kDa yielded three categories of molecules depending on their permeation propensities. Insulin belonged to the category of molecules deliverable across the epithelium only with the assistance of nanowire-coated particles. Other groups of drugs, smaller and bigger, respectively, either did not need the carrier to permeate the epithelium or were not able to cross it even with the support from the nanowire-coated particles. Bioavailability of insulin orally administered to rabbits was also found to be increased when delivered in conjunction with the nanowire-coated planar particles

In vivo delivery of transcription factors with multifunctional oligonucleotides.

Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury

Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo.

Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5 end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5 end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells. Additionally, chemical modifications on the extended 5 end of the crRNA result in enhanced serum stability. Also, extending the 5 end of the crRNA by 59 nucleotides increases the delivery efficiency of Cpf1 RNP in cells and in vivo cationic delivery vehicles including polymer nanoparticle. Thus, 5 extension and chemical modification of the Cpf1 crRNA is an effective method for enhancing the gene editing efficiency of Cpf1 and its delivery in vivo

Inhibition of methane and natural gas hydrate formation by altering the structure of water with amino acids

Natural gas hydrates are solid hydrogen-bonded water crystals containing small molecular gases. The amount of natural gas stored as hydrates in permafrost and ocean sediments is twice that of all other fossil fuels combined. However, hydrate blockages also hinder oil/gas pipeline transportation, and, despite their huge potential as energy sources, our insufficient understanding of hydrates has limited their extraction. Here, we report how the presence of amino acids in water induces changes in its structure and thus interrupts the formation of methane and natural gas hydrates. The perturbation of the structure of water by amino acids and the resulting selective inhibition of hydrate cage formation were observed directly. A strong correlation was found between the inhibition efficiencies of amino acids and their physicochemical properties, which demonstrates the importance of their direct interactions with water and the resulting dissolution environment. The inhibition of methane and natural gas hydrate formation by amino acids has the potential to be highly beneficial in practical applications such as hydrate exploitation, oil/gas transportation, and flow assurance. Further, the interactions between amino acids and water are essential to the equilibria and dynamics of many physical, chemical, biological, and environmental processes.11Ysciescopu

Proceedings of IMECE2008

ABSTRACT Several healthcare products have been developed and marketed in recent times as a result of people's growing interest in personal health. Unstable shoes have been introduced to revitalize the muscles of the lower limbs and to modify the gait posture while walking. However, healthcare products for people should first be proved functional and safe, as some of those can sometimes result in severe injuries and side effects. Certification is, therefore, necessary in the case of unstable shoes. In this study, the functionality of unstable shoes was analyzed; it was proved that difference in pressure distribution resulting from the shape of the unstable shoes helps strengthen the muscles of the lower limbs. These analyses focused on the activation of the muscles by employing EMG (Electromyography). However, the approach involving EMG cannot carry out measurements on hidden muscles, and the noise involved is a source of potential error; therefore, this study utilizes the simulation software SIMM (Software for Interactive Musculoskeletal Modeling) for this purpose. We performed a biomechanical study using a full-body musculoskeletal model. Using the captured 3D motion data and ground reaction forces data, kinetic data was calculated in order to determine its influence on the adjacent segments. We captured the movements of six volunteers, all males in their twenties. The volunteers wore both unstable and normal shoes during each trial. This study focuses on the activation of muscles of the lower limbs when wearing unstable shoes. We inspected the muscles and analyzed the disparities between unstable and normal shoes. We observed from experimental results that most muscles of the lower limbs were revitalized. Further, we observed an improvement in the gait posture after unstable shoes were used for a period of 12 weeks. This analysis of inner muscles that cannot be examined by direct methods can help consumers make informed choices regarding healthcare products. Such analysis is made possible by simulation programs such as SIMM