Protein Transduction Research Articles

Tissue and Cell Dual‐Penetrating Dendritic Lipopeptide Liposomes for Hypertrophic Scar Treatment

AbstractHypertrophic scar is a complex global disease, which can lead to remarkable functional impairment, pruritus, and pain. Treating local skin diseases needs a transdermal drug delivery system that can simultaneously promote cell and tissue penetration within the hypertrophic scar. Herein, a tissue and cell dual‐penetrating liposomal delivery platform consisting of arginine‐based dendritic lipopeptide mimicking the pivotal amino acid sequence in viral protein transduction domains and virion‐like nanostructure to enhance hypertrophic scar tissue deep penetration and cellular internalization is reported. This dendritic lipopeptide liposomal delivery platform can overcome corneum barriers to the punch on hypertrophic scar fibroblast membranes, as are natural viruses. It also can deeply deliver drugs to promote hypertrophic scar fibroblast apoptosis and collagen fiber remodeling within thickened dermis and epidermis, and finally remove the raised scars.

Recruiting In Vitro Transcribed mRNA against Cancer Immunotherapy: A Contemporary Appraisal of the Current Landscape.

Over 100 innovative in vitro transcribed (IVT)-mRNAs are presently undergoing clinical trials, with a projected substantial impact on the pharmaceutical market in the near future. Τhe idea behind this is that after the successful cellular internalization of IVT-mRNAs, they are subsequently translated into proteins with therapeutic or prophylactic relevance. Simultaneously, cancer immunotherapy employs diverse strategies to mobilize the immune system in the battle against cancer. Therefore, in this review, the fundamental principles of IVT-mRNA to its recruitment in cancer immunotherapy, are discussed and analyzed. More specifically, this review paper focuses on the development of mRNA vaccines, the exploitation of neoantigens, as well as Chimeric Antigen Receptor (CAR) T-Cells, showcasing their clinical applications and the ongoing trials for the development of next-generation immunotherapeutics. Furthermore, this study investigates the synergistic potential of combining the CAR immunotherapy and the IVT-mRNAs by introducing our research group novel, patented delivery method that utilizes the Protein Transduction Domain (PTD) technology to transduce the IVT-mRNAs encoding the CAR of interest into the Natural Killer (NK)-92 cells, highlighting the potential for enhancing the CAR NK cell potency, efficiency, and bioenergetics. While IVT-mRNA technology brings exciting progress to cancer immunotherapy, several challenges and limitations must be acknowledged, such as safety, toxicity, and delivery issues. This comprehensive exploration of IVT-mRNA technology, in line with its applications in cancer therapeutics, offers valuable insights into the opportunities and challenges in the evolving landscape of cancer immunotherapy, setting the stage for future advancements in the field.

TFP5, a Peptide Derived from Cdk5 Activator p35, Protects Pancreatic β Cells from Glucose Toxicity.

We studied the effect of TFP5 on MIN6 cells (cultured mouse islet β cells) treated with different concentrations of glucose (5 or 25 mM). The results were verified in C57BL/6J mice (control; n=12) and db/db mice with type 2 diabetes mellitus (n=12). To synthesize TFP5, peptide p5 (a derivative of p35 protein, activator of cyclin-dependent kinase 5, Cdk5) was conjugated with a FITC tag at the N-terminus and an 11-amino acid TAT protein transduction domain at the C-terminus. TFP5 was employed to inhibit Cdk5 activity and then to evaluate its efficiency in treating experimental type 2 diabetes mellitus. TFP5 effectively inhibited the pathological hyperactivity of Cdk5, enhanced insulin secretion, and protected pancreatic β cells from apoptosis in vitro and in vivo. In addition, TFP5 inhibited inflammation in pancreatic islets by reducing the expression of inflammatory cytokines TGF-β1, TNFα, and IL-1β. These novel data indicates that TFP5 is a promising candidate for treatment of type 2 diabetes mellitus.

Direct Estimation of Parameters in ODE Models Using WENDy: Weak-Form Estimation of Nonlinear Dynamics

We introduce the Weak-form Estimation of Nonlinear Dynamics (WENDy) method for estimating model parameters for non-linear systems of ODEs. Without relying on any numerical differential equation solvers, WENDy computes accurate estimates and is robust to large (biologically relevant) levels of measurement noise. For low dimensional systems with modest amounts of data, WENDy is competitive with conventional forward solver-based nonlinear least squares methods in terms of speed and accuracy. For both higher dimensional systems and stiff systems, WENDy is typically both faster (often by orders of magnitude) and more accurate than forward solver-based approaches. The core mathematical idea involves an efficient conversion of the strong form representation of a model to its weak form, and then solving a regression problem to perform parameter inference. The core statistical idea rests on the Errors-In-Variables framework, which necessitates the use of the iteratively reweighted least squares algorithm. Further improvements are obtained by using orthonormal test functions, created from a set of C^{infty } bump functions of varying support sizes.We demonstrate the high robustness and computational efficiency by applying WENDy to estimate parameters in some common models from population biology, neuroscience, and biochemistry, including logistic growth, Lotka-Volterra, FitzHugh-Nagumo, Hindmarsh-Rose, and a Protein Transduction Benchmark model. Software and code for reproducing the examples is available at https://github.com/MathBioCU/WENDy.

In vivo direct cell-penetrating peptide mediated protein transduction system in Acyrthosiphon pisum

ObjectiveThe principal delivery method for CRISPR-based genome editing in insects is now based on microinjection into single cells or embryos. The direct protein transduction systems cannot be employed in aphids because oogenesis occurs without apparent vitellogenesis. Given the limited timing of injection into the embryonic stage in oviparous aphids, a protein delivery system from the hemolymph to the germline and embryos would be a useful tool for genome editing. This study reports a newly developed direct protein delivery system for aphids using cell-penetrating peptides (CPPs). CPPs are short peptides that translocate across the plasma membrane when bound to cargo proteins.ResultsPenetratin (PEN), a widely conserved CPP among insects, was identified in this study. We used mVenus, a recombinant fluorescent protein, as a visual marker for CPP availability assessments, and fused it with PEN by bacterial protein expression. The mVenus-PEN recombinant proteins were introduced into the hemolymph of adult unwinged Acyrthosiphon pisum females using a nanoinjector. Fluorescence emitted by mVenus-PEN was observed in various tissues, such as the gut, trachea, bacteriocytes, and their progeny. This study shows that PEN can deliver exogenously expressed proteins into tissues in vivo, indicating that CPPs are powerful tools for protein transduction.

Micellized protein transduction domain-bone morphogenetic protein-2 accelerates bone healing in a rat tibial distraction osteogenesis model

The clinical application of growth factors such as recombinant human bone morphogenetic protein-2 (rh-BMP-2), for functional bone regeneration remains challenging due to limited in vivo efficacy and adverse effects of previous modalities. To overcome the instability and short half-life of rh-BMP-2 in vivo, we developed a novel osteogenic supplement by fusing a protein transduction domain (PTD) with BMP-2, effectively creating a prodrug of BMP-2. In this study, we first created an improved PTD-BMP-2 formulation using lipid nanoparticle (LNP) micellization, resulting in downsizing from micrometer to nanometer scale and achieving a more even distribution. The micellized PTD-BMP-2 (mPTD-BMP-2) demonstrated improved distribution and aggregation profiles. As a prodrug of BMP-2, mPTD-BMP-2 successfully activated Smad1/5/8 and induced mineralization with osteogenic gene induction in vitro. In vivo pharmacokinetic analysis revealed that mPTD-BMP-2 had a much more stable pharmacokinetic profile than rh-BMP-2, with a 7.5-fold longer half-life. The in vivo BMP-responsive element (BRE) reporter system was also successfully activated by mPTD-BMP-2. In the in vivo rat tibia distraction osteogenesis (DO) model, micro-computed tomography (micro-CT) scan findings indicated that mPTD-BMP-2 significantly increased bone volume, bone surface, axis moment of inertia (MOI), and polar MOI. Furthermore, it increased the expression of osteogenesis-related genes, and induced bone maturation histologically. Based on these findings, mPTD-BMP-2 could be a promising candidate for the next-generation osteogenesis drug to promote new bone formation in DO surgery. Statement of SignificanceThis study introduces micellized bone morphogenetic protein-2 (mPTD-BMP-2), a next-generation osteogenic supplement that combines protein transduction domain (PTD) and nano-sized micelle formulation technique to improve transduction efficiency and stability. The use of PTD represents a novel approach, and our results demonstrate the superiority of mPTD-BMP-2 over rh-BMP-2 in terms of in vivo pharmacokinetic profile and osteogenic potential, particularly in a rat tibial model of distraction osteogenesis. These findings have significant scientific impact and potential clinical applications in the treatment of bone defects that require distraction osteogenesis. By advancing the field of osteogenic supplements, our study has the potential to contribute to the development of more effective treatments for musculoskeletal disorders.

Administration of recombinant FOXN1 protein attenuates Alzheimer’s pathology in mice

BackgroundAlzheimer’s disease (AD) is the most common cause of dementia in older adults and characterized by progressive loss of memory and cognitive functions that are associated with amyloid-beta (Aβ) plaques and neurofibrillary tangles. Immune cells play an important role in the clearance of Aβ deposits and neurofibrillary tangles. T cells are the major component of the immune system. The thymus is the primary organ for T cell generation. T cell development in the thymus depends on thymic epithelial cells (TECs). However, TECs undergo both qualitative and quantitative loss over time. We have previously reported that a recombinant (r) protein containing FOXN1 and a protein transduction domain can increase the number of TECs and subsequently increases the number of T cells in mice. In this study we determined the ability of rFOXN1 to affect cognitive performance and AD pathology in mice. MethodsAged 3xTg-AD and APP/PS1 AD mice were injected with rFOXN1 or control protein. Cognitive performance, AD pathology, the thymic microenvironment and immune cells were then analyzed. ResultsAdministration of rFOXN1 into AD mice improves cognitive performance and reduces Aβ plaque load and phosphorylated tau in the brain. This is related to rejuvenating the aged thymic microenvironment, which results in enhanced T cell generation in the thymus, leading to increased number of T cells, especially IFNγ-producing T cells, in the spleen and the choroid plexus (CP), enhanced expression of immune cell trafficking molecules in the CP, and increased migration of monocyte-derived macrophages into the brain. Furthermore, the production of anti-Aβ antibodies in the serum and the brain, and the macrophage phagocytosis of Aβ are enhanced in rFOXN1-treated AD mice. ConclusionsOur results suggest that rFOXN1 protein has the potential to provide a novel approach to treat AD patients.

Upregulation of mesencephalic astrocyte-derived neurotrophic factor (MANF) expression offers protection against alcohol neurotoxicity.

Alcohol exposure has detrimental effects on both the developing and mature brain. Endoplasmic reticulum (ER) stress is one of the mechanisms that contributes to alcohol-induced neuronal damages. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-responsive protein and is neuroprotective in multiple neuronal injury and neurodegenerative disease models. MANF deficiency has been shown to exacerbate alcohol-induced ER stress and neurodegeneration. However, it is unknown whether MANF supplement is sufficient to protect against alcohol neurotoxicity. Alcohol alters MANF expression in the brain, but the mechanisms underlying alcohol modulation of MANF expression remain unclear. This study was designed to determine how alcohol alters MANF expression in neuronal cells and whether exogeneous MANF can alleviate alcohol neurotoxicity. We showed that alcohol increased MANF transcription and secretion without affecting MANF mRNA stability and protein degradation. ER stress was necessary for alcohol-induced MANF upregulation, as pharmacological inhibition of ER stress by 4-PBA diminished alcohol-induced MANF expression. In addition, the presence of ER stress response element II (ERSE-II) was required for alcohol-stimulated MANF transcription. Mutations or deletion of this sequence abolished alcohol-regulated transcriptional activity. We generated MANF knockout (KO) neuronal cells using CRISPR/Cas9. MANF KO cells exhibited increased unfolded protein response (UPR) and were more susceptible to alcohol-induced cell death. On the other hand, MANF upregulation by the addition of recombinant MANF protein or adenovirus gene transduction protected neuronal cells against alcohol-induced cell death. Further studies using early postnatal mouse pups demonstrated that enhanced MANF expression in the brain by intracerebroventricular (ICV) injection of MANF adeno-associated viruses ameliorated alcohol-induced cell death. Thus, alcohol increased MANF expression through inducing ER stress, which could be a protective response. Exogenous MANF was able to protect against alcohol-induced neurodegeneration.

Pharmacokinetics, biodistribution and toxicology of novel cell-penetrating peptides

Cell-penetrating peptides (CPPs) have been used in basic and preclinical research in the past 30 years to facilitate drug delivery into target cells. However, translation toward the clinic has not been successful so far. Here, we studied the pharmacokinetic (PK) and biodistribution profiles of Shuttle cell-penetrating peptides (S-CPP) in rodents, combined or not with an immunoglobulin G (IgG) cargo. We compared two enantiomers of S-CPP that contain both a protein transduction domain and an endosomal escape domain, with previously shown capacity for cytoplasmic delivery. The plasma concentration versus time curve of both radiolabelled S-CPPs required a two-compartment PK analytical model, which showed a fast distribution phase (t1/2α ranging from 1.25 to 3 min) followed by a slower elimination phase (t1/2β ranging from 5 to 15 h) after intravenous injection. Cargo IgG combined to S-CPPs displayed longer elimination half-life, of up to 25 h. The fast decrease in plasma concentration of S-CPPs was associated with an accumulation in target organs assessed at 1 and 5 h post-injection, particularly in the liver. In addition, in situ cerebral perfusion (ISCP) of L-S-CPP yielded a brain uptake coefficient of 7.2 ± 1.1 µl g−1 s−1, consistent with penetration across the blood–brain barrier (BBB), without damaging its integrity in vivo. No sign of peripheral toxicity was detected either by examining hematologic and biochemical blood parameters, or by measuring cytokine levels in plasma. In conclusion, S-CPPs are promising non-toxic transport vectors for improved tissue distribution of drug cargos in vivo.

Protein Transduction System Based on Tryptophan-zipper against Intracellular Infections via Inhibiting Ferroptosis of Macrophages.

Cells penetrating molecules in living systems hold promise of capturing and eliminating threats and damage that can plan intracellular fate promptly. However, it remains challenging to construct cell penetration systems that are physiologically stable with predictable self-assembly behavior and well-defined mechanisms. In this study, we develop a core-shell nanoparticle using a hyaluronic acid (HA)-coated protein transduction domain (PTD) derived from the human immunodeficiency virus (HIV). This nanoparticle can encapsulate pathogens, transporting the PTD into macrophages via lipid rafts. PTD forms hydrogen bonds with the components of the membrane through TAT, which has a high density of positive charges and reduces the degree of membrane order through Tryptophan (Trp)-zipper binding to the acyl tails of phospholipid molecules. HA-encapsulated PTD increases the resistance to trypsin and proteinase K, thereby penetrating macrophages and eliminating intracellular infections. Interestingly, the nonagglutination mechanism of PTD against pathogens ensures the safe operation of the cellular system. Importantly, PTD can activate the critical pathway of antiferroptosis in macrophages against pathogen infection. The nanoparticles developed in this study demonstrate safety and efficacy against Gram-negative and Gram-positive pathogens in three animal models. Overall, this work highlights the effectiveness of the PTD nanoparticle in encapsulating pathogens and provides a paradigm for transduction systems-anti-intracellular infection therapy.

Arginine-Rich Cell-Penetrating Peptide-Mediated Transduction of Mouse Nasal Cells with FOXP3 Protein Alleviates Allergic Rhinitis.

Intranasal corticosteroids are effective medications against allergic rhinitis (AR). However, mucociliary clearance promptly eliminates these drugs from the nasal cavity and delays their onset of action. Therefore, a faster, longer-lasting therapeutic effect on the nasal mucosa is required to enhance the efficacy of AR management. Our previous study showed that polyarginine, a cell-penetrating peptide, can deliver cargo to nasal cells; moreover, polyarginine-mediated cell-nonspecific protein transduction into the nasal epithelium exhibited high transfection efficiency with minimal cytotoxicity. In this study, poly-arginine-fused forkhead box P3 (FOXP3) protein, the "master transcriptional regulator" of regulatory T cells (Tregs), was administered into the bilateral nasal cavities of the ovalbumin (OVA)-immunoglobulin E mouse model of AR. The effects of these proteins on AR following OVA administration were investigated using histopathological, nasal symptom, flow cytometry, and cytokine dot blot analyses. Polyarginine-mediated FOXP3 protein transduction induced Treg-like cell generation in the nasal epithelium and allergen tolerance. Overall, this study proposes FOXP3 activation-mediated Treg induction as a novel and potential therapeutic strategy for AR, providing a potential alternative to conventional intranasal drug application for nasal drug delivery.

Demonstration of the Early Cardiac Bioavailability of a Non-Specific Cell-Targeted Peptide Using Radionuclide-Based Imaging In Vivo.

The cardiac bioavailability of peptide drugs that inhibit harmful intracellular protein-protein interactions in cardiovascular diseases remains a challenging task in drug development. This study investigates whether a non-specific cell-targeted peptide drug is available in a timely manner at its intended biological destination, the heart, using a combined stepwise nuclear molecular imaging approach. An octapeptide (heart8P) was covalently coupled with the trans-activator of transcription (TAT) protein transduction domain residues 48-59 of human immunodeficiency virus-1 (TAT-heart8P) for efficient internalization into mammalian cells. The pharmacokinetics of TAT-heart8P were evaluated in dogs and rats. The cellular internalization of TAT-heart8P-Cy(5.5) was examined on cardiomyocytes. The real-time cardiac delivery of 68Ga-NODAGA-TAT-heart8P was tested in mice under physiological and pathological conditions. Pharmacokinetic studies of TAT-heart8P in dogs and rats revealed a fast blood clearance, high tissue distribution, and high extraction by the liver. TAT-heart-8P-Cy(5.5) was rapidly internalized in mouse and human cardiomyocytes. Correspondingly, organ uptake of hydrophilic 68Ga-NODAGA-TAT-heart8P occurred rapidly after injection with an initial cardiac bioavailability already 10 min post-injection. The saturable cardiac uptake was revailed by the pre-injection of the unlabeled compound. The cardiac uptake of 68Ga-NODAGA-TAT-heart8P did not change in a model of cell membrane toxicity. This study provides a sequential stepwise workflow to evaluate the cardiac delivery of a hydrophilic, non-specific cell-targeting peptide. 68Ga-NODAGA-TAT-heart8P showed rapid accumulation in the target tissue early after injection. The implementation of PET/CT radionuclide-based imaging methodology as a means to assess effective and temporal cardiac uptake represents a useful and critical application in drug development and pharmacological research and can be extended to the evaluation of comparable drug candidates.

Bone morphogenetic protein-7 attenuates pancreatic damage under diabetic conditions and prevents progression to diabetic nephropathy via inhibition of ferroptosis.

Approximately 30% of diabetic patients develop diabetic nephropathy, a representative microvascular complication. Although the etiological mechanism has not yet been fully elucidated, renal tubular damage by hyperglycemia-induced expression of transforming growth factor-β (TGF-β) is known to be involved. Recently, a new type of cell death by iron metabolism called ferroptosis was reported to be involved in kidney damage in animal models of diabetic nephropathy, which could be induced by TGF-β. Bone morphogenetic protein-7 (BMP7) is a well-known antagonist of TGF-β inhibiting TGF-β-induced fibrosis in many organs. Further, BMP7 has been reported to play a role in the regeneration of pancreatic beta cells in diabetic animal models. We used protein transduction domain (PTD)-fused BMP7 in micelles (mPTD-BMP7) for long-lasting in vivo effects and effective in vitro transduction and secretion. mPTD-BMP7 successfully accelerated the regeneration of diabetic pancreas and impeded progression to diabetic nephropathy. With the administration of mPTD-BMP7, clinical parameters and representative markers of pancreatic damage were alleviated in a mouse model of streptozotocin-induced diabetes. It not only inhibited the downstream genes of TGF-β but also attenuated ferroptosis in the kidney of the diabetic mouse and TGF-β-stimulated rat kidney tubular cells. BMP7 impedes the progression of diabetic nephropathy by inhibiting the canonical TGF-β pathway, attenuating ferroptosis, and helping regenerate diabetic pancreas.

Biomimetic Liquid Metal Mechatronic Devices

AbstractPerceiving mechanical stimuli and converting them into bioelectric signals to complete sensing information transmission and computation is a fundamental mechanism for survival and evolution of natural animals. This leads to a variety of intelligent behaviors, such as processing, learning, judgment, feedback, and so on when tackling complex and changeable environmental challenges. Until now, the realization of a bionic system to mimic the above activities has long been an important goal which is mainly based on the integration of distributed functional units, lacking the coordination between functional units and the whole systems. Herein, based on the mechano‐electronic coupling effect of liquid metals and by following the basic principle of perception and transduction of piezo proteins, a biomimetic functional liquid metal mechatronic device is proposed with the switching behavior via establishing the reversible charge gradient. Owing to flexible features of liquid metals, this all‐soft mechatronic device exhibits excellent compliance with the whole system. Based on electrochemical characteristics of liquid metals, the passive intelligent device demonstrates versatile behaviors, such as self‐energy supply, encoding, computation, sensing, information identification, communicating, and controlling functions etc. which can only possibly be achieved by living animals. This work opens a new strategy for developing intelligent mechatronic systems.

Lipid transport protein ORP2A promotes glucose signaling by facilitating RGS1 degradation.

Heterotrimeric GTP-binding proteins (G proteins) are a group of regulators essential for signal transmission into cells. Regulator of G protein signaling 1 (AtRGS1) possesses intrinsic GTPase-accelerating protein (GAP) activity and could suppress G protein and glucose signal transduction in Arabidopsis (Arabidopsis thaliana). However, how AtRGS1 activity is regulated is poorly understood. Here, we identified a knockout mutant of oxysterol binding protein-related protein 2A, orp2a-1, which exhibits similar phenotypes to the arabidopsis g-protein beta 1-2 (agb1-2) mutant. Transgenic lines overexpressing ORP2A displayed short hypocotyls, a hypersensitive response to sugar, and lower intracellular AtRGS1 levels than the control. Consistently, ORP2A interacted with AtRGS1 in vitro and in vivo. Tissue-specific expression of 2 ORP2A alternative splicing isoforms implied functions in controlling organ size and shape. Bioinformatic data and phenotypes of orp2a-1, agb1-2, and the orp2a-1 agb1-2 double mutant revealed the genetic interactions between ORP2A and Gβ in the regulation of G protein signaling and sugar response. Both alternative protein isoforms of ORP2A localized in the endoplasmic reticulum (ER), plasma membrane (PM), and ER-PM contact sites and interacted with vesicle-associated membrane protein-associated protein 27-1 (VAP27-1) in vivo and in vitro through their two phenylalanines in an acidic track-like motif. ORP2A also displayed differential phosphatidyl phosphoinositide binding activity mediated by the pleckstrin homology domain in vitro. Taken together, the Arabidopsis membrane protein ORP2A interacts with AtRGS1 and VAP27-1 to positively regulate G protein and sugar signaling by facilitating AtRGS1 degradation.

Protective Effects of a Jellyfish-Derived Thioredoxin Fused with Cell-Penetrating Peptide TAT-PTD on H2O2-Induced Oxidative Damage.

Thioredoxin (Trx) plays a critical role in maintaining redox balance in various cells and exhibits anti-oxidative, anti-apoptotic, and anti-inflammatory effects. However, whether exogenous Trx can inhibit intracellular oxidative damage has not been investigated. In previous study, we have identified a novel Trx from the jellyfish Cyanea capillata, named CcTrx1, and confirmed its antioxidant activities in vitro. Here, we obtained a recombinant protein, PTD-CcTrx1, which is a fusion of CcTrx1 and protein transduction domain (PTD) of HIV TAT protein. The transmembrane ability and antioxidant activities of PTD-CcTrx1, and its protective effects against H2O2-induced oxidative damage in HaCaT cells were also detected. Our results revealed that PTD-CcTrx1 exhibited specific transmembrane ability and antioxidant activities, and it could significantly attenuate the intracellular oxidative stress, inhibit H2O2-induced apoptosis, and protect HaCaT cells from oxidative damage. The present study provides critical evidence for application of PTD-CcTrx1 as a novel antioxidant to treat skin oxidative damage in the future.

Adhesive Hydrogel Patch-Mediated Combination Drug Therapy Induces Regenerative Wound Healing through Reconstruction of Regenerative Microenvironment.

Regenerative wound healing involves the scarless wound healing as observed in fetal skin. Multiple features of regenerative wound healing have been well studied; however, the practical application of pro-regenerative materials to recapitulate the regenerative wound healing in adult skins has not yet been achieved. In this study, the authors identified that their novel pro-regenerative material, pyrogallol-functionalized hyaluronic acid (HA-PG) patches in combination with protein transduction domain-fused Dishevelled (Dvl)-binding motif (PTD-DBM), a peptide inhibiting the CXXC-type zinc finger protein 5 (CXXC5)-Dvl interaction, promoted regenerative wound healing in mice. The HA-PG patches loaded with this competitor peptide and valproic acid (VPA), a glycogen synthase kinase 3β (GSK3β) inhibitor, significantly inhibited scar formation during wound healing. The HA-PG patches with PTD-DBM and/or VPA inhibit the expression of differentiated cell markers such as α-smooth muscle actin (α-SMA) while inducing the expression of stem cell markers such as CD105 and Nestin. Moreover, Collagen III, an important factor for regenerative healing, is critically induced by the HA-PG patches with PTD-DBM and/or VPA, as also seen in VPA-treated Cxxc5-/- mouse fibroblasts. Overall, these findings suggest that the novel regeneration-promoting material can be utilized as a potential therapeutic agent to promote both wound healing and scar attenuation.

PTD-FGF2 Attenuates Elastase Induced Emphysema in Mice and Alveolar Epithelial Cell Injury

Aberrant communication in alveolar epithelium is a major feature of inflammatory response for the airway remodeling leading to chronic obstructive pulmonary disease (COPD). In this study, we investigated the effect of protein transduction domains (PTD) conjugated Basic Fibroblast Growth Factor (FGF2) (PTD-FGF2) in response to cigarette smoke extract (CSE) in MLE-12 cells and porcine pancreatic elastase (PPE)-induced emphysematous mice. When PPE-induced mice were intraperitoneally treated with 0.1–0.5 mg/kg PTD-FGF2 or FGF2, the linear intercept, infiltration of inflammatory cells into alveoli and pro-inflammatory cytokines were significantly decreased. In western blot analysis, phosphorylated protein levels of c-Jun N-terminal Kinase 1/2 (JNK1/2), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPK) were decreased in PPE-induced mice treated PTD-FGF2. In MLE-12 cells, PTD-FGF2 treatment decreased reactive oxygen species (ROS) production and further decreased Interleukin-6 (IL-6) and IL-1b cytokines in response to CSE. In addition, phosphorylated protein levels of ERK1/2, JNK1/2 and p38 MAPK were reduced. We next determined microRNA expression in the isolated exosomes of MLE-12 cells. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, level of let-7c miRNA was significantly increased while levels of miR-9 and miR-155 were decreased in response to CSE. These data suggest that PTD-FGF2 treatment plays a protective role in regulation of let-7c, miR-9 and miR-155 miRNA expressions and MAPK signaling pathways in CSE-induced MLE-12 cells and PPE-induced emphysematous mice.

Identification and Biological Evaluation of a Water-Soluble Fullerene Nanomaterial as BTK Kinase Inhibitor.

Thanks to recent advances in synthetic methodology, water-soluble fullerene nanomaterials that interfere with biomolecules, especially DNA/RNA and selected proteins, have been found with tremendous potential for applications in nanomedicine. Herein, we describe the synthesis and evaluation of a water-soluble glycine-derived [60]fullerene hexakisadduct (HDGF) with T h symmetry, which is a first-in-class BTK protein inhibitor. We synthesized and characterized glycine derived [60]fullerene using NMR, ESI-MS, and ATR-FT-IR. DLS and zeta potential were measured and high-resolution transmission electron microscopy (HRTEM) observations were performed. The chemical composition of the water-soluble fullerene nanomaterial was examined by X-ray photoelectron spectrometry. To observe aggregate formation, the cryo-TEM analysis was carried out. The docking studies and molecular dynamic simulations were performed to determine interactions between HDGF and BTK. The in vitro cytotoxicity was evaluated on RAJI and K562 blood cancer cell lines. Subsequently, we examined the induction of cell death by autophagy and apoptosis by determining the expression levels of crucial genes and caspases. We investigated the direct association of HDGF on inhibition of the BTK signalling pathway by examining changes in the calcium levels in RAJI cells after treatment. The inhibitory potential of HDGF against non-receptor tyrosine kinases was evaluated. Finally, we assessed the effects of HDGF and ibrutinib on the expression of the BTK protein and downstream signal transduction in RAJI cells following anti-IgM stimulation. Computational studies revealed that the inhibitory activity of the obtained [60]fullerene derivative is multifaceted: it hampers the BTK active site, interacting directly with the catalytic residues, rendering it inaccessible to phosphorylation, and binds to residues that form the ATP binding pocket. The anticancer activity of produced carbon nanomaterial revealed that it inhibited the BTK protein and its downstream pathways, including PLC and Akt proteins, at the cellular level. The mechanistic studies suggested the formation of autophagosomes (increased gene expression of LC3 and p62) and two caspases (caspase-3 and -9) were responsible for the activation and progression of apoptosis. These data illustrate the potential of fullerene-based BTK protein inhibitors as nanotherapeutics for blood cancer and provide helpful information to support the future development of fullerene nanomaterials as a novel class of enzyme inhibitors.

Research advances on the role and mechanism of microRNA in hypertrophic scar

Hypertrophic scar (HS) affects the function and beauty of patients, and brings a heavy psychological burden to patients. However, the specific pathogenesis mechanism of HS in molecular biology level is not yet clear, and this disease is still one of the clinical diseases difficult to prevent and cure. MicroRNA (miR) is a family of single-stranded endogenous noncoding RNAs that can regulate gene expression. The abnormal transcription of miR in hypertrophic scar fibroblasts can affect the transduction and expression of downstream signal pathway or protein, and the exploration of miR and its downstream signal pathway and protein helps deeply understand the occurrence and development mechanism of scar hyperplasia. This article summarized and analyzed how miR and multiple signal pathways involve in the formation and development of HS in recent years, and further outlined the interaction between miR and target genes in HS.