Metabolomics and miRNA profiling reveals feature of gallbladder cancer-derived biliary extracellular vesicles.

BACKGROUND: Although there are several studies in the development of various human cancers, the role of exosomes is poorly understood in the progression of gallbladder cancer. This study aims to characterize the metabolic changes occurring in exosomes obtained from patients with gallbladder cancer compared with those from other gallbladder disease groups. METHODS: Biliary exosomes were isolated from healthy donors (n = 3) and from patients with gallbladder cancer (n = 3), gallbladder polyps (n = 4), or cholecystitis (n = 3) using a validated exosome isolation kit. Afterward, we performed miRNA profiling and untargeted metabolomic analysis of the exosomes. The results were validated by integrating the results of the miRNA and metabolomic analyses. RESULTS: The gallbladder cancer group exhibited a significant reduction in the levels of multiple unsaturated phosphatidylethanolamines and phosphatidylcholines compared to the normal group, which resulted in the loss of exosome membrane integrity. Additionally, the gallbladder cancer group demonstrated significant overexpression of miR-181c and palmitic acid, and decreased levels of conjugated deoxycholic acid, all of which are strongly associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: Our findings demonstrate that the contents of exosomes are disease-specific, particularly in gallbladder cancer, and that altered metabolites convey critical information regarding their phenotype. We believe that our metabolomic and miRNA profiling results may provide important insights into the development of gallbladder cancer.

Association of Microbial Dysbiosis with Gallbladder Diseases Identified by Bile Microbiome Profiling.

BACKGROUND: Cholecystitis is an important risk factor for gallbladder cancer, but the bile microbiome and its association with gallbladder disease has not been investigated fully. We aimed to analyze the bile microbiome in normal conditions, chronic cholecystitis, and gallbladder cancer, and to identify candidate bacteria that play an important role in gallbladder carcinogenesis. METHODS: We performed metagenome sequencing on bile samples of 10 healthy individuals, 10 patients with chronic cholecystitis, and 5 patients with gallbladder cancer, and compared the clinical, radiological, and pathological characteristics of the participants. RESULTS: No significant bacterial signal was identified in the normal bile. The predominant dysbiotic bacteria in both chronic cholecystitis and gallbladder cancer were those belonging to the Enterobacteriaceae family. Klebsiella increased significantly in the order of normal, chronic cholecystitis, and gallbladder cancer. Patients with chronic cholecystitis and dysbiotic microbiome patterns had larger gallstones and showed marked epithelial atypia, which are considered as precancerous conditions. CONCLUSION: We investigated the bile microbiome in normal, chronic cholecystitis, and gallbladder cancer. We suggest possible roles of Enterobacteriaceae, including Klebsiella, in gallbladder carcinogenesis. Our findings reveal a possible link between a dysbiotic bile microbiome and the development of chronic calculous cholecystitis and gallbladder cancer.

Subsequent meniscal tears following tibial tuberosity advancement and tibial plateau leveling osteotomy in dogs with cranial cruciate ligament deficiency: An in vivo experimental study.

OBJECTIVE: To evaluate the short- and mid-term effects of tibial tuberosity advancement (TTA) and tibial plateau leveling osteotomy (TPLO) on subsequent meniscal tears. STUDY DESIGN: Experimental in vivo study. ANIMALS: Purpose-bred beagle dogs (n = 15). METHODS: For each dog, the cranial cruciate ligaments were transected; one limb underwent TTA and the other limb underwent TPLO. Orthopedic and radiographic examinations were performed preoperatively and at 12 and 32 weeks postoperatively. Gross evaluation of the stifle joint was performed after euthanasia at 12 (n = 10) and 32 (n = 5) weeks. RESULTS: Lameness scores were not different between TTA and TPLO limbs at any time point. Radiographic osteoarthritis scores of TTA stifles (1.33 ± 0.49) were higher than TPLO stifles (0.67 ± 0.49) (p = .002) at 12 weeks postoperatively, but there was no difference between groups at 32 weeks postoperatively. Subsequent medial meniscal tears occurred in 6/10 TTA stifles, and 0/10 TPLO stifles at 12 weeks postoperatively and in 5/5 TTA stifles, and 1/5 TPLO stifles at 32 weeks postoperatively. Subsequent lateral meniscal tears occurred in 4/5 TTA stifles at 32 weeks postoperatively. Medial meniscal total gross pathology score was higher in TTA than TPLO stifles. TTA stifles had more articular cartilage damage when compared with TPLO stifles at 32 weeks postoperatively. CONCLUSION: In this within-dog experimental comparison, subsequent medial meniscal tears and cartilage injury was more prevalent following TTA when compared to TPLO. CLINICAL SIGNIFICANCE: In an experimental model, TPLO protects the medial meniscus and articular cartilage better than TTA in stifles with complete cranial cruciate ligament deficiency.

Exosomes derived from chemically induced human hepatic progenitors inhibit oxidative stress induced cell death.

The emerging field of regenerative medicine has revealed that the exosome contributes to many aspects of development and disease through intercellular communication between donor and recipient cells. However, the biological functions of exosomes secreted from cells have remained largely unexplored. Here, we report that the human hepatic progenitor cells (CdHs)-derived exosome (EXOhCdHs ) plays a crucial role in maintaining cell viability. The inhibition of exosome secretion treatment with GW4869 results in the acceleration of reactive oxygen species (ROS) production, thereby causing a decrease of cell viability. This event provokes inhibition of caspase dependent cell death signaling, leading to a ROS-dependent cell damage response and thus induces promotion of antioxidant gene expression or repair of cell death of hypoxia-exposed cells. Together, these findings show the effect of exosomes in regeneration of liver cells, and offer valuable new insights into liver regeneration.

Biomechanical analysis of canine medial patellar luxation with femoral varus deformity using a computer model.

BACKGROUND: Femoral varus deformities complicating the realignment of the quadriceps muscles are frequently associated with medial patellar luxation (MPL) in dogs. Therefore, distal femoral osteotomy (DFO) is recommended in dogs affected with severe MPL and a distal femoral varus deformity. The presence of an anatomic lateral distal femoral angle (aLDFA) of ≥ 102° has been anecdotally recommended as an indication for performing corrective DFO in large-breed dogs. However, the effect of a femoral varus deformity on MPL has not been scientifically evaluated. We aimed to evaluate the influence of a femoral varus deformity on MPL using a finite element method based computer model. Three-dimensionally reconstructed computed tomographic images of a normal femur from a Beagle dog were deformed using meshing software to create distal varus deformities. A total of thirteen aLDFAs, including 95°, 98° and 100°-110°, were simulated. The patellar positions and reaction force between the patella and trochlear grooves were calculated for all finite element models under constant rectus femoris muscle activation. RESULTS: The patella was displaced medially from the trochlear groove at an aLDFA of ≥103°. With an aLDFA of 103° to 110°, the reaction force was equal to zero and then decreased to negative values during the simulation, while other models with aLDFAs of 95°, 98°, and 100°-102° had positive reaction force values. The patella began to luxate at 24.90 seconds (sec) with an aLDFA of 103°, 19.80 sec with an aLDFA of 104°, 21.40 sec with an aLDFA of 105°, 20.10 sec with an aLDFA of 106°, 18.60 sec with an aLDFA of 107°, 15.30 sec with an aLDFA of 108°, 16.60 sec with an aLDFA of 109°, and 11.90 sec with an aLDFA of 110°. CONCLUSION: Severe distal femoral varus with an aLDFA of ≥103° caused MPL when other anatomical factors were controlled. Thissimplified computer model provides complementary information to anecdotal cutoffs for DFO, hence it should be applied to clinical patients with caution.

Imaging Hydrogen Sulfide in Hypoxic Tissue with [99mTc]Tc-Gluconate.

Hydrogen sulfide (H2S) is the third gasotransmitter and is generated endogenously in hypoxic or inflammatory tissues and various cancers. We have recently demonstrated that endogenous H2S can be imaged with [99mTc]Tc-gluconate. In the present study, we detected H2S generated in hypoxic tissue, both in vitro and in vivo, using [99mTc]Tc-gluconate. In vitro uptake of [99mTc]Tc-gluconate was measured under hypoxic and normoxic conditions, using the colon carcinoma cell line CT26, and was higher in hypoxic cells than that in normoxic cells. An acute hindlimb ischemia-reperfusion model was established in BALB/c mice by exposing the animals to 3 h of ischemia and 3 h of reperfusion prior to in vivo imaging. [99mTc]Tc-gluconate (12.5 MBq) was intravenously injected through the tail vein, and uptake in the lower limb was analyzed by single-photon emission computed tomography/computed tomography (SPECT/CT). SPECT/CT images showed five times higher uptake in the ischemic limb than that in the normal limb. The standard uptake value (SUVmean) of the ischemic limb was 0.39 ± 0.03, while that of the normal limb was 0.07 ± 0.01. [99mTc]Tc-gluconate is a novel imaging agent that can be used both in vitro and in vivo for the detection of endogenous H2S generated in hypoxic tissue.

Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells.

BACKGROUND & AIMS: Currently, much effort is directed towards the development of new cell sources for clinical therapy using cell fate conversion by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. METHODS: Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by 2 small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. RESULTS: We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of the fate conversion process. Based on functional evidence, activation of the HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through the activation of the ERK pathway. hCdHs expressed hepatic progenitor markers and could self-renew for at least 10 passages while retaining a normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. Gene expression profiling using RNAseq confirmed the transcriptional reprogramming of hCdHs towards a progenitor state and the suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation in several models of therapeutic liver repopulation, hCdHs effectively repopulated the damaged parenchyma. CONCLUSION: Our study is the first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a novel tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and the repair of diseased livers. LAY SUMMARY: Human primary hepatocytes were reprogrammed towards hepatic progenitor cells by a combined treatment with 2 small molecules, A83-01 and CHIR99021, and HGF. Chemically derived hepatic progenitors exhibited a high proliferation potential and the ability to differentiate into hepatocytes and biliary epithelial cells both in vitro and in vivo. This approach enables the generation of patient-specific hepatic progenitors and provides a platform for personal and stem cell-based regenerative medicine.

Improved infrared spectroscopic discrimination between gall bladder (GB) polyps and GB cancer using component-descriptive spectral features of separated phases from bile.

This study demonstrates a unique strategy for enhancing infrared (IR) spectroscopic discrimination between gall bladder (GB) polyps and cancer. This strategy includes the separation of raw bile juice into three sections of organic, aqueous, and amphiphilic phases and a cooperative combination of all IR spectral features of each separated phase for the discrimination. Raw bile juice is viscous and complex in composition because it contains fatty acids, cholesterol, proteins, phospholipids, bilirubin, and other components; therefore, the acquisition of IR spectra providing more component-discernible information is fundamental for improving discrimination. For this purpose, raw bile juice was separated into an aqueous phase, mostly containing bile salts, an organic phase with isolated lipids, and an amphiphilic phase, mainly containing proteins. The subsequent IR spectra of each separated phase were mutually characteristic and complementary to each other. When all the IR spectral features were combined, the discrimination was improved compared to that using the spectra of raw bile juice with no separation. The cooperative integration of more component-specific spectra obtained from each separated phase enhanced the discrimination. In addition, the IR spectra of the major constituents in bile juice, such as bile acids, conjugated bile salts, lecithin, and cholesterol, were recorded to explain the IR features of each separated phase.

Evaluation of a self-retaining distractor for hip joint arthroscopy in toy breed dogs.

BACKGROUND: Hip arthroscopy has become a viable option over the last few years for small animal orthopedic diseases, including hip dysplasia, osteoarthritis, and joint evaluation. However, the narrow joint spaces make it difficult to manipulate the instrument, and depth of tissues make it difficult to distract the joint space. In addition, it is very difficult to maintain consistent distraction over time with a manual distraction due to hand fatigue. To overcome these difficulties, distractors are used in human medicine to improve safety and accuracy of arthroscopy. Therefore, in this study, distractor devices were applied to hip joints in small toy breed dogs to evaluate their technical efficacy. Potential iatrogenic neurovascular and articular damage were also evaluated by comparing two techniques for performing hip joint arthroscopy: the self-retaining distractor and external manipulation. RESULTS: The mean ± SD of the joint distraction distance was 8.88 ± 3.54 mm in the self-retaining distraction group and 2.37 ± 0.82 mm in the manual traction group. As the joint space increased, surgeons could more easily place an arthroscopy portal and more comfortably manipulate the instrument with a distractor device. Furthermore, the acetabular cartilage damage (p = 0.004) was significantly greater in the external manipulation group, but articular damage to the femoral head (p = 0.940) was similar in both groups. CONCLUSION: The results of this study revealed that use of a distractor device can be a viable option for performing hip arthroscopy in small animals. The device significantly improved the surgeon's performance without surgical assistance, and it reduced iatrogenic cartilage damage compared with manual traction. Further study is needed to quantify neurapraxia associated with distractor placement.

Fbxw7ß is an inducing mediator of dexamethasone-induced skeletal muscle atrophy in vivo with the axis of Fbxw7ß-myogenin-atrogenes.

Muscle atrophy is induced by several pathways, e.g., it can be attributed to inherited cachectic symptoms, genetic disorders, sarcopenia, or chronic side effects of treatments. However, the underlying regulatory mechanisms that contribute to muscle atrophy have not been fully elucidated. In this study, we evaluated the role of Fbxw7ß, an ubiquitin E3 ligase, in a dexamethasone-induced muscle atrophy model. In this model, endogenous Fbxw7ß was up-regulated; furthermore, the Fbxw7ß-myogenin-atrogene axis was upregulated, supporting our previous results linking Fbxw7ß to muscle atrophy in vitro. Also, muscle atrophy was associated with the Fbxw7ß-myogenin-atrogene axis and the down-regulation of Dach2, a repressor of myogenin. Taken together, these results suggest that the ubiquitin E3 ligase Fbxw7ß and the Fbxw7ß-myogenin-atrogene axis have important roles in a dexamethasone-induced muscle atrophy model in vivo and in vitro. Additionally, the Fbxw7ß-Dach2-myogenin-atrogene axis is a potential mechanism underlying muscle atrophy in cases of abnormal Fbxw7ß expression-induced muscle atrophy or myogenic degenerative disease.

Skeletal muscle atrophy is induced by Fbxw7ß via atrogene upregulation.

Muscle atrophy decreases skeletal muscle mass and is induced by inherited cachectic symptoms, genetic disorders, and sarcopenia. However, the molecular pathways associated with the onset of muscle atrophy are still unclear. In this study, we evaluated Fbxw7ß, a gene associated with the development of muscle atrophy in vitro and in vivo. Among the three Fbxw7 isoforms, ectopically overexpressed Fbxw7ß induced the expression of myogenin and major atrogene markers (atrogin-1 and MuRF-1) and reduced myoblast differentiation. In addition, endogenous expression of Fbxw7ß was also upregulated by dexamethasone, which mimics muscle atrophy in vitro, accompanied by induction of myogenin and atrogene expression in primary myoblasts. Functional analysis of Fbxw7ß using short hairpin RNA (shRNA) and a dominant-negative mutant (ΔFbox) suggested that Fbxw7ß regulated muscle atrophy in vitro and in vivo. In particular, ΔFbox did not reduce the sizes of muscle fibers and did not induce myogenin and atrogene expression in vivo. Therefore, our findings demonstrated, for the first time, that Fbxw7ß induced muscle atrophic phenotypes via atrogenes in adult muscle precursor cells and myofibers; this mechanism could be a potential therapeutic target for skeletal muscle atrophy.

SIRT1 deacetylates and stabilizes hypoxia-inducible factor-1α (HIF-1α) via direct interactions during hypoxia.

Upon shift to a hypoxic environment, cellular HIF-1α protein is stabilized, with a rapid decline in oxygen-sensitive hydroxylation. Several additional post-translational modifications of HIF-1α are critical in controlling protein stability during hypoxia. In the present study, we showed that SIRT1 stabilizes HIF-1α via direct binding and deacetylation during hypoxia. SIRT1 depletion or inactivation led to reduced hypoxic HIF-1α accumulation, accompanied by an increase in HIF-1α acetylation. Impaired HIF-1α accumulation was recovered upon inhibition of 26S proteasome activity, indicating that SIRT1 is essential for HIF-1α stabilization during hypoxia. Consistently, HIF-1α accumulation was enhanced upon overexpression of wild-type SIRT1, but not its dominant-negative form. SIRT1-mediated accumulation of HIF-1α protein led to increased expression of HIF-1α target genes, including VEGF, GLUT1 and MMP2, and ultimate promotion of cancer cell invasion. These findings collectively imply that hypoxic HIF-1α stabilization requires SIRT1 activation. Furthermore, SIRT1 protection of HIF-1α from acetylation may be a prerequisite for stabilization and consequent enhancement of cell invasion.

Patient-tailored application for Duchene muscular dystrophy on mdx mice based induced mesenchymal stem cells.

Mesenchymal stem cells (MSCs) may be used as powerful tools for the repair and regeneration of damaged tissues. However, isolating tissue specific-derived MSCs may cause pain and increased infection rates in patients, and repetitive isolations may be required. To overcome these difficulties, we have examined alternative methods for MSC production. Here, we show that induced pluripotent stem cells (iPSCs) may be differentiated into mesenchymal stem cells (iMSCs) following exposure to SB431542. Purified iMSCs were administered to mdx mice to study skeletal muscle regeneration in a murine model of muscular dystrophy. Purified iMSCs displayed fibroblast-like morphology, formed three-dimensional spheroid structures, and expressed characteristic mesenchymal stem cell surface markers such as CD29, CD33, CD73, CD90, and CD105. Moreover, iMSCs were capable of differentiating into adipogenic, osteogenic, and chondrogenic lineages. Transplanting iMSC cells to tibialis anterior skeletal muscle tissue in mdx mice lowered oxidative damage as evidenced by a reduction in nitrotyrosine levels, and normal dystrophin expression levels were restored. This study demonstrates the therapeutic potential of purified iMSCs in skeletal muscle regeneration in mdx mice, and suggests that iPSCs are a viable alternate source for deriving MSCs as needed.

Biomechanical Comparison of Double 2.3-mm Headless Cannulated Self-Compression Screws and Single 3.5-mm Cortical Screw in Lag Fashion in a Canine Sacroiliac Luxation Model: A Small Dog Cadaveric Study.

OBJECTIVE: The aim of this study was to evaluate the feasibility of safe positioning of double 2.3-mm headless cannulated self-compression screws (HCS) in a small dog cadaveric sacroiliac luxation model and to compare the static rotational biomechanical properties of fixation repaired using two different screw systems with a minimally invasive osteosynthesis technique: double 2.3-mm HCS and a single 3.5-mm standard cortical screw placed in a lag fashion. STUDY DESIGN: A unilateral small dog sacroiliac luxation model was stabilized using double 2.3-mm HCS (n = 11) or a single 3.5-mm cortical screw (n = 11). Radiographic and computed tomography (CT) imaging analyses and biomechanical testing of rotational force on the sacroiliac joint of both fixations were performed. The maximum load at failure and failure modes of each fixation were recorded and compared. RESULTS: Fluoroscopically guided percutaneous application of double HCS was safe in a unilateral sacroiliac luxation model in small dogs without violation of the vertebral and ventral sacral foramen. Furthermore, resistance to rotational force applied on fixation of the sacroiliac joint repaired with double 2.3-mm HCS estimated by maximum failure load was significantly higher than that of a single 3.5-mm cortical screw (p < 0.001). CONCLUSION: Although this was an experimental cadaveric study, based on our results, the use of smaller double HCS may be beneficial as an alternative to the conventional single lag screw for stabilization of sacroiliac luxation in small dogs.

Reconstruction of Bilateral Chronic Triceps Brachii Tendon Disruption Using a Suture-Mediated Anatomic Footprint Repair in a Dog.

A 2-year-old, intact female Pomeranian presented with bilateral forelimb lameness, characterized by the olecranon making contact with the ground. The patient experienced two separate incidents of falling, occurring four and three weeks before admission, respectively. Following each episode, non-weight-bearing lameness was initially observed in the left forelimb, followed by the development of crouch gait. Based on the physical examination, radiographic, and ultrasonographic findings, bilateral triceps brachii tendon disruption was diagnosed. Intraoperatively, excessive granulation tissue at the distal end of the tendon was excised. The footprint region of each triceps brachii tendon was decorticated with a high-speed burr until bleeding was observed. The triceps brachii tendon was reattached to completely cover its footprint on the olecranon using the Krackow suture technique. This method involves anchoring the suture through bone tunnels in the ulna. Trans-articular external skeletal fixation was applied to both forelimbs to immobile and stabilize the elbow joints for nine weeks. Subsequently, the dog gradually increased its walking activities while on a leash over a six-week period. At the three-year follow-up, the patient exhibited improved forelimb function and maintained a normal gait without signs of lameness. Suture-mediated anatomic footprint repair proved useful in this single case and may be an effective surgical alternative for the management of chronic triceps brachii tendon disruption in dogs.

Laparoscopic treatment of paraprostatic cyst in two dogs - complete resection, and partial resection with omentalization: a case report.

Two intact male dogs were evaluated for symptoms, including hematuria, prostatomegaly, anuria, lethargy, and abdominal mass. Presurgical evaluations, including complete physical examinations, blood examinations, abdominal radiography with contrast (only in Case 2), ultrasonography, and computed tomography and magnetic resonance imaging (only in Case 1), were performed. A paraprostatic cyst was diagnosed initially, and laparoscopic exploration and surgery were performed. Complete resection was performed in case 1, whereas partial resection with omentalization was performed in case 2. Histopathological examination of the tissue samples confirmed the presence of paraprostatic pseudocysts in both cases, with no evidence of an epithelial lining. These two cases represent the first documented instances of laparoscopic treatment for extraparenchymal prostatic cysts. The laparoscopic treatment proved feasible even in the case of a giant cyst causing anuria (Case 2). Paraprostatic cysts should be considered a potential differential diagnosis for abnormal urination accompanied by an abdominal mass, and long-term postoperative follow-up is necessary.

Surgical Correction of a Sinus Venosus Atrial Septal Defect with Partial Anomalous Pulmonary Venous Connections Using Cardiac Computed Tomography Imaging and a 3D-Printed Model.

Sinus venosus atrial septal defects (SVASDs), concurrent with partial anomalous pulmonary venous connections (PAPVCs), are a rare congenital heart disease in dogs. Surgical correction is essential when clinical signs or significant hemodynamic changes are present. We aimed to report on the successful surgical correction of an SVASD with PAPVCs, using a computed tomography (CT)-based customized 3D cardiac model. A 10-month-old male poodle was referred for corrective surgery for an ASD. Echocardiography confirmed a hemodynamically significant left-to-right shunting flow through an interatrial septal defect and severe right-sided heart volume overload. For a comprehensive diagnosis, a CT scan was performed, which confirmed an SVASD with PAPVCs. A customized 3D cardiac model was used for preoperative decision-making and surgical rehearsal. The defect was repaired using an autologous pericardial patch under a cardiopulmonary bypass (CPB). Temporary pacing was applied for sinus bradycardia and third-degree atrioventricular block. The patient recovered from the anesthesia without further complications. The pacemaker was removed during hospitalization and the patient was discharged without complications 2 weeks post-surgery. At the three-month follow-up, there was no shunting flow in the interatrial septum and the right-sided volume overload had been resolved. The cardiac medications were discontinued, and there were no complications. This report indicates the validity of surgical correction under CPB for an SVASD with PAPVCs, and the advantages of utilizing a CT-based 3D cardiac model for preoperative planning to increase the surgical success rate.

Regulation of differentiation potential of human mesenchymal stem cells by intracytoplasmic delivery of coactivator-associated arginine methyltransferase 1 protein using cell-penetrating peptide.

Recent studies suggest that epigenetic modifications, such as DNA methylation and histone modification, can alter the differentiation potential of stem cells or progenitor cells. Specifically, coactivator-associated arginine methyltransferase 1 (CARM1) is known to act as a coactivator for various transcription factors and to regulate gene expression by chromatin remodeling through histone methylation. Here, for the first time, we have used direct protein delivery of CARM1 using cell-penetrating peptide (CPP) to regulate the differentiation potential of human mesenchymal stem cells (hMSCs). Immunofluorescence showed that the CPP-CARM1 protein is successfully delivered into the nuclei of hMSCs. Further experiments using immunofluorescence and Western blotting showed that the delivered CARM1 protein can effectively methylate the arginine 17 residue of histone H3 in both bone marrow (BM)- and adipose-derived (AD)-hMSCs, thus suggesting that the CARM1 protein delivered by the CPP system is biologically active in hMSCs. Chromatin immunoprecipitation (ChIP) assay and genome-wide gene expression profiling supported the result that delivered CARM1 protein can cause chromatin remodeling through histone methylation. Finally, the CPP-CARM1 protein efficiently elevated the differentiation efficiency of BM-hMSCs and AD-hMSCs into adipogenic, osteogenic, and myogenic cell lineages in vitro. Altered expression of critical genes after hMSC differentiation was reconfirmed by real-time reverse transcription polymerase chain reaction (qRT-PCR). Collectively, our results suggest that CPP-CARM1 can elevate the differentiation potential of hMSCs into various cell types, and that this system using CPP is a useful tool for exogenous protein delivery in clinical applications of cell-based therapy.

reliable Synthesis of Monodisperse Microparticles: Prevention of Oxygen Diffusion and Organic Solvents Using Conformal Polymeric Coating onto Poly(dimethylsiloxane) Micromold.

An effective polymeric thin film deposited by initiated chemical vapor deposition (iCVD) process was presented and its application as a barrier film on the PDMS micromold blocking the penetration of oxygen and organic solvents was investigated. With this barrier film, we were able to synthesize monodisperse polymeric particles of sizes down to 3 µm, which has been reported to be extremely challenging with bare PDMS micromold. The polymeric barrier film on the PDMS micromold enabled this successful synthesis of microparticles by effectively blocking the diffusion of oxygen, which is a well-known radical quencher in radical polymerization, through the PDMS micromold. Furthermore, the iCVD barrier film substantially decreased the penetration of various organic solvents such as acetone, tert-butanol, PDMS oil, and decane as well as organic substances including fluorescent molecules like rhodamine B and fluorescein isothiocyanate (FITC). Therefore, the polymeric barrier film coated on PDMS micromold via iCVD process will broaden the application of PDMS to microfluidic area for the synthesis of smaller microparticles with various organic substances.