Hypocapnia Stimuli-Responsive Engineered Exosomes Delivering miR-218 Facilitate Sciatic Nerve Regeneration.

Therapeutic strategies of microRNAs (miRNAs) and exosomes have been systematically explored as an enhancing application by paracrine and modulating cellular activity after internalization of recipient cells in vitro, and progressively developed to meet the requirements of peripheral nerve regeneration in vivo. However, how to obtain exosomes with superior properties and effectively deliver miRNAs becomes a key challenge. Hypocapnia environment might play unexpected outcomes in strengthening exosome function when culturing adipose-derived stem cells (ASCs). Previously, we discovered the intensive regulation of miR-218 on the differentiation of ASCs. In the present study, we analyzed the functional differences of secreted exosomes in response to hypocapnia stimulation, and explored the application in combination with miR-218 to facilitate sciatic nerve regeneration. Our results indicated that the delivery system of engineered exosomes derived from ASCs remarkably loads upregulated miR-218 and promotes cellular activity in the recipient cells (PC12 cells), and hypocapnia stimuli-responsive exosomes exhibit strengthening properties. Furthermore, in a sciatic nerve injury model, exosomes delivering miR-218 combined with engineered scaffold facilitated the regeneration of injured sciatic nerves. In the hypocapnia-stimulated exosome group, more encouraging promotion was revealed on the regeneration of motor and nerve fibers. Hypoc-miR-218-ASC exosomes are suggested as a promising cell-free strategy for peripheral nerve repair.

MSI2 knockdown represses extrahepatic cholangiocarcinoma growth and invasion by inhibiting epithelial-mesenchymal transition.

PURPOSE: To investigate the expression and functional role of Musashi2 (MSI2), an RNA-binding protein, in extrahepatic cholangiocarcinoma (eCCA). PATIENTS AND METHODS: We measured MSI2 expression in human specimens and cell lines using Western blot and quantitative real-time polymerase chain reaction, and we analyzed its association with clinicopathologic features in eCCA patients. Univariate and multivariate analyses were performed to identify risk factors correlated with overall survival and disease-free survival. Functional experiments were used to study the mechanisms of MSI2 in regulating eCCA cell growth, migration, and invasion. RESULTS: MSI2 expression was upregulated significantly in both human specimens and cell lines, and high MSI2 expression was associated with lymph node metastasis, advanced TNM stage, and poor prognosis in eCCA patients. Additionally, MSI2 overexpression promoted eCCA cell growth, migration, and invasion, while MSI2 knockdown repressed eCCA cell migration and invasion by inhibiting epithelial-mesenchymal transition. CONCLUSION: MSI2 is an independent prognostic factor for eCCA patients, and MSI2 downregulation inhibits eCCA cell growth and metastasis. MSI2 may be a potential therapeutic target for eCCA patients.

Decreased Resting-State Interhemispheric Functional Connectivity Correlated with Neurocognitive Deficits in Drug-Naive First-Episode Adolescent-Onset Schizophrenia.

Background: Given that adolescence is a critical epoch in the onset of schizophrenia, studying aberrant brain changes in adolescent-onset schizophrenia, particularly in patients with drug-naive first-episode schizophrenia, is important to understand the biological mechanism of this disorder. Previous resting-state functional magnetic resonance imaging studies have shown abnormal functional connectivity in separate hemispheres in patients with adult-onset schizophrenia. Our aim to study adolescent-onset schizophrenia can provide clues for the early aetiology of schizophrenia. Method: A total of 48 drug-naïve, first-episode, adolescent-onset schizophrenia outpatients and 31 healthy controls underwent resting-state functional magnetic resonance imaging scans. Data were subjected to voxel-mirrored homotopic connectivity and support vector machine analyses. Results: Compared with the healthy controls, the adolescent-onset schizophrenia group showed significantly lower voxel-mirrored homotopic connectivity values in different brain regions, including the fusiform gyrus, superior temporal gyrus/insula, precentral gyrus, and precuneus. Decreased voxel-mirrored homotopic connectivity values in the superior temporal gyrus/insula were significantly correlated with Trail-Making Test: Part A performance (r = -0.437, P = .002). A combination of the voxel-mirrored homotopic connectivity values in the precentral gyrus and precuneus may be used to discriminate patients with adolescent-onset schizophrenia from controls with satisfactory classification results, which showed sensitivity of 100%, specificity of 87.09%, and accuracy of 94.93%. Conclusion: Our findings highlight resting-state interhemispheric FC abnormalities within the sensorimotor network of patients with adolescent-onset schizophrenia and confirm the relationship between adolescent-onset schizophrenia and adult-onset schizophrenia. These findings suggest that reduced interhemispheric connectivity within the sensorimotor network has a pivotal role in the pathogenesis of schizophrenia.

ST37 Klebsiella pneumoniae: development of carbapenem resistance in vivo during antimicrobial therapy in neonates.

AIM: To investigate the mechanism leading to in vivo carbapenem resistance development in Klebsiella pneumoniae. METHODS: Carbapenemase was detected using the modified carbapenem inactivation method. ß-lactamases resistant genes were identified by PCR and sequencing. Clonal relatedness was evaluated by random amplified polymorphic DNA and multiple locus sequence typing. The relationship between sequence typing and resistant genes was analyzed by using the chi-squared test. RESULTS: All ST37 carbapenem-resistant isolates were blaOXA-1 positive and all ST37 carbapenem-sensitive isolates were blaOXA-1 negative at Stage I. A significant relationship between carbapenem resistance and blaOXA-1 was observed. The blaOXA-1 -positive rate was significantly higher in ST37 K. pneumoniae than others. CONCLUSION: This is the first study about the development of carbapenem resistance in vivo potentially mediated by blaOXA-1 in ST37 K. pneumoniae among neonates.

Deep sequencing reveals complex mechanisms of microRNA regulation during retinoic acid-induced neuronal differentiation of mesenchymal stem cells.

Retinoic acid (RA) has an important role in nervous system development; exogenous RA could induce stem cells towards neural lineage cells. However, the miRNA regulation mechanism and biological process of this induction require further exploration. In this study, using high-throughput sequencing results, we evaluated the microRNA profiles of neurally differentiated adipose-derived mesenchymal stem cells (ASCs), summarized several crucial microRNAs that profoundly contributed to the differentiation process, and speculated that several miRNAs were likely to mimic RA or other factors to induce the neuronal differentiation of stem cells. The GO terms and KEGG PATHWAY in the DAVID tool were used to elucidate the biological process of RA induction. Finally, we described a network for clarifying the relationship among the miRNAs, target genes and signaling pathways. These findings will be beneficial for understanding the induction mechanism and supporting the application of RA in stem cell transformation.

Differences in the MicroRNA profiles of subcutaneous adipose-derived stem cells and omental adipose-derived stem cells.

Adipose-derived stem cells (ASCs) isolated from subcutaneous (SC) and omentum (O) share similar characteristics, but the differences in their microRNA profiles are mostly unknown. In this study, besides significant differences in cell morphology and the differentiation ability of the two types of ASCs, the microRNA expression profiles of the cell lines were determined using SOLiD next-generation sequencing. The in-depth analysis found that miR-214, miR-222, miR-181a, miR-26a and miR-23/27/24 clusters and miR-375 act as "markers" to distinguish the different fat deposit-derived ASCs. Additionally, the global miRNA-mRNA interaction differences were revealed, and the results of the GO term enrichment and KEGG pathway in the DAVID tool showed that the molecular function, biological process and signaling pathways showed some different in the two types of ASCs. Our findings provided a clue to a more thorough understanding of the difference between SC-ASCs and O-ASCs and indicate their different potentials for clinical use.

Neuronally differentiated adipose-derived stem cells and aligned PHBV nanofiber nerve scaffolds promote sciatic nerve regeneration.

Through a combination of biomaterials and stem cells, tissue engineering strategies for restoring and regenerating damaged peripheral nerves have recently been used to meet the challenges posed by nerve injury. In a previous study, we revealed a new way to induce neuronal differentiation of stem cells based on the temporally sequential use of miR-218 and Fibroblast Growth Factor 2 (FGF2) in vitro (FGF2-miR-218 induction approach). In the present study, we sought to investigate the application of this novel approach in repairing sciatic nerve damage in vivo. The results showed that compared with randomly oriented nanofibers, nanofibers in an aligned orientation more favored stem cell growth and elongation. Stem cells (neuronally differentiated adipose-derived mesenchymal stem cells (ASCs)) treated with the FGF2-miR-218 induction approach and integrated with 3D aligned orientation nanofibers structures as artificial nerve grafts were implanted into 10 mm transected rat sciatic nerves in vivo. The test results of immunohistochemical staining and motor function restoration indicated that the FGF2-miR-218 induction approach combined with the 3D nanofiber scaffolds facilitated the nerve regeneration. Thus, this approach could be an effective tissue engineering method for recovery of nerve damage.

Exosomes Transfer Among Different Species Cells and Mediating miRNAs Delivery.

Exosomes, the natural vehicles of intercellular communication, transfer proteins, mRNAs, and microRNAs (miRNAs) and mediate many physiological and pathological processes. It is not clear that whether exosomal miRNAs could regulate gene expression across species, though some studies suggest interactions of exosomal miRNAs between cells. In this report, we have isolated exosomes from rat PC12 cells and assessed their internalization by human cancer Hela cells. The internalized exosomes were located in Hela lysosomes. Human PTEN expression was significantly deregulated due to miR-21 delivered by rat cell exosomes. Our results prove that exosomes could incorporate between cells of different species and could regulate the protein expressions in the recipient cells by delivering the enclosed miRNAs. Thus our study foreshadows a futuristic treatment approach of utilizing miRNA enclosed exosome vehicles sans species concerns in combating various diseases/ regulating abnormal proteins. J. Cell. Biochem. 118: 4267-4274, 2017. © 2017 Wiley Periodicals, Inc.

MicroRNA profiling analysis revealed different cellular senescence mechanisms in human mesenchymal stem cells derived from different origin.

Mesenchymal stem cells (MSCs) from human umbilical cord (UC) and cord blood (CB) share many common properties and exhibit promising clinical potential. Cellular senescence, which induces the loss of stem cells characters and disrupts their therapeutic functions, has been demonstrated to be under the regulation of microRNAs (miRNAs). In this study, we compared the miRNA profiles in early and late passage UCMSCs and CBMSCs based on deep sequencing. 224 and 170 miRNAs were significantly altered in UCMSCs and CBMSCs respectively. A functional annotation of the predicted miRNA targets revealed a series of common senescence pathways. However, Functional enrichment analysis revealed different bioprocesses involved in cellular senescence of UC- and CB-MSCs. The common miRNAs shared by the two kinds of MSCs also exert different function in terms of GO enrichment analysis. Our results supported MSCs derived from different origin may undergo senescence through different path.

MiR-218 Induces Neuronal Differentiation of ASCs in a Temporally Sequential Manner with Fibroblast Growth Factor by Regulation of the Wnt Signaling Pathway.

Differentiation of neural lineages from mesenchymal stem cells has raised the hope of generating functional cells as seed cells for nerve tissue engineering. As important gene regulators, microRNAs (miRNAs) have been speculated to play a vital role in accelerating stem cell differentiation and repairing neuron damage. However, miRNA roles in directing differentiation of stem cells in current protocols are underexplored and the mechanisms of miRNAs as regulators of neuronal differentiation remain ambiguous. In this study, we have determined that miR-218 serves as crucial constituent regulator in neuronal differentiation of adipose stem cells (ASCs) through Wnt signaling pathway based on comprehensive annotation of miRNA sequencing data. Moreover, we have also discovered that miR-218 and Fibroblast Growth Factor-2 (FGF2) modulate neuronal differentiation in a sequential manner. These findings provide additional understanding of the mechanisms regulating stem cell neuronal differentiation as well as a new method for neural lineage differentiation of ASCs.

Comparative analysis of microRNA expression in human mesenchymal stem cells from umbilical cord and cord blood.

Human mesenchymal stem cells (MSCs) derived from both umbilical cord (UC) and cord blood (CB) share similar characteristics, and their differences are largely unknown. Besides the significant difference in cell morphology, differentiation ability and development processes of the two different origin MSCs, a different expression pattern of microRNAs between the two kinds of MSCs was also obtained. By comprehensively annotating the differently expressed global microRNAs (miRNAs), a series of biological pathways were predicted. We found that miRNAs significantly repressed insulin signaling in UCMSCs, while neural related processes were more repressed in CBMSCs. Particularly, TGF-ß and Notch signaling were differently activated in both MSCs, unveiling their distinct angiogenesis potentials. Taken together, this study illustrates that MSCs from UC and CB display distinct properties, which indicates different potentials for clinical usage.

A novel type of self-assembled nanoparticles as targeted gene carriers: an application for plasmid DNA and antimicroRNA oligonucleotide delivery.

In this study, a new type of amphiphilic cetylated polyethyleneimine (PEI) was synthesized, and then polylactic-co-glycolic acid (PLGA)/cetylated PEI/hyaluronic acid nanoparticles (PCPH NPs) were developed by self-assembly as a novel type of gene-delivering vehicle. The PCPH NPs showed good DNA-condensation ability by forming polyplexes with small particle size and positive zeta potential. The transfection efficiency and cytotoxicity of PCPH NPs were evaluated as plasmid DNA vectors to transfect HepG2 in vitro. PCPH NPs exhibited much lower cytotoxicity and higher gene-transfection efficiency than PEI (25,000) and commercial transfection reagents. Furthermore, PCPH NPs were used as an anti-miR-221 vector for transfecting HepG2 cells, and anti-miR-221 was effectively transfected into cells and produced a greater inhibitory effect on cancer-cell growth by PCPH NPs. These results demonstrate that PCPH NPs can be a promising nonviral vector for gene-delivery systems.

[Human papillomavirus genotypes in male patients attending the STD clinic in Zhenjiang area].

OBJECTIVE: To investigate the status of human papillomavirus ( HPV) infection and its genotypes in male patients in Zhenjiang area. METHODS: Using PCR and reverse dot blot hybridization, we determined the genotypes of HPV DNA in 245 male patients at our Clinic of Dermatology and STD. RESULTS: The total rate of HPV infection was 43.67% (107/245), and 18 subtypes were detected. Among the 107 HPV-positive cases, low-risk, high-risk, and combined high- and low-risk infections accounted for 39.25% (42/107), 38.32% (41/107), and 22.43% (24/107), respectively. The most notable low-risk HPV types were HPV6 and HPV11, and the most notable high-risk HPV types were HPV16, HPV52, and HPV58. The rates of single infection and multi-infection were 53.27% (57/107) and 46.73% (50/107), respectively. One case had the most types, infected with 8 genotypes. No statistically significant differences were observed in the total rate of HPV infection among different age groups (Χ2 = 7.999, P > 0.05). CONCLUSION: The dominant subtypes of HPV infection in male patients in Zhenjiang area were HPV6, HPV11, and HPV16. The most common subtypes were HPV6 and HPV11 in low-risk infection, and HPV16, HPV52, and HPV58 in high-risk infection.

Exosome uptake through clathrin-mediated endocytosis and macropinocytosis and mediating miR-21 delivery.

Exosomes are nanoscale membrane vesicles secreted from many types of cells. Carrying functional molecules, exosomes transfer information between cells and mediate many physiological and pathological processes. In this report, utilizing selective inhibitors, molecular tools, and specific endocytosis markers, the cellular uptake of PC12 cell-derived exosomes was imaged by high-throughput microscopy and statistically analyzed. It was found that the uptake was through clathrin-mediated endocytosis and macropinocytosis. Furthermore, PC12 cell-derived exosomes can enter and deliver microRNAs (miRNAs) into bone marrow-derived mesenchymal stromal cells (BMSCs), and decrease the expression level of transforming growth factor ß receptor II (TGFßRII) and tropomyosin-1 (TPM1) through miR-21. These results show the pathway of exosome internalization and demonstrate that tumor cell-derived exosomes regulate target gene expression in normal cells.

Comprehensive annotation of microRNA expression profiles.

BACKGROUND: MicroRNAs (miRNAs) regulate many biological processes by post-translational gene silencing. Analysis of miRNA expression profiles is a reliable method for investigating particular biological processes due to the stability of miRNA and the development of advanced sequencing methods. However, this approach is limited by the broad specificity of miRNAs, which may target several mRNAs. RESULT: In this study, we developed a method for comprehensive annotation of miRNA array or deep sequencing data for investigation of cellular biological effects. Using this method, the specific pathways and biological processes involved in Alzheimer's disease were predicted with high correlation in four independent samples. Furthermore, this method was validated for evaluation of cadmium telluride (CdTe) nanomaterial cytotoxicity. As a result, apoptosis pathways were selected as the top pathways associated with CdTe nanoparticle exposure, which is consistent with previous studies. CONCLUSIONS: Our findings contribute to the validation of miRNA microarray or deep sequencing results for early diagnosis of disease and evaluation of the biological safety of new materials and drugs.

Effects of epidermal growth factor and basic fibroblast growth factor on the proliferation and osteogenic and neural differentiation of adipose-derived stem cells.

Stem cells used for clinical tissue regeneration therapy should have the capacity of self-renewal, high proliferation, and differentiation and be able to be transplanted in large numbers. Although high concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) may induce the differentiation of stem cells, these factors have been widely used to enhance the propagation of stem cells, including adipose-derived mesenchymal stem cells (ASCs). However, the effects of low concentrations of EGF and bFGF on stem cells need to be evaluated carefully. This study illustrates that low concentrations of EGF (5 ng/mL) and bFGF (10 ng/mL) increase the proliferative ability of ASCs and induce the typical spindle-shaped cell morphology. EGF and bFGF added to medium promoted neural lineage differentiation and impaired the mesodermal differentiation ability of ASCs. This study demonstrates that even low concentrations of EGF and bFGF may limit the differentiation ability of stem cells during stem cell expansion in vitro. EGF and bFGF supplementation should be carefully considered in stem cells for clinical applications.

Dynamics of exosome internalization and trafficking.

Cells release exosomes into extracellular medium. Although the important roles of exosomes in many physiological and pathological processes are being revealed, the mechanism of exosome-cell interaction remains unclear. In this article, employing real-time fluorescence microscopy, the motion of exosomes on the plasma membrane or in the cytoplasm of recipient PC12 cells was observed directly. In addition, several motion modes of exosomes were revealed by single particle tracking (SPT). The changes between motion modes were also detected, presenting the dynamic courses of exosome attachment onto plasma membrane and exosome uptake. Octadecyl rhodamine B chloride (R18) was found to be useful to distinguish endocytosis from fusion during exosome uptake. Colocalization with organelle markers showed exosomes were sorted to acidic vesicles after internalization. The results provide new sight into the exosome-cell interaction mode and the intercellular trafficking of exosomes. This study will help to understand the roles of exosomes at cell level.