Smart graphene-based hydrogel promotes recruitment and neural-like differentiation of bone marrow derived mesenchymal stem cells in rat skin.

Strategies to direct the differentiation of endogenous bone marrow derived mesenchymal stem cells (BMSCs) in vivo following recruitment to the injured site are critical to realizing the potential of stem cell-based therapies. But the differentiation efficiency of BMSCs remains limited without direction. Here we demonstrated a novel strategy to promote neuronal differentiation of BMSCs using cross-linked polyethylenimine (PEI) grafted graphene oxide (GO) as the enzyme responsive vector for delivering active genes to BMSCs. In vivo, a core-shell microfiber arrayed hydrogel with a chemokine (SDF-1α) and the cross-linked GO-PEI/pDNAs-bFGF microparticles incorporated into the shell and core, respectively, were constructed. The arrayed hydrogel was shown to recruit and stimulate the neural-like differentiation of BMSCs effectively by delivering the CXCL12 and GO-PEI/pDNAs-bFGF in a self-controlled manner. With this strategy, both in vitro and in vivo neuronal differentiation of BMSCs with function were accelerated significantly. The cross-linked GO-PEI mediated gene transfection together with a multi-functional microfiber arrayed hydrogel provide a translatable approach for endogenous stem cell-based regenerative therapy.

Synthesis, characterization and inhibitory effects of crocetin derivative compounds in cancer and inflammation.

Crocetin is a natural product possessing extraordinary therapeutic effects for various diseases. However, its extremely low solubility limits its application greatly. Conjugation of organic compounds containing heteroatoms such as N to poor soluble molecules can help the synthesized derivative to form stable hydrogen bonds by lowering the salvation energy, which will improve the solubility of the synthesized compounds. Herein, crocetin was modified by conjugating with piperidyl, diethylin and benzylamine to improve their solubility and bioactivities. In the present study, the conjugation of crocetin with piperidyl, diethylin and benzylamine and their influence on the solubility and the pharmacological effects of crocetin were investigated. With the described strategy, crocetin derivatives were synthesized and their structures were elucidated by 1H NMR, 13C NMR and UPLC-MS spectroscopic analysis. The solubility of crocetin and its derivatives were identified. Upon that, the pharmacological effects of the crocetin derivatives on the tumor and inflammation treatment were investigated. It was shown that, in contrast to crocetin, of which, the solubility and pharmacological effects were low and limited, the synthesized compounds have significantly higher solubility and possess broad spectrum of anticancer effects in multiple tumor cell lines, including B16F10, MCF-7, A549 and SKOV3, as well as enhanced anti-inflammation efficacy in macrophage (RAW264.7) without causing cells damage. Conjugation of piperidyl, diethylin and benzylamine with the crocetin was demonstrated to be a highly efficient strategy to improve the solubility of crocetin. The synthesized crocetin derivatives were shown the promising therapeutics for the tumor and inflammation treatment with high safety.

[Effect of pinostrobin chalcone on adipogenic differentiation of mesenchymal stem cell C3H10T1/2].

Chalcones is a flavonoid wildly presented in many herbs. It has the effect to inhibit cells adipogenic differentiation. In order to study the effect of pinostrobin chalcone extracted and isolated from leaves of hickoryes on the adipogenic differentiation of murine embryonic mesenchymal stem cell (C3H10T1/2), MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium] method was used to detect the cell proliferation; adipogenic differentiation was characterized by oil red O staining and isopropanol extraction; the triglyceride content was detected by GAP-PAP enzyme method; and the C3H10T1/2 cell differentiation into adipocytes was also examined by the mRNA and protein expression of PPARγ, C/EBPα and FABP4 by RT-PCR and Western blot respectively. Results indicated that pinostrobin chalcone almost had no effect on cell proliferation activity when the concentration was less than or equal to 50 µmol•L⁻¹; the oil red O staining, isopropanol extraction and GAP-PAP enzyme method showed that pinostrobin chalcone significantly decreased the C3H10T1/2 adipogenic differentiation and triglyceride content in the cytoplasm of adipocytes; the RT-PCR and Western blot analysis showed that pinostrobin chalcone can down-regulate the mRNA and protein levels of FABP4, PPARγ and C/EBPα in C3H10T1/2 cells(P<0.05 or P<0.01). The experiment results suggest that pinostrobin chalcone can inhibit C3H10T1/2 adipogenic differentiation.

Evidence based anti-osteoporosis effects of Periplaneta americana L on osteoblasts, osteoclasts, vascular endothelial cells and bone marrow derived mesenchymal stem cells.

BACKGROUND: Kangfuxin (KFX) is the ethanol extract of Periplaneta americana L, which has been widely used in the Traditional Chinese Medicine for the repair and regeneration of injured organ and tissues with long history. This study is to investigate the influence of KFX in the various cellular activities and evaluate the anti-osteoporosis potential of KFX. METHODS: The influence of the KFX in the cellular activities, including: 1) migration, osteocalcin secretion of osteoblasts; 2) apoptosis of osteoclasts; 3) migration and tube formation of human umbilical vein endothelial cell (HUVEC); and 4) proliferation, cell cycle regulation and migration of bone marrow mesenchymal stem cells (BMSCs), were investigated systematically. RESULTS: KFX was shown to significantly 1) Promote of the migration of osteoblasts, HUVEC, and BMSCs; 2) Increase the secretion of osteocalcin and mineralization of osteoblasts; 3) Accelerate the apoptosis of osteoclasts; 4) Stimulate the proliferation and regulate the cell cycle of BMSCs. CONCLUSION: Taken together, these results provide the evidence for the osteogenesis, anti-osteoporosis and angiogenesis effects of KFX, with the mechanism of activating the bone formation through stimulating the osteoblasts and HUVECs, as well as inhibiting the bone absorption by inhibiting the osteoclasts activities. The KFX was definitely shown a promising bone turnover agent with great potential for anti-osteoporosis treatment.

Transdermal Gene Delivery by Functional Peptide-Conjugated Cationic Gold Nanoparticle Reverses the Progression and Metastasis of Cutaneous Melanoma.

Permeability barrier imposed by stratum corneum makes an extreme challenge for the topical delivery of plasmid DNA (pDNA), which is widely used in gene therapy. Existing techniques to overcome the skin barrier for bio-macromolecules delivery rely on sophisticated mechanical devices. It is still a big challenge to treat the skin cancer, for example, melanoma, that initiates in the dermal layer by topical gene therapy. To facilitate the skin penetration of pDNA deeply into the melanoma tissues, we here present a cell-penetrating peptide and cationic poly(ethyleneimine) conjugated gold nanoparticle (AuPT) that can compact the pDNAs into cationic nanocomplexes and penetrate through the intact stratum corneum without any additional enhancement used. Moreover, the AuPT is highly efficient in stimulating the intracellular uptake and nuclear targeting of the pDNAs in cells, which guarantees the effective transfection. This study provides evidence that penetrating peptide conjugated cationic gold nanoparticle offers a promising vehicle for both the skin penetration and transfection of pDNAs, possessing great potential in topical gene therapy.

Effect of bosentan is correlated with MMP-9/TIMP-1 ratio in bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis (PF) is a life-threatening non-tumorous disease characterized by progressive fibrosis and worsening lung function. Various drugs, such as bleomycin, can contribute to lung injury and PF, with lung injury potentially occurring in 10% of bleomycin users. Bleomycin is the most commonly used drug in the establishment of an animal model of PF in rats. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) serve an important role in controlling tissue organization and fibrosis following injury. The present study examined the effect of bosentan on fibrotic lung tissue in rats administrated with bleomycin. In total, 48 Wistar rats were administrated with bleomycin, with or without bosentan, while the control rats received saline. The lung tissues were microscopically examined by staining with hematoxylin and eosin and Masson's trichome. ELISA was also used to detect the MMP-9 and TIMP-1 concentrations in the plasma. The results indicated that the bosentan-treated groups on the next day and the 15th day showed significant reversal of pathological findings. In addition, the concentrations of MMP-9 and TIMP-1 appeared to be altered following bosentan treatment, improving the bleomycin-induced PF. Masson's trichome staining showed high collagen deposition in the lung tissue sections, which may be a direct result of the activity of MMP-9 and TIMP-1. Furthermore, the deposition of collagen was significantly inhibited in bosentan-treated groups. In conclusion, these results demonstrated that bosentan inhibited lung fibrosis induced by bleomycin and it may be used as an inhibitor of PF.

Extrapancreatic solid pseudopapillary neoplasm followed by multiple metastases: Case report.

Solid pseudopapillary neoplasm (SPN), also known as Gruber-Frantz tumor, is a rare form of neoplasm that almost exclusively occurs in the pancreas and in young females. While the potential of malignancy is low for SPN, these tumors can mimic other diseases and require a meticulous investigation and a standard treatment by total surgical resection. We present an unusual case of SPN arising in the mesentery of a 40-year-old man with subsequent multiple metastases. Histopathological examination showed similar properties of the mesenteric neoplasm to those of SPN in pancreas. Although the mass was surgically removed, the patient died of recurrent disease 4 years after the initial presentation. We speculate that SPN originates from pancreatic progenitor cells. Further histopathological analyses are required for the prediction of SPN recurrence after resection.

Clinical metagenomic analysis of bacterial communities in breast abscesses of granulomatous mastitis.

BACKGROUND: Granulomatous mastitis (GM) is a chronic inflammatory breast lesion. Its etiology remains incompletely defined. Although mounting evidence suggests the involvement of Corynebacterium in GM, there has been no systematic study of GM bacteriology using -omics technology. METHODS: The bacterial diversity and relative abundances in breast abscesses from 19 women with GM were investigated using 16S rDNA metagenomic sequencing and Sanger sequencing. A quantitative PCR (qPCR) assay was also developed to identify Corynebacterium kroppenstedtii. RESULTS: A bioinformatic analysis revealed that Corynebacterium was present in the 19 GM patients, with abundances ranging from 1.1% to 58.9%. Of note, Corynebacterium was the most abundant taxon in seven patients (more than a third of the subjects). The predominance of Corynebacterium kroppenstedtii infection (11 of 19 patients, 57.9%) was confirmed with Sanger sequencing and the qPCR assay. CONCLUSIONS: This study profiled the microbiota of patients with GM and indicated an important role for Corynebacterium, and in particular C. kroppenstedtii, in the pathogenesis of this disease.

Porous microspheres as promising vehicles for the topical delivery of poorly soluble asiaticoside accelerate wound healing and inhibit scar formation in vitro &in vivo.

Asiaticoside is a natural compound possessing diverse pharmacological effects with great potential for clinical use. However, the low solubility and oil-water partition coefficient of asiaticoside lead to reduced effect and limited application. This study aims to construct a porous microsphere for the sustained release of asiaticoside to improve its absorption and enhance the therapeutic effects. Parameters of the formulations, including the drug to polymer ratio, solvent amounts of the inner and external phases, the stirring speed for preparation, and the drug entrapment efficiency were investigated and optimized. Particle size, morphology, pores structure, and Fourier transform infrared spectrum of the microsphere were characterized. The release kinetics and cellular uptake profiles of the asiaticoside-microspheres were examined. The therapeutic effects of asiaticoside-microspheres on wound healing and skin appendages regeneration were investigated in vitro & in vivo. Results showed that the optimized asiaticoside-microspheres possess spherical spongy structure with cylindrical holes. Asiaticoside can be loaded in the microsphere with high efficiency and released with sustained manner. The cellular uptake of asiaticoside from the microspheres was increased with 9.1 folds higher than that of free solution. Asiaticoside-microspheres expressed the strong promotion in the proliferation, migration of keratinocytes and wound scratching healing in vitro. More importantly, they significantly accelerated the re-epithelization, collagen synthesis and pro-angiogenesis in the rat full-skin wound healing. Porous microsphere was shown a novel carrier for the sustained delivery of poorly soluble asiaticoside, with absorption and therapeutic effects improved. Asiaticoside-microsphere is a promising topical preparation with excellent regenerative effects for the wound therapy.

Functional properties of DENV EDIII­reactive antibodies in human DENV­1­infected sera and rabbit antiserum to EDIII.

The envelope domain III (EDIII) of the dengue virus (DENV) has been confirmed to be involved in receptor binding. It is the target of specific neutralizing antibodies, and is considered to be a promising subunit dengue vaccine candidate. However, several recent studies have shown that anti­EDIII antibodies contribute little to the neutralizing or enhancing ability of human DENV­infected serum. The present study involved an analysis of the neutralization and antibody­dependent enhancement (ADE) activities of EDIII­reactive antibodies in human convalescent sera from patients with primary DENV­1 infection and rabbit antiserum immunized with recombinant DENV­1 EDIII protein. The results indicated that serum neutralization was not associated with titres of EDIII­binding antibodies in the human DENV­1­infected sera. The depletion of anti­EDIII antibodies from these serum samples revealed that the anti­EDIII antibodies of the patients contributed little to neutralization and ADE. However, the EDIII­reactive antibodies from the rabbit antiserum exhibited protective abilities of neutralization at a high dilution (~1:50,000) and ADE at a low dilution (~1:5,000) for the homotypic DENV infection. Notably, the rabbit antiserum displayed ADE activity only at a dilution of 1:40 for the heterotypic virus infection, which suggests that EDIII­reactive antibodies may be safe in secondary infection with heterotypic viruses. These results suggest that DENV EDIII is not the predominant antigen of the DENV infection process; however, purified or recombinant DENV EDIII may be used as a subunit vaccine to provoke an effective and safe antibody response.

Integration of antimicrobial peptides with gold nanoparticles as unique non-viral vectors for gene delivery to mesenchymal stem cells with antibacterial activity.

Gold nanoparticles (AuNPs) have emerged as attractive non-viral gene vectors. However their application in regenerative medicine is still limited partially due to a lack of an intrinsic capacity to transfect difficult-to-transfect cells such as primary cells or stem cells. In current study, we report the synthesis of antimicrobial peptide conjugated cationic AuNPs (AuNPs@PEP) as highly efficient carriers for gene delivery to stem cells with antibacterial ability. The AuNPs@PEP integrate the advantages of cationic AuNPs and antibacterial peptides: the presence of cationic AuNPs can effectively condense DNA and the antimicrobial peptides are essential for the cellular & nucleus entry enhancement to achieve high transfection efficiency and antibacterial ability. As a result, antimicrobial peptides conjugated AuNPs significantly promoted the gene transfection efficiency in rat mesenchymal stem cells than pristine AuNPs, with a similar extent to those expressed by TAT (a well-known cell-penetrating peptide) modified AuNPs. More interestingly, the combinational system has better antibacterial ability than free antimicrobial peptides in vitro and in vivo, possibly due to the high density of peptides on the surface of AuNPs. Finally we present the concept-proving results that AuPs@PEP can be used as a carrier for in vivo gene activation in tissue regeneration, suggesting its potential as a multifunctional system with both gene delivery and antibacterial abilities in clinic.

[Study on anti-angiogenesis effect of three curcumin pigments and expression of their relevant factors].

To study the in vitro anti-angiogenesis effect of three curcumin pigments (curcumin, demethoxycurcumin, bisdemethoxycurcumin). In the study, the inhibitory effect of the three curcumin pigments on proliferation of HUVEC cells induced by OX-LDL and the effect on migration of HUVEC cells were detected. The effect on neovascularization was observed by chorioallantoic membrane (CAM) test. The effect on cell adhesion factors ICAM-1 and VCAM-1 of HUVECs were tested by Real-time RT-PCR. It was found that the three curcumins could inhibit the proliferation of HUVEC cells induced by OX-LDL within the dosage range 4, 8, 16 mg x L(-1), with a dose-dependence. The proliferative effect of curcumins on HUVECs was greater than the other two derivatives (P < 0.01). All of the three curcumin pigments inhibited the migration of HUVEC cells and the angiogenesis of chick chorioallantoic membrane (CAM). The migration inhibition rate of curcumins at middle and high concentrations was greater than the other two (P < 0.01). All of the three curcumin could down-regulate the expression of VEGF and ICAM-1, and curcumins showed more obvious effect in down-regulating VEGF than demethoxycurcumin and bisdemethoxycurcumin(P < 0.01); Bisdemethoxycurcumin showed the most significant effect in down-regulating ICAM-1 (P < 0.01). All of the three showed no remarkable effect on expression of VCAM-1, and only bisdemethoxycurcumin showed the down-regulating effect (P < 0.05). According to the findings, all of the three curcumin pigments could resist angiogenesis by inhibiting proliferation and migration of endothelial cells and down-regulating the expression of VEGF and adhesion molecules ICAM-1.

Dengue virus envelope domain III immunization elicits predominantly cross-reactive, poorly neutralizing antibodies localized to the AB loop: implications for dengue vaccine design.

Dengue virus (DENV) is a mosquito-borne virus that causes severe health problems. An effective tetravalent dengue vaccine candidate that can provide life-long protection simultaneously against all four DENV serotypes is highly anticipated. A better understanding of the antibody response to DENV envelope protein domain III (EDIII) may offer insights into vaccine development. Here, we identified 25 DENV cross-reactive mAbs from immunization with Pichia pastoris-expressed EDIII of a single or all four serotype(s) using a prime-boost protocol, and through pepscan analysis found that 60 % of them (15/25) specifically recognized the same highly conserved linear epitope aa 309-320 of EDIII. All 15 complex-reactive mAbs exhibited significant cross-reactivity with recombinant EDIII from all DENV serotypes and also with C6/36 cells infected with DENV-1, -2, -3 and -4. However, neutralization assays indicated that the majority of these 15 mAbs were either moderately or weakly neutralizing. Through further epitope mapping by yeast surface display, two residues in the AB loop, Q316 and H317, were discovered to be critical. Three-dimensional modelling analysis suggests that this epitope is surface exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially on the surface of the mature virion. It is concluded that EDIII as an immunogen may elicit cross-reactive mAbs toward an epitope that is not exposed on the virion surface, therefore contributing inefficiently to the mAbs neutralization potency. Therefore, the prime-boost strategy of EDIII from a single serotype or four serotypes mainly elicited a poorly neutralizing, cross-reactive antibody response to the conserved AB loop of EDIII.

Evaluation and analysis of dengue virus enhancing and neutralizing activities using simple high-throughput assays.

The risk of antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is a major obstacle for the development of dengue vaccine candidates. Here, we described a novel approach for assessment of ADE by measuring DENV nonstructural protein 1 (NS1) production in culture supernatants with Fcγ receptor-expressing K562 cells in ELISA format (ELISA-ADE). Enhancing activities quantified by measurement of kinetics of NS1 production were in a good agreement with the results of the virus titration assay. In conjunction with the previously established enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well format, the observable dose-response profiles of enhancing and neutralizing activities against all four DENV serotypes were produced with two flaviviral envelope cross-reactive monoclonal antibodies and four primary DENV-1-infected human sera. The simple high-throughput ELISA-ADE assay offers advantages for quantitative measurement of infection enhancement that can potentially be applied to large-scale seroepidemiological studies of DENV infection and vaccination.

17beta-estradiol inhibits endothelin-1 production and attenuates cerebral vasospasm after experimental subarachnoid hemorrhage.

Though cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) has been recognized for over half a century, it remains a major complication in patients with SAH. Clinical studies have shown that elevated levels of endothelin-1 (ET-1) are present in the cerebrospinal fluid of patients with SAH, suggesting that ET-1-mediated vasoconstriction contributes to vascular constriction after SAH. Administration of estrogen promotes vasodilation in humans and in experimental animals, in part by decreasing the production of ET-1. This study evaluated the influence of 17beta-estradiol (E2) on the production of ET-1 and cerebrovasospasm in an experimental SAH 2-hemorrhage model in rat. A 30-mm Silastic tube filled with E2 in corn oil (0.3 mg/ml) was subcutaneously implanted in male rats just before SAH induction. The degree of vasospasm was determined by averaging the cross-sectional areas of basilar artery 7 days after first SAH. Plasma samples collected before death were assayed for ET-1. The protective effect of E2 in attenuating vasospasm achieved statistical significance when compared with the SAH only or SAH plus vehicle groups (P < 0.01). Concentrations of ET-1 were higher in the SAH only and SAH plus vehicle groups than in controls (P < 0.001). Serum levels of ET-1 in the SAH plus E2 and E2 only groups were significantly lower than those in the SAH only and SAH plus vehicle groups (P < 0.001). There was no significant difference between ET-1 levels in the healthy control and SAH plus E2 groups. A significant correlation was found between the cross-sectional areas of basilar artery and ET-1 levels (P < 0.001). The beneficial effect of E2 in attenuating SAH-induced vasospasm may be due in part to decreasing ET-1 production after SAH. The role of E2 in the treatment of cerebral vasospasm after SAH is promising and is worthy of further investigation.