A signature of saliva-derived exosomal small RNAs as predicting biomarker for esophageal carcinoma: a multicenter prospective study.

BACKGROUND: The tRNA-derived small RNAs (tsRNAs) are produced in a nuclease-dependent manner in responses to variety of stresses that are common in cancers. We focus on a cancer-enriched tsRNA signature to develop a salivary exosome-based non-invasive biomarker for human esophageal squamous cell carcinoma (ESCC). METHODS: Cancer-enriched small RNAs were identified by RNA sequencing of salivary exosomes obtained from ESCC patients (n = 3) and healthy controls (n = 3) in a pilot study and further validated in discovery cohort (n = 66). A multicenter prospective observational study was conducted in two ESCC high-incidence regions (n = 320 and 200, respectively) using the newly developed biomarker signature. RESULTS: The tsRNA (tRNA-GlyGCC-5) and a previously undocumented small RNA were specifically enriched in salivary exosomes of ESCC patients, ESCC tissues and ESCC cells. The bi-signature composed of these small RNAs was able to discriminate ESCC patients from the controls with high sensitivity (90.50%) and specificity (94.20%). Based on the bi-signature Risk Score for Prognosis (RSP), patients with high-RSP have both shorter overall survival (OS) (HR 4.95, 95%CI 2.90-8.46) and progression-free survival (PFS) (HR 3.69, 95%CI 2.24-6.10) than those with low-RSP. In addition, adjuvant therapy improved OS (HR 0.47, 95%CI 0.29-0.77) and PFS (HR 0.36, 95%CI 0.21-0.62) only for patients with high but not low RSP. These findings are consistent in both training and validation cohort. CONCLUSIONS: The tsRNA-based signature not only has the potential for diagnosis and prognosis but also may serve as a pre-operative biomarker to select patients who would benefit from adjuvant therapy. TRIAL REGISTRATION: A prospective study of diagnosis biomarkers of esophageal squamous cell carcinoma, ChiCTR2000031507 . Registered 3 April 2016 - Retrospectively registered.

Elevated levels of circulating histones indicate disease activity in patients with hand, foot, and mouth disease (HFMD).

BACKGROUND: Hand, foot, and mouth disease (HFMD) is a common infectious disease in children, characterized by acute viral infection accompanying acute inflammatory responses. Circulating histones are leading mediators of the inflammatory processes. This study aimed to elucidate whether circulating histones play a contributory role during HFMD. METHODS: We measured plasma levels of histones, myeloperoxidase (MPO), lactate dehydrogenase (LDH), and cytokines in HFMD patients (n = 126) and compared the results with those of a control group (n = 30). RESULTS: Circulating histone levels were significantly increased in HFMD patients (3.794 ± 0.156 µg/ml) compared with healthy controls (0.238 ± 0.023 µg/ml, p < 0.0001). In addition, their levels were remarkably higher in severe HFMD (n = 38) than in mild HFMD patients (n = 88) (5.232 ± 0.246 vs 3.293 ± 0.161 µg/ml, p < 0.0001). As for other inflammatory markers, MPO, LDH, IL-1ß, IL-6, IL-10, MIP-1, and TNF-ɑ were found to be significantly higher in HFMD patients than in healthy subjects. Of these, LDH, IL-6, and TNF-ɑ levels correlated with disease severity (all p < 0.05). In mild HFMD, circulating histones correlated positively with plasma IL-6 and IL-10, whereas in severe HFMD, histones were associated with elevated IL-6 and TNF-ɑ levels. CONCLUSIONS: These data demonstrate that circulating histones are excessively released in patients with HFMD, which may indicate disease severity and contribute to systemic inflammation by promoting cytokine production (e.g. IL-6). We suggest that in mild HFMD, circulating histones may originate largely from neutrophil activation, whereas in severe HFMD, dying tissue cells and neutrophil activation may be synergistically involved in the increased levels of histones.

Photoresponsive Hydrogels for Tissue Engineering.

Hydrophilic and biocompatible hydrogels are widely applied as ideal scaffolds in tissue engineering. The "smart" gelation material can alter its structural, physiochemical, and functional features in answer to various endo/exogenous stimuli to better biomimic the endogenous extracellular matrix for the engineering of cells and tissues. Light irradiation owns a high spatial-temporal resolution, complete biorthogonal reactivity, and fine-tunability and can thus induce physiochemical reactions within the matrix of photoresponsive hydrogels with good precision, efficiency, and safety. Both gel structure (e.g., geometry, porosity, and dimension) and performance (like conductivity and thermogenic or mechanical properties) can hence be programmed on-demand to yield the biochemical and biophysical signals regulating the morphology, growth, motility, and phenotype of engineered cells and tissues. Here we summarize the strategies and mechanisms for encoding light-reactivity into a hydrogel and demonstrate how fantastically such responsive gels change their structure and properties with light irradiation as desired and thus improve their applications in tissue engineering including cargo delivery, dynamic three-dimensional cell culture, and tissue repair and regeneration, aiming to provide a basis for more and better translation of photoresponsive hydrogels in the clinic.

Folate Decoration Supports the Targeting of Camptothecin Micelles against Activated Hepatic Stellate Cells and the Suppression of Fibrogenesis.

As the central cellular player in fibrogenesis, activated hepatic stellate cells (aHSCs) are the major target of antifibrotic nanomedicines. Based on our finding that activated HSCs increase the expression of folate receptor alpha (FRα), we tried to apply folic acid (FA) decoration to generate an active drug-targeting at aHSCs and suppress hepato-fibrogenesis. FA-conjugated poly(ethylene glycol)-poly(ε-caprolactone) copolymers (PEG-PCL) were synthesized and self-assembled into the spherical micelles that owned a uniform size distribution averaging at 60 nm, excellent hemo- and cyto-compatibility, and pH-sensitive stability. These FA-modified micelles were preferentially ingested by aHSCs as expected and accumulated more in acutely CCl4 injured mouse livers compared to nondecorated counterparts. Such an aHSC targetability facilitated the loaded medicinal camptothecin (CPT) to achieve a greater therapeutic efficacy and inhibition of MF phenotypic genes in aHSCs. Encouragingly, though free CPT and nontargeting CPT micelles produced negligible curative outcomes, FA-decorated CPT micelles yielded effectively remedial effects in chronically CCl4-induced fibrotic mice, as represented by a significant shrinkage of aHSC population, suppression of fibrogenesis, and recovery of liver structure and function, clearly indicating the success of the folate decoration-supported aHSC-targeted strategy for antifibrotic nanomedicines in fibrosis resolution.

Novel Biomaterial-Binding/Osteogenic Bi-Functional Peptide Binds to Silk Fibroin Membranes to Effectively Induce Osteogenesis In Vitro and In Vivo.

Peptides can introduce new functions to biomaterials but their immobilization usually relies on inefficient physical adsorption or tedious chemical conjugation. Using the Bombyx mori silk fibroin (SF) membrane (SFm) as a model biomaterial, here, we demonstrate a universal strategy for discovering new peptides that can "stick" to a biomaterial to functionalize it. Specifically, two peptide motifs, one screened by phage display biopanning for binding to the biomaterial (i.e., SF) and another derived from an osteogenic growth factor (i.e., bone morphogenetic protein-2), are fused into a new chimeric peptide that can bind to SFm for more efficient osteogenesis. Theoretical simulations and experimental assays confirm that the chimeric peptide binds to SF with high affinity, facilely achieving its immobilization onto SFm. The peptide enables SFm to effectively induce osteogenic differentiation of human mesenchymal stem cells (MSCs) even without other osteogenic inducers and efficiently stimulate bone regeneration in a subcutaneous rat model in 8 weeks, even without MSC seeding, while not causing inflammatory responses. Since biomaterial-binding peptides can be readily screened using phage display and functional peptides can be generated from growth factors, our work suggests a universal strategy for combining them to seek new peptides for binding and functionalizing biomaterials.

Arginine induces protein self-assembly into nanofibers for triggering osteogenic differentiation of stem cells.

Although silk proteins are considered promising in building a scaffold for tissue engineering, one of the silk proteins, Bombyx mori silk sericin (BS), has limited processability in producing nanofibrous scaffolds because its surface charge anisotropy promotes gelation instead. To overcome this daunting challenge, we developed a mild and simple procedure for assembling BS into nanofibers and nanofibrous scaffolds. Briefly, arginine was added to the aqueous BS solution to reduce the negative charge of BS, thereby inducing BS to self-assemble into nanofibers in the solution. Circular dichroism (CD) and Fourier transform infrared (FT-IR) spectra showed that arginine promoted the formation of ß-sheet conformation in BS and increased its thermal stability. Furthermore, the arginine-induced BS nanofiber solution could be casted into scaffolds made of abundant network-like nanofibrous structures. The BS scaffolds promoted cell adhesion and growth and stimulated osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs) in the absence of differentiation inducers in culture media. Our study presents a new strategy for assembling proteins into osteogenic nanofibrous scaffolds for inducing stem cell differentiation in regenerative medicine.

Magnesium ion leachables induce a conversion of contractile vascular smooth muscle cells to an inflammatory phenotype.

Phenotype switching is a characteristic response of vascular smooth muscle cells (vSMCs) to the dynamic microenvironment and contributes to all stages of atherosclerotic plaque. Here, we immersed pure magnesium and AZ31 alloy in the completed medium under cell culture condition, applied the resultant leaching extracts to the isolated contractile rat aortic vSMCs and investigated how vSMCs phenotypically responded to the degradation of the magnesium-based stent materials. vSMCs became more proliferative and migratory but underwent more apoptosis when exposed to the degradation products of pure magnesium; while the AZ31 extracts caused less cell division but more apoptosis, thus slowing cell moving and growing. Noticeably, both leaching extracts dramatically downregulated the contractile phenotypic genes at mRNA and protein levels while significantly induced the inflammatory adhesive molecules and cytokines. Exogenously added Mg ions excited similar transformations of vSMCs. With the liberation or supplementation of Mg2+ , the expression patterns of the pro-contractile transactivator myocardin and the pro-inflammatory transcriptional factor kruppel-like factor 4 (KLF4) were reversed. Overall, the degradation of the Mg-based materials would evoke a shift of the contractile vSMCs to an inflammatory phenotype via releasing Mg ions to induce a transition from the phenotypic control of vSMCs by the myocardin to that by the KLF4. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 988-1001, 2019.

Polydopamine-Coated Antheraea pernyi (A. pernyi) Silk Fibroin Films Promote Cell Adhesion and Wound Healing in Skin Tissue Repair.

Wound dressings are important materials for the successful recovery of skin trauma. Traditional wound dressings such as gauzes are not efficient in wound healing. Here we show that silk fibroin, spun from a wild silkworm Antheraea pernyi (A. pernyi) and rich in Arg-Gly-Asp (RGD) sequences, can be developed into a wound dressing after proper modification for improving the cell adhesion to accelerate the skin repair. Specifically, polydopamine (PDA) was coated on an A. pernyi silk fibroin (AF) film to form the PAF film to achieve enhanced cell adhesion and would healing. The PDA coating significantly increased the roughness and hydrophilicity of the AF film and thus its protein absorption capability. Furthermore, the PAF films promoted the adhesion and migration of mesenchymal stem cells (MSCs) in the in vitro wound healing assay. In vivo testing confirmed that wound covered with the PAF film was completely healed with the formation of the new skin and hair within 14 days post trauma. Histological examination indicated that, compared to the AF film and gauze control, the PAF film did not cause significant inflammation in the wound but promoted the epithelialization and well-organized collagen deposition in the dermis. This work indicates that AF films coated with PDA are promising wound dressings for skin tissue repair.

Polydopamine modification of silk fibroin membranes significantly promotes their wound healing effect.

Natural polymer-based wound dressings have gained great attention in skin tissue engineering. Silk fibroin (SF) spun from Bombyx mori (B. mori) is a potential wound dressing material due to its outstanding biocompatibility and biodegradability, however, its wound healing effect is still limited. To maximize the wound healing effect of SF-based wound dressing, we first fabricated fibrous electrospun SF (ESF) membranes with large porosity and specific surface area, and then formed polydopamine (PDA) coating on the ESF fibers to form PESF membranes. We found that PDA coating enabled the PESF membranes to outperform the ESF membranes in enhancing the hydrophilicity and protein adsorption ability of the membranes as well as the attachment, spreading and proliferation of fibroblasts on the membranes in vitro. Our further in vivo histological analysis confirmed that the PESF membranes accelerated wound healing in a rat skin wound model more effectively within 2 weeks than both the ESF membrane and a commercial dressing (3M™ Tegaderm™). The enhanced wound healing effect of the PESF membranes was further proved by the increase in the content of hydroxyproline (a constituent in collagen) in the wound treated by the PESF membranes. Therefore, the PESF membranes could be used as a promising wound dressing for wound healing and skin regeneration.

Evaluation of Salivary Exosomal Chimeric GOLM1-NAA35 RNA as a Potential Biomarker in Esophageal Carcinoma.

PURPOSE: Transcriptionally induced chimeric RNAs are an important emerging area of research into molecular signatures for biomarker and therapeutic target development. Salivary exosomes represent a relatively unexplored, but convenient, and noninvasive area of cancer biomarker discovery. However, the potential of cancer-derived exosomal chimeric RNAs in saliva as biomarkers is unknown. Here, we explore the potential clinical utility of salivary exosomal GOLM1-NAA35 chimeric RNA (seG-NchiRNA) in esophageal squamous cell carcinoma (ESCC). EXPERIMENTAL DESIGN: In a retrospective study, the prognostic significance of G-NchiRNA was determined in ESCC tissues. The correlation between seG-NchiRNA and circulating exosomal or tumoral G-NchiRNA was ascertained in cultured cells and mice. In multiple prospective cohorts of patients with ESCC, seG-NchiRNA was measured by qRT-PCR and analyzed for diagnostic accuracy, longitudinal monitoring of treatment response, and prediction of progression-free survival (PFS). RESULTS: Exosomal G-NchiRNA was readily detectable in ESCC cells and nude mouse ESCC xenografts. SeG-NchiRNA levels reflected tumor burden in vivo and correlated with tumor G-NchiRNA levels. In prospective studies of a training cohort (n = 220) and a validation cohort (n = 102), seG-NchiRNA levels were substantially reduced after ESCC resection. Moreover, seG-NchiRNA was successfully used to evaluate chemoradiation responsiveness, as well as to detect disease progression earlier than imaging studies. Changes in seG-NchiRNA levels also predicted PFS of patients after chemoradiation. CONCLUSIONS: SeG-NchiRNA constitutes an effective candidate noninvasive biomarker for the convenient, reliable assessment of therapeutic response, recurrence, and early detection.

Dynamically tunable polymer microwells for directing mesenchymal stem cell differentiation into osteogenesis.

Directing stem cells towards a desired location and function by utilizing the structural cues of biomaterials is a promising approach for inducing effective tissue regeneration. Here, we exploited the dynamic structural signals resulting from the micro-well substrates to guide the rat bone marrow mesenchymal stem cells (rBMSCs) to differentiate along osteogenesis. The micro-well substrates were fabricated via thermal lithography with arrays of triangle-, square-, hexagon- or round-shaped micro-wells, which were made of a biocompatible and biodegradable polymer network containing six-arm poly(ethylene glycol)-poly(ε-caprolactone) (6A PEGPCL) with the excellent thermally activated shape memory function. The dynamically tunable geometric microwells and the resulting mechanical force effectively and significantly regulated the cytoskeletal structure and tension of rBMSC in vitro without induction media when compared with the static patterned. Cellular and molecular analyses revealed that cells cultured in various dynamic micro-wells had disparately differentiated along adipogenesis and osteogenesis pathways. We further implanted these dynamically tunable geometric micro-well substrates into the site of the rabbit mandible defect and found that they prompted differentiation of mesenchymal stem cells into osteogenesis and in turn efficiently repaired the mandible bone defect. Taken together, this work points to the role that dynamic geometric shape cues can orchestrate the mechano-chemical signals thus direct MSCs to appropriate fates.

The control of mesenchymal stem cell differentiation using dynamically tunable surface microgrooves.

Many studies have demonstrated the potential to modulate stem cell differentiation by using static material substrate surfaces. However, cells actually grow in a dynamically diverse microenvironment in vivo. The regulated signals to the differentiation provided by these materials should not be passive or static but be active and dynamic. To mimic the endogenous cell culture microenvironment, a novel system is designed to realize the dynamic change of the surface geometries as well as a resultant mechanical force using a thermally activated four-stage shape memory polymer. The parallel microgroove surface patterns are fabricated via thermal embossing lithography on the polymer substrate surface. The dynamic microgroove surfaces accompanying with the mechanical force can effectively and significantly regulate the shape and the cytoskeletal arrangement of rBMSC compared with the static patterned and non-patterned surfaces. Cellular and molecular analyses reveal that the spatiotemporally programmed regulation of cell shape is more viable to coax lineage-specific differentiation of stem cell in contrast to the general reports with the static surfaces. Therefore, this study provides a facile strategy in designing and manufacturing an artificial substrate with a mimic natural cellular environment to precisely direct the cell differentiation.

[Self-ligating vs. conventional brackets in the treatment of patients with Class II division 2 malocclusion: a clinical trial of dental and cephalometric analysis].

OBJECTIVE: To compare the dental and skeletal changes in patients with Class II division 2 malocclusion treated with self-ligating and conventional bracket systems. METHODS: Forty patients with Class II division 2 malocclusion were treated non-extraction. Half of them were treated with self-ligating brackets and the others were treated with conventional brackets. The dental casts and lateral cephalometric radiophotographs were analyzed before and after treatment. RESULTS: Significant increase in inter premolar widths, arch lengths, U1-NA, L1-MP and arch perimeters were noted after orthodontic treatment in both groups. Statistically greater increase in upper inter-first premolar [(4.45 ± 2.60) mm] was found in self-ligating group than in conventional group [(2.41 ± 2.74) mm, P < 0.05]. Stepwise regression analysis indicated that the increase in arch length and arch width contributed to the increase in arch perimeter. CONCLUSIONS: There were an overall increase in arch widths and lengths in patients treated with self-ligating and conventional bracktets. However, more arch width increase was found in self-ligating group than in conventional group.