Manganese-Based Immunomodulatory Nanocomposite with Catalase-Like Activity and Microwave-Enhanced ROS Elimination Ability for Efficient Rheumatoid Arthritis Therapy.

Rheumatoid arthritis (RA) is a chronic autoimmune disease commonly associated with the accumulation of hyperactive immune cells (HICs), particularly macrophages of pro-inflammatory (M1) phenotype, accompanied by the elevated level of reactive oxygen species (ROS), decreased pH and O2 content in joint synovium. In this work, an immunomodulatory nanosystem (IMN) is developed for RA therapy by modulating and restoring the function of HICs in inflamed tissues. Manganese tetraoxide nanoparticles (Mn3 O4 ) nanoparticles anchored on UiO-66-NH2 are designed, and then the hybrid is coated with Mn-EGCG film, further wrapped with HA to obtain the final nanocomposite of UiO-66-NH2 @Mn3 O4 /Mn-EGCG@HA (termed as UMnEH). When UMnEH diffuses to the inflammatory site of RA synovium, the stimulation of microwave (MW) irradiation and low pH trigger the slow dissociation of Mn-EGCG film. Then the endogenously overexpressed hydrogen peroxide (H2 O2 ) disintegrates the exposed Mn3 O4 NPs to promote ROS scavenging and O2 generation. Assisted by MW irradiation, the elevated O2 content in the RA microenvironment down-regulates the expression of hypoxia-inducible factor-1α (HIF-1α). Coupled with the clearance of ROS, it promotes the re-polarization of M1 phenotype macrophages into anti-inflammatory (M2) phenotype macrophages. Therefore, the multifunctional UMnEH nanoplatform, as the IMN, exhibits a promising potential to modulate and restore the function of HICs and has an exciting prospect in the treatment of RA.

Production of recombinant human long-acting IL-18 binding protein: inhibitory effect on ulcerative colitis in mice.

Interleukin-18 binding protein (IL-18BP) is a natural IL-18 inhibitor in vivo, which can effectively neutralize IL-18 and inhibit the inflammatory signaling pathway induced by IL-18, thus playing an anti-inflammatory role. Traditional production methods primarily rely on eukaryotic animal cell expression systems, which often entail complex processes, lower yields, and increase production costs. In this study, we present a novel approach for expressing IL-18BP fusion protein using the Escherichia coli (E. coli) system. The N-terminal segment of IL-18BP was fused with the small ubiquitin-related modifier (SUMO) tag, enabling soluble expression, while the C-terminal segment was fused with the human IgG1 Fc fragment to prolong its in vivo lifespan. Through screening, we obtained a high-expression engineering strain from a single colony and developed optimized protocols for fermentation and purification of the recombinant SUMO-IL-18BP-Fc protein. The SUMO tag was subsequently cleaved using SUMO protease, and the purified recombinant human IL-18BP-Fc (rhIL-18BP-Fc) exhibited a purity exceeding 90% with a yield of 1 g per liter of bacterial solution. The biological activities and underlying mechanisms of rhIL-18BP-Fc were evaluated using cell lines and a mouse model. Our results demonstrated that rhIL-18BP-Fc effectively inhibited IL-18-stimulated IFN-γ production in KG-1a cells in vitro and ameliorated DSS-induced ulcerative colitis in mice. In conclusion, we successfully employed the SUMO fusion system to achieve high-level production, soluble expression, and prolonged activity of rhIL-18BP-Fc in E. coli. These findings lay the groundwork for future large-scale industrial production and pharmaceutical development of rhIL-18BP-Fc protein. KEY POINTS: • Effective expression, fermentation, and purification of bioactive rhIL-18BP-Fc protein in E. coli. • The rhIL-18BP-Fc protein has a great potential for the therapy of ulcerative colitis by inhibiting the expression of inflammatory factors.

Separating fast and slow exchange transfer and magnetization transfer using off-resonance variable-delay multiple-pulse (VDMP) MRI.

PURPOSE: To develop a method that can separate and quantify the fast (>1 kHz) and slow exchange transfer and magnetization transfer components in Z-spectra. METHODS: Z-spectra were recorded as a function of mixing time using a train of selective pulses providing variable-delay multipulse build-up curves. Fast and slow transfer components in the Z-spectra were separated and quantified on a voxel-by-voxel basis by fitting the mixing time-dependent CEST signal using a 3-pool model. RESULTS: Phantom studies of glutamate solution, bovine serum albumin solution, and hair conditioner showed the capability of the proposed method to separate fast and slow transfer components. In vivo mouse brain studies showed a strong contrast between white matter and gray matter in the slow-transferring map, corresponding to an asymmetric component of the conventional semisolid magnetization transfer contrast. In addition, a fast-transferring proton map was found that was homogeneous across the brain and attributed to the total contributions of the fast-exchanging protons from proteins, metabolites, and a symmetric magnetization transfer contrast component. CONCLUSIONS: This new method provides a simple way to extract fast and slow transfer components from the Z-spectrum, leading to novel MRI contrasts, and providing insight into the different magnetization transfer contrast contributions.

Investigation of the contribution of total creatine to the CEST Z-spectrum of brain using a knockout mouse model.

The current study aims to assign and estimate the total creatine (tCr) signal contribution to the Z-spectrum in mouse brain at 11.7 T. Creatine (Cr), phosphocreatine (PCr) and protein phantoms were used to confirm the presence of a guanidinium resonance at this field strength. Wild-type (WT) and knockout mice with guanidinoacetate N-methyltransferase deficiency (GAMT-/-), which have low Cr and PCr concentrations in the brain, were used to assign the tCr contribution to the Z-spectrum. To estimate the total guanidinium concentrations, two pools for the Z-spectrum around 2 ppm were assumed: (i) a Lorentzian function representing the guanidinium chemical exchange saturation transfer (CEST) at 1.95 ppm in the 11.7-T Z-spectrum; and (ii) a background signal that can be fitted by a polynomial function. Comparison between the WT and GAMT-/- mice provided strong evidence for three types of contribution to the peak in the Z-spectrum at 1.95 ppm, namely proteins, Cr and PCr, the latter fitted as tCr. A ratio of 20 ± 7% (protein) and 80 ± 7% tCr was found in brain at 2 µT and 2 s saturation. Based on phantom experiments, the tCr peak was estimated to consist of about 83 ± 5% Cr and 17 ± 5% PCr. Maps for tCr of mouse brain were generated based on the peak at 1.95 ppm after concentration calibration with in vivo magnetic resonance spectroscopy.

Anatomic Study of Ophthalmic Artery Embolism Following Cosmetic Injection.

INTRODUCTION: Cosmetic injections of dermal fillers or fat could cause ophthalmic artery embolism and even blindness, the high-risk regions of which are considered glabellar, nasal dorsum, and nasolabial fold. Understanding anatomy of the related arteries is important for a physician to safely perform filler injections. To investigate the mechanisms of ophthalmic artery embolism following the injections, cadaver anatomy was studied. METHODS: Ophthalmic artery, facial artery, their branches, and anastomoses among them were anatomized in 12 fresh cadavers. Mimetic injections of hyaluronic acid were performed in glabellar region, nasal dorsum, and nasolabial fold, the relationships between injected filler and related arteries were then investigated. RESULTS: It was clearly found that 4 arteries were located in common injection regions and connected to ophthalmic artery: supratrochlear artery, supraorbital artery, dorsal nasal artery, and angular artery. In the glabellar region, the deep injection on the periosteum will be risky to injure supratrochlear artery and supraorbital artery, whereas in nasal dorsum and nasolabial fold, the sub- superficial musculo aponeurotic system layer injection has the possibility to injure dorsal nasal artery, angular artery, and facial artery. CONCLUSION: The anatomic mechanism of ophthalmic artery embolism is the anastomoses among the related arteries and ophthalmic artery. Based on the findings of the study, injections in periosteum layer at glabellar region or sub-superficial musculo aponeurotic system layer of nasal dorsum and nasolabial fold are not advised.

Synthesis of Europium-Doped Fluorapatite Nanorods and Their Biomedical Applications in Drug Delivery.

Europium (Eu)-doped fluorapatite (FA) nanorods have a biocompatibility similar to that of hydroxyapatite (HA) for use as cell imaging biomaterials due to their luminescent property. Here, we discuss the new application of europium-doped fluorapatite (Eu-FA) nanorods as an anticancer drug carrier. The Eu-FA nanorods were prepared by using a hydrothermal method. The morphology, crystal structure, fluorescence, and composition were investigated. The specific crystal structure enables the effective loading of drug molecules. Doxorubicin (DOX), which was used as a model anticancer drug, effectively loaded onto the surface of the nanorods. The DOX release was pH-dependent and occurred more rapidly at pH 5.5 than at pH 7.4. The intracellular penetration of the DOX-loaded Eu-FA nanorods (Eu-FA/DOX) can be imaged in situ due to the self-fluorescence property. Treatment of melanoma A375 cells with Eu-FA/DOX elicited a more effective apoptosis rate than direct DOX treatment. Overall, Eu-FA exhibits potential for tracking and treating tumors and may be potentially useful as a multifunctional carrier system to effectively load and sustainably deliver drugs.

(15)N Heteronuclear Chemical Exchange Saturation Transfer MRI.

A two-step heteronuclear enhancement approach was combined with chemical exchange saturation transfer (CEST) to magnify (15)N MRI signal of molecules through indirect detection via water protons. Previous CEST studies have been limited to radiofrequency (rf) saturation transfer or excitation transfer employing protons. Here, the signal of (15)N is detected indirectly through the water signal by first inverting selectively protons that are scalar-coupled to (15)N in the urea molecule, followed by chemical exchange of the amide proton to bulk water. In addition to providing a small sensitivity enhancement, this approach can be used to monitor the exchange rates and thus the pH sensitivity of the participating (15)N-bound protons.

Magnetization transfer contrast-suppressed imaging of amide proton transfer and relayed nuclear overhauser enhancement chemical exchange saturation transfer effects in the human brain at 7T.

PURPOSE: To use the variable delay multipulse (VDMP) chemical exchange saturation transfer (CEST) approach to obtain clean amide proton transfer (APT) and relayed Nuclear Overhauser enhancement (rNOE) CEST images in the human brain by suppressing the conventional magnetization transfer contrast (MTC) and reducing the direct water saturation contribution. METHODS: The VDMP CEST scheme consists of a train of RF pulses with a specific mixing time. The CEST signal with respect to the mixing time shows distinguishable characteristics for protons with different exchange rates. Exchange rate filtered CEST images are generated by subtracting images acquired at two mixing times at which the MTC signals are equal, while the APT and rNOE-CEST signals differ. Because the subtraction is performed at the same frequency offset for each voxel and the CEST signals are broad, no B0 correction is needed. RESULTS: MTC-suppressed APT and rNOE-CEST images of human brain were obtained using the VDMP method. The APT-CEST data show hyperintensity in gray matter versus white matter, whereas the rNOE-CEST images show negligible contrast between gray and white matter. CONCLUSION: The VDMP approach provides a simple and rapid way of recording MTC-suppressed APT-CEST and rNOE-CEST images without the need for B0 field correction.

Detection of living anionic species in polymerization reactions using hyperpolarized NMR.

Intermediates during the anionic polymerization of styrene were observed using hyperpolarized NMR. Dissolution dynamic nuclear polarization (DNP) of monomers provides a sufficient signal-to-noise ratio for detection of (13)C NMR signals in real time as the reaction progresses. Because of its large chemical shift dispersion, (13)C is well-suited to distinguish and characterize the chemical species that arise during the reaction. At the same time, incorporation of hyperpolarized small-molecule monomers is a unique way to generate polymers that exhibit a transient signal enhancement at the active site. This strategy is applicable despite the decay of the hyperpolarization of the polymer due to rapid spin-lattice relaxation. Real-time measurements on polymerization reactions provide both mechanistic and kinetic information without the need for stable isotope labeling of the molecules of interest. These capabilities are orthogonal to currently established methods that separate synthesis and analysis into two steps, making dissolution DNP an attractive method to study polymerization reactions.

Systematic analysis of clinical outcomes of anterior maxillary and mandibular subapical osteotomy with preoperative modeling in the treatment of bimaxillary protrusion.

The purpose of this study was to determine the changes in teeth and hard tissues after preoperative modeling and bimaxillary anterior subapical osteotomy for the treatment of bimaxillary protrusion. Cephalometric analysis was used to evaluate the aesthetic effects and occlusal relationships obtained. The subjects included 19 women and 1 man (aged 19-41 years; average, 29 years) with bimaxillary protrusion who underwent anterior subapical osteotomy of both the maxilla and mandible, with simultaneous genioplasty, if required. Based on a preoperative computer-aided manufacturing/design-assisted and model surgical design and an occlusal guide plate, new occlusal relationships were established for the patients. In addition, the preoperative and postoperative cephalometric radiographs were systematically analyzed. In all patients, the surgical incisions underwent primary healing, with no infection or osteonecrosis. Significant differences were observed in the preoperative and postoperative values of all hard tissue and teeth parameters, except for SGn-FH degrees and Co-MP. The most obvious significant differences were seen in L1-OP°, Id-Pog-Go°, IIA°, U1E-Apog, L1E-Apog, U1E-NA, and L1-NA° (P < 0.001). Postoperative follow-up lasted for 12 to 36 months. All patients eventually achieved normal jaw relationships, tooth arch forms, and Spee curves. No evident irregularities of teeth arrangement or abnormal occlusal relationships were observed. All patients were satisfied with their postoperative facial appearance, except for 1 patient, who underwent repeat surgery because of relapse. With the use of a precise preoperative model surgical design, orthognathic surgery, a simple and time-saving technique, can be used to correct bimaxillary protrusion with satisfactory postoperative occlusal relationship and facial aesthetic appearance and minimal postoperative complications.

Surgical treatment of craniomaxillofacial fibrous dysplasia: functionally or aesthetically?

BACKGROUND: Fibrous dysplasia (FD) is a tumor-like growth that consists of replacement of the medullary bone with fibrous tissue, causing the expansion and weakening of the areas of bone involved. The most commonly affected bones are facial bones, causing a number of facial cosmetic and functional problems. METHODS: From December 2008 to July 2012, 10 patients with craniomaxillofacial fibrous dysplasia were treated by conservative resection and local recontouring. The patients were followed up yearly, with an average of 3 years; the longest follow-up period was 5 years. RESULTS: All the 10 patients received appropriate treatment and histopathological examinations were performed to confirm the diagnosis of FD. Four patients with zygoma involved had received partial zygoma osteoectomy and 2 patients received mandibular partial osteoectomy. Average time of follow-up was 3 years, with a range from 1 to 5 years, and all patients obtained satisfactory aesthetic and functional results. CONCLUSION: In most patients, a conservative surgery will achieve good functional and aesthetic results. For patients with mild symptoms, the aesthetic effect should be given priority while for the heavier patients the restoration of function and aesthetic effects should all be taken into account.

Current Strategies for Exosome Cargo Loading and Targeting Delivery.

Extracellular vesicles (EVs) such as ectosomes and exosomes have gained attention as promising natural carriers for drug delivery. Exosomes, which range from 30 to 100 nm in diameter, possess a lipid bilayer and are secreted by various cells. Due to their high biocompatibility, stability, and low immunogenicity, exosomes are favored as cargo carriers. The lipid bilayer membrane of exosomes also offers protection against cargo degradation, making them a desirable candidate for drug delivery. However, loading cargo into exosomes remains to be a challenge. Despite various strategies such as incubation, electroporation, sonication, extrusion, freeze-thaw cycling, and transfection that have been developed to facilitate cargo loading, inadequate efficiency still persists. This review offers an overview of current cargo delivery strategies using exosomes and summarizes recent approaches for loading small-molecule, nucleic acid, and protein drugs into exosomes. With insights from these studies, we provide ideas for more efficient and effective delivery of drug molecules by using exosomes.

A competing risk-based nomogram to predict cancer-specific survival in patients with retroperitoneal leiomyosarcoma.

Considering the rarity and aggressive nature of retroperitoneal leiomyosarcoma (RLMS), several prognostic factors might contribute to the cancer-specific mortality of these patients. This study aimed to construct a competing risk-based nomogram to predict cancer-specific survival (CSS) for patients with RLMS. In total, 788 cases from the Surveillance, Epidemiology, and End Results (SEER) database (2000-2015) were included. Based on the Fine & Gray's method, independent predictors were screened to develop a nomogram for predicting 1-, 3-, and 5-year CSS. After multivariate analysis, CSS was found significantly associated with tumor characteristics (tumor grade, size, range), as well as surgery status. The nomogram showed solid prediction power and was well calibrated. Through decision curve analysis (DCA), a favorable clinical utility of the nomogram was demonstrated. Additionally, a risk stratification system was developed and distinctive survival between risk groups was observed. In summary, this nomogram showed a better performance than the AJCC 8th staging system and can assist in the clinical management of RLMS.

Nuclear magnetic resonance of hyperpolarized fluorine for characterization of protein-ligand interactions.

Fluorine NMR spectroscopy is widely used for detection of protein-ligand interactions in drug discovery because of the simplicity of fluorine spectra combined with a relatively high likelihood for a drug molecule to include at least one fluorine atom. In general, an important limitation of NMR spectroscopy in drug discovery is its sensitivity, which results in the need for unphysiologically high protein concentrations and large ligand:protein ratios. An enhancement in the (19)F signal of several thousand fold by dynamic nuclear polarization allows for the detection of submicromolar concentrations of fluorinated small molecules. Techniques for exploiting this gain in signal to detect ligands in the strong-, intermediate-, and weak-binding regimes are presented. Similar to conventional NMR analysis, dissociation constants are determined. However, the ability to use a low ligand concentration permits the detection of ligands in slow exchange that are not easily amenable to drug screening by traditional NMR methods. The relative speed and additional information gained may make the hyperpolarization-based approach an interesting alternative for use in drug discovery.

Single-Cell RNA-Sequencing Reveals the Cellular and Genetic Heterogeneity of Skin Scar to Verify the Therapeutic Effects and Mechanism of Action of Dispel-Scar Ointment in Hypertrophic Scar Inhibition.

Hypertrophic scarring (HS), caused by excessive fibrosis of injured skin, imposes a psychological burden and creates a source of distress that impairs the quality of life of affected individuals. However, the gold standard for HS treatment has not yet been determined due to the complicated and difficult nature of the routines and procedures involved. Previous studies have indicated that the topical application of certain active components found in traditional Chinese medicines shows potential as a therapeutic alternative for scars. Here, single-cell RNA-sequencing was performed to determine cellular heterogeneity and identify marker genes and mechanisms associated with HS. It was found that fibroblasts comprise the largest proportion of HS cell types. The marker genes that were highly expressed in fibroblasts were extracellular matrix (ECM)-related, whereas ECM-receptor interactions and the transforming growth factor (TGF)-ß signalling pathway were also found to be active. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, which was applied to identify the molecular compounds of Dispel-Scar Ointment (DSO), revealed 74 effective chemical components belonging to 14 types of constituents, such as flavonoids, tanshinones, salvianolic acids, glycosides, and phthalides. Furthermore, in vivo studies using rat scar models showed that the topical application of Salvia miltiorrhiza, Ligusticum chuanxiong, peach kernel, safflower, and motherwort exerted beneficial effects on fibroblasts. DSO promoted scar maturation and reduced scar areas, its efficacy being similar to that of topically applied silicone. Functional studies using immunofluorescence staining, western blotting, and quantitative real-time polymerase chain reaction demonstrated that DSO may target the TGF-ß/Smad pathway to inhibit collagen synthesis and promote ECM remodelling. However, further in vitro mechanistic research and single-drug prescription studies may be required to identify the specific effective compound or active ingredient of DSO, which would provide more substantial evidence regarding the potential therapeutic value of traditional herbs in HS.

Upregulation of Stress-Induced Protein Kinase CK1 Delta is associated with a Poor Prognosis for patients with Hepatocellular Carcinoma.

Objective: This study was designed to analyze the expression of CSNK1D in hepatocellular carcinoma (HCC) and investigate the relationship between the expression of CSNK1D and the prognosis of HCC patients. Methods: The CSNK1D and alpha-fetoprotein (AFP) expression levels in patients with HCC and their corresponding clinical data were downloaded from The Cancer Genome Atlas (TCGA) and sorted with a Perl program. CSNK1D and AFP expression differences in liver tissue and liver cancer were compared and analyzed, based on the online database human cancer metastasis database, the relationships between the expression levels of CSNK1D and AFP and the proliferation and metastases of HCC were explored. The immunohistochemical data obtained from the Human Protein Atlas Database further verified the differences in the expression levels of CSNK1D and AFP in liver tissues and liver cancer tissues. Through Kaplan-Meier survival analysis, the effects of CSNK1D and AFP expression levels on the prognosis of patients with HCC were investigated, and the influences of and patients' gender, age and grades of cancer cells, tumor size, the status of lymph node metastasis, distant metastasis, and tumor stage on the expression of CSNK1D were analyzed with R language. The influence of differential expressions of CSNK1D on survival time was compared and the prognostic factors influencing the survival of HCC patients were statistically explored by univariate analysis and multivariate analysis. The potential influencing mechanism of CSNK1D on the prognosis of HCC patients was explored by Gene Set Enrichment Analysis (GSEA) enrichment. Results: The expression level of CSNK1D and AFP in cancer foci was significantly higher than that in normal tissues, However, in the same patient, the expression levels of AFP in paracarcinoma tissues and cancer tissues showed no significant difference. The expression level of CSNK1D in HCC with distant metastases was higher than that in those without metastasis, but the expression level of AFP in metastatic HCC was lower than that in those HCC without metastases. In immunohistochemical tests, CSNK1D was moderately positive in normal liver tissues, slightly positive in normal bile duct tissues, and highly positive in HCC. AFP was slightly positive in normal liver tissues and negative in HCC, but it was not detected in normal intrahepatic bile duct tissue. Survival analysis results suggested that the higher expression level of CSNK1D corresponded to the shorter survival period, whereas the expression level of AFP showed no significant influence on survival time. The expression level of CSNK1D was not correlated with gender, age, the status of lymph node metastasis status, or distant metastasis of patients. The main factors influencing the expression level of CSNK1D included tumor size, cancer cell grade, and tumor stage. The expression levels of CSNK1D in T2 and T3 were higher than that in T1. The expression levels of CSNK1D in G3 and G4 were higher than that in G1. The expression levels of CSNK1D in Stage II and Stage III were higher than that in Stage I. Univariate analysis suggested that tumor size, cell grade, distant metastasis, clinical stage, and CSNK1D expression level were the prognostic factors influencing the survival of patients. Multivariate analysis suggested that CSNK1D expression level was an independent factor influencing the prognosis of HCC patients. GSEA enrichment analysis indicated that CSNK1D mainly affected the prognosis of HCC patients through cell cycle, WNT signaling pathway, amino acid degradation metabolism, and other pathways. Conclusion: CSNK1D is an independent influencing factor for the prognosis of HCC patients and has the potential to be developed as a potential therapeutic target for HCC, and better than AFP in predicting the prognosis of HCC.

Quantitative rate determination by dynamic nuclear polarization enhanced NMR of a Diels-Alder reaction.

Emerging techniques for hyperpolarization of nuclear spins, foremost dynamic nuclear polarization (DNP), lend unprecedented sensitivity to nuclear magnetic resonance spectroscopy. Sufficient signal can be obtained from a single scan, and reactions even far from equilibrium can be studied in real-time. When following the progress of a reaction by nuclear magnetic resonance, however, spin relaxation occurs concomitantly with the reaction to alter resonance line intensities. Here, we present a model for accounting for spin-relaxation in such reactions studied by hyperpolarized NMR. The model takes into account auto- and cross-relaxation in dipole-dipole coupled spin systems and is therefore applicable to NMR of hyperpolarized protons, the most abundant NMR-active nuclei. Applied to the Diels-Alder reaction of 1,4-dipheneylbutadiene (DPBD) with 4-phenyl-1,2,4-triazole-3,5-dione (PTD), reaction rates could be obtained accurately and reproducibly. Additional parameters available from the same experiment include relaxation rates of the reaction product, which may yield further information about the molecular properties of the product. The method presented is also compatible with an experiment where a single spin in the reactant is labeled in its spin-state by a selective radio frequency pulse for subsequent tracking through the reaction, allowing the unambiguous identification of its position in the product molecule. In this case, the chemical shift specificity of high-resolution NMR can allow for the simultaneous determination of reaction rates and mechanistic information in one experiment.

[Blocking of Notch signaling decreased scar formation in rabbit ear hypertrophic scar model].

OBJECTIVE: To investigate the effects of Notch signaling on scars in a rabbit ear model of hypertrophic scarring. METHODS: The hypertrophic scar of rabbits' ears was reproduced. The left rabbit's ear wounds as the N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT) treated group were treated intradermally with the gamma-secretase inhibitor DAPT to inhibit the activation of Notch at 1, 3, 7 and 14 day time points. The right ears as the control group were treated with normal saline at the same time points. Experimental and control wounds were harvested on days 14, 21, 28 and 35 post wounding, and then examined histologically to quantify hypertrophic index and fibroblasts. The expression of epidermal differentiation markers-keratin 14 (K14), keratin 19 (K19), Involucrin and Notch downstream molecules-P21, P63 were examined and analyzed with immunohistochemistry staining. RESULTS: Both hypertrophic index (1.93 +/- 0.32, 1.82 +/- 0.36, 1.79 +/- 0.25) and number of fibroblasts [(4.08 +/- 0.88), (3.30 +/- 0.53), (3.19 +/- 0.73) x 10(3)/mm(2)] in the DAPT treated group were significantly reduced on days 21, 28 and 35, compared with the control group [2.56 +/- 0.29, 2.61 +/- 0.30, 2.58 +/- 0.39, and (5.45 +/- 0.99), (4.80 +/- 1.13), (4.43 +/- 1.17) x 10(3)/mm(2), all P < 0.01)]. The K19, K14 and P63 increased their expression in the DAPT treated group (28.6% +/- 5.7%, 53.1% +/- 4.5%, 57.0% +/- 5.8%) relative to the control group (10.1% +/- 2.8%, 30.8% +/- 4.9%, 16.5% +/- 2.2%, all P < 0.01) on day 14 post wounding, while the Involucrin and P21 decreased their expression in the DAPT treated group (12.3% +/- 1.9%, 11.0% +/- 1.7%) relative to the control group (29.3% +/- 4.6%, 44.3% +/- 3.5%, both P < 0.01). CONCLUSION: Inactivation of Notch signaling will inhibit scar epidermis to over-differentiation, and thereby inhibit proliferation of hypertrophic scars in the rabbit ears.

Chemical shift correlations from hyperpolarized NMR by off-resonance decoupling.

Nuclear magnetic resonance, through observation of chemical shift, allows the separate identification of each atom in a molecule. Thus, NMR spectra impart an often unrivaled wealth of information on molecular structure. A particular advantage of NMR spectroscopy is the ability to record multidimensional spectra, which provide correlations between atoms. When compared to other techniques, such as optical spectroscopy, the acquisition of NMR spectra is however an insensitive process, requiring samples of high concentration and long acquisition times. Recently, it has been demonstrated that dynamic nuclear polarization, a hyperpolarization technique, can increase the NMR signal by several orders of magnitude. Here, we present a robust method that allows recording two-dimensional chemical shift correlations from such hyperpolarized molecules. The method makes use of an apparent scaling of the scalar coupling observed on one type of atom, when an off-resonance decoupling field is applied to another type of atom. Thus, two-dimensional chemical shift correlations can be read directly from a small number of scans acquired using a hyperpolarized sample. Due to the ease of implementing this technique on commercial hyperpolarization and NMR equipment, it appears ideally suited for routine application, for example, to obtain carbon-proton chemical shift correlations in organic molecules.