Auricle shaping using 3D printing and autologous diced cartilage.

ObjectiveTo reconstruct the auricle using a porous, hollow, three-dimensional (3D)-printed mold and autologous diced cartilage mixed with platelet-rich plasma (PRP).MethodsMaterialise Magics v20.03 was used to design a 3D, porous, hollow auricle mold. Ten molds were printed by selective laser sintering with polyamide. Cartilage grafts were harvested from one ear of a New Zealand rabbit, and PRP was prepared using 10 mL of auricular blood from the same animal. Ear cartilage was diced into 0.5- to 2.0-mm pieces, weighed, mixed with PRP, and then placed inside the hollow mold. Composite grafts were then implanted into the backs of respective rabbits (n = 10) for 4 months. The shape and composition of the diced cartilage were assessed histologically, and biomechanical testing was used to determine stiffness.ResultsThe 3D-printed auricle molds were 0.6-mm thick and showed connectivity between the internal and external surfaces, with round pores of 0.1 to 0.3 cm. After 4 months, the diced cartilage pieces had fused into an auricular shape with high fidelity to the anthropotomy. The weight of the diced cartilage was 5.157 ± 0.230 g (P > 0.05, compared with preoperative). Histological staining showed high chondrocyte viability and the production of collagen II, glycosaminoglycans, and other cartilaginous matrix components. In unrestricted compression tests, auricle stiffness was 0.158 ± 0.187 N/mm, similar to that in humans.ConclusionAuricle grafts were constructed successfully through packing a 3D-printed, porous, hollow auricle mold with diced cartilage mixed with PRP. The auricle cartilage contained viable chondrocytes, appropriate extracellular matrix components, and good mechanical properties.Levels of evidenceNA. Laryngoscope, 129:2467-2474, 2019

Serum miR-195-5p Exhibits Clinical Significance in the Diagnosis of Essential Hypertension with Type 2 Diabetes Mellitus by Targeting DRD1

OBJECTIVES: Diagnosis and management of essential hypertension (EH) or type 2 diabetes mellitus (T2DM) by combining comprehensive treatment and classificatory diagnosis have been continuously improved. However, understanding the pathogenesis of EH patients with concomitant T2DM and subsequent treatment remain the major challenges owing to the lack of non-invasive biomarkers and information regarding the underlying mechanisms. METHODS: Herein, we collected 200 serum samples from EH and/or T2DM patients and healthy donors (N). Gene-expression profiling was conducted to identify candidate microRNAs with clinical significance. Then, a larger cohort of the aforementioned patients and 50 N were used to identify the correlation between the tumor suppressor miR-195-5p and EH and/or T2DM. The dual-luciferase reporter assay was used to explore the target genes of miR-195-5p. The suppressive effects of miR-195-5p on the 3′-UTR of the dopamine receptor D1 (DRD1) transcript in EH patients with concomitant T2DM were verified as well. RESULTS: Compared with that in other groups, serum miR-195-5p was highly downregulated in EH patients with concomitant T2DM. miR-195-5p overexpression efficiently suppressed DRD1 expression by binding to the two 3′-UTRs. Additionally, two single nucleotide polymorphisms, including 231T-A and 233C-G, in the miR-195-5p binding sites of the DRD1 3′-UTR were further identified. Collectively, we identified the potential clinical significance of DRD1 regulation by miR-195-5p in EH patients with concomitant T2DM. CONCLUSIONS: Our data suggested that miR-195-5p circulating in the peripheral blood served as a novel biomarker and therapeutic target for EH and T2DM, which could eventually help address major challenges during the diagnosis and treatment of EH and T2DM

Thermal-Enhanced bri1-301 Instability Reveals a Plasma Membrane Protein Quality Control System in Plants

Brassinosteroids (BRs) are essential phytohormones mainly perceived by a single-pass transmembrane receptor-like protein kinase (RLK), BRASSINOSTEROID INSENSITIVE 1 (BRI1). bri1-5 and bri1-9, two distinct mutants with point mutations in the extracellular domain of BRI1, show weak defective phenotypes. Previous studies indicated that bri1-5 and bri1-9 mutated proteins can be recognized and eliminated via an endoplasmic reticulum quality control (ERQC) mechanism. Most of these two proteins, therefore, cannot reach their destination, plasma membrane. Here, we report our functional characterization of bri1-301, another BRI1 mutant protein with an amino acid substitution in the cytoplasmic kinase domain. bri1-301 is a partially functional BR receptor with significantly decreased protein abundance. Interestingly, protein stability and subcellular localization of bri1-301 are temperature-sensitive. At 22°C, an optimal temperature for indoor Arabidopsis growth, bri1-301 shows a weak defective phenotype. At a lower temperature condition such as 18°C, bri1-301 exhibits subtle morphological defects. At a higher temperature condition such as 28°C, on the other hand, bri1-301 displays an extremely severe phenotype reminiscent to that of a null bri1 mutant due to greatly increased bri1-301 internalization and degradation. Our detailed analyses suggest that bri1-301 stability is controlled by ERQC and plasma membrane quality control (PMQC) systems. Since PMQC has not been well studied in plants, bri1-301 can be used as a model mutant for future genetic dissection of this critical process

Identification of Key Gene Networks Associated With Cell Wall Components Leading to Flesh Firmness in Watermelon

Flesh firmness of watermelon is an important quality trait for commercial fruit values, including fruit storability, transportability, and shelf life. To date, knowledge of the gene networks underlying this trait is still limited. Herein, we used weighted genes co-expression network analysis (WGCNA) based on correlation and the association of phenotypic data (cell wall contents) with significantly differentially expressed genes between two materials, a near isogeneic line “HWF” (with high average flesh firmness) and inbred line “203Z” (with low average flesh firmness), to identify the gene networks responsible for changes in fruit flesh firmness. We identified three gene modules harboring 354 genes; these gene modules demonstrated significant correlation with water-soluble pectin, cellulose, hemicellulose, and protopectin. Based on intramodular significance, eight genes involved in cell wall biosynthesis and ethylene pathway are identified as hub genes within these modules. Among these genes, two genes, Cla012351 (Cellulose synthase) and Cla004251 (Pectinesterase), were significantly correlated with cellulose (r2 = 0.83) and protopectin (r2 = 0.81); three genes, Cla004120 (ERF1), Cla009966 (Cellulose synthase), and Cla006648 (Galactosyltransferase), had a significant correlation with water-soluble pectin (r2 = 0.91), cellulose (r2 = 0.9), and protopectin (r2 = 0.92); and three genes, Cla007092 (ERF2a), Cla004119 (probable glycosyltransferase), and Cla018816 (Xyloglucan endotransglucosylase/hydrolase), were correlated with hemicellulose (r2 = 0.85), cellulose (r2 = 0.8), and protopectin (r2 = 0.8). This study generated important insights of biosynthesis of a cell wall structure and ethylene signaling transduction pathway, the mechanism controlling the flesh firmness changes in watermelon, which provide a significant source to accelerate future functional analysis in watermelon to facilitate crop improvement

Derivation of a Triple Mosaic Adenovirus for Cancer Gene Therapy

A safe and efficacious cancer medicine is necessary due to the increasing population of cancer patients whose particular diseases cannot be cured by the currently available treatment. Adenoviral (Ad) vectors represent a promising therapeutic medicine for human cancer therapy. However, several improvements are needed in order for Ad vectors to be effective cancer therapeutics, which include, but are not limited to, improvement of cellular uptake, enhanced cancer cell killing activity, and the capability of vector visualization and tracking once injected into the patients. To this end, we attempted to develop an Ad as a multifunctional platform incorporating targeting, imaging, and therapeutic motifs. In this study, we explored the utility of this proposed platform by generating an Ad vector containing the poly-lysine (pK), the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK), and the monomeric red fluorescent protein (mRFP1) as targeting, tumor cell killing, and imaging motifs, respectively. Our study herein demonstrates the generation of the triple mosaic Ad vector with pK, HSV-1 TK, and mRFP1 at the carboxyl termini of Ad minor capsid protein IX (pIX). In addition, the functionalities of pK, HSV-1 TK, and mRFP1 proteins on the Ad vector were retained as confirmed by corresponding functional assays, indicating the potential multifunctional application of this new Ad vector for cancer gene therapy. The validation of the triple mosaic Ad vectors also argues for the ability of pIX modification as a base for the development of multifunctional Ad vectors

Characterization of transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles

<p>Abstract</p> <p>Background</p> <p>Cultivated watermelon [<it>Citrullus lanatus </it>(Thunb.) Matsum. & Nakai var. <it>lanatus</it>] is an important agriculture crop world-wide. The fruit of watermelon undergoes distinct stages of development with dramatic changes in its size, color, sweetness, texture and aroma. In order to better understand the genetic and molecular basis of these changes and significantly expand the watermelon transcript catalog, we have selected four critical stages of watermelon fruit development and used Roche/454 next-generation sequencing technology to generate a large expressed sequence tag (EST) dataset and a comprehensive transcriptome profile for watermelon fruit flesh tissues.</p> <p>Results</p> <p>We performed half Roche/454 GS-FLX run for each of the four watermelon fruit developmental stages (immature white, white-pink flesh, red flesh and over-ripe) and obtained 577,023 high quality ESTs with an average length of 302.8 bp. <it>De novo </it>assembly of these ESTs together with 11,786 watermelon ESTs collected from GenBank produced 75,068 unigenes with a total length of approximately 31.8 Mb. Overall 54.9% of the unigenes showed significant similarities to known sequences in GenBank non-redundant (nr) protein database and around two-thirds of them matched proteins of cucumber, the most closely-related species with a sequenced genome. The unigenes were further assigned with gene ontology (GO) terms and mapped to biochemical pathways. More than 5,000 SSRs were identified from the EST collection. Furthermore we carried out digital gene expression analysis of these ESTs and identified 3,023 genes that were differentially expressed during watermelon fruit development and ripening, which provided novel insights into watermelon fruit biology and a comprehensive resource of candidate genes for future functional analysis. We then generated profiles of several interesting metabolites that are important to fruit quality including pigmentation and sweetness. Integrative analysis of metabolite and digital gene expression profiles helped elucidating molecular mechanisms governing these important quality-related traits during watermelon fruit development.</p> <p>Conclusion</p> <p>We have generated a large collection of watermelon ESTs, which represents a significant expansion of the current transcript catalog of watermelon and a valuable resource for future studies on the genomics of watermelon and other closely-related species. Digital expression analysis of this EST collection allowed us to identify a large set of genes that were differentially expressed during watermelon fruit development and ripening, which provide a rich source of candidates for future functional analysis and represent a valuable increase in our knowledge base of watermelon fruit biology.</p

Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including “pK7” and/or “RGD” motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber “knob” domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both αvβ3/αvβ5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI

Genetic Evidence for an Indispensable Role of Somatic Embryogenesis Receptor Kinases in Brassinosteroid Signaling

The authors are grateful to the Arabidopsis Biological Resource Center for providing the T-DNA insertion lines discussed in this work. We thank Dr. Yanhai Yin (Iowa State University) for providing anti-BES1 antibody, Dr. Jiayang Li (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for bri1-301 seeds, and Dr. Xing-wang Deng (Yale University) for cop1-4 and cop1-6 seeds as controls.Author Summary Brassinosteroids (BRs) are a group of plant hormones critical for plant growth and development. BRs are perceived by a cell-surface receptor complex including two distinctive receptor kinases, BRI1 and BAK1. Whereas BRI1 is a true BR-binding receptor, BAK1 does not appear to have BR-binding activity. Therefore, BAK1 is likely a co-receptor in BR signal transduction. The genetic significance of BAK1 was not clearly demonstrated in previous studies largely due to functional redundancy of BAK1 and its closely related homologues. It was not clear whether BAK1 plays an essential role or only an enhancing role in BR signaling. In this study, we identified all possible BAK1 redundant genes in the Arabidopsis thaliana genome and generated single, double, triple, and quadruple mutants. Detailed analysis indicated that, without BAK1 and its functionally redundant proteins, BR signaling is completely disrupted, largely because BRI1 has lost its ability to activate downstream components. These studies provide the first piece of loss-of-functional genetic evidence that BAK1 is indispensable to the early events of the BR signaling pathway.Yeshttp://www.plosgenetics.org/static/editorial#pee

Effect of interface structure regulation caused by variation of imidization rate on conduction current characteristics of PI/nano-Al2O3 three-layer composite films

A series of sandwich structure PI films were prepared by different imidization process, with pure PI film as the interlayer and PI/Al2O3 composite films as outer layers. The imidization rate of the film with different cured processes was calculated by characterizing by infrared spectrum (FT-IR), and the morphology of interlayer interface with different imidization rates by scanning electron microscope (SEM). When the imidization conditions of the first and second films were 260 °C/120 min, the composite films displayed better interface structure and higher imidization rate (ID) than others. Moreover, results also showed that the conduction current of three-layer composite film steadily improved with increased ID and temperature, and was higher than that of the pure film. At the temperature of 30 °C, the electrical aging threshold at different ID was obtained. When the ID reached the maximum value of 78.9%, the electrical aging threshold reached the maximum 41.69 kV/mm. Keywords: Polyimides, Composite films, Interfaces, Conduction current, Electrical degradation threshol

Characterization of the Volatile Compounds of Onion with Different Fresh-Cut Styles and Storage Temperatures

The flavor of fresh onion and its processed products is an important index with which to evaluate its quality. In this study, the highly volatile compounds of onion with different fresh-cut styles (bulb, ring, and square) and different storage temperatures (4 °C, 20 °C, and 25 °C) were characterized at the molecular level, focusing in particular on the volatile sulfur compounds. Headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) and headspace solid-phase microextraction-gas chromatography−mass spectrometry (HS-SPME-GC-MS) were employed. A total of 14 highly volatile compounds were identified in onion samples by HS-GC-IMS, and the square sample contained more volatile components. (E,E)-2,4-heptadianal, ethyl acetate, 2-methyl-1-pentanol, 2-pentylfuran, propyl acetate, and 2,6-dimethylpyrazine were produced in the ring and square samples when stored at higher temperatures, while pentanal, 2-heptenal, hexanal were decreased after cutting. Simultaneously, 16 sulfur compounds were identified in onions by HS-SPME-GC-MS. The sulfur compounds profile of the bulbs was significantly different from that of the rings and squares at any temperature. When stored at a low temperature (4 °C), cutting onions into a ring or square shape produced more sulfur. However, at higher temperatures (20 °C and 25 °C), fresh-cutting decreased the sulfur concentration. The total content of sulfur compounds was higher in the same cut style stored at higher temperatures (20 °C or 25 °C). 2-Mercapto-3,4-dimethyl-2,3-dihydrothiophene and 2,4-dimethylthiophene were formed during storage; however, (E)-1-(prop-1-en-1-yl)-3-propyltrisulfane, 1-(1-(methylthio)propyl)-2-propyldisulfane, (Z)-1-(1-propenyldithio)propyl disulfide, dipropyl trisulfide, and methyl 1-(1-propenylthio)propyl disulfide were lost from all samples after storage