The Synthesis of a Coumarin Carbohydrazide Dinuclear Copper Complex Based Fluorescence Probe and Its Detection of Thiols

Small-molecule thiols, such as cysteine (CYS) and glutathione (GSH), are essential for maintaining the cellular redox environment and play important roles in regulating various cellular physiological functions. A fluorescence probe (compound 1-Cu²⁺) for thiols based on coumarin carbohydrazide dinuclear copper complex was developed. Compound 1 was synthesized from the reaction of 7-(diethylamino)-2-oxo-2H-chromene-3-carbohydrazide with 4-tert-butyl-2,6- diformylphenol. Accordingly, the copper complex (compound 1-Cu²⁺) was prepared by mixing compound 1 with 2 equivalents copper ions. Compound 1 had strong fluorescence while compound 1-Cu²⁺ hardly possessed fluorescence owing to the quenching nature of paramagnetism Cu²⁺ to the fluorescence molecule excited state. However, the fluorescence intensity of compound 1-Cu²⁺ was increased dramatically after the addition of thiol-containing amino acids, but not the other non-sulfhydryl amino acids. UV-vis absorption and fluorescence spectra indicated that compound 1-Cu²⁺ had good selectivity and sensitivity for thiols such as glutathione in CH₃CN:H₂O (3:2, v/v) PBS solution. The fluorescence imaging experiments implied that compound 1-Cu²⁺ has potential application in thiol-containing amino acids detection in living cells.     

The systematic value of pollen morphology in Smilacaceae

Smilacaceae are a small family of dioecious, mostly climbing, net-veined monocotyledons with a cosmopolitan distribution. Relatively little is known about the variation of pollen morphology within the family. For this reason, and to investigate the systematic value of palynology in Smilacaceae, pollen from 125 species of Smilax, Heterosmilax, and Ripogonum was examined using light and scanning electron microscopy. Ten of these were examined further by transmission electron microscopy. Four distinct pollen types grouped into two major pollen classes were distinguished: Class 1, represented by the pollen of all Smilax and Heterosmilax species, is mostly spheroidal, inaperturate, and spinulate or microspinulate, with a thin, fragile exine of varied sculpturing; three pollen types are represented within this class. Class 2 is found only in Ripogonum and contains a single pollen type with prolate, monosulcate, reticulately-sculptured pollen. The unique pollen morphology of Ripogonum supports its removal from Smilacaceae. In contrast, the characteristics of Heterosmilax pollen intergrade with those seen in Smilax, suggesting that the former might be better reduced to synonymy with the latter. A key to the identification of these pollen types is presented along with a discussion of geographic and possible evolutionary trends among them.     

Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements

Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix. Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum (ER) to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site (ERES) protein Tango1. Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4αEFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco (tbc), encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.     

Effect of a subset of adipose-derived stem cells isolated with liposome magnetic beads to promote cartilage repair

This study aimed to investigate the ability of CD146⁺ subset of ADSCs to repair cartilage defects. In this study, we prepared CD146⁺ liposome magnetic beads (CD146⁺LMB) to isolate CD146⁺ADSCs. The cells were induced for chondrogenic differentiation and verified by cartilage-specific mRNA and protein expression. Then a mouse model of cartilage defect was constructed and treated by filling the induced cartilage cells into the damaged joint, to evaluate the function of such cells in the cartilage microenvironment. Our results demonstrated that the CD146⁺LMBs we prepared were uniform, small and highly stable, and cell experiments showed that the CD146⁺LMB has low cytotoxicity to the ADSCs. ADSCs isolated with CD146⁺LMB were all CD146⁺, CD105⁺, CD166⁺ and CD73⁺. After chondrogenic induction, the cells showed significantly increased expression of cartilage markers Sox9, collagen Ⅱ and aggrecan at protein level and significantly increased Sox9, collagen Ⅱ and aggrecan at mRNA level, and the protein expression and mRNA expression of CD146⁺ADSCs group were higher than those of ADSCs group. The CD146⁺ADSCs group showed superior tissue repair ability than the ADSCs group and blank control group in the animal experiment, as judged by gross observation, histological observation and histological scoring. The above results proved that CD146⁺LMB can successfully isolate the CD146⁺ADSCs, and after chondrogenic induction, these cells successfully promoted repair of articular cartilage defects, which may be a new direction of tissue engineering.     

A 30-residue fragment of the carp granulin-1 protein folds into a stack of two beta-hairpins similar to that found in the native protein.

Upon air oxidation, a peptide corresponding to the 30-residue N-terminal subdomain of carp granulin-1 spontaneously formed the disulfide pairing observed in the native protein. Structural characterization using NMR showed the presence of a defined secondary structure within this peptide. The chemical shifts for most of the alphaCH protons of the peptide and the protein are very similar, and the observed NOE contacts of the peptide strongly resemble those in the protein. A structure calculation of the peptide using NOE distance constraints indicates that the peptide fragment adopts the same conformation as formed within the native protein. The 30-residue N-terminal peptide of carp granulin-1 is the first example of an independently folded stack of two beta-hairpins reinforced by two interhairpin disulfide bonds. Two key areas of the structure show a clustering of hydrophobic residues that may account for its exceptional conformational stability.     

Identification and characterization of a new naringinase-producing strain, Williopsis californica Jmudeb007

A new naringinase-producing strain, Jmudeb007 was indentified by means of morphological observation, gene homogeneous analysis and physiological and biochemical test. Its characteristics in expressing naringinase were investigated by batch culture in 7 L fermentors. Jmudeb007 grown white, circular, convex, and smooth edged colonies, and single, oval-shaped and nucleus contained cells. Its gene sequences of 26S rDNA D1/D2 region and 5.8S rDNA-ITS region both showed homogeneities at 99% to Williopsis californica. It appeared positive in glucose fermentation test, negative in urease test and diazo blue B (DBB) test. Strain Jmudeb007 was identified to W. californica. Cultivation of Jmudeb007 with three media containing different amount of glucose showed it was capable of expressing naringinase which hydrolyze naringin to naringenin. The naringinase synthesis of Jmudeb007 was regulated by glucose which depended on the concentration. Jumdeb007 could express naringinase in medium containing glucose no more than 4 g/L. The present work provides a new source of naringinase, W. californica Jmudeb007, which is non-pathogenic, convenient to conduct breeding operation, easy to develop fermentation process. It is the first report about yeast that can produce naringinase, which help to explore naringinase and other glycosidase from yeast.     

S1PR3 deficiency alleviates radiation-induced pulmonary fibrosis through the regulation of epithelial–mesenchymal transition by targeting miR-495-3p

Radiation-induced pulmonary fibrosis (RIPF) is a life-threatening complication of thoracic radiotherapy, which contributes to continued deterioration in pulmonary function. Sphingosine-1 phosphate receptor 3 (S1PR3) has been identified as a crucial molecule in fibrosis. Accumulating evidence indicated that the inhibition of the S1PRs ameliorates fibrogenesis. Thus, this study aims to explore whether S1PR3 participates in RIPF and elucidates the molecular mechanisms underlying S1PR3-modulated epithelial–mesenchymal transition (EMT) in transforming growth factor-β1-induced pulmonary epithelia. A recombinant adeno-associated viral-mediated S1PR3 (AAV-S1PR3) gene therapy analyzed the effect of S1PR3 gene deficiency on the altered histology structure and molecular mechanisms in the lung of mice with whole-lung irradiation. Compared with the AAV-negative control mice, AAV-mediated S1PR3 knockdown in the lung of mice attenuated pulmonary fibrosis induced by the radiation, as indicated by the alleviation of collagen accumulation, lessened histopathological alterations, and the suppression of inflammatory cells infiltration. S1PR3 deficiency reversed the RIPF concomitantly with abrogated EMT-related protein (α-smooth muscle actin). Consistently, S1PR3-deficient pulmonary epithelia inhibited the EMT process changes and fibrosis formation. Furthermore, S1PR3 was designated as one of the target genes for microRNA-495-3p (miR-495-3p). The inhibition of miR-495-3p promoted the expression of S1PR3 in pulmonary epithelia, whereas the overexpression of miR-495-3p inhibited the S1PR3/SMAD2/3 pathway and suppressed the EMT process. Collectively, miR-495-3p might be a negative regulator of the EMT process in fibrosis formation by inhibiting the targeted S1PR3 gene. These results established a link between the S1PR3 gene, the EMT process, and the fibrosis, suggesting the pharmacological blockage of S1PR3 as a potential therapeutic strategy for RIPF.