Search

Of the known regulators of atherosclerosis, miRNAs have been demonstrated to play critical roles in lipoprotein homeostasis and plaque formation. Here, we generated a novel animal model of atherosclerosis by knocking in LDLR(W483X) in C57BL/6 mice, as the W483X mutation in LDLR is considered the most common newly identified pathogenic mutation in Chinese familial hypercholesterolemia (FH) individuals. Using the new in vivo mouse model combined with a well-established atherosclerotic in vitro human cell model, we identified a novel atherosclerosis-related miRNA, miR-23a-3p, by microarray analysis of mouse aortic tissue specimens and human aortic endothelial cells (HAECs). miR-23a-3p was consistently downregulated in both models, which was confirmed by qPCR. Bioinformatics analysis and further validation experiments revealed that the TNFα-induced protein 3 (TNFAIP3) gene was the key target of miR-23a-3p. The miR-23a-3p-related functional pathways were then analyzed in HAECs. Collectively, the present results suggest that miR-23a-3p regulates inflammatory and apoptotic pathways in atherogenesis by targeting TNFAIP3 through the NF-κB and p38/MAPK signaling pathways.

Ming-Rui Tang, Jia-Yan Guo, Di Wang, Nan Xu Department of Plastic Surgery, The First Hospital of China Medical University, Shenyang 110001, People’s Republic of China Objective: Cutaneous melanoma (CM) is a common skin cancer. Surgery is still the primary treatment for CM, as melanoma is resistant to chemotherapy. In the recent years, it has been found that cancer stem-like cells (CSCs) are responsible for this drug resistance. CD24 is a widely used marker to isolate CSCs. In this study, we aimed to analyze the properties of CD24+ and CD24− subpopulation of melanoma cells. Materials and methods: We isolated CD24+ cells CSCs using magnetic-activated cell sorting system. We extracted total RNA and carried out reverse transcription polymerase chain reaction analysis. We counted the cell colonies using soft agar assay and assessed the cell invasion using cell migration assay. We implanted CD24+ or CD24− cells into the flank of non-obese diabetic severe combined immunodeficiency mice, and measured the tumor volumes every 5 days until the end of the experiment. We carried out immunohistochemical analysis to study the tissue sections. Results: We demonstrated that the CD24+ subpopulation has self-renewal properties in vitro and in vivo by using soft agar assay and xenograft tumor model. Furthermore, we confirmed that CD24 expression is accompanied by activation of Notch1 signaling pathway. Conclusion: This study provides new knowledge on the role of CD24 in the tumorigenic ability of melanoma. Keywords: melanoma, CD24, apoptosis, migration, therapy

Microtubules are a major component of the neuronal cytoskeleton. Tubulin, the subunit protein of microtubules, is an α/β heterodimer. Both α and β exist as families of isotypes, whose members are encoded by different genes and have different amino acid sequences. The βII and βIII isotypes are very prominent in the nervous system. Our previous work has suggested that βII may play a role in neuronal differentiation, but the role of βIII in neurons is not well understood. In the work reported here, we examined the roles of the different β-tubulin isotypes in response to glutamate/glycine treatment, and found that both βII and βIII bind to glutathione in the presence of ROS, especially βIII. In contrast, βI did not bind to glutathione. Our results suggest that βII and βIII, but especially βIII, may play an important role in the response of neuronal cells to stress. In view of the high levels of βII and βIII expressed in the nervous system it is conceivable that these tubulin isotypes may use their sulfhydryl groups to scavenge ROS and protect neuronal cells against oxidative stress.

Minimally invasive aesthetic and cosmetic procedures have increased in popularity. Injectable dermal fillers provide soft tissue augmentation, improve facial rejuvenation and wrinkles, and correct tissue defects. To investigate the use of adipose‑derived stem cells integrated with a hyaluronic acid based gel as a dermal filler, the present study used cytotoxicity studies, proliferation studies, adipogenic and osteogenic differentiation, apoptosis assays and scanning electron microscopy. Although hyaluronic acid induced low levels of apoptosis in adipose‑derived stem cells, its significantly promoted proliferation of adipose‑derived stem cells. Hyaluronic acid demonstrates little toxicity against adipose‑derived stem cells. Adipose‑derived stem cells were able to differentiate into adipocytes and osteoblasts. Furthermore, scanning electron microscopy revealed that adipose‑derived stem cells maintained intact structures on the surface of hyaluronic acid as well as in it, and demonstrated abundant cell attachments. The present study demonstrated the compatibility of adipose‑derived stem cells and hyaluronic acid based gels in vitro.

Adipose-derived stem cells (ADSCs) and their derivatives have aroused intense interest in fields of dermatological and aesthetic medicine. As a major component detected in ADSCs secretome, platelet-derived growth factor AA (PDGF-AA) has been reported mediating extracellular matrix deposition and remodeling, thus might contribute to its anti-aging effect. On the basis of establishing an experimental model that simulate actual skin aging by exposing HDFs to both intrinsic and extrinsic aging factors, we pretreated human dermal fibroblasts (HDFs) with ADSC-conditioned medium (ADSC-CM) before being irradiated, aiming at exploring preventive effects of ADSCs secretome against aging damages. 48 h after irradiation, we detected cellular proliferation; β-galactosidase stain; mRNA expressions of MMP-1, MMP-9, and TIMP-1; and protein expressions of collagen I, collagen III, and elastin. Moreover, we detected related protein expression of PI3K/Akt signal pathway, which can be activated by PDGF-AA and was newly found to promote extracellular matrix protein synthesis. Concentration of PDGF-AA in the prepared ADSC-CM decreased over time and maintained excellent bioactivity at low temperature until the 11th week. ADSC-CM pretreatment can slightly or significantly improve cellular proliferative activity and reduce cellular senescence in irradiated HDFs. Besides, ADSC-CM pretreatment increased collagen I, collagen III, elastin, and TIMP-1 expressions but decreased MMP-1 and MMP-9 expressions both in irradiated and nonirradiated HDFs. ADSC-CM pretreatment significantly increased pAkt protein expression, and ECM protein expression greatly decreased in case of LY294002 application. The results were similar in three generations of HDFs, yet varied with different degrees. Generally, ADSC-CM we prepared demonstrates a certain degree of positive role in preventing HDFs from intrinsic and extrinsic aging damages and that PDGF-AA may contribute to making it become effective with some other components in ADSC-CM.

Background Gossypium barbadense (Sea Island, Egyptian or Pima cotton) cotton has high fiber quality, however, few studies have investigated the genetic basis of its traits using molecular markers. Genome complexity reduction approaches such as genotyping-by-sequencing have been utilized to develop abundant markers for the construction of high-density genetic maps to locate quantitative trait loci (QTLs). Results The Chinese G. barbadense cultivar 5917 and American Pima S-7 were used to develop a recombinant inbred line (RIL) population with 143 lines. The 143 RILs together with their parents were tested in three replicated field tests for lint yield traits (boll weight and lint percentage) and fiber quality traits (fiber length, fiber elongation, fiber strength, fiber uniformity and micronaire) and then genotyped using GBS to develop single-nucleotide polymorphism (SNP) markers. A high-density genetic map with 26 linkage groups (LGs) was constructed using 3557 GBS SNPs spanning a total genetic distance of 3076.23 cM at an average density of 1.09 cM between adjacent markers. A total of 42 QTLs were identified, including 24 QTLs on 12 LGs for fiber quality and 18 QTLs on 7 LGs for lint yield traits, with LG1 (9 QTLs), LG10 (7 QTLs) and LG14 (6 QTLs) carrying more QTLs. Common QTLs for the same traits and overlapping QTLs for different traits were detected. Each individual QTLs explained 0.97 to 20.7% of the phenotypic variation. Conclusions This study represents one of the first genetic mapping studies on the fiber quality and lint yield traits in a RIL population of G. barbadense using GBS-SNPs. The results provide important information for the subsequent fine mapping of QTLs and the prediction of candidate genes towards map-based cloning and marker-assisted selection in cotton.

Background and aims Familial hypercholesterolemia (FH) is a an autosomal dominant disorder characterized by very high levels of low-density lipoprotein cholesterol (LDL-C). It is estimated that >85% of all FH-causing mutations involve genetic variants in the LDL receptor (LDLR). To date, 795 single amino acid LDLR missense mutations have been reported in the Leiden Open Variation Database (LOVD). However, the functional impact of these variants on the LDLR pathway has received little attention and remains poorly understood. We aim to establish a systematic functional prediction model for LDLR single missense mutations. Methods Using a combined structural modeling and bioinformatics algorithm, we developed an in silico prediction model called “Structure-based Functional Impact Prediction for Mutation Identification” (SFIP-MutID) for FH with LDLR single missense mutations. We compared the pathogenicity and functional impact predictions of our model to those of other conventional tools with experimentally validated variants, as well as in vitro functional test results for patients with LDLR variants. Results Our SFIP-MutID model systematically predicted 13,167 potential LDLR single amino acid missense substitutions with biological effects. The functional impact of 52 out of 54 specific mutations with reported in vitro experimental data was predicted correctly. Further functional tests on LDLR variants from patients were also consistent with the prediction of our model. Conclusions Our LDLR structure-based computational model predicted the pathogenicity of LDLR missense mutations by linking genotypes with LDLR functional phenotypes. Our model complements other prediction tools for variant interpretation and facilitates the precision diagnosis and treatment of FH and atherosclerotic cardiovascular diseases.

The ultimate goal of treating peripheral nerve defects is reconstructing continuity of the nerve stumps to regain nerve conduction and functional recovery. Clinically, autologous nerve grafts and Schwann cell (SC) therapy have limitations, such as the need for secondary surgery, sacrifice of another nerve and donor site complication. Adipose derived stem cells (ADSCs) may promise to be ideal alternative cells of SCs. To explore the potential of ADSCs promoting peripheral nerve regeneration, the present study investigated the influences of ADSCs on proliferation and neurotrophic function of SCs using co‑culture model in vitro. Western blot analysis, immunocytochemistry, a cell viability assay, reverse transcription‑polymerase chain reaction (RT‑PCR) and ELISA were applied for examining the interaction of ADSCs and SCs in a co‑culture model in vitro. Western blot analysis and immunocytochemistry demonstrated that protein expression levels of glial filament acidic protein (GFAP) and S100 in ADSCs co‑cultured with SCs for 14 days were significantly higher compared with cells cultured alone. Cell viability assay indicated that the cell viability of SCs co‑cultured with ADSCs for 3, 4, 5, 6 and 7 days was significantly higher than those cultured alone. RT‑PCR showed that expression levels of neurotrophic factors [nerve growth factor (NGF) and glial cell line‑derived neurotrophic factor (GDNF)] and extracellular matrix components [fibronectin (FN) and laminin (LN)] in SCs co‑cultured with ADSCs for 14 days were significantly higher than those in SCs cultured alone. NGF, GDNF, FN and LN in the supernatants of co‑culture system were significantly higher than cells cultured alone, as ELISA revealed. The results of this study suggested that the transplantation of ADSCs may have a promoting potential to the peripheral nerve regeneration as undifferentiated state.

Tubulin, the subunit protein of microtubules, is an α/β heterodimer. Both α- and β-tubulin exist as numerous isotypes, differing in their amino acid sequences and encoded by different genes. The differences are highly conserved in evolution, suggesting that they are functionally significant. Neurons are a potentially very useful system for elucidating this significance, because they are highly differentiated cells and rich in tubulin isotypes. We have examined the distribution of β-tubulin isotypes in mouse primary cultured cortical neurons from embryonic fetus, newborn pups and adults. Neurons from both embryonic and adult mouse brains express the βI, βII, and βIII isotypes, but apparently not βIV or βV. βI, βII, and βIII are found in both cell bodies and neurites. However, the situation is different in newborn mice. Although βI and βIII are present in these neurons in both cell bodies and neurites and βIV is absent, just like in embryonic and adult mice, two striking differences were noted in the neurons from newborn mice. First, βV is apparently present evanescently in the neurons in both cell bodies and neurites. Interestingly, the βV was expressed strongly in newborn neurons after one day of culture; expression became much weaker after 3 days, and almost disappeared after 5 days. Second, the distribution of βII is different from other isotypes. After newborn mouse neurons were cultured for 3 days, βII started to disappear partly from the cell bodies; this was much more pronounced after five days in culture. Our findings suggest that βII's major function may involve the neurites and not the cell body. They also raise the possibility that βV has a unique role in the neurons of newborn mice.

The differences among the vertebrate beta isotypes of tubulin are highly conserved in evolution, suggesting that they have functional significance. To address this, we have used differentiating neuroblastoma cells as a model system. These cells express the betaI, betaII, and betaIII isotypes. Although there is no difference prior to differentiation, a striking difference is seen after differentiation. Both betaI and betaIII occur in cell bodies and neurites, while betaII occurs mostly in neurites. Knocking down betaI causes a large decrease in cell viability while silencing betaII and betaIII does not. Knocking down betaII causes a large decrease in neurite outgrowth without affecting viability. Knocking down betaIII has little effect on neurite outgrowth and only decreases viability if cells are treated with glutamate and glycine, a combination known to generate free radicals and reactive oxygen species. It appears, therefore, that betaI is required for cell viability, betaII for neurite outgrowth and betaIII for protection against free radicals and reactive oxygen species.

A large proportion of protein-protein interactions is mediated by families of peptide-binding domains. Comprehensive characterization of each of these domains is critical for understanding the mechanisms and networks of protein interaction at the domain level. However, existing methods are all based on large scale screenings for each domain that are inefficient to deal with hundreds of members in major domain families. We developed a systematic strategy for efficient binding property characterization of peptide-binding domains based on high throughput validation screening of a specialized candidate ligand library using yeast two-hybrid mating array. Its outstanding feature is that the overall efficiency is dramatically improved compared with that of traditional screening, and it will be higher as the system cycles. PDZ domain family was first used to test the strategy. Five PDZ domains were rapidly characterized. Broader binding properties were identified compared with other methods, including novel recognition specificities that provided the basis for major revision of conventional PDZ classification. Several novel interactions were discovered, serving as significant clues for further functional investigation. This strategy can be easily extended to a variety of peptide-binding domains as a powerful tool for comprehensive analysis of domain binding property in proteomic scale.

Melanoma is an aggressive cutaneous malignancy. In this study, we demonstrated that the levels of the programmed cell death 4 (PDCD4) protein and mRNA were lower in tumor tissues compared with normal tissues. In order to further investigate the effects of PDCD4 and its fragments in B16 melanoma cells, we established B16 clones with expression of different PDCD4 fragments. Intact PDCD4, PDCD4∆164‑469 and PDCD4∆327-440 expression, respectively, decreased proliferation and migration and increased apoptosis in B16 cells in vitro. We found that intact PDCD4, PDCD4∆164-469 or PDCD4∆327-440 can inhibit the activity of MMP-2 and the expression of CXCR4. However, PDCD4∆164-275 showed no effects on B16 cells. These results may prove helpful for the development of novel therapies for melanoma treatment.