Nuclear ßII-Tubulin and its Possible Utility in Cancer Diagnosis, Prognosis and Treatment.

Microtubules are organelles that usually occur only in the cytosol. Walss et al. (1999) discovered the ßII isotype of tubulin, complexed with α, in the nuclei of certain cultured cells, in non-microtubule form. When fluorescently labeled tubulins were microinjected into the cells, only αßII appeared in the nucleus, and only after one cycle of nuclear disassembly and reassembly. It appeared as if αßII does not cross the nuclear envelope but is trapped in the nucleus by the re-forming nuclear envelope in whose reassembly ßII may be involved. ßII is present in the cytoplasm and nuclei of many tumor cells. With some exceptions, normal tissues that expressed ßII rarely had ßII in their nuclei. It is possible that ßII is involved in nuclear reassembly and then disappears from the nucleus. Ruksha et al. (2019) observed that patients whose colon cancer cells in the invasive front showed no ßII had a median survival of about 5.5 years, which was more than halved if they had cytosolic ßII and further lessened if they had nuclear ßII, suggesting that the presence and location of ßII in biopsies could be a useful prognostic indicator and also that ßII may be involved in cancer progression. Yeh and Ludueña. (2004) observed that many tumors were surrounded by non-cancerous cells exhibiting cytosolic and nuclear ßII, suggesting a signaling pathway that causes ßII to be synthesized in nearby cells and localized to their nuclei. ßII could be useful in cancer diagnosis, since the presence of ßII in non-cancerous cells could indicate a nearby tumor. Investigation of this pathway might reveal novel targets for chemotherapy. Another possibility would be to combine αßII with CRISPR-Cas9. This complex would likely enter the nucleus of a cancer cell and, if guided to the appropriate gene, might destroy the cancer cell or make it less aggressive; possible targets will be discussed here. The possibilities raised here about the utility of ßII in cancer diagnosis, prognosis, biology and therapy may repay further investigation.

Hsa-miRNA-23a-3p promotes atherogenesis in a novel mouse model of atherosclerosis.

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 LDLRW483X 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.

Systematic prediction of familial hypercholesterolemia caused by low-density lipoprotein receptor missense mutations.

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.

Identification of CD24 as a marker for tumorigenesis of melanoma.

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.

Interactions of ß tubulin isotypes with glutathione in differentiated neuroblastoma cells subject to oxidative stress.

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

Promoting potential of adipose derived stem cells on peripheral nerve regeneration.

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 co­culture model in vitro. Western blot analysis, immunocytochemistry, a cell viability assay, reverse transcription­polymerase chain reaction (RT­PCR) and ELISA were applied for examining the interaction of ADSCs and SCs in a co­culture model in vitro. Western blot analysis and immunocytochemistry demonstrated that protein expression levels of glial filament acidic protein (GFAP) and S100 in ADSCs co­cultured with SCs for 14 days were significantly higher compared with cells cultured alone. Cell viability assay indicated that the cell viability of SCs co­cultured with ADSCs for 3, 4, 5, 6 and 7 days was significantly higher than those cultured alone. RT­PCR showed that expression levels of neurotrophic factors [nerve growth factor (NGF) and glial cell line­derived neurotrophic factor (GDNF)] and extracellular matrix components [fibronectin (FN) and laminin (LN)] in SCs co­cultured with ADSCs for 14 days were significantly higher than those in SCs cultured alone. NGF, GDNF, FN and LN in the supernatants of co­culture 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.

Distinct roles of different fragments of PDCD4 in regulating the metastatic behavior of B16 melanoma cells.

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∆164­469 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.

Endogenous PGE(2) induces MCP-1 expression via EP4/p38 MAPK signaling in melanoma.

It has been demonstrated that cyclooxygenase-2 (COX-2) is expressed in melanoma tissues and prostaglandin E(2) (PGE(2)) is produced by melanoma cells in vitro. However, the roles of COX-2/PGE(2) in melanoma are largely unknown. In the present study, we set out to analyze the correlation of endogenous PGE(2) with the expression of macrophage chemoattractant protein-1 (MCP-1) and to identify the signaling pathway involved. It was found that MCP-1 mRNA was heterogeneously expressed in 18 melanoma tissue specimens, and the levels of MCP-1 mRNA were positively correlated with those of COX-2 mRNA. Inhibition of endogenous PGE(2) production by a COX-2 inhibitor, COX-2 siRNA or an NFκB inhibitor suppressed MCP-1 expression, whereas treatment with TNF-α (to stimulate endogenous PGE(2) production) or exogenous PGE(2) enhanced MCP-1 expression in melanoma cells. Both the EP4 antagonist and the p38 MAPK inhibitor reduced MCP-1 production in melanoma cells, and abrogated the increased MCP-1 secretion induced by TNF-α or exogenous PGE(2). Conditioned medium from melanoma cells promoted macrophage migration, which was blocked by inhibitors of the PGE(2)/EP4/p38 MAPK signaling pathway. These results indicate that endogenous PGE(2) induces MCP-1 expression via EP4/p38 MAPK signaling in an autocrinal manner in melanoma, and melanoma cell-derived PGE(2) may be involved in macrophage recruitment in the melanoma microenvironment.

[One-staged correction of lip and nose deformities in complete unilateral cleft lip].

OBJECTIVE: To investigate the influence of timing, operative method and perioperative sequence treatment on the therapeutic effect of one-staged correction of lip and nose deformities in complete unilateral cleft lip. METHODS: Seventy-six patients with unilateral complete cleft lip, aged from 3 to 12 months, underwent one-staged correction of lip and nose deformities, based on the Salyer, Noordhoff method. Sequence treatment was performed before and after operation. RESULTS: The patients were followed up for 3 months to 20 years with excellent results in 39 cases (51.3%), good in 33 patients (43.4%), unsatisfied in 4 patients (5.3%). CONCLUSIONS: Good results can be achieved when the one-staged correction of lip and nose deformities, combined with perioperative sequence treatment, is performed. The operative technique of plastic surgeon or maxillofacial surgeon is also important for less morbidity and scar.

The beta isotypes of tubulin in neuronal differentiation.

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 high efficiency strategy for binding property characterization of peptide-binding domains.

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.