Suppression of cracking in drying colloidal suspensions with chain-like particles.

The prevention of drying-induced cracking is crucial in maintaining the mechanical integrity and functionality of colloidal deposits and coatings. Despite exploring various approaches, controlling drying-induced cracking remains a subject of great scientific interest and practical importance. By introducing chain-like particles composed of the same material and with comparable size into commonly used colloidal suspensions of spherical silica nanoparticles, we can significantly reduce the cracks formed in dried particle deposits and achieve a fivefold increase in the critical cracking thickness of colloidal silica coatings. The mechanism underlying the crack suppression is attributed to the increased porosity and pore sizes in dried particle deposits containing chain-like particle, which essentially leads to reduction in internal stresses developed during the drying process. Meanwhile, the nanoindentation measurements reveal that colloidal deposits with chain-like particles exhibit a smaller reduction in hardness compared to those reported using other cracking suppression approaches. This work demonstrates a promising technique for preparing colloidal coatings with enhanced crack resistance while maintaining desirable mechanical properties.

Interplay of Consolidation Fronts and Cracks in Drying Colloidal Coatings and Its Application in Controlling Crack Pattern Formation.

Cracks are frequently observed in drying colloidal coatings. Although a rich collection of crack patterns has been reported, the systematic study on how cracks grow into the final morphology during the drying process remains elusive. In this work, we use directional drying channels with wedge-shaped edges of different angles to study the interplay of advancing consolidation fronts and propagating cracks. We found that although the shape of the advancing consolidation fronts is altered by the drying edge, the growth direction of the following cracks remains perpendicular to the consolidation fronts during the whole drying process, resulting in cracks with a large curvature. We rationalize the evolution of consolidation fronts with the distribution of capillary pressure revealed by a Laplace model. Further, the growth direction of cracks can be explained by the fracture mechanics mechanism that the main orientation of internal tensile stresses developed during the consolidation determines the crack growth direction. Utilizing this understanding, wavy crack patterns are generated in rectangular drying channels with an alternating temperature field, demonstrating a feasible method of designing and controlling drying-induced crack patterns for micro-/nano-fabrication applications.

Upregulation of TLR9 may contribute to activation of microglia and painful diabetic neuropathy via the p38 MAPK pathway in rats.

Painful diabetic neuropathy is a common chronic complication of diabetes, and the underlying mechanism remains largely elusive. A rat model of painful diabetic neuropathy was established via streptozotocin (STZ) injection and assessed as increased heat and mechanical hypersensitivity. An upregulation of TLR9 was observed in the spinal cords of rats injected with STZ and rat microglia (primary microglia and immortalized microglia HAPI) treated with high glucose. To investigate the role of TLR9 in high glucose-induced microglia activation, short hairpin RNAs targeting TLR9 were used in vitro to knock down TLR9 in HAPI cells. TLR9 interference suppressed the high glucose-induced expression and secretion of inflammatory cytokines (TNF-α, IL-1ß, and IL-6), IBA-1 expression and the chemotaxis of HAPI microglia. Similar results were obtained when HAPI microglia were incubated with a p38 inhibitor (SB203580). P38 and ERK were downstream of TLR9 because TLR9 ablation markedly inhibited the phosphorylation of p38 and ERK. TLR9 was also knocked down in vivo via the injection of shTLR9 lentiviral vector into the rat spinal cord. Relief of STZ-induced heat and mechanical hypersensitivity was observed in rats with TLR9 interference, and TLR9 knockdown prevented STZ-induced inflammatory cytokine secretion and microglial and MAPK signaling activation. Our study revealed the participation of TLR9 in microglial activation and diabetes-induced hyperalgesia likely via the MAPK pathway. The targeting of TLR9 may be an effective strategy for the treatment of painful diabetic neuropathy.

A chalcone inhibits the growth and metastasis of KYSE-4 esophageal cancer cells.

OBJECTIVE: To investigate the in vitro and in vivo anticancer effects of a chalcone against KYSE-4 esophageal cancer cells. METHODS: A chalcone was synthesized via the molecular hybridization strategy based on the anticancer activity of chalcone and dithiocarbamate scaffolds. The anticancer effects of different concentrations of the chalcone derivative were compared in esophageal cancer cells. RESULTS: This chalcone displayed strong inhibitory effects on esophageal cancer cell growth with an IC50 of 1.06 µM in KYSE-4 cells. Analysis of the mechanism revealed that the derivative obviously inhibited KYSE-4 cell growth, migration, and invasion in a concentration-dependent manner. Furthermore, the compound regulated migration-related biomarkers (E-cadherin, N-cadherin, and Slug) and inhibited the Wnt/ß-catenin pathway. According to western blotting, this chalcone suppressed the expression of proline-rich protein 11 (PRR11) in a concentration- and time-dependent manner. CONCLUSIONS: This chalcone might be a leading candidate for suppressing the growth and metastasis of esophageal cancer by downregulating PRR11 expression and inhibiting Wnt/ß-catenin signaling.

Betulinic acid enhanced the chemical sensitivity of esophageal cancer cells to cisplatin by inducing cell pyroptosis and reducing cell stemness.

BACKGROUND: Betulinic acid (BA) is a lupine pentacyclic triterpene compound derived from the bark of the white mulberry tree, which has a variety of pharmacological properties. The purpose of this study was to investigate the effects of BA combined with cisplatin on the proliferation, stemness, pyroptosis, and xenograft growth of esophageal carcinoma cells in a nude mouse xenograft model. METHODS: The cell survival rate was detected by CCK-8 method. TE-11 cells were treated with 3µM of BA and 15 µM of cisplatin. The cells were randomly divided into four groups: the control group, the BA group, the cisplatin group, and the BA + Cisplatin group. Western blotting was used to detect the expression levels of Ki67, PCNA, SOX2, OCT4, ASC, and Caspase-1. The xenograft model of nude mice was constructed to detect tumor volume, and the positive expression rates of Ki67 and Caspase-1 were detected by immunohistochemistry. RESULTS: Compared with the control group, Ki67, PCNA, SOX2, and OCT4 levels in the BA and cisplatin groups were significantly lower (P<0.05), while ASC and Caspase-1 levels were significantly higher (P<0.05). Compared with the BA group, Ki67, PCNA, SOX2, and OCT4 levels in the BA + cisplatin group were significantly lower (P<0.05), while ASC and Caspase-1 levels were significantly higher (P<0.05). In the nude mouse xenograft model, compared with the control group, the tumor volume of the BA and cisplatin groups was significantly decreased (P<0.05), the expression rate of Ki67 was significantly decreased (P<0.05), and the expression rate of Caspase-1 was significantly increased (P<0.05). Compared with the BA group, the levels of ASC and Caspase-1 in the BA + cisplatin group were significantly lower (P<0.05), the positive expression rate of Ki67 was significantly lower (P<0.05), and the positive expression rate of Caspase-1 was significantly higher (P<0.05). CONCLUSIONS: BA enhances the chemical sensitivity of esophageal cancer cells to cisplatin by inhibiting cell proliferation, reducing cell stemness, and inducing pyroptosis.

Knockdown of c-Myc inhibits cell proliferation by negatively regulating the Cdk/Rb/E2F pathway in nasopharyngeal carcinoma cells.

The proto-oncogene c-Myc encodes a transcription factor that is involved in the regulation of cellular proliferation, differentiation, and apoptosis. Several studies indicate that the over-expression of c-Myc is a frequent genetic abnormality in nasopharyngeal carcinoma (NPC). Therefore, specifically reducing its level by genetic means in established NPC cell lines helps to better understand its role in the pathogenesis of NPC. In this study, for the first time, we successfully established and characterized NPC 5-8F cell line with stably suppressed c-Myc expression by employing a DNA-based RNA interference approach. The suppression of c-Myc resulted in reduced cell growth, colony formation, and cell cycle progression in 5-8F cells. In vivo tumor formation assays revealed that the knockdown of c-Myc reduced the tumorigenic potential of 5-8F cells in nude mice. At the molecular level, we found that the knockdown of c-Myc could decrease the expression of several critical molecules involved in the Cdk/Rb/E2F pathway, including CDK4, cyclin D1, CDK2, pRb, E2F3, and DP2, and significantly reduced the promoter activity of cyclin D1. Taken together, these findings provide valuable mechanistic insights into the role of c-Myc in nasopharyngeal carcinogenesis and suggest that the knockdown of c-Myc may be a potential therapeutic approach for the treatment of NPC.

microRNA-141 is involved in a nasopharyngeal carcinoma-related genes network.

microRNAs (miRNAs) are small non-coding RNAs and have been implicated in the pathology of various diseases, including cancer. Here we report that the miRNA profiles have been changed after knockdown of one of the most important oncogene c-MYC or re-expression of a candidate tumor suppressor gene SPLUNC1 in nasopharyngeal carcinoma (NPC) cells. Both c-MYC knockdown and SPLUNC1 re-expression can down-regulate microRNA-141 (miR-141). miR-141 is up-regulated in NPC specimens in comparison with normal nasopharyngeal epithelium. Inhibition of miR-141 could affect cell cycle, apoptosis, cell growth, migration and invasion in NPC cells. We found that BRD3, UBAP1 and PTEN are potential targets of miR-141, which had been confirmed following luciferase reporter assays and western blotting. BRD3 and UBAP1 are both involved in NPC carcinogenesis as confirmed through our previous studies and PTEN is a crucial tumor suppressor in many tumor types. BRD3 is involved in the regulation of the Rb/E2F pathway. Inhibition of miR-141 could affect some important molecules in the Rb/E2F, JNK2 and AKT pathways. It is well known that carcinogenesis of NPC is involved in the networks of genetic and epigenetic alteration events. We propose that miR-141- and tumor-related genes c-MYC, SPLUNC1, BRD3, UBAP1 and PTEN may constitute a gene-miRNA network to contribute to NPC development.

Transcriptional regulation of BRD7 expression by Sp1 and c-Myc.

BACKGROUND: Bromodomain is an evolutionally conserved domain that is found in proteins strongly implicated in signal-dependent transcriptional regulation. Genetic alterations of bromodomain genes contributed to the development of many human cancers and other disorders. BRD7 is a recently identified bromodomain gene. It plays a critical role in cellular growth, cell cycle progression, and signal-dependent gene expression. Previous studies showed that BRD7 gene exhibited much higher-level of mRNA expression in normal nasopharyngeal epithelia than in nasopharyngeal carcinoma (NPC) biopsies and cell lines. However, little is known about its transcriptional regulation. In this study, we explored the transcriptional regulation of BRD7 gene. METHOD: Potential binding sites of transcription factors within the promoter region of BRD7 gene were predicted with MatInspector Professional http://genomatix.de/cgi-bin/matinspector_prof/mat_fam.pl. Mutation construct methods and luciferase assays were performed to define the minimal promoter of BRD7 gene. RT-PCR and western blot assays were used to detect the endogenous expression of transcription factor Sp1, c-Myc and E2F6 in all cell lines used in this study. Electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) were used to detect the direct transcription factors that are responsible for the promoter activity of BRD7 gene. DNA vector-based siRNA technology and cell transfection methods were employed to establish clone pools that stably expresses SiRNA against c-Myc expression in nasopharyngeal carcinoma 5-8F cells. Real-time PCR was used to detect mRNA expression of BRD7 gene in 5-8F/Si-c-Myc cells. RESULTS: We defined the minimal promoter of BRD7 gene in a 55-bp region (from -266 to -212bp), and identified that its promoter activity is inversely related to c-Myc expression. Sp1 binds to the Sp1/Myc-Max overlapping site of BRD7 minimal promoter, and slightly positively regulate its promoter activity. c-Myc binds to this Sp1/Myc-Max overlapping site as well, and negatively regulates the promoter activity and endogenous mRNA expression of BRD7 gene. Knock-down of c-Myc increases the promoter activity and mRNA level of BRD7 gene. The luciferase activity of the mutated promoter constructs showed that Sp1/Myc-Max overlapping site is a positive regulation element of BRD7 promoter. CONCLUSION: These studies provide for the first time the evidence that c-Myc is indeed a negative regulator of BRD7 gene. These findings will help to further understand and uncover the bio-functions of BRD7 gene involved in the pathogenesis of NPC.

Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription. METHODS: The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter. RESULTS: We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals. CONCLUSION: BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC.

Preparation of polyclonal antibody specific for BRD7 and detection of its expression pattern in the human fetus.

BRD7 is a novel bromodomain gene. It plays critical role in cell growth, cell cycle progression, and signal-dependent gene expression. Overexpression of the BRD7 gene in nasopharyngeal carcinoma cells is effective to inhibit cell growth and cell cycle progression from G1 to S phase. However, little is known about its bio-functions because of the unavailability of a specific BRD7 antibody. In this study, for the first time, we generated a highly specific BRD7 antibody. It is able to specifically recognize recombinant GST-BRD7N protein with a molecular mass of 65 kDa and recognize BRD7-Myc and endogenously expressed BRD7 protein with an approximate molecular mass of 75 kDa, which corresponds well with the calculated molecular mass of the BRD7 protein. More importantly, with these antisera, we analyzed BRD7 distribution in the human fetus by Western blot and immunohistochemistry assays. Obvious nuclear expression of BRD7 protein presents in human cerebellum, pancreas, intestines, liver, and kidney. Cardiomyocyte shows high cytoplasm expression of the BRD7 protein. Weak nuclear expression of the BRD7 protein is found in human cerebrum, lung, and stomach. These data may help to further study the cellular role of the BRD7 gene. In particular, the prepared BRD7 antibody will be helpful for studying the bio-functions of endogenously expressed BRD7 protein.

Cloning and characterization of the BRD7 gene promoter.

BRD7, a novel bromodomain gene, encodes a protein that inhibits cell growth and cell cycle progression by transcriptional regulation of some cell cycle-related genes. Its transcriptional down-expression has been shown to be critical to the pathogenesis of Nasopharyngeal carcinoma (NPC). Little is known about the transcriptional mechanisms controlling BRD7 gene expression. In this paper, we have characterized the 5' regulatory region of the BRD7 gene in order to understand the molecular mechanisms regulating its expression. Transient transfection results suggested that the analyzed upstream sequences of the BRD7 gene might contain some important but not sufficient sequence information to confer the cell-type specificity of BRD7 gene expression. Further analysis with a series of deletions demonstrated that a 125-bp region was required for the basal promoter activity of the BRD7 gene. Results from ChIP and EMSA indicated that the promoter was responsive to Sp1, E2F, and E2F6. All of these suggest a possible mechanism that transcriptional factor Sp1, E2F, and E2F-6 are associated in the BRD7 promoter region and regulate BRD7 promoter activity. Taken together, these results will help to better understand the role of the BRD7 gene in signal-dependent transcriptional regulation, and to develop new reagents for therapeutic upregulation of the BRD7 gene in NPC.