Chip Analysis Research Articles

Tumor budding of cervical squamous cell carcinoma: epithelial-mesenchymal transition-like cancer stem cells?

Recent evidence indicates that cancer stem cells (CSCs) are the origin of cancers. Scientists have identified CSCs in various tumors and have suggested the existence of a variety of states of CSCs. The existence of epithelial–mesenchymal transition (EMT)-like CSCs has been confirmed in vitro, but they have not been identified in vivo. Tumor budding was defined as single cell or clusters of ≤ 5 cells at the invasive front of cancers. Such tumor budding is hypothesized to be closely related to EMT and linked to CSCs, especially to those migrating at the invasive front. Therefore, tumor budding has been proposed to represent EMT-like stem cells. However, this hypothesis has not yet been proven. Thus, we studied the expression of EMT markers, certain CSC markers of tumor budding, and the tumor center of cervical squamous cell carcinoma (CxSCC). We performed tissue chip analyses of 95 primary CxSCCs from patients. Expression of EMT and CSC markers (E-cadherin, β-catenin, vimentin, Ki67, CD44, SOX2 , and ALDH1A1) in a set of tumor samples on tissue chips (87 cases of tumor budding/the main tumor body) were evaluated by immunohistochemistry. We found that the cell-membranous expression of β-catenin was stronger in the main tumor body than in tumor buds. Compared with the main tumor body, tumor buds had reduced proliferative activity as measured by Ki67. Moreover, vimentin expression was high and E-cadherin expression was low in tumor buds. Expression of EMT-related markers suggested that tumor buds were correlated with EMT. We noted that CxSCC tumor buds had a CD44negative/low/SOX2high/ALDH1A1high staining pattern, indicating that tumor buds of CxSCC present CSC-like immunophenotypic features. Taken together, our data indicate that tumor buds in CxSCC may represent EMT-like CSCs in vivo.

Nrf2 overexpression increases the resistance of acute myeloid leukemia to cytarabine by inhibiting replication factor C4

Drug resistance is a key factor in the treatment failure of acute myeloid leukemia (AML). Nuclear factor E2-related factor 2 (Nrf2) plays a crucial role in tumor chemotherapy resistance. However, the potential mechanism of Nrf2 regulating DNA mismatch repair (MMR) pathway to mediate gene-instability drug resistance in AML is still unclear. Here, it was found that Nrf2 expression was closely related to the disease progression of AML as well as highly expressed in AML patients with poor prognostic gene mutations. Meanwhile, it was also found that the expression of Nrf2 was significantly negatively correlated with DNA MMR gene replication factor C4 (RFC4) in AML. CHIP analysis combined with luciferase reporter gene results further showed that Nrf2 may inhibit the expression of RFC4 by its interaction with the RFC4 promoter. In vitro and vivo experiments showed that the overexpression of Nrf2 decreased the killing effect of chemotherapy drug cytarabine (Ara-C) on leukemia cells and inhibited the expression of RFC4. Mechanistically, The result that Nrf2-RFC4 axis mediated AML genetic instability drug resistance might be received by activating the JNK/NF-κB signaling pathway. Taken together, these findings may provide a new idea for improving AML drug resistance.

ATF2 loss promotes tumor invasion in colorectal cancer cells via upregulation of cancer driver TROP2

In cancer, the activating transcription factor 2 (ATF2) has pleiotropic functions in cellular responses to growth stimuli, damage, or inflammation. Due to only limited studies, the significance of ATF2 in colorectal cancer (CRC) is not well understood. We report that low ATF2 levels correlated with worse prognosis and tumor aggressiveness in CRC patients. NanoString gene expression and ChIP analysis confirmed trophoblast cell surface antigen 2 (TROP2) as a novel inhibitory ATF2 target gene. This inverse correlation was further observed in primary human tumor tissues. Immunostainings revealed that high intratumoral heterogeneity for ATF2 and TROP2 expression was sustained also in liver metastasis. Mechanistically, our in vitro data of CRISPR/Cas9-generated ATF2 knockout (KO) clones revealed that high TROP2 levels were critical for cell de-adhesion and increased cell migration without triggering EMT. TROP2 was enriched in filopodia and displaced Paxillin from adherens junctions. In vivo imaging, micro-computer tomography, and immunostainings verified that an ATF2KO/TROP2high status triggered tumor invasiveness in in vivo mouse and chicken xenograft models. In silico analysis provided direct support that ATF2low/TROP2high expression status defined high-risk CRC patients. Finally, our data demonstrate that ATF2 acts as a tumor suppressor by inhibiting the cancer driver TROP2. Therapeutic TROP2 targeting might prevent particularly the first steps in metastasis, i.e., the de-adhesion and invasion of colon cancer cells.

Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice.

Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. During these processes, studying the mechanical properties of the cells is often challenging. Conventional measurement methods based on contact such as compression or stretching are prone to cause cell damage, affecting measurement accuracy and subsequent cell culture. Measurements in adherent state can also affect accuracy, especially after irradiation since ionizing radiation will flatten cells and enhance adhesion. Here, a cell mechanics measurement system based on acoustofluidic method has been developed. The cell compressibility can be obtained by recording the cell motion trajectory under the action of the acoustic force, which can realize fast and non-destructive measurement in suspended state. This paper reports in detail the protocols for chip design, sample preparation, trajectory recording, parameter extraction and analysis. The compressibility of different types of tumor cells was measured based on this method. Measurement of the compressibility of nucleus was also achieved by adjusting the resonance frequency of the piezoelectric ceramic and the width of the microchannel. Combined with the molecular level verification of immunofluorescence experiments, the cell compressibility before and after drug-induced epithelial to mesenchymal transition (EMT) were compared. Further, the change of cell compressibility after X-ray irradiation with different doses was revealed. The cell mechanics measurement method proposed in this paper is universal and flexible and has broad application prospects in scientific research and clinical practice.

The effect of AKT in extracellular matrix stiffness induced osteogenic differentiation of hBMSCs

Extracellular matrix (ECM) stiffness is an important biophysical factor in human bone marrow mesenchymal stem cells (hBMSCs) differentiation. Although there is evidence that Yes-associated protein (YAP) plays an important role in ECM elasticity induced osteogenesis, but the regulatory mechanism and signaling pathways have not been distinctly uncovered. In this study, hBMSCs were cultured on collagen-coated polydimethylsiloxane hydrogels with stiffness corresponding to Young's moduli of 0.5 kPa and 32 kPa, and gene chip analyses revealed the phosphoinositide 3-kinase (PI3K)-AKT pathway was highly correlated with ECM stiffness. Following western blots indicated that AKT phosphorylation was evidently affected in 5th–7th days after ECM stiffness stimulation, while PI3K showed little difference. The AKT activator SC79 and inhibitor MK2206 were utilized to modulate AKT phosphorylation. SC79 and MK2206 caused alteration in the mRNA expression and protein level of alkaline phosphatase (ALP), collagen type I alpha 1 (COL1A1) and runt related transcription factor 2 (RUNX2). On 32 kPa substrates, YAP enrichment in nucleus were significantly promoted by SC79 and remarkably decreased by MK2206. Besides, the ratio of YAP/p-YAP is upregulated by SC79 on both 32 kPa and 0.5 kPa substrates. In conclusion, these findings suggest that AKT is involved in the modulation of ECM stiffness induced osteogenesis, and AKT phosphorylation also influences the subcellular localization and activation of YAP.

Therapeutic targeting of RBPJ, an upstream regulator of ETV6 gene, abrogates ETV6-NTRK3 fusion gene transformations in glioblastoma

Fusion genes are abnormal genes resulting from chromosomal translocation, insertion, deletion, inversion, etc. ETV6, a rather promiscuous partner forms fusions with several other genes, most commonly, the NTRK3 gene. This fusion leads to the formation of a constitutively activated tyrosine kinase which activates the Ras-Raf-MEK and PI3K/AKT/MAPK pathways, leading the cells through cycles of uncontrolled division and ultimately resulting in cancer. Targeted therapies against this ETV6-NTRK3 fusion protein are much needed. Therefore, to find a targeted approach, a transcription factor RBPJ regulating the ETV6 gene was established and since the ETV6-NTRK3 fusion gene is downstream of the ETV6 promoter/enhancer, this fusion protein is also regulated. The regulation of the ETV6 gene via RBPJ was validated by ChIP analysis in human glioblastoma (GBM) cell lines and patient tissue samples. This study was further followed by the identification of an inhibitor, Furamidine, against transcription factor RBPJ. It was found to be binding with the DNA binding domain of RBPJ with antitumorigenic properties and minimal organ toxicity. Hence, a new target RBPJ, regulating the production of ETV6 and ETV6-NTRK3 fusion protein was found along with a potent RBPJ inhibitor Furamidine.

2D FEM simulation and experimental verification of Al 7075‐T6 during turning process

AbstractIn this work, temperature distribution, effective stress and strain distribution in Al 7075‐T6 material and plastic flow of materials during turning process have been studied using Deform 3D v11.0 software. The four different points (P1, P2, P3, and P4) were chosen at the different locations in the cylindrical workpiece and the graph has been plotted with different parameters namely, temperature distribution, effective stress and strain. For temperature distribution, a good agreement has been found between simulation and experimental results at P4. The following results obtained from the simulation: In tertiary zone, the maximum temperature of specimen reaches upto 142 °C at points P1 due to friction between the tool rack face and the lower surface of chip. Maximum effective stress value reaches upto 650 MPa and 704 MPa at P1 and P4 respectively in the first sticking zone due to the combined effect of high normal force by tool edge and ploughing force. The effective strain value at P1 and P4 obtained, strain at P4 reaches maximum compare to P1 due to friction and unven temperature distribution. Additionally, chip analysis has been performed with microscopic image.

Introduction of a long synthetic repetitive DNA sequence into cultured tobacco cells.

Genome information has been accumulated for many species, and these genes and regulatory sequences are expected to be applied in plants by enhancing or creating new metabolic pathways. We hypothesized that manipulating a long array of repetitive sequences using tethered chromatin modulators would be effective for robust regulation of gene expression in close proximity to the arrays. This approach is based on a human artificial chromosome made of long synthetic repetitive DNA sequences in which we manipulated the chromatin by tethering the modifiers. However, a method for introducing long repetitive DNA sequences into plants has not yet been established. Therefore, we constructed a bacterial artificial chromosome-based binary vector in Escherichia coli cells to generate a construct in which a cassette of marker genes was inserted into 60-kb synthetic human centromeric repetitive DNA. The binary vector was then transferred to Agrobacterium cells and its stable maintenance confirmed. Next, using Agrobacterium-mediated genetic transformation, this construct was successfully introduced into the genome of cultured tobacco BY-2 cells to obtain a large number of stable one-copy strains. ChIP analysis of obtained BY-2 cell lines revealed that the introduced synthetic repetitive DNA has moderate chromatin modification levels with lower heterochromatin (H3K9me2) or euchromatin (H3K4me3) modifications compared to the host centromeric repetitive DNA or an active Tub6 gene, respectively. Such a synthetic DNA sequence with moderate chromatin modification levels is expected to facilitate manipulation of the chromatin structure to either open or closed.

Ras suppressor-1 (RSU1) exerts a tumor suppressive role with prognostic significance in lung adenocarcinoma.

Ras suppressor-1 (RSU1), originally described as a suppressor of Ras oncogenic transformation, localizes to focal adhesions interacting with the ILK-PINCH-PARVIN (IPP) complex that exerts a well-established oncogenic role in cancer. However, RSU1 implication in lung cancer is currently unknown. Our study aims to address the role of RSU1 in lung adenocarcinoma (LUADC). We here show that RSU1 protein expression by immunohistochemistry is downregulated in LUADC human tissue samples and represents a significant prognostic indicator. In silico analysis of gene chip and RNA seq data validated our findings. Depletion of RSU1 by siRNA in lung cancer cells promotes anchorage-independent cell growth, cell motility and epithelial to mesenchymal transition (EMT). Silencing of RSU1 also alters IPP complex expression in lung cancer cells. The p29 RSU1 truncated isoform is detected in lung cancer cells, and its expression is downregulated upon RSU1 silencing, whereas it is overexpressed upon ILK overexpression. These findings suggest that RSU1 exerts a tumor suppressive role with prognostic significance in LUADC.

Abstract 5807: MDM2 mitochondrial translocation mediates metabolic reprogramming towards OXPHOS in TKI-resistant oncogene-addicted cancer

Abstract There is mounting evidence of oxidative phosphorylation (OXPHOS) dependency in cancers resistant to tyrosine kinase inhibitors (TKI), but the cause of this metabolic switch remains elusive. Mitochondria-bound MDM2 (mtMDM2), the fraction of MDM2 oncoprotein which is actively imported into the mitochondria, has been found to dysregulate electron transport chain (ETC) complex I function and OXPHOS. AKT is known to be a regulator of MDM2 protein stability. Given that AKT phosphorylation is commonly detected in oncogene-addicted tumours, we investigated the role of mtMDM2 and AKT in promoting TKI resistance through reprogrammed cellular metabolism. EGFR+ NSCLC [HCC827 and HCC827-GR (gefitinib-resistant)] and BRAF+ melanoma [A375 and A375-VR (vemurafenib-resistant)] cells were established via repetitive pulsed strategy. Translocation of MDM2 was examined through subcellular fractionation and Western Blotting. To investigate the role of MDM2 in mediating OXPHOS, genetic knockdown of MDM2 was performed via siRNA followed by measurement of oxygen consumption rate (Seahorse Analyzer); mRNA expression of mtDNA-encoded ETC subunits by real-time PCR (qPCR). ChIP-qPCR analysis was performed to examine the binding affinity of MDM2 and TFAM (mitochondrial transcription factor A) to mitochondrial DNA (mtDNA). Mitochondrial translocation of MDM2 was predominant in TKI-sensitive cells; MDM2 silencing upregulated OXPHOS and induced mRNA expression of mtDNA-encoded ETC complex I subunits, suggesting that mtMDM2 inhibits OXPHOS. ChIP analysis revealed competitive binding of MDM2 and TFAM at the LSP region of mtDNA. Conversely, MDM2 was primarily localized the cytoplasm in TKI-resistant cells and was associated with increased OXPHOS in TKI-resistant cells. Mechanistically, both pAKT and pMDM2 (Ser166/Ser186) were higher in TKI-resistant cells. Collectively, we describe a novel observation that OXPHOS upregulation underpinning TKI resistance in cancer cells could be mediated by AKT upregulation and MDM2 localisation. Citation Format: Jie Qing Eu, Li Ren Kong, Jayshree Hirpara, Boon Cher Goh, Andrea LA Wong, Shazib Pervaiz. MDM2 mitochondrial translocation mediates metabolic reprogramming towards OXPHOS in TKI-resistant oncogene-addicted cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5807.

Abstract 5837: A truncated nucleus-located derivative of FGFR1 contributes to the transformation of leukemia stem cell in leukemogenesis driven by FGFR1 fusion kinases

Abstract Myeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated as a result of chromosome translocations, which lead to acquisition of dimerization motifs in the chimeric proteins. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated. Stem cell transduction and transplantation in syngeneic mice were used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and the results showed that tnFGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells. These leukemia cells show a mixed immunophenotype with a B-cell B220+Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. RPPA and RNA-Seq examination of the related signaling pathways and target genes revealed that tnFGFR1, however, does not activate classic FGFR1 downstream signaling pathways but induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. De novo human AML also expressed tnFGFR1 which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but treatment of these cells with the Quizartinib (AC220) FLT3 inhibitor, suppresses in vitro growth and development of leukemia in vivo suggesting an alternative approach for treatment of SCLL. This study demonstrates a novel model for transformation of hematopoietic stem cells by chimeric FGFR1 kinases results with the combined effects of direct protein activation by the full-length kinases and gene regulation by the truncated nucleus derivative tnFGFR1, which is associated with GZMB expression levels. Genes significantly upregulated by tnFGFR1 include Flt3 and Kit which promote a leukemia stem cell phenotype. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile. Citation Format: Baohuan Cai, Yun Liu, Yating Chong, Hualei Zhang, Stephanie Mori, Atsuko Matsunaga, Xuexiu Fang, John Cowell, Tianxiang Hu. A truncated nucleus-located derivative of FGFR1 contributes to the transformation of leukemia stem cell in leukemogenesis driven by FGFR1 fusion kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5837.

Abstract 1535: Novel lncRNA ENSG00000254887 is a key factor for MYC-driven cell proliferation

Abstract Long non-coding RNAs (lncRNAs) are widely expressed, and many of them are involved in gene regulation. In a CRISPRi study, we looked for lncRNAs required for MYC-driven proliferation in human lymphoid cells and found a novel lncRNA, ENSG00000254887, among the top candidates. In the genome, ENSG00000254887 is head-to-head with a coding gene, POU2F2. To our knowledge, ENSG00000254887 has not been described in the literature yet. We aim to characterize the importance of ENSG00000254887 in cancer development and its mechanisms of action. We performed a ChIP analysis to confirm the direct interaction of MYC with ENSG00000254887 in Ramos cells, a Burkitt’s lymphoma cell line. To knock down the expression, we generated a library of single guide RNAs (sgRNAs) covering the whole length of ENSG00000254887. We transduced Cas9-expressing Ramos cells with this library and cultured them for 14 days. We then measured the differential depletion of guides using next generation sequencing. To validate these results, we performed CRISPR growth competition assays with individual GFP-expressing sgRNA constructs, and the cell growth was measured by flow cytometry. We corroborated the importance of ENSG00000254887-regulated expression by two additional approaches: downregulation by small-hairpin RNA (shRNA) and upregulation by expression of an ectopic copy of the gene. We have been able to retrieve the genomic sequence of ENSG00000254887 using an anti-MYC antibody, thus confirming the presence of binding sites for this transcription factor. Using CRISPR, we have mapped several sites along the ENSG00000254887 sequence where Cas9-introduced changes lead to loss of function and reduce the ability of the cells to proliferate. We selected three of the most efficient sgRNAs used for CRISPR for further analysis and observed that they induced a reduction in the expression of both ENSG00000254887 and the nearby coding gene, POU2F2. This significant result was validated using shRNA targeting the sequence of the lncRNA. On the contrary, the overexpression of ENSG00000254887 from an inducible ectopic copy has no effect on the expression of POU2F2. In conclusion, the expression of ENSG00000254887 is critical for MYC-driven cell proliferation and can be altered through Cas9-introduced changes. A downregulation of this lncRNA is always accompanied by a reduction in the expression of the nearby coding gene, POU2F2. The absence of any effect on POU2F2 when we overexpress ENSG00000254887 from an ectopic copy indicates that this lncRNA might be acting in cis. However, further research is needed to explain this fact and establish the molecular mechanisms. Acknowledgments: This work was supported by the National Cancer Institute of the National Institutes of Health under award R35 CA197582 (PKV). Citation Format: Daniel García-Caballero, Jonathan R. Hart, Peter K. Vogt. Novel lncRNA ENSG00000254887 is a key factor for MYC-driven cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1535.

Screening and Identification of Hub Genes in the Development of Early Diabetic Kidney Disease Based on Weighted Gene Co-Expression Network Analysis.

ObjectiveThe study aimed to screen key genes in early diabetic kidney disease (DKD) and predict their biological functions and signaling pathways using bioinformatics analysis of gene chips interrelated to early DKD in the Gene Expression Omnibus database.MethodsGene chip data for early DKD was obtained from the Gene Expression Omnibus expression profile database. We analyzed differentially expressed genes (DEGs) between patients with early DKD and healthy controls using the R language. For the screened DEGs, we predicted the biological functions and relevant signaling pathways by enrichment analysis of Gene Ontology (GO) biological functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways. Using the STRING database and Cytoscape software, we constructed a protein interaction network to screen hub pathogenic genes. Finally, we performed immunohistochemistry on kidney specimens from the Beijing Hospital to verify the above findings.ResultsA total of 267 differential genes were obtained using GSE142025, namely, 176 upregulated and 91 downregulated genes. GO functional annotation enrichment analysis indicated that the DEGs were mainly involved in immune inflammatory response and cytokine effects. KEGG pathway analysis indicated that C-C receptor interactions and the IL-17 signaling pathway are essential for early DKD. We identified FOS, EGR1, ATF3, and JUN as hub sites of protein interactions using a protein–protein interaction network and module analysis. We performed immunohistochemistry (IHC) on five samples of early DKD and three normal samples from the Beijing Hospital to label the proteins. This demonstrated that FOS, EGR1, ATF3, and JUN in the early DKD group were significantly downregulated.ConclusionThe four hub genes FOS, EGR1, ATF3, and JUN were strongly associated with the infiltration of monocytes, M2 macrophages, and T regulatory cells in early DKD samples. We revealed that the expression of immune response or inflammatory genes was suppressed in early DKD. Meanwhile, the FOS group of low-expression genes showed that the activated biological functions included mRNA methylation, insulin receptor binding, and protein kinase A binding. These genes and pathways may serve as potential targets for treating early DKD.

SIRT1 regulates mitotic catastrophe via autophagy and BubR1 signaling.

Mitotic catastrophe (MC) is a suppressive mechanism that mediates the elimination of mitosis-deficient cells through apoptosis, necrosis or senescence after M phase block. SIRT1 is involved in the regulation of several cellular processes, including autophagy. However, the relationship between SIRT1 and MC has been largely obscure. Our study highlights that SIRT1 might be involved in the regulation of MC. We have shown that degradation of the SIRT1 protein via proteasome and lysosomal pathway was accompanied by MC induced via BMH-21. Overexpression of SIRT1 alleviated MC by decreasing the proportion of apoptotic and multinuclear cells induced by G2/M block and triggered autophagy whereas knockdown of SIRT1 aggravated MC and repressed autophagy. Furthermore, we found that serum starvation triggered autophagy evidently generated lower MC whereas siRNA of ATG5/7 suppressed autophagy leading to higher MC. ChIP analysis revealed that SIRT1 could bind to the promoter of BubR1, a component of spindle assembly checkpoint (SAC), to upregulate its expression. Overexpression of BubR1 decreased MC whereas knockdown of BubR1 increased it. These results reveal that SIRT1 regulates MC through autophagy and BubR1 signaling, and provide evidence for SIRT1, autophagy and BubR1 being the potential cancer therapeutic targets.

Octacosanol Modifies Obesity, Expression Profile and Inflammation Response of Hepatic Tissues in High-Fat Diet Mice.

The incidence of obesity has increased significantly on account of the alterations of living habits, especially changes in eating habits. In this study, we investigated the effect of octacosanol on lipid lowering and its molecular mechanism. High-fat diet (HFD)-induced obesity mouse model was used in the study. Thirty C57BL/6J mice were divided into control, HFD, and HFD+Oct groups randomly, and every group included ten mice. The mice of HFD+Oct group were intragastrically administrated 100 mg/kg/day of octacosanol. After 10 weeks for treatment, our results indicated that octacosanol supplementation decreased the body, liver, and adipose tissues weight of HFD mice; levels of TC, TG, and LDL-c were reduced in the plasma of HFD mice; and level of HDL-c were increased. H&E staining indicated that octacosanol supplementation reduces the size of fat droplets of hepatic tissues and adipose cells comparing with the HFD group. Gene chip analysis found that octacosanol regulated 72 genes involved in lipid metabolism in the tissues of liver comparing to the HFD group. IPA pathway network analysis indicated that PPAR and AMPK may play a pivotal role in the lipid-lowering function of octacosanol. Real-time quantitative PCR and Western blot showed that the octacosanol supplementation caused change of expression levels of AMPK, PPARs, FASN, ACC, SREBP-1c, and SIRT1, which were closely related to lipid metabolism. Taken together, our results suggest that octacosanol supplementation exerts a lipid-decreasing effect in the HFD-fed mice through modulating the lipid metabolism-related signal pathway.

Vascular toxicity of multi-walled carbon nanotubes targeting vascular endothelial growth factor

Multiwalled carbon nanotubes (MWCNTs) are currently widely used and are expected to be used as drug carriers and contrast agents in clinical practice. Previous studies mainly focused on their lung toxicity; therefore, their effects on the vascular endothelium are unclear. In this study, a human angiogenesis array was used to determine the effect of MWCNTs on the expression profile of angiogenic factors in endothelial cells and to clarify the role of vascular endothelial growth factor (VEGF) in MWCNT-induced endothelial cell injury at the cellular and animal levels. The results indicated that MWCNTs (20–30 nm and 30–50 nm) could enter endothelial cells and disrupt human umbilical vein endothelial cell (HUVECs) activity in a concentration-dependent manner. MWCNTs disrupted the tube formation ability and cell migration function of HUVECs. The results from a Matrigel Plug experiment in mice showed that angiogenesis in the MWCNT experimental group was significantly reduced. The results of a protein chip analysis indicated that VEGF expression in the MWCNT treatment group was decreased, a finding that was validated by ELISA results. The protein expression levels of AKT and eNOS in the MWCNT treatment group were significantly decreased; the administration of recombinant VEGF significantly alleviated the migration ability and tube formation ability of endothelial cells injured by MWCNTs, upregulated the protein expression of AKT and eNOS, and increased the number of neovascularization in mice in the MWCNT treatment group. This study demonstrated that MWCNTs affect angiogenesis via the VEGF-Akt-eNOS axis which can be rescued by VEGF endothelial treatment.

The micro-fabrication and performance analysis of non-magnetic heating chip for miniaturized SERF atomic magnetometer

The elevated temperature field is one of the fundamental conditions for the spin-exchange relaxation-free (SERF) regime, which can be accomplished by electric heating chip without introducing stray field. Therefore, a non-magnetic heating chip composed of double identical ring-structure layers with a silicon oxide insulating layer between them is carefully designed and fabricated with the material of nichrome alloy on a borosilicate glass substrate by Micro Electro-Mechanical Systems (MEMS) technology. What’s more, the influence of annealing treatment process on the microstructure of the heating chip is characterized and explicated utilizing modern surface science technique, including four-probe meter, X-ray diffraction (XRD) and atomic force microscope (AFM). Then, the integrated miniature single-beam SERF atomic magnetometer is developed based on the microfabricated heating chip. The temperature stability of heating chip is tested with the value less than ± 10 mK at 120 °C by the proportional integral derivative (PID) control program, and the magnetic current coefficient is determined of only 0.506 nT/mA by the spin resonance response of the SERF atomic magnetometer near the zero field. Finally, the developed SERF atomic magnetometer achieves a high measuring sensitivity of 70 fT/Hz1/2. These experimental results provide a new avenue for the wafer-level microfabrication of the high-performance non-magnetic heating chip. More importantly, the presented work paves the way towards chip-scale atomic magnetometers with high sensitivity and mm-scale spatial resolution in the applications of magnetic source imaging and magnetic anomaly detection.

Genome-wide analysis of the apple family 1 glycosyltransferases identified a flavonoid-modifying UGT, MdUGT83L3, which is targeted by MdMYB88 and contributes to stress adaptation

The plant family 1 UDP-glycosyltransferases (UGTs) are increasingly being investigated because of their contribution to plant secondary metabolism and other diverse biological roles. The apple (Malus domestica) is one of the most widely cultivated fruit trees with great economic importance. However, little is known regarding the apple UGTs. In this study, we identified 229 members of family 1 through a genome-wide analysis of the apple UGTs, which were clustered into 18 groups, from A to R. We also performed detailed analysis of 34 apple UGTs by quantitative RT-PCR, and discovered a number of stress-regulated UGTs. Among them, we characterized the role of MD09G1064900, also named MdUGT83L3, which was significantly induced by salt and cold. In vivo analysis showed that it has high activity towards cyanidin, and moderate activity towards quercetin and keampferol. Transgenic callus and regenerated apple plants overexpressing MdUGT83L3 showed enhanced tolerance to salt and cold treatments. Overexpression of MdUGT83L3 also increased anthocyanin accumulation in the callus tissues and enhanced ROS clearing upon exposure to salt and cold stresses. Furthermore, via yeast-one-hybrid assay, EMSA and CHIP analyses, we also found that MdUGT83L3 could be directly regulated by MdMYB88. Our study indicated that MdUGT83L3, under the regulation of MdMYB88, plays important roles in salt and cold stress adaptation via modulating flavonoid metabolism in apple.

Mitotic genome bookmarking by nuclear receptor VDR advocates transmission of cellular transcriptional memory to progeny cells

Mitosis is an essential process for the self-renewal of cells that is accompanied by dynamic changes in nuclear architecture and chromatin organization. Despite all the changes, the cell manages to re-establish all the parental epigenetic marks, post-mitotically. Recent reports suggest that some sequence-specific transcription factors remain attached to mitotic chromatin during cell division to ensure timely reactivation of a subset of transcription factors necessary to maintain cell identity. These mitotically associated factors are suggested to act as ‘genome bookmarking factors' and the phenomenon is termed ‘genome bookmarking’. Here, we studied this phenomenon with Vitamin D Receptor (VDR), a key regulator of calcium and phosphate homeostasis and a member of the nuclear receptor superfamily. This study, for the first time, has confirmed VDR as a mitotic bookmarking factor that may be playing a crucial role in the maintenance of cell identity and genome bookmarking. Full ‘DNA binding domain (DBD)’ present in VDR was identified as essential for enrichment of VDR on mitotic chromatin. Furthermore, the study also demonstrates that VDR evokes mitotic chromatin binding behaviour in its heterodimeric partner Retinoid X receptor (RXR). Interestingly, for promoting bookmarking behaviour in RXR, both DBD and/or ligand-binding domain (LBD) in conjunction with hinge region of VDR were required. Additionally, ChIP analysis showed that VDR remains associated with DR3 (direct repeat 3) region of its specific target gene promoter CYP24A1(Cytochrome P450 family 24 subfamily A member1), during mitosis. Altogether, our study illustrates a novel function of VDR in the epigenetic transmission and control of expression of target proteome for maintenance of cell identity and traits in progeny cells.

MiR-320b and miR-320d as Biomarkers to Predict and Participate in the Formation of Platinum Resistance in Ovarian Cancer Patients.

Patients with ovarian cancer who receive platinum-based chemotherapy typically develop platinum resistance, which leads to tumor recurrence and mortality. Therefore, finding the underlying mechanisms and biomarkers is critical. A total of 51 platinum-resistant and 70 platinum-sensitive ovarian cancer patients were enrolled in this study. We examined the GSE131978 dataset in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database for differentially expressed long non-coding RNAs and messenger RNAs (mRNAs) between platinum-resistant and platinum-sensitive patients and completed a microRNA chip analysis. After filtering by Pearson correlation analysis, the competitive endogenous RNA (ceRNA) networks were subsequently constructed. Then, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology enrichment analyses about mRNAs in ceRNA networks were accomplished. More crucially, we demonstrated the differentially expressed microRNAs using quantitative real-time PCR and fluorescence in situ hybridization. The feasibility of microRNAs as biomarkers to predict platinum resistance and tumor recurrence was assessed using the receiver operating characteristic curve and survival analysis. MiR-320b and miR-320d exhibited high area under the curve values of 0.757 and 0.702, respectively. In our study, ceRNA networks including miR-320b and miR-320d probably provided novel insights for platinum resistance in ovarian cancer patients.