Electrochemical detection of SARS-CoV-2 based on copper nanoflower-triggered in situ growth of electroactive polymers.

SARS-CoV-2, the pathogen of COVID-19, has introduced massive confirmed cases and millions of deaths worldwide, which poses a serious public health threat. For the early diagnosis of COVID-19, we have constructed an electrochemical biosensor-combined magnetic separation system with copper nanoflower-triggered cascade signal amplification strategy. In the proposed system, magnetic beads were utilized to fabricate the recognition element for capturing the conserved sequence of SARS-CoV-2. As the copper ions source, oligonucleotides modified copper nanoflowers with special layered structure provide numerous catalysts for click chemistry reaction. When target sequence RdRP_SARSr-P2 appears, copper nanoflowers will be bound with magnetic beads, thus prompting the Cu(I)-catalyzed azide-alkyne cycloaddition reaction through the connection of the SARS-CoV-2 conserved sequence. Then, a large number of signal molecules FMMA can be grafted onto the modified electrode surface by electrochemically mediated atom-transfer radical polymerization to amplify the signal for the quantitative analysis of SARS-CoV-2. Under optimal conditions, a linear range from 0.1 to 103 nM with a detection limit of 33.83 pM is obtained. It provides a powerful tool for the diagnosis of COVID-19, which further benefits the early monitoring of other explosive infectious diseases effectively, thus guaranteeing public health safety.

Pentraxin 3 promotes oxLDL uptake and inhibits cholesterol efflux from macrophage-derived foam cells.

BACKGROUND: The objective of this study was to determine the effects of pentraxin3 (PTX3) on human oxidized low density lipoprotein (oxLDL) uptake and cholesterol efflux from human macrophage foam cells, which may play a critical role in atherogenesis. METHODS: The effects of PTX3 on oxLDL uptake and cholesterol efflux were determined after transfection of human THP-1 macrophages with pSG5hPTX3 or PTX3siRNA plasmids. To evaluate the role of specific signaling pathways, human THP-1 cells were pre-treated with inhibitors of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), phosphatidylinositide 3-kinases (PI3-K), and p38 mitogen-activated protein kinase (MAPK) pathways (PD98059, LY294002, and SB203580, respectively), and then exposed to oxLDL for the uptake assay or oxLDL and [(3)H]-cholesterol and apolipoprotein A-I (apoA-I) for the cholesterol efflux assay. RESULTS: PTX3 overexpression not only promoted oxLDL uptake but also significantly reduced cholesterol efflux to apoA-I; it also significantly decreased the expression of peroxisome proliferator-activated receptor-γ (PPARγ), liver X receptor alpha (LXRα) and ATP-binding membrane cassette transporter A-1 (ABCA1), which was increased with PTX3 silencing. Furthermore, PTX3 significantly increased p-ERK1/2 levels in THP-1-derived foam cells, and inhibition of ERK1/2 by PD98059 significantly reduced the oxLDL uptake and promoted the cholesterol efflux induced by PTX3 overexpression. CONCLUSION: Here, we demonstrate that PTX3 affects lipid accumulation in human macrophages, increasing oxLDL uptake and inhibiting cholesterol efflux. That is the underlying possible mechanisms of PTX3 contribution to the progression of atherosclerosis.

Correlation of Forkhead Box c2 with subtypes and invasive ability of invasive breast cancer.

Forkhead Box c2 (FOXC2) is a member of forkhead/winged-helix family of transcription factors. The relationship between FOXC2 and invasive breast cancers, including basal-like breast cancer (BLBC, a subtype of breast cancer), remains to be elucidated. In this study, immunohistochemistry was used to detect the expression of FOXC2 in samples from 103 cases of invasive breast cancers and 15 cases of normal mammary glands. The relationship between FOXC2 and clinical parameters of invasive breast cancers such as patient's age, tumor size, lymph node metastasis, tumor grade, the expression of ER, PR, HER-2 and p53, and Ki-67 labeling index (LI) was evaluated. The expression of FOXC2 was detected in parent MCF7 cells, MCF cells transfected with FOXC2 expression vectors and MDA-MB-435 cells by immunohistochemistry and Western blotting. Transwell assay was used to determine the invasive ability of these cells. The results showed that FOXC2 was strongly expressed in basal epithelial cells in normal mammary glands and weakly expressed or even not expressed in glandular epithelial cells. The majority of invasive breast cancers (71.8%, 74/103) had negative or weak expression of FOXC2. However, FOXC2 was strongly expressed in 60.7% of BLBCs. Moreover, FOXC2 was related with tumor grade, p53 expression, ki-67 LI and lymph nodes metastasis. It was expressed in FOXC2-transfected MCF cells and MDA-MB-435 cells but not in parent MCF cells. Transwell assay revealed that MCF cells transfected with FOXC2 expression vectors were more aggressive than the parent MCF cells, suggesting a positive correlation between FOXC2 and the invasion of breast cancer. It was concluded that there is a significant association between FOXC2 and the metastasis of invasive breast cancer. FOXC2 may be used as a new marker for the diagnosis and prognosis prediction of different subtypes of invasive breast cancers.

Longitudinal change of six common inflammatory cytokines and their relationship to anxiety, depression, and cognitive impairment in acute ischemic stroke patients.

Inflammatory cytokines are known to be involved in acute ischemic stroke (AIS), while the relationship of multiple inflammatory cytokines with mental disorders in AIS is less reported. This research intended to explore the longitudinal variation of common inflammatory cytokines and their correlation with anxiety, depression, and cognitive impairment in AIS patients. Six inflammatory cytokines were detected by enzyme-linked immunosorbent assay among 175 AIS patients at admission (baseline) and on the day (D)1, D3, and D7 after admission. Anxiety, depression, and cognition were evaluated using the Hospital Anxiety and Depression Scale and Mini-Mental State Examination at discharge, respectively. Anxiety, depression, and cognitive impairment rates were 32.6, 39.4, and 19.4%, respectively. Tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, and IL-17A increased from baseline to D1, then decreased from D1 to D7 (all P<0.001), while IL-10 presented an opposite trend (P<0.001). Interestingly, TNF-α on D1 and D3, IL-6 on D3, IL-8 on D3 and D7, and IL-17A on D1, D3, and D7 correlated with higher anxiety rate (all P<0.05). TNF-α on D1, D3, and D7, IL-8 at baseline, D1, D3, and D7, IL-17A on D1 and D7 correlated with increased depression rate (all P<0.05). In addition, IL-1ß on D1 and IL-17 at baseline, D1, D3, and D7 correlated with elevated cognitive-impairment rate (all P<0.05). Inflammatory cytokines were dysregulated after disease onset, and their longitudinal change correlated with psychological issues in AIS patients.

TGF-ß1-induced LPP expression dependant on Rho kinase during differentiation and migration of bone marrow-derived smooth muscle progenitor cells.

Lipoma preferred partner (LPP) has been identified as a protein which is highly selective for smooth muscle progenitor cells (SMPCs) and regulates differentiation and migration of SMPCs, but mechanisms of LPP expression are not elucidated clearly. The aim of the present study was to discuss the mechanisms by which LPP expression is regulated in the differentiation and migration of SMPCs induced by TGF-ß1. It was found that TGF-ß1 could significantly increase the expression of LPP, smooth muscle α-actin, smooth muscle myosin heavy chain (SM-MHC), and smoothelin in SMPCs. Moreover, inactivation of Rho kinase (ROK) with ROK inhibitors significantly inhibited LPP mRNA expression in TGF-ß1-treated SMPCs and mouse aortic smooth muscle cells (MAoSMCs). At the same time, LPP silencing with short interfering RNA significantly decreased SMPCs migration. In conclusion, LPP appears to be a ROK-dependant SMPCs differentiation marker that plays a role in regulating SMPCs migration.

INSM1 Expression in Mesenchymal Tumors and Its Clinicopathological Significance.

Background: Insulinoma-associated protein 1 (INSM1) has been identified as a nuclear marker of neuroendocrine tumors. Although INSM1 appears to be a subtle and specific biomarker for neuroendocrine tumor, its expression and clinicopathological significance in mesenchymal tumors remain unclear. Methods: We analyzed INSM1 mRNA level in GEO database and conducted immunohistological staining to detect the expression of INSM1 on 576 mesenchymal tumors from pathology department of Tongji Hospital. Results: At transcription level, INSM1 expression in AITL (angioimmunoblastic T-cell lymphoma) was higher than their adjacent normal tissues as well as Hodgkin's lymphoma. Moreover, INSM1 expression in well-differentiated liposarcoma (WDLPS) was significantly higher than normal fat (P = 0.014) and dedifferentiated liposarcoma (DDLPS) (P = 0.0248). At protein level, the positive rate of INSM1 in AITL was 18/48 (47.4%), while in DDLPS was 9/20 (45%). INSM1 expression in AITL was significantly higher than Hodgkin's lymphoma (P = 0.008). And INSM1 expression in WDLPS was significantly lower than DDLPS (P = 0.015). Conclusion: The combination of GEO data and immunohistochemistry data indicated that the expression level of INSM1 was higher in AITL compared with normal control, suggesting that INSM1 may be involved in pathogenesis of AITL. The abnormal expression of INSM1 was found in WDLPS, and the positive rate of INSM1 was higher in DDLPS than in WDLPS. INSM1 may be involved in the regulation of liposarcoma development. There were significant differences in the expression of INSM1 between AITL and Hodgkin's lymphoma and WDLPS and DDLPS. These findings may assist in the differential diagnosis of these tumors when common markers are difficult to identify, enriching the diagnostic index system of mesenchymal tumors.

Foxc2 overexpression enhances benefit of endothelial progenitor cells for inhibiting neointimal formation by promoting CXCR4-dependent homing.

OBJECTIVE: Endothelial progenitor cells (EPCs) are capable of enhancing re-endothelialization and attenuating neointimal formation. However, inefficient homing limits the therapeutic efficacy of EPCs transplantation. CXCR4 plays a critical role in regulating EPCs homing. Here, we studied the effect of Foxc2 overexpression on CXCR4 expression and the homing capacity of EPCs as well as the EPCs-mediated therapeutic benefit after artery injury. METHODS: Bone marrow-derived EPCs were transfected with Foxc2 expression vector (Foxc2-EPCs) or empty control vector (Ctrl-EPCs) and examined 48 hours later. CXCR4 expression of EPCs was detected by flow cytometry and quantitative reverse transcriptase-polymerase chain reaction. The migration of EPCs toward SDF-1α was evaluated in a transwell migration assay, and the adhesion to fibronectin was determined using a static adhesion assay. For in vivo studies, EPCs were injected intravenously into the mice subjected to carotid injury. At 3 days after green fluorescent protein (GFP)/EPCs delivery, the recruited cells to the injury sites were detected by fluorescent microscopy. Re-endothelialization and neointimal formation were, respectively, assessed by Evans blue dye at 7 days and by the morphometric analysis for neointima and media area ratio (N/M) at 28 days after EPCs transfusion. RESULTS: Foxc2 overexpression significantly increased the surface expression of CXCR4 on EPCs (about 1.9-fold of Ctrl-EPCs, P < .05). Foxc2-EPCs showed an increased migration toward SDF-1α (P < .05); Foxc2 overexpression increased also the adhesion capacity of EPCs (P < .05). In vivo, the number of recruited GFP cells was significantly higher in the mice transfused with Foxc2-GFP/EPCs compared with Ctrl-GFP/EPCs (about 2-fold of Ctrl-GFP/EPCs). The degree of re-endothelialization was higher in mice transfused with Foxc2-EPCs compared with Ctrl-EPCs (90.3% ± 1.6% vs 57.2% ± 1.3%; P < .05). Foxc2-EPCs delivery resulted in a greater inhibition of neointimal hyperplasia than Ctrl-EPCs administration (N/M: 0.38 ± 0.03 vs 0.67 ± 0.05, P < .05). Preincubation with CXCR4-Ab, AMD3100, or LY294002 significantly attenuated the enhanced in vitro and in vivo effects of Foxc2-EPCs. CONCLUSIONS: Our findings indicate that Foxc2 overexpression increases CXCR4 expression of EPCs and efficiently enhances the homing potential of EPCs, thereby improving EPCs-mediated therapeutic benefit after endothelial injury. Foxc2 may be a novel molecular target for improving the therapeutic efficacy of EPCs transplantation.

Macrophages overexpressing VEGF, transdifferentiate into endothelial-like cells in vitro and in vivo.

Vascular endothelial growth factor (VEGF) induces endothelial cell differentiation. To investigate the role of VEGF overexpression in regulating the phenotype of macrophages, we transfected mouse macrophages with human VEGF(165) and examined the expression of the genes and proteins for various endothelial markers. Macrophages overexpressing VEGF significantly expressed fetal liver kinase 1 (FLK-1), vascular endothelial cadherin (VE-cadherin), CD31, Von Willebrand factor (vWF), endothelial nitric oxide synthase (eNOS) and CD105. Furthermore, in a model of myocardial infarction (MI), macrophages overexpressing VEGF incorporated into blood vessels. Thus macrophages overexpressing VEGF were transdifferentiate into endothelial-like cells (ELCs) both in vitro and in vivo.

Oxidized low density lipoprotein-induced transdifferentiation of bone marrow-derived smooth muscle-like cells into foam-like cells in vitro.

Oxidized-low density lipoprotein (ox-LDL) is believed to contribute to atherogenesis in part by being taken up into smooth muscle cells (SMC) via specific scavenger receptors; however, it is not clear whether ox-LDL receptor(s) are expressed in bone marrow-derived smooth muscle-like cells (SMLCs) and whether they play a role in the process of SMLC development. Therefore, we examined the ox-LDL-induced transdifferentiation of SMLCs that is mediated by lectin-like ox-LDL receptor-1 (LOX-1). Smooth muscle progenitor cells (SMPCs) from bone marrow mesenchymal stem cells (BMSCs) were isolated using a tissue-specific promoter sorting method with a mouse SM22_ promoter (_480 bp)/green fluorescent protein recombinant plasmid. The SMPCs were myocardin+CD105+KDR+CD45(-)CD34(-), but did not express SMC-specific markers alpha-smooth muscle actin (alpha-SMA), SM22, smooth muscle myosin heavy chain (SM-MHC) and smoothelin. After long-term culture with platelet-derived growth factor-BB (PDGF-BB), SMPCs expressed alpha-SMA, SM22 and SM-MHC and differentiated into SMLCs. When SMLCs were incubated with different concentrations of ox-LDL, LOX-1 expression on the surface of SMLCs gradually increased with the increase of the ox-LDL concentration, but myocardin and SMC-specific marker genes decreased, accordingly. Furthermore, receptor-mediated endocytosis was enhanced and lipid droplets accumulated in the cytoplasm of SMLCs. A subpopulation of myocardin+CD105+KDR+CD45(-)CD34(-) SMPCs exist in BMSCs that can differentiate into SMLCs under induction with PDGF-BB. Moreover, LOX-1 contributes to the ox-LDL-induced transdifferentiation of bone marrow-derived SMLCs into foam-like cells.

CXCR4 positive bone mesenchymal stem cells migrate to human endothelial cell stimulated by ox-LDL via SDF-1alpha/CXCR4 signaling axis.

BACKGROUND: Bone mesenchymal stem cells (BMSCs) are attractive candidates for cell based therapies to cardiovascular disease such as infarction and atherosclerosis; however, the mechanisms responsible for stem cell chemotaxis and homing remain unknown. Chemokine stromal cell-derived factor 1 (SDF-1alpha) is involved in the process of atherogenesis. This study was aimed at investigating whether the SDF-1alpha of human umbilical vein endothelial cells (HUVECs) plays a role in migration of BM-derived CXCR4(+)(receptor for SDF-1alpha) stem cells. METHODS: HUVECs were cultured from human umbilical cords and was treated with ox-LDL. The mRNA and protein expression of SDF-1alpha was detected in HUVECs. CXCR4(+)BMSCs from bone marrow were isolated and were tested by migration and adhesion assays. RESULTS: It was found that ox-LDL induced HUVECs to increase the mRNA and protein expression of SDF-1alpha. Ox-LDL increased the migratory and adhesion response of CXCR4(+)BMSCs. When the neutralizing SDF-1alpha antibody abrogated the secreted SDF-1alpha, the migration and adhesion response of CXCR4(+)BMSCs markedly decreased. CONCLUSIONS: Our data indicated that the endothelial cells (ECs) stimulated by ox-LDL could increase the BMSCs migratory response via SDF-1alpha/CXCR4 signaling axis. These findings provide a new paradigm for biological effects of ox-LDL and have implications for novel stem cell therapeutic strategies for atherosclerosis.

SDF-1/CXCR4-mediated migration of transplanted bone marrow stromal cells toward areas of heart myocardial infarction through activation of PI3K/Akt.

Stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, are crucial for homing and migration of multiple stem cell types. Their potential role in mediating bone marrow-derived mesenchymal stem cell (BMSC) migration in areas of myocardial infarction (MI) has not been demonstrated. In this study, rat heart MI was created by left coronary artery ligation, and green fluorescent protein-labeled BMSCs were directly infused into the left ventricular cavity. Reverse transcriptase-polymerase chain reaction and Western blot analysis showed that SDF-1 was predominantly localized in the MI lesion, and its levels peaked by 3 to 7 days and were maintained at least 14 days. Additionally, this was matched with increased accumulation of BMSCs and an improvement in cardiac function. Furthermore, this effect was blocked by the phosphoinositide 3-kinase inhibitor, LY294002. In vitro experiments showed that CXCR4 expression by BMSCs was elevated during hypoxia and SDF-1 induced a concentration-dependent migration of BMSCs. This migration was CXCR4-dependent as confirmed by its total inhibition by AMD3100, a CXCR4-specific antagonist. Migration was also almost completely blocked by LY294002. Analysis showed that phosphorylated Akt was highly increased in SDF-1-treated BMSCs. Together these results demonstrated that SDF-1/CXCR4 may mediate the migration of BMSCs toward heart MI through activation of PI3K/Akt.

[Expression of myocardin in differentiation of bone marrow-derived mesenchymal stem cells to smooth muscle cells].

OBJECTIVE: To investigate the expression profiles of myocardin gene during the differentiation of bone marrow-derived mesenchymal stem cell to smooth muscle cells in the conditional medium combined with a high concentration of fetal bovine serum (FBS). METHODS: Marrow-derived mesenchymal stem cells were isolated and purified from mouse femoral bone and shinbones using differential adherent methods. Cells at the third passage were induced by 20% FBS in conditioned medium, conditioned medium alone, 20% FBS or 10% FBS alone respectively. Mouse aortic smooth muscle cells were cultured as the positive control. Levels of mRNA and protein expression of myocardin and several smooth muscle cells marker genes were determined by immunofluorescence, RT-PCR and Western blot before and 3, 7, 10, 14 d after the induction. The presence of smooth muscle myofilaments was detected by using transmission electron microscope. RESULTS: Naive bone marrow-derived mesenchymal stem cells displayed multiple morphological forms including fusiform, polygon, oval, and micro-spherical, as compared to the single macro-spindle form after the induction. Typical appearance of peak valley was displayed on the 21st day after induction. At the same time, the expression of smooth muscle marker genes was reinforced along with an up-regulation of myocardin expression. Immunofluorescence study showed that the cells expressing myocardin and smooth muscle marker genes such as alpha-SMA and SM-MHC increased. Fluorescence domain of myocardin translocated from cytoplasm to nucleus and the amounts of double positive cells for myocardin with alpha-SMA or SM-MHC also increased. RT-PCR confirmed that the mRNA expression of myocardin increased gradually and remained stabilized after achieving its peak on the 7th day after induction. The expression of smooth muscle marker genes, alpha-SMA and SM22alpha, remained stable on the 10th day of induction. It was also confirmed by Western blot that the protein expression of both myocardin and alpha-SMA were markedly increased during the induction. Finally, transmission electron microscopy revealed the presence of myofilament on the 21st day after induction. CONCLUSIONS: Bone marrow-derived mesenchymal stem cells can be effectively induced into smooth muscle-like cells by conditioned medium combined with 20% FBS. Myocardin plays an important role in the differentiation process of bone marrow-derived mesenchymal stem cells to the peripheral smooth muscle cells.

Longitudinal change of six common inflammatory cytokines and their relationship to anxiety, depression, and cognitive impairment in acute ischemic stroke patients

Inflammatory cytokines are known to be involved in acute ischemic stroke (AIS), while the relationship of multiple inflammatory cytokines with mental disorders in AIS is less reported. This research intended to explore the longitudinal variation of common inflammatory cytokines and their correlation with anxiety, depression, and cognitive impairment in AIS patients. Six inflammatory cytokines were detected by enzyme-linked immunosorbent assay among 175 AIS patients at admission (baseline) and on the day (D)1, D3, and D7 after admission. Anxiety, depression, and cognition were evaluated using the Hospital Anxiety and Depression Scale and Mini-Mental State Examination at discharge, respectively. Anxiety, depression, and cognitive impairment rates were 32.6, 39.4, and 19.4%, respectively. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, and IL-17A increased from baseline to D1, then decreased from D1 to D7 (all P<0.001), while IL-10 presented an opposite trend (P<0.001). Interestingly, TNF-α on D1 and D3, IL-6 on D3, IL-8 on D3 and D7, and IL-17A on D1, D3, and D7 correlated with higher anxiety rate (all P<0.05). TNF-α on D1, D3, and D7, IL-8 at baseline, D1, D3, and D7, IL-17A on D1 and D7 correlated with increased depression rate (all P<0.05). In addition, IL-1β on D1 and IL-17 at baseline, D1, D3, and D7 correlated with elevated cognitive-impairment rate (all P<0.05). Inflammatory cytokines were dysregulated after disease onset, and their longitudinal change correlated with psychological issues in AIS patients.

Tetramethylpyrazine inhibits agiontensin II-induced nuclear factor-kappaB activation and bone morphogenetic protein-2 downregulation in rat vascular smooth muscle cells.

Tetramethylpyrazine (TMP), an effective component of traditional Chinese medicine Chuanxiong, is commonly used to resolve embolism. Its possible therapeutic effect against atherosclerosis has received considerable attention recently. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMCs), resulting in atherosclerosis. The mechanisms of TMP in the proliferation of VSMCs induced by Ang II remain to be defined. The present study was aimed to study the effect of TMP on Ang II-induced VSMC proliferation through detection of nuclear factor-kappaB (NF-kappaB) activity and bone morphogenetic protein-2 (BMP-2) expression. Primary cultured rat aortic smooth muscle cells were divided into the control group, Ang II group, Ang II + TMP group and TMP group. Cells in each group were harvested at different time points (15, 30 and 60 min for detection of NF-kappaB activity; 6, 12 and 24 h for measurement of BMP-2 expression). NF-kappaB activation was identified as nuclear staining by immunohistochemistry. BMP-2 expression was observed through Western blot, immunohistochemistry and in situ hybridization. The results showed that: (1) Ang II stimulated the activation of NF-kappaB. Translocation of NF-kappaB p65 subunit from cytoplasm to nucleus appeared as early as 15 min, peaked at 30 min (P<0.01) and declined after 1 h. (2) TMP inhibited Ang II-induced NF-kappaB activation (P<0.01). (3) Ang II increased BMP-2 expression at 6 h but declined it significantly at 12 and 24 h (P<0.01). (4) BMP-2 expression was also kept at high level at 6 h in Ang II + TMP group but maintained at the normal level at 12 and 24 h. (5) There was no significant difference in NF-kappaB activation and BMP-2 expression between the control group and TMP group. These results indicate that TMP inhibits Ang II-induced VSMC proliferation through repression of NF-kappaB activation and BMP-2 reduction, and BMP-2 expression is independent of the NF-kappaB pathway. In conclusion, TMP has therapeutic potential for the treatment of atherosclerosis.

Novel Use for DOG1 in Discriminating Breast Invasive Carcinoma from Noninvasive Breast Lesions.

AIMS: DOG1 has proven to be a useful marker of gastrointestinal stromal tumors (GISTs). Recently, DOG1 expression has also been reported in some non-GIST malignant tumors, but the details related to DOG1 expression in breast tissue remain unclear. The aim of this study was to detect the expression of DOG1 in the human breast and to evaluate the feasibility of using DOG1 to discriminate between invasive breast carcinoma and noninvasive breast lesions. METHODS AND RESULTS: A total of 210 cases, including both invasive and noninvasive breast lesions, were collected to assess DOG1 expression immunohistochemically. DOG1 expression was consistently positive in breast myoepithelial cells (MECs), which was similar to the results obtained for three other MEC markers: calponin, smooth muscle myosin heavy chain (SMMHC), and P63 (P > 0.05 in all). Importantly, DOG1 was useful in discriminating invasive breast carcinoma from noninvasive breast lesions (P < 0.05). CONCLUSIONS: DOG1 is a useful marker of breast MECs, and adding DOG1 to the MEC identification panel will provide more sophisticated information when diagnosing uncertain cases in the breast.

Triptolide has anticancer and chemosensitization effects by down-regulating Akt activation through the MDM2/REST pathway in human breast cancer.

Triptolide has been shown to exhibit anticancer activity. However, its mechanism of action is not clearly defined. Herein we report a novel signaling pathway, MDM2/Akt, is involved in the anticancer mechanism of triptolide. We observed that triptolide inhibits MDM2 expression in human breast cancer cells with either wild-type or mutant p53. This MDM2 inhibition resulted in decreased Akt activation. More specifically, triptolide interfered with the interaction between MDM2 and the transcription factor REST to increase expression of the regulatory subunit of PI3-kinase p85 and consequently inhibit Akt activation. We further showed that, regardless of p53 status, triptolide inhibited proliferation, induced apoptosis, and caused G1 phase cell cycle arrest. Triptolide also enhanced the cytotoxic effect of doxorubicin. MDM2 inhibition plays a causative role in these effects. The inhibitory effect of triptolide on MDM2-mediated Akt activation was eliminated with MDM2 overexpression. MDM2-overexpressing tumor cells, in turn, were less susceptible to the anticancer and chemosensitization effects of triptolide than control cells. Triptolide also exhibited anticancer and chemosensitization effects in nude mouse xenograft model. When it was administered to tumor-bearing nude mice, triptolide inhibited tumor growth and enhanced the antitumor effects of doxorubicin. In summary, triptolide has anticancer and chemosensitization effects by down-regulating Akt activation through the MDM2/REST pathway in human breast cancer. Our study helps to elucidate the p53-independent regulatory function of MDM2 in Akt signaling, offering a novel view of the mechanism by which triptolide functions as an anticancer agent.

[Homocysteine induces macrophage inflammatory protein-1alpha expression by activating NF-kappaB in THP-1 monocytes].

OBJECTIVE: To investigate the effect of homocysteine (HCY) on activation of nuclear factor (NF-kappaB) and inhibitory factor IkappaB-alpha in human monocyte cell line THP-1, as well as its association with macrophage inflammatory protein (MIP-1alpha) upregulation. METHODS: THP-1 monocytes were incubated with HCY, with and without NF-kappaB inhibitor pyrolidine dithiocarbamate (PDTC) pretreatment. Northern blot analysis and flow cytometry were used to detect MIP-1alpha mRNA and protein respectively. The nuclear protein NF-kappaB P65 subunit and the inhibitory protein IkappaB-alpha were analyzed by Western blotting. RESULTS: Compared with controls, HCY, at a concentration of 0.1 mmol/L, was able to enhance the expression of MIP-1alpha mRNA (up to 3.69-fold) and protein (1.16-fold) in THP-1 monocytes, as well as enhance NF-kappaB P65 transcription to nuclear proteins. These actions were significantly suppressed after pretreatment with 100 micromol/L PDTC for 30 minutes before HCY incubation; whereas incubation of THP-1 monocytes with PDTC only had no effect on both the expression of MIP-1alpha and nuclear transcription of NF-kappaB P65. Moreover, the level of IkappaB-alpha protein in THP-1 monocytes decreased after a 30-minute incubation with HCY, which gradually increased after 120 minutes. CONCLUSIONS: Homocysteine at a pathologic concentration stimulates MIP-1alpha expression in THP-1 monocytes, probably via NF-kappaB activation. Such activation may be caused by enhanced phosphorylation and degradation of the inhibitor protein IkappaB-alpha.

Optomizing Transfection Efficiency of Cervical Cancer Cells Transfected by Cationic Liposomes LipofectamineTM2000.

BACKGROUND: Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines. MATERIALS AND METHODS: Lipofectamine(TM)2000 as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA , miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents. RESULTS: The seeding density of Hela cell line and Siha are 1.5 x 10(4) (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (P<0.01).MTT assay showed that according to the above conditions which has the lowest cytotoxicity. CONCLUSIONS: The method of Liposome to transfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.

Construction of eukaryotic expression plasmid of human PRX3 and its expression in HEK-293FT cells.

To construct the eukaryotic expression plasmid of human PRX3 and measure its expression in the HEK-293FT cells, the full-length coding region of human PRX3 was cloned by PCR and inserted into the eukaryotic expression vector pcDNA4-Xpress (A). HEK-293FT cells were transiently transfected with the recombinant plasmid. Western blot and immuofluorescence were used to detect the expression of the fusion protein. In the experiment, restriction analysis identified the construction of the recombinant plasmid and the inserted sequence was identical with that published on GenBank. Western blot and immunofluorescence confirmed the expression of the recombinant protein in transfected HEK-293FT cells. It was concluded that the eukaryotic expression plasmid of human PRX3 was constructed successfully and the recombinant could be expressed efficiently in HEK-293FT cells, which provides a sound basis for the further study on human PRX3.

[Lipopolysaccharide induces expression of macrophage inflammatory protein-1alpha in human umbilical vein endothelial cells].

OBJECTIVE: To understand whether endotoxin lipopolysaccharide (LPS) is able to induce the expression of macrophage inflammatory protein-1alpha (MIP-1alpha) mRNA and protein in human umbilical vein endothelial cells (HUVECs). METHODS: The expression of MIP-1alpha mRNA was determined by dot blotting analysis and by in situ hybridization using a digoxigenin-labeled MIP-1alpha cDNA probe after exposure of the cultured HUVECs to LPS at different concentrations. The expression of MIP-1alpha mRNA was determined by RT-PCR as well. In addition, the expression of MIP-1alpha protein was tested by cell enzyme-linked immunosorbent assay (ELISA) using a goat anti-human monoclonal MIP-1alpha antibody. RESULTS: Dot blotting showed that the absorbance values of the dots on the nitrocellulose membrane were 1.490 and 3.310 when exposed to LPS at the concentrations of 1 micro g/ml and 10 micro g/ml which were 1.97- and 4.38-fold over that of the control group (0.775), respectively. In situ hybridization revealed that exposure to LPS at a concentration of 1 micro g/ml led to a significant increase in the MIP-1alpha mRNA expression in HUVECs as compared to the control group (F = 142.83, P < 0.01), whereas the MIP-1alpha mRNA in HUVECs was somewhat decreased when exposed to LPS at a concentration of 10 micro g/ml. RT-PCR revealed that the expression of MIP-1alpha mRNA in HUVECs were 1.65-, 2.86- and 1.26-fold over that of the control group when exposed to LPS at the concentrations of 1 micro g/ml, 5 micro g/ml and 10 micro g/ml respectively. Cell ELISA showed that after exposure of the HUVECs to LPS at the concentrations mentioned above, the expression of MIP-1alpha protein was strongly increased, especially in the 5 micro g/ml LPS group. Analysis of variance showed that there was a significant difference between groups (F = 15.36, P < 0.05). CONCLUSIONS: LPS may induce a high level of MIP-1alpha mRNA and protein expression in HUVECs, and it might, hereby, play an important role in the recruitment of the monocytes/macrophages into the arterial intima.