A hydrodynamic-based dual-function microfluidic chip for high throughput discriminating tumor cells.

A hydrodynamic-based microfluidic chip consisted of two function units that could not only separate tumor cells (TCs) from whole blood but also remove residual blood cells was designed. The separation of TCs was achieved by a straight contraction-expansion array (CEA) microchannel on the front end of the chip. The addition of contractive structure brought a micro-vortex like Dean vortex that promoted cell focusing in the channel, while when cells entered the dilated region, the wall-induced lift force generated by the channel wall gave cells a push away from the wall. As the wall-induced lift force is proportional to the third power of the cell diameter, TCs with larger diameter will have a larger lateral migration under the wall-induced lift force, realizing the separation of TCs from blood sample. Fluorescent particles with diameters of 19.3 µm and 4.5 µm were used to simulate TCs and red blood cells, respectively, to verify the separation capacity of the proposed CEA microchannel for particles with different diameter. And a separation efficiency 98.7% for 19.3 µm particles and a removal rate 96.2% for 4.5 µm particles was observed at sample flow rate of 10 µL min-1 and sheath flow rate of 190 µL min-1. In addition, a separation efficiency about 96.1% for MCF-7 cells (stained with DiI) and removal rates of 96.2% for red blood cells (RBCs) and 98.7% for white blood cells (WBCs) were also obtained under the same condition. However, on account of the large number of blood cells in the blood, there will be a large number of blood cells remained in the isolated TCs, so a purification unit based on hydrodynamic filtration (HDF) was added after the separation microchannel. The purification channel is a size-dictated cell filter that can remove residual blood cells but retain TCs, thus achieving the purification of TCs. Combined the CEA microchannel and the purifier, the microchip facilitates sorting of MCF-7 cells from whole blood with a separation rate about 95.3% and a removal rate over 99.99% for blood cells at a sample flow rate of 10 µL min-1, sheath flow rate of 190 µL min-1 and washing flow rate of 63 µL min-1.

Tracking and imaging nano-plastics in fresh plant using cryogenic laser ablation inductively coupled plasma mass spectrometry.

Tracking and imaging of nano-plastics are extremely challenging, especially in fresh biological samples. Here, we propose a new strategy in which polystyrene (PS) was doped with the europium chelate Eu (DBM)3bpy to quantify, track, and in situ image nano-plastics in fresh cucumber based on inherent metals using cryogenic laser ablation inductively coupled plasma mass spectrometry (cryo-LA-ICP-MS). The cryogenic conditions provide a stable condition for imaging fresh cucumber, suppressing the evaporation of water in fresh plants, and maintaining the original structure of plants with respect to room temperature imaging in LA-ICP-MS. The plants were cultivated in two types of nano-plastics solutions with low (50 mg/L) and high (200 mg/L) concentrations for 9 days. The results showed that nano-plastics mainly enrich the roots and have negative effects, which decrease the trace elements of Zn, Mn, and Cu in cucumber. Smaller PS particles are able to penetrate the plant more easily and inflict serious damage. Novel imaging method provides a novel insight into the tracking and imaging of nano-plastics in fresh plant samples. The results illustrated that nano-plastics deposition on plants has the potential to have direct ecological effects as well as consequences for potential health.

Interaction of Cellular Uptake of Nanosilver and Metallothionein Stress Expression Elucidated by 2D Single-Cell Analyses Based on LIF and ICP-MS.

The exploration of cytology mechanisms of nanosilver uptake, toxicity, and detoxification has become an important issue due to its widespread applications. Previous studies have shown differences in the toxic response of mammalian cells to nanosilver. However, the analysis results based on cell populations ignore the impact of cell uptake heterogeneity on the expression of associated stress proteins and cellular physiological activities. In this respect, this work investigated the interaction between silver uptake and metallothionein (MT) expression in individual cells. In addition, we have also preliminarily elucidated the sensitivity variation to AgNPs by using five cell lines, e.g., LX-2, HepG-2, SK-HEP-1, Huh-7, and MDA-MB-231, by adopting a two-dimensional (2D) high-throughput single-cell analysis platform coupling laser-induced fluorescence (LIF) and inductively coupled plasma mass spectrometry (ICP-MS). We developed a 2D data analysis method for one-to-one unification of fluorescence-mass spectrometry signals corresponding to a specific single cell. It indicated that there is no obvious correlation between cellular silver uptake and cell size, and the low MT expression of cells is more sensitive to silver nanoparticles. For each cell line, significant heterogeneity in MT expression was observed. This provides important information for understanding the potential heterogeneous effects of nanosilver on mammalian biological systems. Overall, detoxified cells are more tolerant to nanosilver and normal cells are more tolerant than cancer cells.

Elevated Expression of TRPC4 in Cortical Lesions of Focal Cortical Dysplasia II and Tuberous Sclerosis Complex.

Focal cortical dysplasia type II (FCD II) and tuberous sclerosis complex (TSC) are well-known causes of chronic refractory epilepsy in children. Canonical transient receptor potential channels (TRPCs) are non-selective cation channels that are commonly activated by phospholipase C (PLC) stimulation. Previous studies found that TRPC4 may participate in the process of epileptogenesis. This study aimed to examine the expression and distribution of TRPC4 in FCD II (n = 24) and TSC (n = 11) surgical specimens compared with that in age-matched autopsy control samples (n = 12). We found that the protein levels of TRPC4 and its upstream factor, PLC delta 1 (PLCD1), were elevated in FCD II and TSC samples compared to those of control samples. Immunohistochemistry assays revealed that TRPC4 staining was stronger in malformed cells, such as dysmorphic neurons, balloon cells and giant cells. Moderate-to-strong staining of the upstream factor PLCD1 was also identified in abnormal neurons. Moreover, double immunofluorescence staining revealed that TRPC4 was colocalised with glutamatergic and GABAergic neuron markers. Taken together, our results indicate that overexpression of TRPC4 protein may be involved in the epileptogenesis of FCD II and TSC.

Epidemiology and Management of Acute Kidney Injury in Hepatocellular Carcinoma Patients Undergoing Transcatheter Arterial Chemoembolization.

Transcatheter arterial chemoembolization (TACE) is an effective therapy for hepatocellular carcinoma (HCC). However, acute kidney injury (AKI) may occur after TACE due to the contrast agent and cytotoxic anticancer drugs used in this procedure. Post-TACE AKI is not an unusual event, and may adversely affect patient outcome. Coexisting situations including cirrhosis, renal insufficiency, diabetes and hypertension play a role in the development of HCC, and may predispose patients to AKI after TACE. Most post-TACE are transient and reversible, while prolonged AKI may predict a decreased survival. The best strategy to manage post-TACE AKI is prevention. Patients, before undergoing TACE, should be carefully assessed. In this study, we reviewed the current literature published in English about the incidence rate, risk factors, management and prognosis of AKI in patients with HCC undergoing TACE for a better understanding of this complication.

Downregulation of CD47 and CD200 in patients with focal cortical dysplasia type IIb and tuberous sclerosis complex.

BACKGROUND: Focal cortical dysplasia type IIb (FCD IIb) and tuberous sclerosis complex (TSC) are well-recognized causes of chronic intractable epilepsy in children. Accumulating evidence suggests that activation of the microglia/macrophage and concomitant inflammatory response in FCD IIb and TSC may contribute to the initiation and recurrence of seizures. The membrane glycoproteins CD47 and CD200, which are highly expressed in neurons and other cells, mediate inhibitory signals through their receptors, signal regulatory protein α (SIRP-α) and CD200R, respectively, in microglia/macrophages. We investigate the levels and expression pattern of CD47/SIRP-α and CD200/CD200R in surgically resected brain tissues from patients with FCD IIb and TSC, and the potential effect of soluble human CD47 Fc and CD200 Fc on the inhibition of several proinflammatory cytokines associated with FCD IIb and TSC in living epileptogenic brain slices in vitro. The level of interleukin-4 (IL-4), a modulator of CD200, was also investigated. METHODS: Twelve FCD IIb (range 1.8-9.5 years), 13 TSC (range 1.5-10 years) patients, and 6 control cases (range 1.5-11 years) were enrolled. The levels of CD47/SIRP-α and CD200/CD200R were assessed by quantitative real-time polymerase chain reaction and western blot. The expression pattern of CD47/SIRP-α and CD200/CD200R was investigated by immunohistochemical analysis, and the cytokine concentrations were measured by enzyme-linked immune-sorbent assays. RESULTS: Both the messenger RNA and protein levels of CD47, SIRP-α, and CD200, as well as the mRNA level of IL-4, were downregulated in epileptogenic lesions of FCD IIb and TSC compared with the control specimens, whereas CD200R levels were not significantly changed. CD47, SIRP-α, and CD200 were decreasingly expressed in dysmorphic neuron, balloon cells, and giant cells. CD47 Fc and CD200 Fc could inhibit IL-6 release but did not suppress IL-1ß or IL-17 production. CONCLUSIONS: Our results suggest that microglial activation may be partially caused by CD47/SIRP-α- and CD200/CD200R-mediated reductions in the immune inhibitory pathways within FCD IIb and TSC cortical lesions where chronic neuroinflammation has been established. Upregulation or activation of CD47/SIRP-α and CD200/CD200R may have therapeutic potential for controlling neuroinflammation in human FCD IIb and TSC.

Increased Expression of Transient Receptor Potential Vanilloid 4 in Cortical Lesions of Patients with Focal Cortical Dysplasia.

AIM: Focal cortical dysplasia (FCD) represents a well-known cause of medically intractable epilepsy. Studies found that transient receptor potential vanilloid receptor 4 (TRPV4) may participate in the occurrence of seizures. This study investigated the expression patterns of TRPV4 in FCD and the cascade that regulate functional state of TRPV4 in cortical neurons. METHODS: Thirty-nine surgical specimens from FCD patients and 10 age-matched control samples from autopsies were included in this study. Protein expression and distribution were detected by Western blot, immunohistochemistry, and immunofluorescence staining. Calcium imaging was used to detect the TRPV4-mediated Ca(2+) influx in cortical neurons. RESULTS: (1) The protein levels of TRPV4 and of an upstream factor, protein kinase C (PKC), were markedly elevated in FCD. (2) TRPV4 staining was stronger in the dysplastic cortices of FCD and mainly observed in neuronal microcolumns and malformed cells. (3) The activation of TRPV4 was central for [Ca(2+)]i elevation in cortical neurons, and this activity of TRPV4 in cortical neurons was regulated by the PKC, but not the PKA, pathway. CONCLUSION: The overexpression and altered cellular distribution of TRPV4 in FCD suggest that TRPV4 may potentially contribute to the epileptogenesis of FCD.

Increased Expression and Cellular Localization of P2X7R in Cortical Lesions of Patients With Focal Cortical Dysplasia.

Focal cortical dysplasias (FCDs) are major brain malformations that commonly lead to medically intractable epilepsy. The purinergic ionotropic P2X7 receptor (P2X7R) is an atypical P2X subtype that gates calcium and sodium ions. Previous animal studies have suggested that P2X7R is a contributing factor in epileptogenesis. This study aimed to define the distribution and expression of P2X7R in 35 FCD patient-surgical-resection specimens relative to autopsy control samples (n = 8). Immunohistochemical colocalization assays revealed that P2X7R was primarily expressed in neurons, astrocytes, and microglia. In FCD samples, P2X7R protein levels were increased in abnormal cell types such as dysmorphic neurons and balloon cells, which are characteristic of FCD. By real-time PCR and Western blotting, P2X7R mRNA and protein expression levels were elevated in FCD patient samples vs control samples; P2X7R expression was also higher in FCDII vs FCDIa patient samples. Because interleukin-1ß is a downstream factor of the P2X7R signaling pathway, we determined that there was also moderate-to-strong interleukin-1ß expression in the dysmorphic neurons, balloon cells, and microglia in FCD patient lesions. These results indicate that increasing P2X7R levels may contribute to the pathogenesis of human FCD and that P2X7R represents a potential anti-epileptogenic target.

Down-regulated expression of acid-sensing ion channel 1a in cortical lesions of patients with focal cortical dysplasia.

Focal cortical dysplasia (FCD) represents a well-recognized cause of medically intractable epilepsy. Previous studies have indicated that seizures can reduce brain pH and then eliminate seizure discharges. Acid-sensing ion channels (ASICs) are H(+)-gated cation channels that are widely expressed in the central and peripheral nervous systems. To understand the potential roles of ASIC1a in the epileptogenesis of FCD, we investigated the expression and distribution patterns of ASIC1a in surgical specimens from patients with FCD and age-matched normal cortices (CTX). Decreased ASIC1a messenger RNA (mRNA) and protein expression were detected in FCD compared with CTX. Moreover, the expression of ASIC1a was significantly lower in FCD type II than FCD type I. Immunohistochemistry results indicated that the overall immunoreactivity of the ASIC1a staining was diminished in the dysplastic cortices of FCD compared to the CTX samples. In FCD, ASIC1a immunoreactivity was mainly observed in reactive astrocytes and a minority of malformed cells, including hypertrophic neurons, dysmorphic neurons, and balloon cells. Confocal analysis demonstrated that most malformed cells expressing ASIC1a were co-labeled with neuronal rather than astrocytic markers, indicating a neuronal lineage. In conclusion, the downregulation and altered cellular distribution of ASIC1a in FCD suggest that ASIC1a may potentially contribute to the epileptogenesis of FCD.

Using CD133 positive U251 glioblastoma stem cells to establish nude mice model of transplanted tumor.

This study tried to use CD133 positive U251 glioblastoma stem cells to establish nude mice model of transplanted tumor. CD133 positive U251 stem cells were isolated and identified. Twenty-five male nude mice were divided into three groups: U251 cell group, U251 stem cell group and normal control group. U251 cells and stem cells were respectively inoculated into mouse brain to establish model of transplanted tumor. Mice growing condition and behavior were observed. Magnetic resonance imaging (MRI) was performed to detect tumor growth in brain. HE staining assay, immunohistochemical assay and CD34 marked microvascular density (MVD) test were performed. As a result, the successful rates of both model groups were 100%. Growing condition and behavior in U251 stem cell group was more significantly exaggerated and tumor growth and image in magnetic resonance in U251 stem cell group was more significantly malignant than that in U251 cell group. It could be found that models in U251 stem cell group showed stronger pathogolical malignance features than that in U251 cell group. Glial fibrillary acidic protein (GFAP) expression in U251 cell and stem cell group showed the transplanted tumor originated from astrocytes. CD34 in U251 stem cell group was highest and significantly higher than that in the other two groups. In conclusion, use of U251 stem cells to establish transplanted tumor model in nude mice is an excellent method and the model is more likely and malignant than the one established by cancer cells, which showed a new animal model to research glioma.

Influence of Etoposide on anti-apoptotic and multidrug resistance-associated protein genes in CD133 positive U251 glioblastoma stem-like cells.

It has been hypothesized that cancer stem cell is responsible for the refractoriness of glioblastoma therapy. This study is to observe the influence of Etoposide on anti-apoptotic and multidrug resistance-associated protein genes in glioblastoma stem-like cells. U251 glioblastoma cells were cultured and CD133 positive cancer stem-like cells were isolated and identified. Cell counting kit-8 assay, cell morphology and flow cytometry were employed for assaying cell survival condition. Real-time quantitative PCR was chosen for detecting mRNA expression of livin, livinalpha, livinbeta, survivin, MRP1 and MRP3. As results, after Etoposide intervention, the U251 stem-like cells showed more resistant property, more intact morphology and lower apoptotic rate than that in U251 cells (p<0.05). It could be found that the expression of livinbeta in U251 stem-like cells was significantly higher (p<0.05). After Etoposide intervention, only livinalpha was suppressed markedly (p<0.05), while livin expression was not notably decreased with livinbeta increased on the contrary (p<0.05). MRP1 and MRP3 in U251 stem-like cells were significantly higher than that in cancer cells, and after chemotherapy, the expression of MRP1 increased notably (p<0.05). But the expression of survivin and MRP3 did not show these features. In conclusion, after Etoposide intervention glioblastoma stem-like cells showed a stronger resistance to apoptosis and death, and the anti-apoptotic gene livinbeta was more related with the high survival rate and MRP1 appeared to be more related with transporting chemotherapeutics out of glioblastoma stem-like cells.

[Expression of anti-apoptotic and multi-drug resistance-associated protein genes in cancer stem cell isolated from TJ905 glioblastoma multiforme cell line].

OBJECTIVE: To isolate cancer stem cells glioblastoma cells and detect the expression of anti-apoptotic and multi-drug resistance-associated protein (MRP) genes thereof. METHODS: CD133 positive cells were isolated from human glioblastoma multiforme cells of the line TJ905 by immunomagnetic beads technique and nestin, beta-tubulin and GFAP expression were examined by Immunofluorescence staining. RT-PCR was used to detect the expression of livin, livinalpha, Livinbeta, Survivin, MRP1, and MRP3. RESULTS: Only 0.21% of the TJ905 cells maintained in serum was CD133(+) and showed characteristics of cancer stem cells, positive in nestin. These cells maintained a sphere-like growth status in serum-free medium in vitro, and could self-renew, proliferate, conditionally differentiate into tubulin-beta(+) and GFAP(+) cells, and produce neurons as well as glial cells. The mRNA expression levels of livin, livinalpha, survivin, MRP1, and MRP3 of the TJ905 tumor stem cells were significantly lower than those f the of TJ905 cells. CONCLUSION: Cancer stem cells can be isolated from TJ905 glioblastoma multiforme cells. However, the generating rate of the tumor stem cells is lower than that of the TJ905 cells, and the expression levels of anti-apoptotic and MRP genes are lower than those of the progenitor cells. Showing that cancer stem cells are not the solo factor to maintain tumor growth and resist apoptosis and to pump the anti-tumor drugs out of cells.

Paradoxical expression of anti-apoptotic and MRP genes on cancer stem-like cell isolated from TJ905 glioblastoma multiforme cell line.

Existence of cancer stem cell is regarded as a main reason for metastasis and/or recurrences. In this study, the cancer stem-like cell derived from TJ905 glioblastoma multiforme cell line was isolated successfully. However, it was observed that generating rate of the cancer stem-like cells was lower than that of TJ905 cells, that expression of the anti-apoptotic and multidrug resistance-associated protein (MRP) genes were paradoxical to the literatures, which showed the uncertainty of cancer stem cells, and that some stem cell was not the solo factor to maintain tumor growth and resist apoptosis and anti-tumor drugs.