Electrochemiluminescence Analysis of Multiple Glycans on Single Living Cell with a Closed Bipolar Electrode Array Chip.

The profiling of multiple glycans on a single cell is important for elucidating glycosylation mechanisms and accurately identifying disease states. Herein, we developed a closed bipolar electrode (BPE) array chip for live single-cell trapping and in situ galactose and sialic acid detection with the electrochemiluminescence (ECL) method. Methylene blue-DNA (MB-DNA) as well as biotin-DNA (Bio-DNA) codecorated AuNPs were prepared as nanoprobes, which were selectively labeled on the cell surface through chemoselective labeling techniques. The individual cell was captured and labeled in the microtrap of the cathodic chamber, under an appropriate potential, MB molecules on the cellular membrane underwent oxidation, triggering the reduction of [Ru(bpy)3]2+/TPA and consequently generating ECL signals in the anodic chamber. The abundance of MB groups on the single cell enabled selective monitoring of both sialic acid and galactosyl groups with high sensitivity using ECL. The sialic acid and galactosyl content per HepG2 cell were detected to be 0.66 and 0.82 fmol, respectively. Through comprehensive evaluation of these two types of glycans on a single cell, tumor cells, and normal cells could be effectively discriminated and the accuracy of single-cell heterogeneous analysis was improved. Additionally, dynamic monitoring of variations in galactosyl groups on the surface of the single cell was also achieved. This work introduced a straightforward and convenient approach for heterogeneity analysis among single cells.

Ultrasound-responsive glycopolymer micelles for targeted dual drug delivery in cancer therapy.

Controlled drug release of nanoparticles was achieved by irreversibly disrupting polymer micelles through high-intensity focused ultrasound (HIFU) induction. An ultrasound-responsive block copolymer was synthesized, comprising an end-functional Eosin Y fluorophore, 2-tetrahydropyranyl acrylate (THPA), and acrylate mannose (MAN). The block copolymer was then self-assembled to produce micelles. The chemotherapy drug dasatinib (DAS) and the sonodynamic therapy agent methylene blue (MB) were encapsulated by the self-assembly of the block copolymer. This targeted nanoparticle enables sonodynamic therapy through high-intensity focused ultrasound while triggering nanoparticle disassembly for controlled drug release. The ultrasound-mediated, non-invasive strategy provides external spatiotemporal control for targeted tumour treatment.

A nanomedicine enables synergistic chemo/photodynamic therapy for pancreatic cancer treatment.

Pancreatic cancer is one of the leading causes of cancer-related deaths worldwide. Gemcitabine (Gem) has been a key chemotherapy agent for pancreatic cancer treatment by suppressing cell proliferation and inducing apoptosis. However, the overexpression of inhibitors of apoptosis (IAP) family of proteins during the carcinogenesis of pancreatic cancer can develop resistance to chemotherapy treatment and result in poor efficacy. To achieve the synergistic combinations of multiple strategies for this dismal disease, we developed a robust nanomedicine system, consisting of a photodynamic therapeutic agent (chlorine e6, Ce6) and a pro-apoptotic peptide-Gem conjugate. To have spatiotemporally controlled drug release, the pro-apoptotic peptide-Gem conjugate was designed to have a vinyldithioether linker that was sensitive to reactive oxygen species (ROS). The nanomedicine was fabricated by the direct self-assembly of the pro-apoptotic peptide-Gem conjugate with Ce6. After being delivered into tumors, the nanomedicine disassembled and rapidly released Gem, Ce6, and the pro-apoptotic peptide upon light illumination (660 nm). Both in vitro and in vivo studies in pancreatic cancer models confirmed the tumor inhibition efficacy with low systemic toxicity to animals.

Verticillium dahliae secreted protein Vd424Y is required for full virulence, targets the nucleus of plant cells, and induces cell death.

Fungal pathogens secrete effector proteins that regulate host immunity and can suppress basal defence mechanisms against colonization in plants. Verticillium dahliae is a widespread and destructive soilborne fungus that can cause vascular wilt disease and reduces plant yields. However, little is currently known about how the effectors secreted by V. dahliae function. In this study, we analysed and identified 34 candidate effectors in the V. dahliae secretome and found that Vd424Y, a glycoside hydrolase family 11 protein, was highly upregulated during the early stages of V. dahliae infection in cotton plants. This protein was located in the nucleus and its deletion compromised the virulence of the fungus. The transient expression of Vd424Y in Nicotiana benthamiana induced BAK1- and SOBIR1-dependent cell death and activated both salicylic acid and jasmonic acid signalling. This enhanced its resistance to the oomycetes Phytophthora capsici in a way that depended on its nuclear localization signal and signal peptides. Our results demonstrate that Vd424Y is an important effector protein targeting the host nucleus to regulate and activate effector-triggered immunity in plants.

Second sacral sacralalar-iliac (S2AI) screw placement in adult degenerative scoliosis (ADS) patients: an imaging study.

BACKGROUND: The imaging characteristics of sacral sacralalar-iliac (S2AI) screw trajectory in adult degenerative scoliosis (ADS) patients will be determined. METHODS: S2AI screw trajectories were mapped on three-dimensional computed tomography (3DCT) reconstructions of 40 ADS patients. The starting point, placement plane, screw template, and a circle centered at the lowest point of the ilium inner cortex were set on these images. A tangent line from the starting point to the outer diameter of the circle was selected as the axis of the screw trajectory. The related parameters in different populations were analyzed and compared. RESULTS: The trajectory length of S2AI screws in ADS patients was 12.00 ± 0.99 cm, the lateral angle was 41.24 ± 3.92°, the caudal angle was 27.73 ± 6.45°, the distance from the axis of the screw trajectory to the iliosciatic notch was 1.05 ± 0.81 cm, the distance from the axis of the screw trajectory to the upper edge of the acetabulum was 1.85 ± 0.33 cm, and the iliac width was 2.12 ± 1.65 cm. Compared with females, the lateral angle of male ADS patients was decreased, but the trajectory length was increased (P < 0.05). Compared to patients without ADS in previous studies, the lateral angle of male patients was larger, the lateral angle of female patients was increased, and the caudal angle was decreased (P < 0.05). CONCLUSIONS: There is an ideal trajectory of S2AI screws in ADS patients. A different direction should be noticed in the placement of S2AI screws, especially in female patients.

Plasma claudin-3 is associated with tumor necrosis factor-alpha-induced intestinal endotoxemia in liver disease.

OBJECTIVE: To investigate intestinal endotoxemia (IETM), intestinal permeability (IP) and cytokine activity in patients with liver cirrhosis (LC). MATERIALS AND METHODS: Twenty-nine patients with chronic hepatitis B (CHB), 28 with compensated LC, 33 with decompensated LC, 24 with spontaneous bacterial peritonitis (SBP), 26 with acute-on-chronic liver failure (ACLF), and 24 with decompensated LC complicated by hepatocellular carcinoma (HCC) were recruited. Thirty-one healthy people were included as a control group. Plasma tumor necrosis factor (TNF)-α, interferon (IFN)-γ, D-lactate, endotoxin, and claudin-3 levels were assayed. Data were compared using Pearson correlation testing and analysis of variance, with P < 0.05 considered significant. RESULTS: TNF-α, claudin-3, and endotoxin levels were significantly increased (P < 0.05) in the plasma of all patients with liver disease compared with that of controls, particularly in patients with decompensated LC, SBP, ACLF, or HCC (P < 0.01). IFN-γ was significantly higher in HCC than in other liver diseases (P < 0.01). Plasma D-lactate was significantly decreased in all liver diseases, except SBP (P < 0.01). TNF-α, endotoxin, and claudin-3 levels were positively correlated (P < 0.01), but correlations of IFN-γ with endotoxin or claudin-3 were not significant. The plasma D-lactate level did not significantly correlate with either TNF-α, endotoxin, or claudin-3 levels. CONCLUSION: Plasma claudin-3, but not D-lactate, was found to be a marker of IP in patients with liver diseases. Elevated plasma TNF-α in such patients was likely to have injured the intestinal barrier, leading to IETM, especially in end-stage LC.

Effect of tumor necrosis factor-α on the expression of the ammonia transporter Rhcg in the brain in mice with acute liver failure.

BACKGROUND: Ammonia and tumor necrosis factor-alpha (TNF-α) play important roles in the mechanisms of hepatic encephalopathy (HE). Rhesus glycoprotein C (Rhcg) is important for ammonia transport especially in the kidney. The aim of the present study was to investigate the role of Rhcg in the brain in acute liver failure (ALF) and the effect of TNF-α on Rhcg expression. METHODS: ALF mouse models were generated by treatment with D-galactosamine (D-GalN) and lipopolysaccharide (LPS), or D-GalN and TNF-α. ALF induction was blocked by pretreatment with anti-TNF-α IgG. The levels of serum TNF-α were determined by ELISA. Blood ammonia and brain ammonia concentrations were detected using an ammonia assay kit. The expression and distribution of Rhcg in the brain tissues of ALF mice were examined by western blotting, real-time PCR, immunohistochemical, and immunofluorescence analyses. RESULTS: Serum TNF-α levels were increased in the LPS/D-GalN group. Blood and brain ammonia were increased in the LPS/D-GalN- and TNF-α/D-GalN-induced ALF groups. Rhcg mRNA and protein levels were elevated in both ALF groups, consistent with the increase in blood and brain ammonia. Rhcg was mainly expressed in vascular endothelial cells and astrocytes. Pretreatment with anti-TNF-α IgG antibody downregulated Rhcg in brain tissues in the LPS/D-GalN group, prevented the occurrence of ALF, and reduced blood and brain ammonia levels in the LPS/D-GalN group. CONCLUSION: TNF-α promoted the transport of ammonia from the blood to brain tissues and exacerbated the toxic effects of ammonia by upregulating Rhcg.

Clinical characteristics and diagnosis of a rare case of systemic AL amyloidosis: a descriptive study.

Systemic amyloidosis is a rare disease involving multiple organs. It is difficult to establish diagnosis as the symptoms is diverse and non-specific. And without specific therapy the prognosis is very poor. We analyzed detailed clinical and laboratorial data of a 53-year-old male patient. The characteristic features included refractory pleural effusion, extraordinary hepatomegaly and cardiac failure. The illness lasted 9 months and therapy period spanned 4 months. Fine needle biopsy of liver, lung, heart, pancreas and kidney was performed. Immunohistochemistry, immunofluorescence, Congo staining and hematoxylin and eosin staining were performed. All specimens were stained pink with haematoxylin and eosin staining. Amorphous deposits of eosinophilic material were visible within the Congo red dye stained liver tissue whereas under cross-polarized light pathognomonic apple-green birefringence of amyloid deposits was visible. At last systemic AL amyloidosis diagnosis was confirmed. The report showed an unusual AL amyloidosis case in detail which would be helpful for physician in clinical work.

Optimal Pelvic Incidence Minus Lumbar Lordosis Mismatch after Long Posterior Instrumentation and Fusion for Adult Degenerative Scoliosis.

OBJECTIVE: To evaluate the influence of Scoliosis Research Society (SRS)-Schwab sagittal modifiers of pelvic incidence minus lumbar lordosis mismatch (PI-LL) on clinical outcomes for adult degenerative scoliosis (ADS) after long posterior instrumentation and fusion. METHODS: This was a single-institute, retrospective study. From 2012 to 2014, 44 patients with ADS who underwent posterior instrumentation and fusion treatment were reviewed. Radiological evaluations were investigated by standing whole spine (posteroanterior and lateral views) X-ray and all radiological measurements, including Cobb's angle, LL, PI, and the grading of vertebral rotation, were performed by two experienced surgeons who were blind to the operations. The patients were divided into three groups based on postoperative PI-LL and the classification of the SRS-Schwab: 0 grade PI-LL (<10°, n = 13); + grade PI-LL (10°-20°, n = 19); and ++ grade PI-LL (>20°, n = 12). The clinical outcomes were assessed according to Japanese Orthopaedic Association (JOA) score, Oswestry Disability Index (ODI), Visual Analog Scale (VAS), Lumbar Stiffness Disability Index (LSDI), and complications. Other characteristic data of patients were also collected, including intraoperative blood loss, operative time, length of hospital stay, complications, number of fusion levels, and number of decompressions. RESULTS: The mean operative time, blood loss, and hospital stay were 284.5 ± 30.2 min, 1040.5 ± 1207.6 mL, and 14.5 ± 1.9 day. At the last follow-up (2.6 ± 0.6 years), the radiological and functional parameters, except the grading of vertebral rotation, were all significantly improved in comparison with preoperative results (P < 0.05), but it was obvious that an ideal PI-LL (≤10°) was not achieved in some patients. Significant differences were only observed among the three groups in the ODI and LSDI. Patients with + grade PI-LL seemed to have the best surgical outcome compared to those with 0 and ++ grade PI-LL, with the lowest ODI score (+ grade vs 0 grade, 17.3 ± 4.9 vs 26.0 ± 5.4; + grade vs ++ grade, 17.3 ± 4.9 vs 32.4 ± 7.3; P < 0.05) and lower LSDI (+ grade vs 0 grade, 1.6 ± 1.0 vs 3.5 ± 0.5, P < 0.05; + grade vs ++ grade, 1.6 ± 1.0 vs 0.6 ± 0.5, P > 0.05). A Pearson correlation analysis further demonstrated that LSDI was negatively associated with PI-LL. Furthermore, the incidence rate of postoperative complications was lower in patients with + grade PI-LL (1/19, 5.26%) than that in patients with 0 (2/13, 15.4%) and ++ grade PI-LL (3/12, 25%). CONCLUSION: Our present study suggest that the ideal PI-LL may be between 10° and 20° in ADS patients after long posterior instrumentation and fusion.

Tumor necrosis factor-alpha-induced reduction of glomerular filtration rate in rats with fulminant hepatic failure.

The mechanism of renal failure during fulminant hepatic failure (FHF) or end-stage of liver disease is not fully understood. The present study aims to delineate the mechanisms of decreased glomerular filtration rate (GFR) in acute hepatic failure. A rat model of renal insufficiency in severe liver injury was established by lipopolysaccharide (LPS) plus D-galactosamine (GalN) exposure. GFR was evaluated by continuous infusion of fluorescein isothiocyanate-inulin with implanted micro-osmotic pumps. GalN/LPS intoxication resulted in severe hepatocyte toxicity as evidenced by liver histology and biochemical tests, whereas renal morphology remained normal. GFR was reduced by 33% of the controls 12 h after GalN/LPS exposure, accompanied with a decreased serum sodium levels, a marked increase in serum TNF-α and ET-1 levels as well as significantly upregulated renal type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) expression. The upregulated IP3R1 expression was abrogated by the treatment of anti-TNF-α antibodies, but not by 2-aminoethoxydiphenylborate (2-APB), which blocks the inositol 1,4,5-trisphosphate signaling pathway. Treatments with either TNF-α antibodies or 2-APB also significantly improved the compromised GFR, elevated serum urea nitrogen and creatinine levels, and reversed the decrease in glomerular inulin space and the increase in glomerular calcium content in GalN/LPS-exposed rats. The extent of acute liver injury as reflected by serum ALT levels was much more attenuated by anti-TNF-α antibodies than by 2-APB. Liver histology further confirmed that anti-TNF-α antibodies conferred better protection than 2-APB in GalN/LPS-exposed rats. LPS-elicited TNF-α over-production is responsible for decreased GFR through IP3R1 overexpression, and the compromised GFR resulted in the development of acute renal failure in rats with FHF.

Tumor necrosis factor alpha increases intestinal permeability in mice with fulminant hepatic failure.

AIM: To determine the effect of tumor necrosis factor alpha (TNF-α) on intestinal permeability (IP) in mice with fulminant hepatic failure (FHF), and the expression of tight junction proteins. METHODS: We selected D-lactate as an index of IP, induced FHF using D-galactosamine/lipopolysaccharide and D-galactosamine/TNF-α, assessed the results using an enzymatic-spectrophotometric method, transmission electron microscopy, immunohistochemistry, Western blotting and real-time quantitative polymerase chain reaction. The effect of the administration of anti-TNF-α immunoglobulin G (IgG) antibody, before the administration of D-galactosamine/lipopolysaccharide, on TNF-α was also assessed. RESULTS: IP was significantly increased in the mouse model of FHF 6 h after injection (13.57 ± 1.70 mg/L, 13.02 ± 1.97 mg/L vs 3.76 ± 0.67 mg/L, P = 0.001). Electron microscopic analysis revealed tight junction (TJ) disruptions, epithelial cell swelling, and atrophy of intestinal villi. Expression of occludin and claudin-1 mRNA was significantly decreased in both FHF models (occludin: 0.57 ± 0.159 fold vs baseline, P = 0.000; claudin-1: 0.3067 ± 0.1291 fold vs baseline, P = 0.003), as were the distribution density of proteins in the intestinal mucosa and the levels of occludin and claudin-1 protein (occludin: 0.61 ± 0.0473 fold vs baseline, P = 0.000; claudin-1: 0.6633 ± 0.0328 fold vs baseline, P = 0.000). Prophylactic treatment with anti-TNF-α IgG antibody prevented changes in IP (4.50 ± 0.97 mg/L vs 3.76 ± 0.67 mg/L, P = 0.791), intestinal tissue ultrastructure, and the mRNA levels of occludin and claudin-1 expression (occludin: 0.8865 ± 0.0274 fold vs baseline, P = 0.505; claudin-1: 0.85 ± 0.1437 fold vs baseline, P = 0.1), and in the protein levels (occludin: 0.9467 ± 0.0285 fold vs baseline, P > 0.05; claudin-1: 0.9533 ± 0.0186 fold vs baseline, P = 0.148). CONCLUSION: Increased in IP stemmed from the downregulation of the TJ proteins occludin and claudin-1, and destruction of the TJ in the colon, which were induced by TNF-α in FHF mice.

TNFα-induced IP3R1 expression through TNFR1/PC-PLC/PKCα and TNFR2 signalling pathways in human mesangial cell.

BACKGROUND: Little information is available regarding the mechanisms involved in cytokine-induced type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) expression in human mesangial cells (HMCs) in the occurrence of hepatorenal syndrome (HRS). Over-expression of IP(3)R1 would enhance both IP(3)-binding activity and sensitivity. We hypothesize that it is possible that increased IP(3)R1, induced by TNFα, would lead to increased IP(3) sensitivity in response to a variety of vasoconstrictors, and promote HMC contraction and thus lead to reduced GFP, promoting HRS occurrence and development. METHODS: Quantitative real-time polymerase chain reaction and immunoblot assay were used to examine the effects of TNFα on IP(3)R1 mRNA and protein expression. Several inhibitors of kinases, depletion PKC, over-expression of dominant-negative mutant of PKC and non-radioactive PKC assay were used to examine the mechanism of signal transduction of TNFα-regulated IP(3)R1 in HMCs. RESULTS: TNFα increased IP(3)R1 mRNA and protein expression in HMCs, an effect that was blocked by prolonged incubated chronic PMA, D609, safingol and also by transfection with domain-negative PKCα construct. TNFα activated and promoted autophosphorylation of the PKCα. In addition, both anti-TNFR1 and anti-TNFR2 antibodies blocked TNFα-induced IP(3)R1 protein expression, while only anti-TNFR1 antibodies but not anti-TNFR2 antibodies attenuated TNFα-induced PKCα activity. CONCLUSIONS: TNFα increased the expression of IP(3)R1, and this was mediated, at least in part, through the TNFR1/PC-PLC/PKCα and TNFR2 signalling pathways in HMCs.