Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE)

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl₂ differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.     

A Unidirectional Cell Switching Gate by Engineering Grating Length and Bending Angle

On a microgrooved substrate, cells migrate along the pattern, and at random positions, reverse their directions. Here, we demonstrate that these reversals can be controlled by introducing discontinuities to the pattern. On “V-shaped grating patterns”, mouse osteogenic progenitor MC3T3-E1 cells reversed predominately at the bends and the ends. The patterns were engineered in a way that the combined effects of angle- and length-dependence could be examined in addition to their individual effects. Results show that when the bend was placed closer to one end, migration behaviour of cells depends on their direction of approach. At an obtuse bend (135°), more cells reversed when approaching from the long segment than from the short segment. But at an acute bend (45°), this relationship was reversed. Based on this anisotropic behaviour, the designed patterns effectively allowed cells to move in one direction but blocked migrations in the opposing direction. This study demonstrates that by the strategic placement of bends and ends on grating patterns, we can engineer effective unidirectional switching gates that can control the movement of adherent cells. The knowledge developed in this study could be utilised in future cell sorting or filtering platforms without the need for chemotaxis or microfluidic control.     

Preparation and Characterization of Biochars from Eichornia crassipes for Cadmium Removal in Aqueous Solutions

The study investigated the preparation and characterization of biochars from water hyacinth at 300°C to 700°C for cadmium (Cd) removal from aqueous solutions. The adsorption process was dominated by oxygen-containing functional groups with irregular surfaces via esterification reactions. Furthermore, the mineral components in the biochars also contributed to Cd absorption through precipitation. Parameters such as the effects of solution pH, contact time, and initial concentration were studied. The optimum pH value was observed at 5.0, in which nearly 90% of Cd was removed. The maximum Cd adsorption capacities based on the Langmuir isotherm were calculated at 49.837, 36.899, and 25.826 mg g⁻¹. The adsorption processes of the biochars followed the pseudo-second-order kinetics, with the equilibrium achieved around 5 h. The biochar from E. crassipes is a promising adsorbent for the treatment of wastewater, which can in turn convert one environmental problem to a new cleaning Technology.     

Decreased TUSC3 Promotes Pancreatic Cancer Proliferation,Invasion and Metastasis

Pancreatic cancer is an aggressive disease with dismal prognosis. It is of paramount importance to understand the underlying etiological mechanisms and identify novel, consistent, and easy-to-apply prognostic factors for precision therapy. TUSC3 (tumor suppressor candidate 3) was identified as a potential tumor suppressor gene and previous study showed TUSC3 is decreased in pancreatic cancer at mRNA level, but its putative tumor suppressor function remains to be verified. In this study, TUSC3 expression was found to be suppressed both at mRNA and protein levels in cell line models as well as in clinical samples; decreased TUSC3 expression was associated with higher pathological TNM staging and poorer outcome. In three pairs of cell lines with different NF-κB activity, TUSC3 expression was found to be reversely correlated with NF-κB activity. TUSC3-silenced pancreatic cancer cell line exhibited enhanced potential of proliferation, migration and invasion. In an orthotopic implanted mice model, TUSC3 silenced cells exhibited more aggressive phenotype with more liver metastasis. In conclusion, the current study shows that decreased immunological TUSC3 staining is a factor prognostic of poor survival in pancreatic cancer patients and decreased TUSC3 promotes pancreatic cancer cell proliferation, invasion and metastasis. The reverse correlation between NF-κB activity and TUSC3 expression may suggest a novel regulation pattern for this molecule.     

Factors that Affect Pancreatic Islet Cell Autophagy in Adult Rats: Evaluation of a Calorie-Restricted Diet and a High-Fat Diet

Aging may be a risk factor for type 2 diabetes in the elderly. Dietary intervention can affect glucose tolerance in adults, which may be due to body composition and islet cell autophagy. The aim of this study was to determine the effects of various dietary interventions on islet cell autophagy. Pancreatic tissue and blood samples were collected from Sprague Dawley rats (14–16 months old, n = 15 for each group) that received a normal diet (ND), a high-fat diet (HFD), or a calorie-restricted diet (CRD). The body weight (BW), visceral fat, serum lipid levels, fasting serum glucose, insulin levels, and β/α cell area were determined in 14-16-(0-w), 16-18-(8-w), and 18-20(16-w)-month-old rats. Pancreatic islet autophagy (LC3B and LAMP2), AP (Acid Phosphatase) and apoptosis (apoptosis index, AI (TUNEL assay) and cleaved caspase-3) were detected using immunohistochemistry, ELISA and western blot. At 16 weeks, the expressions of LC3B, LAMP2 and AP markedly increased in both the HFD (P<0.01) and CRD (P<0.05) groups; however, an increase in the AI (P<0.05), cleaved caspase-3 and Beclin1 expression and a decrease in the expressions of BCL2 and BCLXL (P<0.05) were observed in only the HFD group. FFA, triglyceride levels, HOMA-IR, insulin levels and glucagon levels were significantly increased in the HFD group but decreased in the CRD group at 16 weeks (P<0.05). The degree of islet cell autophagy was potentially regulated by the levels of FFA and islet cell insulin and glucagon, which may have been due to the effects of Beclin1/BCL2.     

The MIF Antagonist ISO-1 Attenuates Corticosteroid-Insensitive Inflammation and Airways Hyperresponsiveness in an Ozone-Induced Model of COPD

Introduction

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with acute and chronic inflammatory disorders and corticosteroid insensitivity. Its expression in the airways of patients with chronic obstructive pulmonary disease (COPD), a relatively steroid insensitive inflammatory disease is unclear, however.

Methods

Sputum, bronchoalveolar lavage (BAL) macrophages and serum were obtained from non-smokers, smokers and COPD patients. To mimic oxidative stress-induced COPD, mice were exposed to ozone for six-weeks and treated with ISO-1, a MIF inhibitor, and/or dexamethasone before each exposure. BAL fluid and lung tissue were collected after the final exposure. Airway hyperresponsiveness (AHR) and lung function were measured using whole body plethysmography. HIF-1α binding to the Mif promoter was determined by Chromatin Immunoprecipitation assays.

Results

MIF levels in sputum and BAL macrophages from COPD patients were higher than those from non-smokers, with healthy smokers having intermediate levels. MIF expression correlated with that of HIF-1α in all patients groups and in ozone-exposed mice. BAL cell counts, cytokine mRNA and protein expression in lungs and BAL, including MIF, were elevated in ozone-exposed mice and had increased AHR. Dexamethasone had no effect on these parameters in the mouse but ISO-1 attenuated cell recruitment, cytokine release and AHR.

Conclusion

MIF and HIF-1α levels are elevated in COPD BAL macrophages and inhibition of MIF function blocks corticosteroid-insensitive lung inflammation and AHR. Inhibition of MIF may provide a novel anti-inflammatory approach in COPD.     

Probing the polarity and water environment at the protein-peptide binding interface using tryptophan analogues

7-Azatryptophan and 2,7-diazatryptophan are sensitive to polarity changes and water content, respectively, and should be ideal for studying protein-protein and protein-peptide interactions. In this study, we replaced the tryptophan in peptide Baa (LKWKKLLKLLKKLLKLG-NH₂) with 7-azatryptophan or 2,7-diazatryptophan, forming (7-aza)Trp-Baa and (2,7-aza)Trp-Baa, to study the calmodulin (CaM)-peptide interaction. Dramatic differences in the (7-aza)Trp-Baa and (2,7-aza)Trp-Baa fluorescence properties between free peptide in water and calmodulin-bound peptide were observed, showing a less polar and water scant environment at the binding interface of the peptide upon calmodulin binding. The affinity of the peptides for binding CaM followed the trend Baa (210±10 pM)<(7-aza)Trp-Baa (109±5 pM)<(2,7-aza)Trp-Baa (45±2 pM), showing moderate increase in binding affinity upon increasing the number of nitrogen atoms in the Trp analogue. The increased binding affinity may be due to the formation of more hydrogen bonds upon binding CaM for the Trp analogue with more nitrogen atoms. Importantly, the results demonstrate that (7-aza)Trp and (2,7-aza)Trp are excellent probes for exploring the environment at the interface of protein-peptide interactions.     

Lysosomal membrane permeabilization is involved in oxidative stress-induced apoptotic cell death in LAMP2-deficient iPSCs-derived cerebral cortical neurons

Patients with Danon disease may suffer from severe cardiomyopathy, skeletal muscle dysfunction as well as varying degrees of mental retardation, in which the primary deficiency of lysosomal membrane-associated protein-2 (LAMP2) is considerably associated. Owing to the scarcity of human neurons, the pathological role of LAMP2 deficiency in neural injury of humans remains largely elusive. However, the application of induced pluripotent stem cells (iPSCs) may shed light on overcoming such scarcity.In this study, we obtained iPSCs derived from a patient carrying a mutated LAMP2 gene that is associated with Danon disease. By differentiating such LAMP2-deficient iPSCs into cerebral cortical neurons and with the aid of various biochemical assays, we demonstrated that the LAMP2-deficient neurons are more susceptible to mild oxidative stress-induced injury.The data from MTT assay and apoptotic analysis demonstrated that there was no notable difference in cellular viability between the normal and LAMP2-deficient neurons under non-stressed condition. When exposed to mild oxidative stress (10 μM H₂O₂), the LAMP2-deficient neurons exhibited a significant increase in apoptosis. Surprisingly, we did not observe any aberrant accumulation of autophagic materials in the LAMP2-deficient neurons under such stress condition.Our results from cellular fractionation and inhibitor blockade experiments further revealed that oxidative stress-induced apoptosis in the LAMP2-deficient cortical neurons was caused by increased abundance of cytosolic cathepsin L. These results suggest the involvement of lysosomal membrane permeabilization in the LAMP2 deficiency associated neural injury.     

包埋法固定化对硫氧化微生物菌群结构和功能的影响

【目的】为探讨包埋法固定化过程对硫氧化菌群硫化物去除能力及菌群微生物群落结构的影响,【方法】以聚乙烯醇-海藻酸钠-活性炭为载体,对硫氧化菌群进行了固定化,并采用富含硫化物的无机盐培养基,对比固定化与非固定化硫氧化菌群对硫化物的氧化去除能力。同时,利用PCR-DGGE技术,探讨硫氧化菌群在固定化前后以及在硫化物氧化去除过程中微生物群落结构变化。【结果】在对硫氧化菌群进行固定化之后,12 h之内对硫化物的最大去除能力从1000 mg/L下降为600 mg/L。硫氧化菌群的微生物群落结构发生了明显变化,但菌群中的硫氧化菌Catenococcus thiocycli未受影响,硫氧化菌Thioclava pacifica在菌群中的地位反而得到了强化。【结论】受制于底物在载体材料中的扩散迁移效率,硫氧化菌群对硫化物的氧化去除能力在固定化之后有所下降。由于不同微生物对固定化形成的微环境的适应能力以及对载体附着能力的不同,固定化对硫氧化菌群的微生物群落结构产生较大影响。     

表面展示表达果胶酯酶的重组酿酒酵母构建及乙醇发酵

【目的】以淀粉为原料的乙醇发酵工艺仍然是当前燃料乙醇的主要生产方式。然而,一些原料中含有的果胶物质不仅降低了乙醇产率,而且会导致醪液粘度增大,从而会进一步影响传质和传热、增加设备负担等。构建能够自主降解果胶质的重组酿酒酵母并应用于燃料乙醇生产是值得探索的领域。【方法】论文将来源于黑曲霉的果胶酯酶基因克隆于α因子信号肽下游并通过酵母α-凝集素C-端蛋白的介导构建了在细胞表面锚定表达果胶酯酶的重组酿酒酵母PE。【结果】重组酵母的果胶酯酶表达水平达到2.6 U/g(菌体湿重),并进一步鉴定了重组果胶酯酶性质。以甘薯粉为原料的同步糖化发酵实验中,重组酵母PE的乙醇浓度和乙醇转化率分别达到95 g/L和88.1%,与出发菌株相比提高了2.2%。更重要的是,表面展示果胶酯酶能够显著降低发酵过程中的发酵液粘度。【结论】通过在工业酿酒酵母表面展示表达果胶酯酶不仅能够提高糖化酶等的作用效果和酿酒酵母的代谢能力,而且能够显著降低乙醇生产过程中发酵液的粘度,将对工业规模乙醇生产在降低设备负担、节约能耗方面具有一定的潜在价值。