scholarly journals A LCMS Metabolomic Workflow to Investigate Metabolic Patterns in Human Intestinal Cells Exposed to Hydrolyzed Crab Waste Materials

2021 ◽  
Vol 9 ◽  
Author(s):  
Fionn Ó Fearghail ◽  
Patrice Behan ◽  
Niklas Engström ◽  
Nathalie Scheers
Keyword(s):  
Sample Preparation ◽  
Cell Cultures ◽  
Cell Response ◽  
Cost Effective ◽  
Waste Materials ◽  
Intestinal Cells ◽  
The Novel ◽  
Data Pipeline ◽  
Anti Inflammatory ◽  
Human Intestinal Cells

We have developed a LCMS metabolomic workflow to investigate metabolic patterns from human intestinal cells treated with simulated gastrointestinal-digested hydrolyzed crab waste materials. This workflow facilitates smart and reproducible comparisons of cell cultures exposed to different treatments. In this case the variable was the hydrolysis methods, also accounting for the GI digestion giving an output of direct correlation between cellular metabolic patterns caused by the treatments. In addition, we used the output from this workflow to select treatments for further evaluation of the Caco-2 cell response in terms of tentative anti-inflammatory activity in the hopes to find value in the crab waste materials to be used for food products. As hypothesized, the treatment identified to change the cellular metabolomic pattern most readily, was also found to cause the greatest effect in the cells, although the response was pro-inflammatory rather than anti-inflammatory, it proves that changes in cellular metabolic patterns are useful predictors of bioactivity. We conclude that the developed workflow allows for cost effective, rapid sample preparation as well as accurate and repeatable LCMS analysis and introduces a data pipeline specifically for probe the novel metabolite patterns created as a means to assess the performing treatments.

A Sample Preparation on Decapsulation Methodology for Effective Failure Analysis on Thin Small Leadless (TSLP) Flip Chip Package with Copper Pillar (CuP) Bump Interconnect Technology

2014 ◽  
Author(s):  
Gwee Hoon Yen ◽  
Ng Kiong Kay
Keyword(s):  
Sample Preparation ◽  
Failure Analysis ◽  
Flip Chip ◽  
Fault Localization ◽  
Cost Effective ◽  
Fault Isolation ◽  
Voltage Contrast ◽  
Interconnect Technology

Abstract Today, failure analysis involving flip chip [1] with copper pillar bump packaging technologies would be the major challenges faced by analysts. Most often, handling on the chips after destructive chemical decapsulation is extremely critical as there are several failure analysis steps to be continued such as chip level fault localization, chip micro probing for fault isolation, parallel lapping [2, 3, 4] and passive voltage contrast. Therefore, quality of sample preparation is critical. This paper discussed and demonstrated a quick, reliable and cost effective methodology to decapsulate the thin small leadless (TSLP) flip chip package with copper pillar (CuP) bump interconnect technology.

CADHERIN-11, AN ESSENTIAL REGULATOR OF CELL-CELL ADHESION UPREGULATED IN IBD PATIENTS, PLAYS ESSENTIAL ROLES IN PROMOTING FIBROBLAST ACTIVATION AND DEVELOPMENT OF INTESTINAL INFLAMMATION AND FIBROSIS

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S34-S35
Author(s):  
Jiannan Li ◽  
Ilyssa Gordon ◽  
Dina Dejanovic ◽  
Sinan Lin ◽  
Jie Wang ◽  
...  
Keyword(s):  
Weight Loss ◽  
Smooth Muscle ◽  
Knockout Mice ◽  
Clinical Severity ◽  
Intestinal Cells ◽  
Full Thickness ◽  
Chronic Colitis ◽  
Expression Levels ◽  
Clinical Scores ◽  
Human Intestinal Cells

Abstract Background Intestinal fibrosis is a severe complication of inflammatory bowel diseases (IBD) leading to intestinal strictures and need for surgery. No effective anti-fibrotic therapy is available. Cadherin-11 (Cad-11) is an adherens junction protein, which is upregulated in rheumatoid arthritis (RA), idiopathic pulmonary fibrosis (IPF) and skin fibrosis. Inhibition of cadherin-11 has shown beneficial effects in RA and IPF animal models. A phase II clinical trial of cadherin-11 inhibition in RA has shown a good safety profile. Our aim was to evaluate the expression levels and function of Cad-11 in IBD patients using intestinal tissues, primary human intestinal cells, and the murine dextran sulfate sodium (DSS)-induced chronic colitis model. Methods IBD (Crohn’s disease (CD) n=20; Ulcerative colitis (UC) (n=10) and control (n=10) full thickness resected intestinal tissues were procured from adults in accordance with IRB approval. Protein and mRNA were extracted for western blot (WB) and quantitative polymerase chain reaction (qPCR). Distribution of Cad-11 was evaluated by immunofluorescence (IF) and RNA hybridization in frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections, respectively. Primary human intestinal myofibroblasts (HIMF) were used in functional experiments. Recombinant human Fc and Cad-11 extracellular domain (hCAD-11-Fc) was used as activator and siRNA as inhibitor of Cad-11 in HIMF. Murine chronic colitis was induced in wildtype BALB/c mice and cadherin-11 knockout mice by DSS. Anti-Cad-11 monoclonal antibody (H1M1) was used for the treatment of BALB/c mouse colitis. Results Increased gene and protein expression levels of Cad-11 were found in intestinal full thickness IBD tissue compared to controls (45-fold, p<0.01). Cad-11 colocalized with alpha smooth muscle actin (α-SMA) (Figure 1), indicating that Cad-11 is selectively expressed in intestinal myofibroblasts and smooth muscle cells. Among all the primary human intestinal cells, Cad-11 was expressed exclusively in HIMF and HIMC cells. Level of Cad-11 was increased in IBD HIMFs compared to non-IBD controls, and increased upon stimulation with TNF-α, IL-1β, b-FGF and TGF-β (all p<0.01). Knocking down Cad-11 with siRNA decreased FN expression, while hCAD-11-Fc increased the expression FN in a dose- and time-dependent manner as well as the proliferation of HIMF. Upon treatment with H1M1 antibody, DSS-treated mice showed lower clinical scores and weight loss compared to control mice (p<0.001. Figure 2), as well as less FN expression (p<0.001). Cadherin-11 knockout mice also showed lower clinical scores and weight loss compared to wild type mice (p<0.001). Conclusions Cad-11 expression is increased in CD stricture tissues and its blockade reduces profibrotic effects in HIMF in vitro. Inhibition of Cad-11 in vivo reduces clinical severity and fibrosis of experimental colitis.

scholarly journals Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion

2021 ◽  
Vol 18 (4) ◽  
pp. 1380
Author(s):  
Neusa Figueiredo ◽  
Beatriz Matos ◽  
Mário Diniz ◽  
Vasco Branco ◽  
Marta Martins
Keyword(s):  
Cell Viability ◽  
Cell Cultures ◽  
Marine Fish ◽  
Sparus Aurata ◽  
Cost Effective ◽  
Primary Hepatocyte ◽  
Digestion Method ◽  
Trypan Blue Exclusion ◽  
And Function

Primary cell cultures from wild organisms have been gaining relevance in ecotoxicology as they are considered more sensitive than immortalized cell lines and retain the biochemical pathways found in vivo. In this study, the efficacy of two methods for primary hepatocyte cell isolation was compared using liver from two marine fish (Sparus aurata and Psetta maxima): (i) two-step collagenase perfusion and (ii) pancreatin digestion with modifications. Cell cultures were incubated in L-15 medium at 17 ± 1 °C and monitored for up to six days for cell viability and function using the trypan blue exclusion test, MTT test, lactate dehydrogenase (LDH) activity, and ethoxyresorufin O-deethylase (EROD) activity after Benzo[a]Pyrene exposure. The results showed significant differences between the number of viable cells (p < 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 ± 1.9 × 107 cells). Moreover, the hepatocytes showed solid adherence to the culture plate and the rounded shape, changing into a triangular/polygonal shape. The cell viability and function obtained by pancreatin digestion were maintained for five days, and the EROD induction after exposure to the B[a]P showed that cells were metabolically active. This study shows that the optimized pancreatin digestion method is a valid, cost-effective, and simple alternative to the standard perfusion method for the isolation of primary hepatocytes from fish and is suitable for ecotoxicological studies involving marine pollutants, such as PAHs.

scholarly journals Chronic exposure to short-chain fatty acids modulates transport and metabolism of microbiome-derived phenolics in human intestinal cells

2017 ◽  
Vol 39 ◽  
pp. 156-168 ◽  
Author(s):  
Evelien Van Rymenant ◽  
László Abrankó ◽  
Sarka Tumova ◽  
Charlotte Grootaert ◽  
John Van Camp ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Chronic Exposure ◽  
Short Chain Fatty Acids ◽  
Short Chain ◽  
Intestinal Cells ◽  
Human Intestinal Cells ◽  
Chain Fatty Acids

scholarly journals Human milk glycosaminoglycans inhibit in vitro the adhesion of Escherichia coli and Salmonella fyris to human intestinal cells

2015 ◽  
Vol 79 (4) ◽  
pp. 603-607 ◽  
Author(s):  
Giovanni V. Coppa ◽  
Bruna Facinelli ◽  
Gloria Magi ◽  
Emanuela Marini ◽  
Lucia Zampini ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Human Milk ◽  
Intestinal Cells ◽  
Human Intestinal Cells

scholarly journals Clozapine Prevents Poly (I:C) Induced Inflammation by Modulating NLRP3 Pathway in Microglial Cells

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 577
Author(s):  
Vijayasree V. Giridharan ◽  
Giselli Scaini ◽  
Gabriela D. Colpo ◽  
Tejaswini Doifode ◽  
Omar F. Pinjari ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Nlrp3 Inflammasome ◽  
Antipsychotic Drugs ◽  
Poly I:C ◽  
Tnf Α ◽  
Cytokine Levels ◽  
Primary Microglial Cell ◽  
Anti Inflammatory ◽  
The Brain ◽  
Nlrp3 Expression

Schizophrenia is a complex psychiatric disorder that exhibits an interconnection between the immune system and the brain. Experimental and clinical studies have suggested the presence of neuroinflammation in schizophrenia. In the present study, the effect of antipsychotic drugs, including clozapine, risperidone, and haloperidol (10, 20 and 20 μM, respectively), on the production of IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, IL-18, INF-γ, and TNF-α was investigated in the unstimulated and polyriboinosinic-polyribocytidilic acid [poly (I:C)]-stimulated primary microglial cell cultures. In the unstimulated cultures, clozapine, risperidone, and haloperidol did not influence the cytokine levels. Nevertheless, in cell cultures under strong inflammatory activation by poly (I:C), clozapine reduced the levels of IL-1α, IL-1β, IL-2, and IL-17. Risperidone and haloperidol both reduced the levels of IL-1α, IL-1β, IL-2, and IL-17, and increased the levels of IL-6, IL-10, INF-γ, and TNF-α. Based on the results that were obtained with the antipsychotic drugs and observing that clozapine presented with a more significant anti-inflammatory effect, clozapine was selected for the subsequent experiments. We compared the profile of cytokine suppression obtained with the use of NLRP3 inflammasome inhibitor, CRID3 to that obtained with clozapine, to test our hypothesis that clozapine inhibits the NLRP3 inflammasome. Clozapine and CRID3 both reduced the IL-1α, IL-1β, IL-2, and IL-17 levels. Clozapine reduced the level of poly (I:C)-activated NLRP3 expression by 57%, which was higher than the reduction thay was seen with CRID3 treatment (45%). These results suggest that clozapine might exhibit anti-inflammatory effects by inhibiting NLRP3 inflammasome and this activity is not typical with the use of other antipsychotic drugs under the conditions of strong microglial activation.

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