scholarly journals An in situ activity assay for lysyl oxidases

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Huilei Wang ◽  
Alan Poe ◽  
Lydia Pak ◽  
Kavitha Nandakumar ◽  
Sandeep Jandu ◽  
...  
Keyword(s):  
Lysyl Oxidase ◽  
Activity Assay ◽  
Strong Reaction ◽  
Tissue Samples ◽  
Lysyl Oxidases ◽  
Catalytic Function ◽  
Elevated Expression ◽  
Representative Member

AbstractThe lysyl oxidase family of enzymes (LOXs) catalyze oxidative deamination of lysine side chains on collagen and elastin to initialize cross-linking that is essential for the formation of the extracellular matrix (ECM). Elevated expression of LOXs is highly associated with diverse disease processes. To date, the inability to detect total LOX catalytic function in situ has limited the ability to fully elucidate the role of LOXs in pathobiological mechanisms. Using LOXL2 as a representative member of the LOX family, we developed an in situ activity assay by utilizing the strong reaction between hydrazide and aldehyde to label the LOX-catalyzed allysine (-CHO) residues with biotin-hydrazide. The biotinylated ECM proteins are then labeled via biotin-streptavidin interaction and detected by fluorescence microscopy. This assay detects the total LOX activity in situ for both overexpressed and endogenous LOXs in cells and tissue samples and can be used for studies of LOXs as therapeutic targets.

scholarly journals A Novel In Situ Activity Assay for Lysyl Oxidases

2021 ◽  
Author(s):  
Huilei Wang ◽  
Alan Poe ◽  
Lydia Pak ◽  
Sandeep Jandu ◽  
Kavitha Nandakumar ◽  
...  
Keyword(s):  
Lysyl Oxidase ◽  
Activity Assay ◽  
Strong Reaction ◽  
Tissue Samples ◽  
Lysyl Oxidases ◽  
Catalytic Function ◽  
Elevated Expression ◽  
Representative Member

AbstractThe lysyl oxidase family of enzymes (LOXs) catalyze oxidative deamination of lysine side chains on collagen and elastin to initialize cross-linking that is essential for the formation of the extracellular matrix (ECM). Elevated expression of LOXs is highly associated with diverse disease processes. To date, the inability to detect total LOX catalytic function in situ has limited the ability to fully elucidate the role of LOXs in pathobiological mechanisms. Using LOXL2 as a representative member of the LOX family, we developed an in situ activity assay by utilizing the strong reaction between hydrazide and aldehyde to label the LOX-catalyzed allysine (-CHO) residues with biotin-hydrazide. The biotinylated ECM proteins are then labeled via biotin-streptavidin interaction and detected by fluorescence microscopy. This assay detects the total LOX activity in situ for both overexpressed and endogenous LOXs in cells and tissue samples and can be used for studies of LOXs as therapeutic targets.

scholarly journals Author Correction: An in situ activity assay for lysyl oxidases

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Huilei Wang ◽  
Alan Poe ◽  
Lydia Pak ◽  
Kavitha Nandakumar ◽  
Sandeep Jandu ◽  
...  
Keyword(s):  
Activity Assay ◽  
Lysyl Oxidases

scholarly journals Development of a Novel In Situ Activity Assay for Lysyl Oxidase Like‐2 (LOXL2)

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Huilei Wang ◽  
Mahmoud Abouelkheir ◽  
Alan Poe ◽  
Yurie Hong ◽  
Sandeep Jandu ◽  
...  
Keyword(s):  
Lysyl Oxidase ◽  
Activity Assay

scholarly journals Inflammasome gene expression alterations in Staphylococcus aureus biofilm-associated chronic rhinosinusitis

2013 ◽  
Vol 51 (4) ◽  
pp. 315-322
Author(s):  
C. Jardeleza ◽  
D. Miljkovic ◽  
L. Baker ◽  
S. Boase ◽  
N.C.W. Tan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Chronic Rhinosinusitis ◽  
Treatment Modalities ◽  
Patient Stratification ◽  
Tissue Samples ◽  
Double Stranded Dna ◽  
The Subject ◽  
Gene Expression Alterations

Background: The role of inflammasomes in chronic inflammation has been the subject of intense research in recent years. Chronic rhinosinusitis (CRS), a persistent inflammatory disease, continues to be investigated hoping that a clearer pathophysiologic description will guide discovery of future treatment modalities. This study investigates the role of inflammasome complexes in CRS patients with Staphylococcus aureus biofilm infection, a key culprit associated with disease severity and recalcitrance. Methodology: Sinonasal tissue samples were collected from CRS patients with (P+) and without (P-) polyps and controls. S. aureus biofilm status was obtained using fluorescence in situ hybridization and classified as biofilm positive (B+) or negative (B-). RNA was analysed using a Human Inflammasome PCR array, profiling the expression of 84 genes involved in inflammasome function. Results: Sixteen samples were obtained: 5 B+P+, 5 B-P- and 6 controls. Comparing B+P+ vs. controls showed the greatest number of differentially expressed genes. In particular, Absent in Melanoma 2 (AIM2) was consistently and significantly up-regulated in the B+P+ vs. B-P- and controls. In contrast, when comparing the B-P- vs. controls, no genes showed significant changes. Conclusion: Our results indicate the involvement of inflammasome complexes and their signalling pathways in CRS patients with polyps and S. aureus biofilms. In particular, AIM2, activated by intracellular double-stranded DNA, is up-regulated in this group, implying that S. aureus may play a role in intracellular triggering of the inflammasome response. Studies with further patient stratification and assessing corresponding protein expression are needed to further characterize the role of inflammasomes in CRS.

scholarly journals Mycoplasma hyorhinis infection in early cases of mycoplasmal pneumonia in swine and evaluation of diagnostic assays

2017 ◽  
Vol 37 (10) ◽  
pp. 1057-1063
Author(s):  
Carlos E.R. Pereira ◽  
Fabio A. Vannucci ◽  
Michelle de P. Gabardo ◽  
Lucas F. dos Santos ◽  
Nelson Mores ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Influenza A ◽  
Mycoplasma Hyorhinis ◽  
Tissue Samples ◽  
Nursery Pigs ◽  
Diagnostic Assays ◽  
Mycoplasmal Pneumonia ◽  
Group 2

ABSTRACT: Mycoplasmal pneumonia is an important disease in the pig industry. Due to the controversial role of Mycoplasma hyorhinis in this disease, confirmation of the presence of this bacterium and the identification of its roles in respiratory disease remain major challenges. The objectives of this study were to evaluate the presence of M. hyorhinis in early cases of mycoplasmal pneumonia and to determine the usefulness of fluorescent in situ hybridization (FISH) for the diagnosis of respiratory mycoplasmosis in naturally infected pigs. Ninety M. hyopneumoniae and/or M. hyorhinis-infected lung tissue samples based on diagnostic mosaic (DM) were used. The average age of the animals was 116 and 57 days (P<0.01) for groups 1 (positive-M. hyopneumoniae only) and 2 (positive-M. hyorhinis only), respectively. These findings suggest that development of lesions caused by M. hyorhinis occurs earlier than for M. hyopneumoniae. Using the DM as the gold standard, the sensitivity and specificity of FISH for M. hyopneumoniae were 75 and 100%, respectively, and were 40 and 73.3% for the immunohistochemistry (IHC). The sensitivity and specificity of FISH for M. hyorhinis were 76.7 and 100%, respectively. These findings demonstrate that FISH can be a useful tool for diagnosing mycoplasmosis. Viral antigens (PCV2 or influenza A) were detected in 53.3% (16/30) of the samples in group 2 (M. hyorhinis-PCR positive) and 13.3% (4/30) of the samples in group 1 (M. hyopneumoniae-PCR positive). This finding indicates that the association of M. hyorhinis and viral infection in nursery pigs likely starts due to a viral immunosuppressive condition.

scholarly journals Expression Analysis of FGF/FGFR and FOX Family Proteins in Mucosal Tissue Obtained from Orofacial Cleft-Affected Children

Biology ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 423
Author(s):  
Māra Pilmane ◽  
Nityanand Jain ◽  
Zane Vitenberga-Verza
Keyword(s):  
Endothelial Cells ◽  
Epithelial Cells ◽  
Cellular Proliferation ◽  
Local Site ◽  
Cell Tissue ◽  
Elevated Expression ◽  
Correction Surgery ◽  
Underlying Mechanisms

Orofacial clefts affect hundreds of thousands of children worldwide annually and are usually corrected by a series of surgeries extending to childhood. The underlying mechanisms that lead to clefts are still unknown, mainly because of the multifactorial etiology and the myriad of interactions between genes and environmental factors. In the present study, we investigated the role and expression of candidate genes belonging to the FGF/FGFR signaling pathway and FOX family in tissue material obtained from 12 pediatric patients undergoing cleft correction surgery. The expression was investigated using immunohistochemistry (IHC) and chromogenic in-situ hybridization (CISH) in three cell/tissue types—epithelial cells, connective tissue, and endothelial cells. We found elevated expression of FGFR1 in epithelial cells while no expression was observed in endothelial cells. Further, our results elucidate the potential pathogenetic role of FGFR1 in cellular proliferation, local site inflammation, and fibrosis in cleft patients. Along with bFGF (also called FGF2), FGFR1 could play a pro-inflammatory role in clefts. Over-amplification of FGFR2 in some patients, along with bFGF, could potentially suggest roles for these genes in angiogenesis. Additionally, increased expression of FOXE1 (also called TTF2) contributes to local site inflammation. Finally, zero to low amplification of FOXO1 could suggest its potential role in inducing oxidative stress in the endothelium along with reduced epithelial apoptosis.

scholarly journals Integrative Metabolomics Reveals Deep Tissue and Systemic Metabolic Remodeling in Glioblastoma

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5157
Author(s):  
Vianney Gilard ◽  
Justine Ferey ◽  
Florent Marguet ◽  
Maxime Fontanilles ◽  
Franklin Ducatez ◽  
...  
Keyword(s):  
Deep Tissue ◽  
Tissue Samples ◽  
Diagnostic Strategies ◽  
Metabolic Remodeling ◽  
Progressive Neurological Deficit ◽  
Underlying Mechanisms ◽  
Functional Analyses ◽  
Tumoral Cells

(1) Background: Glioblastoma is the most common malignant brain tumor in adults. Its etiology remains unknown in most cases. Glioblastoma pathogenesis consists of a progressive infiltration of the white matter by tumoral cells leading to progressive neurological deficit, epilepsy, and/or intracranial hypertension. The mean survival is between 15 to 17 months. Given this aggressive prognosis, there is an urgent need for a better understanding of the underlying mechanisms of glioblastoma to unveil new diagnostic strategies and therapeutic targets through a deeper understanding of its biology. (2) Methods: To systematically address this issue, we performed targeted and untargeted metabolomics-based investigations on both tissue and plasma samples from patients with glioblastoma. (3) Results: This study revealed 176 differentially expressed lipids and metabolites, 148 in plasma and 28 in tissue samples. Main biochemical classes include phospholipids, acylcarnitines, sphingomyelins, and triacylglycerols. Functional analyses revealed deep metabolic remodeling in glioblastoma lipids and energy substrates, which unveils the major role of lipids in tumor progression by modulating its own environment. (4) Conclusions: Overall, our study demonstrates in situ and systemic metabolic rewiring in glioblastoma that could shed light on its underlying biological plasticity and progression to inform diagnosis and/or therapeutic strategies.

scholarly journals Application of a Novel In Situ Activity Assay to Detect Lysyl Oxidase Like‐2 (LOXL2)

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Huilei Wang ◽  
Alan Poe ◽  
Sandeep Jandu ◽  
Kavitha Nandakumar ◽  
Lakshmi Santhanam
Keyword(s):  
Lysyl Oxidase ◽  
Activity Assay

scholarly journals Role of Intraoperative Frozen Section Diagnosis in the Management of Patients with CNS Lesions: Evaluation of 60 Consecutive Intraoperative Frozen Sections from 1991 to 1993

2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Seyed Mirsattari ◽  
Fraser W. Saunders
Keyword(s):  
Frozen Section ◽  
Imaging Techniques ◽  
Frozen Sections ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Intraoperative Management ◽  
Clinical Diagnoses ◽  
Cns Lesions

To determine the role of intraoperative frozen sections (FSs) in the management of patients with central nervous system (CNS) lesions, 60 consecutive intraoperative clinical diagnoses of CNS lesions were presented and compared with concomitantly obtained FS diagnoses. Clinical diagnoses were established byhistory, physical examination, imaging techniques, and gross appearance of the abnormal tissue in situ. Tissue samples were obtained intraoperatively and processed for FS diagnoses. The findings of the FS diagnoses were reported to the operating room and compared with the clinical diagnoses. The remainingbiopsy samples were used to prepare paraffin-embedded tissue sections from which the definitive diagnoses were made. Comparison of the clinical and FS diagnoses, using paraffin-embedded tissue as the true diagnosis, shows that FS diagnosis has a limited contribution to intraoperative patient management by the neurosurgeon. The rate of diagnostic failures between the two techniques was very similar; clinical diagnoses and FSs were misinterpreted in 12 and 11 of the 60 cases, respectively. Compared to a clinical diagnosis, the intraoperative FS technique provided no significant improvement in diagnosis and management; it altered the intraoperative management of the patients in 2 of 60 cases.

Targeting the lysyl oxidases in tumour desmoplasia

2019 ◽  
Vol 47 (6) ◽  
pp. 1661-1678 ◽  
Author(s):  
Jessica L. Chitty ◽  
Yordanos F.I. Setargew ◽  
Thomas R. Cox
Keyword(s):  
Small Molecule ◽  
Active Sites ◽  
Small Molecule Inhibitors ◽  
Amine Oxidase ◽  
Lysyl Oxidase ◽  
Lysyl Oxidases ◽  
Assembly Mechanism ◽  
Cancer Field ◽  
Fibrillar Collagens

The extracellular matrix (ECM) is a fundamental component of tissue microenvironments and its dysregulation has been implicated in a number of diseases, in particular cancer. Tumour desmoplasia (fibrosis) accompanies the progression of many solid cancers, and is also often induced as a result of many frontline chemotherapies. This has recently led to an increased interest in targeting the underlying processes. The major structural components of the ECM contributing to desmoplasia are the fibrillar collagens, whose key assembly mechanism is the enzymatic stabilisation of procollagen monomers by the lysyl oxidases. The lysyl oxidase family of copper-dependent amine oxidase enzymes are required for covalent cross-linking of collagen (as well as elastin) molecules into the mature ECM. This key step in the assembly of collagens is of particular interest in the cancer field since it is essential to the tumour desmoplastic response. LOX family members are dysregulated in many cancers and consequently the development of small molecule inhibitors targeting their enzymatic activity has been initiated by many groups. Development of specific small molecule inhibitors however has been hindered by the lack of crystal structures of the active sites, and therefore alternate indirect approaches to target LOX have also been explored. In this review, we introduce the importance of, and assembly steps of the ECM in the tumour desmoplastic response focussing on the role of the lysyl oxidases. We also discuss recent progress in targeting this family of enzymes as a potential therapeutic approach.

Sign in / Sign up

Export Citation Format

Share Document