Factors Influencing the Formation of Chemical–Hemoglobin Adducts

Hemoglobin (Hb) adducts have been used as biomarkers for the internal exposure to chemicals. Simultaneous exposure to chemicals that bond with the N-terminal valine of Hb to form adducts, such as glycidol, acrylamide, and glucose, may affect the formation of the individual Hb adducts. In this study, various factors influencing the formation of chemical–Hb adducts were analyzed using in vitro and in vivo systems. In the in vitro assays, the formation of glycidol– and acrylamide–Hb adducts was altered in the presence of glucose, serum albumin, and other chemicals. In contrast, in the in vivo experiments, glycidol– and acrylamide–Hb adduct formation was unchanged in mice exposed to glycidol and acrylamide. The interaction between glycidol and acrylamide with residues other than the N-terminal valine of Hb was analyzed using the protein thermal shift assay. Glycidol and acrylamide also interacted with amino acid residues other than the N-terminal valine of Hb. The presence of other blood components, such as amino acids, may affect the formation of chemical–Hb adducts. Further research is expected to elucidate the remaining unknown factors that affect the formation of chemical–Hb adducts.

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Antibiotic loaded β-tricalcium phosphate/calcium sulfate for antimicrobial potency, prevention and killing efficacy of Pseudomonas aeruginosa and Staphylococcus aureus biofilms

Scientific Reports ◽

10.1038/s41598-020-80764-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nan Jiang ◽

Devendra H. Dusane ◽

Jacob R. Brooks ◽

Craig P. Delury ◽

Sean S. Aiken ◽

...

Keyword(s):

Tricalcium Phosphate ◽

Calcium Sulfate ◽

Bone Graft Substitute ◽

In Vitro Assays ◽

In Vivo Experiments ◽

Orthopaedic Infections ◽

Clinical Investigations ◽

Antimicrobial Potency

AbstractThis study investigated the efficacy of a biphasic synthetic β-tricalcium phosphate/calcium sulfate (β-TCP/CS) bone graft substitute for compatibility with vancomycin (V) in combination with tobramycin (T) or gentamicin (G) evidenced by the duration of potency and the prevention and killing efficacies of P. aeruginosa (PAO1) and S. aureus (SAP231) biofilms in in vitro assays. Antibiotic loaded β-TCP/CS beads were compared with antibiotic loaded beads formed from a well characterized synthetic calcium sulfate (CS) bone void filler. β-TCP/CS antibiotic loaded showed antimicrobial potency against PAO1 in a repeated Kirby-Bauer like zone of inhibition assay for 6 days compared to 8 days for CS. However, both bead types showed potency against SAP231 for 40 days. Both formulations loaded with V + T completely prevented biofilm formation (CFU below detection limits) for the 3 days of the experiment with daily fresh inoculum challenges (P < 0.001). In addition, both antibiotic loaded materials and antibiotic combinations significantly reduced the bioburden of pre-grown biofilms by between 3 and 5 logs (P < 0.001) with V + G performing slightly better against PAO1 than V + T. Our data, combined with previous data on osteogenesis suggest that antibiotic loaded β-TCP/CS may have potential to stimulate osteogenesis through acting as a scaffold as well as simultaneously protecting against biofilm infection. Future in vivo experiments and clinical investigations are warranted to more comprehensively evaluate the use of β-TCP/CS in the management of orthopaedic infections.

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Potential Anti-inflammatory Sesquiterpene Lactones from Eupatorium lindleyanum

Planta Medica ◽

10.1055/s-0043-117742 ◽

2017 ◽

Vol 84 (02) ◽

pp. 123-128 ◽

Cited By ~ 10

Author(s):

Fang Wang ◽

Huanhuan Zhong ◽

Shiqi Fang ◽

Yunfeng Zheng ◽

Cunyu Li ◽

...

Keyword(s):

Sesquiterpene Lactones ◽

Folk Medicine ◽

2D Nmr ◽

In Vitro Assays ◽

Raw 264.7 Cells ◽

Therapeutic Effects ◽

In Vivo Experiments ◽

Anti Inflammatory

Abstract Eupatorium lindleyanum has traditionally been used as folk medicine in Asian countries for its therapeutic effects on tracheitis and tonsillitis. Investigation of the anti-inflammatory active constituents from E. lindleyanum led to the isolation of two novel sesquiterpene lactones, named eupalinolide L (1) and eupalinolide M (2), and seven known sesquiterpene lactones (3–9). The structures and configurations of the new compounds were determined on the basis of spectroscopic analysis, especially 2D NMR techniques. In vivo experiments showed that the sesquiterpenes fraction significantly reduced mouse ear edema induced by xylene (18.6%, p < 0.05). In in vitro assays, compounds 1–9 showed excellent anti-inflammatory activities, as they lowered TNF-α and IL-6 levels in lipopolysaccharide-stimulated murine macrophage RAW 264.7 cells (p < 0.001). The above results suggest that the sesquiterpene lactones from E. lindleyanum can be developed as novel potential natural anti-inflammatory agents.

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Agent-based modeling of cancer stem cell driven solid tumor growth

10.1101/035162 ◽

2015 ◽

Author(s):

Jan T Poleszczuk ◽

Paul Macklin ◽

Heiko Enderling

Keyword(s):

Stem Cell ◽

Cancer Stem Cell ◽

Tumor Growth ◽

Population Level ◽

In Vitro Assays ◽

Agent Based ◽

In Vivo Experiments ◽

Behavioral Rules

Computational modeling of tumor growth has become an invaluable tool to simulate complex cell-cell interactions and emerging population-level dynamics. Agent-based models are commonly used to describe the behavior and interaction of individual cells in different environments. Behavioral rules can be informed and calibrated by in vitro assays, and emerging population-level dynamics may be validated with both in vitro and in vivo experiments. Here, we describe the design and implementation of a lattice-based agent-based model of cancer stem cell driven tumor growth.

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Host Defence Cryptides from Human Apolipoproteins: Applications in Medicinal Chemistry

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200427091454 ◽

2020 ◽

Vol 20 (14) ◽

pp. 1324-1337 ◽

Cited By ~ 1

Author(s):

Rosa Gaglione ◽

Elio Pizzo ◽

Eugenio Notomista ◽

Cesar de la Fuente-Nunez ◽

Angela Arciello

Keyword(s):

Bioactive Peptides ◽

Biological Activities ◽

Host Defence ◽

New Drugs ◽

In Vitro Assays ◽

Protein Cleavage ◽

Anticancer Activities ◽

In Vivo Experiments

Several eukaryotic proteins with defined physiological roles may act as precursors of cryptic bioactive peptides released upon protein cleavage by the host and/or bacterial proteases. Based on this, the term “cryptome” has been used to define the unique portion of the proteome encompassing proteins with the ability to generate bioactive peptides (cryptides) and proteins (crypteins) upon proteolytic cleavage. Hence, the cryptome represents a source of peptides with potential pharmacological interest. Among eukaryotic precursor proteins, human apolipoproteins play an important role, since promising bioactive peptides have been identified and characterized from apolipoproteins E, B, and A-I sequences. Human apolipoproteins derived peptides have been shown to exhibit antibacterial, anti-biofilm, antiviral, anti-inflammatory, anti-atherogenic, antioxidant, or anticancer activities in in vitro assays and, in some cases, also in in vivo experiments on animal models. The most interesting Host Defence Peptides (HDPs) identified thus far in human apolipoproteins are described here with a focus on their biological activities applicable to biomedicine. Altogether, reported evidence clearly indicates that cryptic peptides represent promising templates for the generation of new drugs and therapeutics against infectious diseases.

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Paired-Type Homeodomain Transcription Factors Are Imported into the Nucleus by Karyopherin 13

Molecular and Cellular Biology ◽

10.1128/mcb.24.11.4824-4834.2004 ◽

2004 ◽

Vol 24 (11) ◽

pp. 4824-4834 ◽

Cited By ~ 34

Author(s):

Jonathan E. Ploski ◽

Monee K. Shamsher ◽

Aurelian Radu

Keyword(s):

Transcription Factor ◽

In Vitro Assays ◽

Nuclear Localization Sequence ◽

Amino Acid Residues ◽

Homeodomain Transcription Factor ◽

Permeabilized Cells ◽

Localization Sequence ◽

Direct Binding

ABSTRACT We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin β family member Karyopherin 13 (Kap13). Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen. Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation. Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay. Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain. Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted. The paired-type homeodomain transcription factor family includes more than 20 members. All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13. We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13.

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Bacillus-based products for management of kiwifruit bacterial canker

Phytopathologia Mediterranea ◽

10.36253/phyto-12184 ◽

2021 ◽

Vol 60 (2) ◽

pp. 215-228

Author(s):

Enrico BIONDI ◽

Lorenzo GALLIPOLI ◽

Angelo MAZZAGLIA ◽

Set Perez FUENTEALBA ◽

Nemanja KUZMANOVIĆ ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Control Strategies ◽

In Vitro Assays ◽

Bacterial Canker ◽

Streptomycin Sulphate ◽

In Vivo Experiments ◽

Ca 50 ◽

Pathogen Populations

Pseudomonas syringae pv. actinidiae is an important pathogen of kiwifruit (Actinidia deliciosa), and bacterial canker of this host is managed by monitoring and chemical control strategies. The efficacy of the bio-pesticides Amylo-X® (based on Bacillus amyloliquefaciens subsp. plantarum strain D747) and Serenade Max® (strain QST713 of B. subtilis) was evaluated by in vitro and in vivo experiments. Both antagonists inhibited different biovars of the pathogen in in vitro assays; QST713 was more efficient than D747. The two Bacillus strains also colonized A. deliciosa flowers (c. 105-7 cfu per flower) up to 96 h after inoculation. D747 persisted on leaves (c. 104-6 cfu cm-2) up to 4 weeks after inoculation, during 2 years in Emilia Romagna and Latium regions of Italy. On flowers, the antagonists reduced pathogen populations, compared to untreated (control) flowers. On A. deliciosa and A. chinensis plants under controlled conditions, Amylo-X® reduced severity of bacterial canker, providing ca. 50% relative protection on A. deliciosa and 70% on A. chinensis. Serenade Max® was less effective, giving 0% relative protection on A. deliciosa and 40% on A. chinensis. In a field trial, on A. deliciosa plants, Amylo-X® reduced the severity of bacterial canker on leaves, providing ca. 40% relative protection. The sensitivity of both antagonistic strains to streptomycin sulphate was confirmed by testing the most used concentration where antibiotics are approved for management of bacterial pathogens.

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Chemoattraction of Neoplastic Glial Cells with CXCL10, CCL2 and CCL11 as a Paradigm for a Promising Therapeutic Approach for Primary Brain Tumors

International Journal of Molecular Sciences ◽

10.3390/ijms222212150 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12150

Author(s):

Laurence Déry ◽

Gabriel Charest ◽

Brigitte Guérin ◽

Mohsen Akbari ◽

David Fortin

Keyword(s):

Brain Tumors ◽

Tumor Cells ◽

Therapeutic Approach ◽

Brain Parenchyma ◽

In Vitro Assays ◽

Primary Brain Tumors ◽

In Vivo Experiments ◽

Promising Therapeutic Approach

Chemoattraction is a normal and essential process, but it can also be involved in tumorigenesis. This phenomenon plays a key role in glioblastoma (GBM). The GBM tumor cells are extremely difficult to eradicate, due to their strong capacity to migrate into the brain parenchyma. Consequently, a complete resection of the tumor is rarely a possibility, and recurrence is inevitable. To overcome this problem, we proposed to exploit this behavior by using three chemoattractants: CXCL10, CCL2 and CCL11, released by a biodegradable hydrogel (GlioGel) to produce a migration of tumor cells toward a therapeutic trap. To investigate this hypothesis, the agarose drop assay was used to test the chemoattraction capacity of these three chemokines on murine F98 and human U87MG cell lines. We then studied the potency of this approach in vivo in the well-established syngeneic F98-Fischer glioma-bearing rat model using GlioGel containing different mixtures of the chemoattractants. In vitro assays resulted in an invasive cell rate 2-fold higher when chemokines were present in the environment. In vivo experiments demonstrated the capacity of these specific chemoattractants to strongly attract neoplastic glioblastoma cells. The use of this strong locomotion ability to our end is a promising avenue in the establishment of a new therapeutic approach in the treatment of primary brain tumors.

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PIAS1 alleviates diabetic peripheral neuropathy through SUMOlation of PPAR-γ and miR-124-induced downregulation of EZH2/STAT3

Cell Death Discovery ◽

10.1038/s41420-021-00765-w ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Zixin Hou ◽

Ji Chen ◽

Huan Yang ◽

Xiaoling Hu ◽

Fengrui Yang

Keyword(s):

Peripheral Neuropathy ◽

Schwann Cells ◽

Diabetic Peripheral Neuropathy ◽

Nerve Tissue ◽

In Vitro Assays ◽

In Vivo Experiments ◽

Ppar Γ ◽

Gene Reporter Assay

AbstractDiabetic peripheral neuropathy (DPN) is a frequently occurring chronic complication of diabetes. In this study, we aim to explore the regulatory mechanism of protein inhibitor of activated STAT1 (PIAS1) in DPN in terms of autophagy and apoptosis of Schwann cells. The SUMOlation of PPAR-γ by PIAS1 was examined, and ChIP was performed to verify the binding of PPAR-γ to miR-124 promoter region. Dual-luciferase gene reporter assay was used to validate the binding affinity between miR-124 and EZH2/STAT3. Following loss‐ and gain‐of-function experiments, in vitro assays in high glucose-treated Schwann cells (SC4) and in vivo assays in db/db and ob/ob mice were performed to detect the effects of PIAS1 on autophagy and apoptosis of Schwann cells as well as symptoms of DPN by regulating the PPAR-γ-miR-124-EZH2/STAT3. The expression of PIAS1, PPAR-γ, and miR-124 was downregulated in the sciatic nerve tissue of diabetic mice. PIAS1 enhanced the expression of PPAR-γ through direct binding and SUMOlation of PPAR-γ. PPAR-γ enhanced the expression of miR-124 by enhancing the promoter activity of miR-124. Furthermore, miR-124 targeted and inversely modulated EZH2 and STAT3, promoting the autophagy of Schwann cells and inhibiting their apoptosis. In vivo experiments further substantiated that PIAS1 could promote the autophagy and inhibit the apoptosis of Schwann cells through the PPAR-γ-miR-124-EZH2/STAT3 axis. In conclusion, PIAS1 promoted SUMOlation of PPAR-γ to stabilize PPAR-γ expression, which upregulated miR-124 to inactivate EZH2/STAT3, thereby inhibiting apoptosis and promoting autophagy of Schwann cells to suppress the development of DPN.

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A in Vivo Assay for Standardizing Heparin Preparations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646810 ◽

1979 ◽

Vol 41 (03) ◽

pp. 576-582

Author(s):

A R Pomeroy

Keyword(s):

In Vitro Assays ◽

British Pharmacopoeia ◽

In Vitro Method ◽

Standard Preparation ◽

Reliable Estimation ◽

Assay Procedure ◽

Accuracy And Precision ◽

Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

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Potentially Thrombogenic Materials in Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647857 ◽

1975 ◽

Vol 33 (03) ◽

pp. 617-631 ◽

Cited By ~ 47

Author(s):

H. S Kingdon ◽

R. L Lundblad ◽

J. J Veltkamp ◽

D. L Aronson

Keyword(s):

Human Plasma ◽

Antithrombin Iii ◽

Factor Ix ◽

In Vitro Assays ◽

Substantial Agreement ◽

Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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