scholarly journals Macrophage Identification In Situ

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1393
Author(s):  
Krisztina Nikovics ◽  
Anne-Laure Favier
Keyword(s):  
Tissue Regeneration ◽  
Reporter Genes ◽  
Cellular Mechanisms ◽  
Comprehensive Overview ◽  
Distinctive Features ◽  
Long Time

Understanding the processes of inflammation and tissue regeneration after injury is of great importance. For a long time, macrophages have been known to play a central role during different stages of inflammation and tissue regeneration. However, the molecular and cellular mechanisms by which they exert their effects are as yet mostly unknown. While in vitro macrophages have been characterized, recent progress in macrophage biology studies revealed that macrophages in vivo exhibited distinctive features. Actually, the precise characterization of the macrophages in vivo is essential to develop new healing treatments and can be approached via in situ analyses. Nowadays, the characterization of macrophages in situ has improved significantly using antigen surface markers and cytokine secretion identification resulting in specific patterns. This review aims for a comprehensive overview of different tools used for in situ macrophage identification, reporter genes, immunolabeling and in situ hybridization, discussing their advantages and limitations.

scholarly journals Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick—Pathogen Interactions

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 70
Author(s):  
Lourdes Mateos-Hernández ◽  
Natália Pipová ◽  
Eléonore Allain ◽  
Céline Henry ◽  
Clotilde Rouxel ◽  
...  
Keyword(s):  
Cell Line ◽  
Peripheral Nerves ◽  
Ixodes Scapularis ◽  
Embryonic Cell ◽  
Cell Subpopulation ◽  
In Vivo Experiments

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick–pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick–pathogen interactions.

The effect of modifying the nanostructure of gelatin fiber scaffolds on early angiogenesis in vitro and in vivo

2021 ◽  
Author(s):  
Yanyi Liu ◽  
Xiaoxue Wang ◽  
Fei Hu ◽  
Xiaohui Rausch-fan ◽  
Thorsten Steinberg ◽  
...  
Keyword(s):  
Tissue Regeneration ◽  
Fiber Diameter ◽  
Early Stage ◽  
Fiber Membrane ◽  
Soft Tissue Regeneration ◽  
Two Factors ◽  
Fiber Scaffolds

Abstract Early angiogenesis is one of the key challenges in tissue regeneration. Crosslinking mode and fiber diameter are critical factors to affect the adhesion and proliferation of cells. However, whether and how these two factors affect early angiogenesis remain largely unknown. To address the issue, the optimal crosslinking mode and fiber diameter of gelatin fiber membrane for early angiogenesis in vivo and in vitro were explored in this work. Compared with the post crosslinked gelatin fiber membrane with the same fiber diameter, the 700 nm diameter in situ crosslinked gelatin fiber membrane was found to have smaller roughness (230.67 ± 19 nm) and stronger hydrophilicity (54.77 ± 1.2°), which were suitable for cell growth and adhesion. Moreover, the in situ crosslinked gelatin fiber membrane with a fiber diameter of 1000 nm had significant advantages in early angiogenesis over the two with fiber diameters of 500 and 700 nm by up-regulating the expression of Ang1, VEGF, and integrin-β1. Our findings indicated that the in situ crosslinked gelatin fiber membrane with a diameter of 1000 nm might solve the problem of insufficient blood supply in the early stage of soft tissue regeneration and has broad clinical application prospects in promoting tissue regeneration.

Platelet-rich gel-incorporated silk scaffold promotes meniscus regeneration in a rabbit total meniscectomy model

2019 ◽  
Vol 14 (8) ◽  
pp. 753-768 ◽  
Author(s):  
Yifan Wu ◽  
Jianqiao Hong ◽  
Guangyao Jiang ◽  
Sihao Li ◽  
Shiming Chen ◽  
...  
Keyword(s):  
Cartilage Degeneration ◽  
Functional Regeneration ◽  
Scaffold Materials ◽  
Silk Scaffold ◽  
Meniscus Regeneration ◽  
Tgf Β1

Aim: To investigate whether platelet-rich gel (PRG) incorporation could promote meniscal regeneration of the silk scaffold. Materials & methods: A PRG-incorporated silk sponge was fabricated for reconstruction of the meniscus in a rabbit meniscectomy model. Subsequently, characterization of the scaffold, as well as the in vitro cytocompatibility and in vivo function was evaluated. Results: Our results showed that the PRG-incorporated silk scaffold provided a sustained release of TGF-β1 over 1 week. The PRG enhanced the cytocompatibility in vitro and cell infiltration in vivo of the silk sponge. Meanwhile, the implantation of the composite in situ ameliorated the cartilage degeneration in knee at 3 months. Conclusion: These findings indicated that PRG-incorporated silk scaffold could promote functional regeneration of the meniscus and effectively prevented subsequent osteoarthritis after meniscectomy.

scholarly journals Truncation of poly-N-acetylglucosamine (PNAG) polymerization with N-acetylglucosamine analogues

2021 ◽  
Author(s):  
Zachary Morrison ◽  
Alexander Eddenden ◽  
Adithya S Subramanian ◽  
P. Lynne Howell ◽  
mark nitz
Keyword(s):  
Biofilm Formation ◽  
Chain Termination ◽  
Salvage Pathway ◽  
Biofilm Inhibition ◽  
E Coli ◽  
Substrate Analogues

Bacteria require polysaccharides for structure, survival, and virulence. Despite the central role these structures play in microbiology few tools are available to manipulate their production. In E. coli the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N3, SH, NH2) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro the inhibitors cause PNAG chain termination; consistent with the mechanism of PNAG polymerization from the non-reducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

scholarly journals In Vivo Imaging-Driven Approaches to Study Virus Dissemination and Pathogenesis

2019 ◽  
Vol 6 (1) ◽  
pp. 501-524 ◽  
Author(s):  
Pradeep D. Uchil ◽  
Kelsey A. Haugh ◽  
Ruoxi Pi ◽  
Walther Mothes
Keyword(s):  
Cell Types ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Causative Agents ◽  
Long Time ◽  
Approaches To Study ◽  
Living Organisms

Viruses are causative agents for many diseases and infect all living organisms on the planet. Development of effective therapies has relied on our ability to isolate and culture viruses in vitro, allowing mechanistic studies and strategic interventions. While this reductionist approach is necessary, testing the relevance of in vitro findings often takes a very long time. New developments in imaging technologies are transforming our experimental approach where viral pathogenesis can be studied in vivo at multiple spatial and temporal resolutions. Here, we outline a vision of a top-down approach using noninvasive whole-body imaging as a guide for in-depth characterization of key tissues, physiologically relevant cell types, and pathways of spread to elucidate mechanisms of virus spread and pathogenesis. Tool development toward imaging of infectious diseases is expected to transform clinical diagnosis and treatment.

scholarly journals In Situ Characterization of Hfq Bacterial Amyloid: A Fourier-Transform Infrared Spectroscopy Study

Pathogens ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 36 ◽  
Author(s):  
David Partouche ◽  
Valeria Militello ◽  
Andrea Gomez-Zavaglia ◽  
Frank Wien ◽  
Christophe Sandt ◽  
...  
Keyword(s):  
Self Assembly ◽  
Transcriptional Level ◽  
In Situ Characterization ◽  
Bacterial Adaptation ◽  
Amyloid A

Hfq is a bacterial protein that regulates gene expression at the post-transcriptional level in Gram-negative bacteria. We have previously shown that Escherichia coli Hfq protein, and more precisely its C-terminal region (CTR), self-assembles into an amyloid-like structure in vitro. In the present work, we present evidence that Hfq unambiguously forms amyloid structures also in vivo. Taking into account the role of this protein in bacterial adaptation and virulence, our work opens possibilities to target Hfq amyloid self-assembly and cell location, with important potential to block bacterial adaptation and treat infections.

scholarly journals Success Criteria for Preclinical Testing of Cell-Instructive Hydrogels for Tendon Regeneration

2020 ◽  
Author(s):  
Ryan C. Locke ◽  
Eden M. Ford ◽  
Karin G. Silbernagel ◽  
April M. Kloxin ◽  
Megan L. Killian
Keyword(s):  
Structural Properties ◽  
Tissue Regeneration ◽  
Tendon Repair ◽  
Tendon Healing ◽  
Preclinical Research ◽  
Success Criteria ◽  
Synthetic Hydrogels

ABSTRACTTendon injuries are difficult to heal in part because intrinsic tendon healing, which is dominated by scar tissue formation, does not effectively regenerate the native structure and function of healthy tendon. Further, many current treatment strategies also fall short of producing regenerated tendon with the native properties of healthy tendon. There is increasing interest in the use of cell-instructive strategies to limit the intrinsic fibrotic response following injury and improve the regenerative capacity of tendon in vivo. We have established multi-functional, cell-instructive hydrogels for treating injured tendon that afford tunable control over the biomechanical, biochemical, and structural properties of the cell microenvironments. Specifically, we incorporated integrin-binding domains (RGDS) and assembled multi-functional collagen mimetic peptides (mfCMPs) that enable cell adhesion and elongation of stem cells within synthetic hydrogels of designed biomechanical properties and evaluated these materials using targeted success criteria developed for testing in mechanically-demanding environments like tendon healing. The in vitro and in situ success criteria were determined based on systematic reviews of the most commonly reported outcome measures of hydrogels for tendon repair and established standards for testing of biomaterials. We then showed, using validation experiments, that multi-functional and synthetic hydrogels meet these criteria. Specifically, these hydrogels have mechanical properties comparable to developing tendon; are non-cytotoxic both in 2D bolus exposure (hydrogel components) and 3D encapsulation (full hydrogel); are formed, retained, and visualized within tendon defects over time (two-weeks); and provide mechanical support to tendon defects at the time of injection and in situ formation. Ultimately, the in vitro and in situ success criteria evaluated in this study were designed for preclinical research to rigorously test the potential to achieve successful tendon repair prior to in vivo testing and indicate the promise of multi-functional and synthetic hydrogels for continued translation.IMPACT STATEMENTTendon healing results in a weak scar that forms due to poor cell-mediated repair of the injured tissue. Treatments that tailor the instructions experienced by cells during healing afford opportunities to regenerate the healthy tendon. Engineered cell-instructive cues, including the biomechanical, biochemical, and structural properties of the cell microenvironment, within multi-functional synthetic hydrogels are promising therapeutic strategies for tissue regeneration. In this paper, the preclinical efficacy of multi-functional synthetic hydrogels for tendon repair is tested against rigorous in vitro and in situ success criteria. This study indicates the promise for continued preclinical translation of synthetic hydrogels for tissue regeneration.

scholarly journals Characterization of Three XylT-Like [2Fe-2S] Ferredoxins Associated with Catabolism of Cresols or Naphthalene: Evidence for Their Involvement in Catechol Dioxygenase Reactivation

2000 ◽  
Vol 182 (19) ◽  
pp. 5580-5585 ◽  
Author(s):  
N. Hugo ◽  
C. Meyer ◽  
J. Armengaud ◽  
J. Gaillard ◽  
K. N. Timmis ◽  
...  
Keyword(s):  
Biochemical Properties ◽  
Ring Cleavage ◽  
Redox Potentials ◽  
Catechol Dioxygenase ◽  
Distinctive Features ◽  
Wild Type Phenotype ◽  
Spectroscopic Characteristics

ABSTRACT The xylT gene product, a component of the xylene catabolic pathway of Pseudomonas putida mt2, has been recently characterized as a novel [2Fe-2S] ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in Escherichia coli, purified, and compared with respect to their biochemical properties and interaction with XylE. The three XylT analogues show general spectroscopic characteristics common to plant-type [2Fe-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were −286 mV and −323 mV, respectively. Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT. The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. A polypeptide band with an M r of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases. The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenase-ferredoxin interaction. In complementation experiments, plasmids expressing xylT or its analogues were introduced into an XylT-null mutant of P. putida which is unable to grow on p-methylbenzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.

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