scholarly journals Ratiometric bioluminescent sensors towards in vivo imaging of bacterial signaling

2019 ◽  
Author(s):  
A. B Dippel ◽  
W. A. Anderson ◽  
J. H. Park ◽  
F. H. Yildiz ◽  
M.C. Hammond
Keyword(s):  
High Throughput Screening ◽  
High Sensitivity ◽  
Second Messenger ◽  
Live Cells ◽  
Diagnostic Tools ◽  
Signaling Networks ◽  
Mammalian Host ◽  
Host Infection

ABSTRACTSecond messenger signaling networks allow cells to sense and adapt to changing environmental conditions. In bacteria, the nearly ubiquitous second messenger molecule cyclic di-GMP coordinates diverse processes such as motility, biofilm formation, and virulence. In bacterial pathogens, these signaling networks allow the bacteria to survive changing environment conditions that are experienced during infection of a mammalian host. While studies have examined the effects of cyclic di-GMP levels on virulence in these pathogens, it has previously not been possible to visualize cyclic di-GMP levels in real time during the stages of host infection. Towards this goal, we generate the first ratiometric, chemiluminescent biosensor scaffold that selectively responds to c-di-GMP. By engineering the biosensor scaffold, a suite of Venus-YcgR-NLuc (VYN) biosensors is generated that provide extremely high sensitivity (KD < 300 pM) and large BRET signal changes (up to 109%). As a proof-of-concept that VYN biosensors can image cyclic di-GMP during host infection, we show that the VYN biosensors function in the context of a tissue phantom model, with only ∼103-104 biosensor-expressing cells required for the measurement. Furthermore, the stable BRET signal suggests that the sensors could be used for long-term imaging of cyclic di-GMP dynamics during host infection. The VYN sensors developed here can serve as robust in vitro diagnostic tools for high throughput screening, as well as genetically encodable tools for monitoring the dynamics of c-di-GMP in live cells, and lay the groundwork for live cell imaging of c-di-GMP dynamics in bacteria during host infection, and other complex environments.

scholarly journals Inhibition of Plasmodium Liver Infection by Ivermectin

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
António M. Mendes ◽  
Inês S. Albuquerque ◽  
Marta Machado ◽  
Joana Pissarra ◽  
Patrícia Meireles ◽  
...  
Keyword(s):  
Control Strategies ◽  
Parasite Development ◽  
Mammalian Host ◽  
Mosquito Vector ◽  
Content Type ◽  
Vector Borne Diseases ◽  
Host Infection ◽  
Liver Infection

ABSTRACT Avermectins are powerful endectocides with an established potential to reduce the incidence of vector-borne diseases. Here, we show that several avermectins inhibit the hepatic stage of Plasmodium infection in vitro. Notably, ivermectin potently inhibits liver infection in vivo by impairing parasite development inside hepatocytes. This impairment has a clear impact on the ensuing blood stage parasitemia, reducing disease severity and enhancing host survival. Ivermectin has been proposed as a tool to control malaria transmission because of its effects on the mosquito vector. Our study extends the effect of ivermectin to the early stages of mammalian host infection and supports the inclusion of this multipurpose drug in malaria control strategies.

scholarly journals Drug Efficacy Comparison of 3D Forming and Preforming Sphere Models with a Micropillar and Microwell Chip Platform

2019 ◽  
Vol 24 (4) ◽  
pp. 476-483 ◽  
Author(s):  
Il Doh ◽  
Yong-Jun Kwon ◽  
Bosung Ku ◽  
Dong Woo Lee
Keyword(s):  
High Throughput Screening ◽  
Drug Efficacy ◽  
High Sensitivity ◽  
Chemotherapeutic Agents ◽  
Cell Culture Model ◽  
Drug Candidates ◽  
Tumor Microenvironments ◽  
Culture Model

Hepatocellular carcinoma (HCC), a major histological subtype of liver cancer, is the third most common cause of cancer-related death worldwide. Currently, many curative standard treatments using target-specific chemotherapeutic agents are being developed. However, drug efficacy tests based on the 2D monolayer cell culture model do not effectively screen the best drug candidates because they do not accurately reflect in vivo tumor microenvironments. Thus, to select the best drug candidates or repositioning drugs, we developed new 3D in vitro hepatic tumor models, including 3D forming and preformed sphere models. A micropillar and microwell chip platform was used for the 3D in vitro liver cell-based model for high-throughput screening. We measured the efficacy of 60 drugs and sorted the most efficacious drugs by comparing the drug response of the 2D monolayer model with the 3D forming and preformed sphere models. Among the 60 drugs, 17 drugs (28.3%) showed a significant high efficacy in the 3D preformed sphere model, while 45 drugs (75%) showed an efficacy in the 2D model. We also calculated the IC50 values of the 17 drugs and found that 7 drugs exhibited a high sensitivity in HCC, which was in agreement with previous studies.

scholarly journals Using Biosensors to Study Protein–Protein Interaction in the Hippo Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Alexander Pipchuk ◽  
Xiaolong Yang
Keyword(s):  
Protein Interactions ◽  
High Throughput Screening ◽  
Cellular Proliferation ◽  
Affinity Purification ◽  
Hippo Pathway ◽  
High Sensitivity ◽  
Live Cells ◽  
Hippo Signaling ◽  
The Hippo Pathway

The Hippo signaling network is dependent on protein–protein interactions (PPIs) as a mechanism of signal transduction to regulate organ size, cellular proliferation and differentiation, tumorigenesis, and other cellular processes. Current efforts aim to resolve the complex regulation of upstream Hippo components or focus on identifying targeted drugs for use in cancer therapy. Despite extensive characterization of the Hippo pathway interactome by affinity purification mass spectrometry (AP-MS) and other methodologies, previous research methods have not been sufficient to achieve these aims. In this review, we describe several recent studies that make use of luciferase-based biosensors as a new approach to study the Hippo Pathway. These biosensors serve as powerful tools with which to study PPIs both in vitro using purified biosensor proteins, and in real time in live cells. Notably, luciferase biosensors have excellent sensitivity and have been used to screen for upstream kinase regulators of the Hippo pathway. Furthermore, the high sensitivity and stability of these biosensors enables their application in high throughput screening for Hippo-targeted chemotherapeutics. Finally, we describe the strengths and weaknesses of this method relative to AP-MS and discuss potential future directions for using biosensors to study Hippo signaling.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

Polyamines and Related Nitrogen Compounds in the Chemotherapy of Neglected Diseases Caused by Kinetoplastids

2018 ◽  
Vol 18 (5) ◽  
pp. 321-368 ◽  
Author(s):  
Juan A. Bisceglia ◽  
Maria C. Mollo ◽  
Nadia Gruber ◽  
Liliana R. Orelli
Keyword(s):  
Drug Targets ◽  
Redox Homeostasis ◽  
Cost Effective ◽  
Structural Features ◽  
Mammalian Host ◽  
Neglected Diseases ◽  
Nitrogen Compounds ◽  
Chemical Structures

Neglected diseases due to the parasitic protozoa Leishmania and Trypanosoma (kinetoplastids) affect millions of people worldwide, and the lack of suitable treatments has promoted an ongoing drug discovery effort to identify novel nontoxic and cost-effective chemotherapies. Polyamines are ubiquitous small organic molecules that play key roles in kinetoplastid parasites metabolism, redox homeostasis and in the normal progression of cell cycles, which differ from those found in the mammalian host. These features make polyamines attractive in terms of antiparasitic drug development. The present work provides a comprehensive insight on the use of polyamine derivatives and related nitrogen compounds in the chemotherapy of kinetoplastid diseases. The amount of literature on this subject is considerable, and a classification considering drug targets and chemical structures were made. Polyamines, aminoalcohols and basic heterocycles designed to target the relevant parasitic enzyme trypanothione reductase are discussed in the first section, followed by compounds directed to less common targets, like parasite SOD and the aminopurine P2 transporter. Finally, the third section comprises nitrogen compounds structurally derived from antimalaric agents. References on the chemical synthesis of the selected compounds are reported together with their in vivo and/or in vitro IC50 values, and structureactivity relationships within each group are analyzed. Some favourable structural features were identified from the SAR analyses comprising protonable sites, hydrophobic groups and optimum distances between them. The importance of certain pharmacophoric groups or amino acid residues in the bioactivity of polyamine derived compounds is also discussed.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

scholarly journals Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Pia Montanucci ◽  
Silvia Terenzi ◽  
Claudio Santi ◽  
Ilaria Pennoni ◽  
Vittorio Bini ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Chemical Properties ◽  
Live Cells ◽  
High Performing ◽  
Physical Chemical ◽  
Degenerative Disorders ◽  
Selection Of ◽  
Better Than

Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation,in vitroandin vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules’ basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

scholarly journals Biological nanoscale fluorescent probes: From structure and performance to bioimaging

2020 ◽  
Vol 39 (1) ◽  
pp. 209-221
Author(s):  
Jiafeng Wan ◽  
Xiaoyuan Zhang ◽  
Kai Zhang ◽  
Zhiqiang Su
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Biological Imaging ◽  
Fluorescent Materials ◽  
And Performance

Abstract In recent years, nanomaterials have attracted lots of attention from researchers due to their unique properties. Nanometer fluorescent materials, such as organic dyes, semiconductor quantum dots (QDs), metal nano-clusters (MNCs), carbon dots (CDs), etc., are widely used in biological imaging due to their high sensitivity, short response time, and excellent accuracy. Nanometer fluorescent probes can not only perform in vitro imaging of organisms but also achieve in vivo imaging. This provides medical staff with great convenience in cancer treatment. Combined with contemporary medical methods, faster and more effective treatment of cancer is achievable. This article explains the response mechanism of three-nanometer fluorescent probes: the principle of induced electron transfer (PET), the principle of fluorescence resonance energy transfer (FRET), and the principle of intramolecular charge transfer (ICT), showing the semiconductor QDs, precious MNCs, and CDs. The excellent performance of the three kinds of nano fluorescent materials in biological imaging is highlighted, and the application of these three kinds of nano fluorescent probes in targeted biological imaging is also introduced. Nanometer fluorescent materials will show their significance in the field of biomedicine.

scholarly journals Streptococcal Adhesion and Colonization

1997 ◽  
Vol 8 (2) ◽  
pp. 175-200 ◽  
Author(s):  
H.F. Jenkinson ◽  
RJ Lamont
Keyword(s):  
Immune Modulation ◽  
Rational Design ◽  
Repeated Sequence ◽  
Host Cells ◽  
Mammalian Host ◽  
Related Factors ◽  
Bacterial Populations ◽  
Acellular Vaccines

Streptococci express arrays of adhesins on their cell surfaces that facilitate adherence to substrates present in their natural environment within the mammalian host. A consequence of such promiscuous binding ability is that streptococcal cells may adhere simultaneously to a spectrum of substrates, including salivary glycoproteins, extracellular matrix and serum components, host cells, and other microbial cells. The multiplicity of streptococcal adherence interactions accounts, at least in part, for their success in colonizing the oral and epithelial surfaces of humans. Adhesion facilitates colonization and may be a precursor to tissue invasion and immune modulation, events that presage the development of disease. Many of the streptococcal adhesins and virulence-related factors are cell-wall-associated proteins containing repeated sequence blocks of amino acids. Linear sequences, both within the blocks and within non-repetitive regions of the proteins, have been implicated in substrate binding. Sequences and functions of these proteins among the streptococci have become assorted through gene duplication and horizontal transfer between bacterial populations. Several adhesins identified and characterized through in vitro binding assays have been analyzed for in vivo expression and function by means of animal models used for colonization and virulence. Information on the molecular structure of adhesins as related to their in vivo function will allow for the rational design of novel acellular vaccines, recombinant antibodies, and adhesion agonists for the future control or prevention of streptococcal colonization and streptococcal diseases.

scholarly journals Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2157-2169 ◽  
Author(s):  
Sudarson Sundarrajan ◽  
Junjappa Raghupatil ◽  
Aradhana Vipra ◽  
Nagalakshmi Narasimhaswamy ◽  
Sanjeev Saravanan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mechanism Of Action ◽  
Catalytic Domain ◽  
Antibiotic Sensitivity ◽  
High Sensitivity ◽  
Common Mechanism ◽  
Cross Bridge ◽  
Sensitivity Pattern

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

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