Optical Nanomotion Detection to Rapidly Discriminate between Fungicidal and Fungistatic Effects of Antifungals on Single-Cell .

is an emerging pathogen that poses a significant challenge due to its multidrug-resistant nature. There are two types of antifungal agents, fungicidal and fungistatic, with distinct mechanisms of action against fungal pathogens. Fungicidal agents kill fungal pathogens, whereas fungistatic agents inhibit their growth.

A simplified version of rapid susceptibility testing of bacteria and yeasts using optical nanomotion detection.

We present a novel optical nanomotion-based rapid antibiotic and antifungal susceptibility test. The technique consisted of studying the effects of antibiotics or antifungals on the nanometric scale displacements of bacteria or yeasts to assess their sensitivity or resistance to drugs. The technique relies on a traditional optical microscope, a video camera, and custom-made image analysis software.

Machine learning method for the classification of the state of living organisms' oscillations.

The World Health Organization highlights the urgent need to address the global threat posed by antibiotic-resistant bacteria. Efficient and rapid detection of bacterial response to antibiotics and their virulence state is crucial for the effective treatment of bacterial infections. However, current methods for investigating bacterial antibiotic response and metabolic state are time-consuming and lack accuracy.

Simple optical nanomotion method for single-bacterium viability and antibiotic response testing.

Antibiotic resistance is nowadays a major public health issue. Rapid antimicrobial susceptibility tests (AST) are one of the options to fight this deadly threat. Performing AST with single-cell sensitivity that is rapid, cheap, and widely accessible, is challenging.

Mitochondrial nanomotion measured by optical microscopy.

Nanometric scale size oscillations seem to be a fundamental feature of all living organisms on Earth. Their detection usually requires complex and very sensitive devices. However, some recent studies demonstrated that very simple optical microscopes and dedicated image processing software can also fulfill this task.

Modulation of the nanoscale motion rate of by X-rays.

Introduction: Patients undergoing cancer treatment by radiation therapy commonly develop infections (candidiasis). Such infections are generally treated by antifungals that unfortunately also induce numerous secondary effects in the patient. Additional to the effect on the immune system, ionizing radiation influences the vital activity of cells themselves; however, the reaction of to ionizing radiation acting simultaneously with antifungals is much less well documented.

Fast Self-Assembly Dynamics of a β-Sheet Peptide Soft Material.

Peptide-based hydrogels are promising biocompatible materials for wound healing, drug delivery, and tissue engineering applications. The physical properties of these nanostructured materials depend strongly on the morphology of the gel network. However, the self-assembly mechanism of the peptides that leads to a distinct network morphology is still a subject of ongoing debate, since complete assembly pathways have not yet been resolved.

High-Speed Atomic Force Microscopy Visualization of Protein-DNA Interactions Using DNA Origami Frames.

Direct, live imaging of protein-DNA interactions under physiological conditions is invaluable for understanding the mechanism and kinetics of binding and understanding the topological changes of the DNA strand. The DNA origami technology allows for precise placement of target molecules in a designed nanostructure. Here, we describe a protocol for the self-assembly of DNA origami frames with 2 stretched DNA sequences containing the binding site of a transcription factor, i.

Development of bone cell microarrays in microfluidic chips for studying osteocyte-osteoblast communication under fluid flow mechanical loading.

Bone tissue remodels throughout life in response to mechanical loads. Impaired activities of bone cells (osteocytes, osteoblasts and osteoclasts) result in a disruption of the bone remodelling cycle, which eventually leads to bone disorders such as osteoporosis. To develop efficient therapeutic strategies against bone disorders, new tools are needed to unravel the bone remodelling cycle at the molecular level.

The Flo Adhesin Family.

The first step in the infection of fungal pathogens in humans is the adhesion of the pathogen to host tissue cells or abiotic surfaces such as catheters and implants. One of the main players involved in this are the expressed cell wall adhesins. Here, we review the Flo adhesin family and their involvement in the adhesion of these yeasts during human infections.

Nanomotion Detection-Based Rapid Antibiotic Susceptibility Testing.

Rapid antibiotic susceptibility testing (AST) could play a major role in fighting multidrug-resistant bacteria. Recently, it was discovered that all living organisms oscillate in the range of nanometers and that these oscillations, referred to as nanomotion, stop as soon the organism dies. This finding led to the development of rapid AST techniques based on the monitoring of these oscillations upon exposure to antibiotics.

Single yeast cell nanomotions correlate with cellular activity.

Living single yeast cells show a specific cellular motion at the nanometer scale with a magnitude that is proportional to the cellular activity of the cell. We characterized this cellular nanomotion pattern of nonattached single yeast cells using classical optical microscopy. The distribution of the cellular displacements over a short time period is distinct from random motion.

Adhesins of Yeasts: Protein Structure and Interactions.

The ability of yeast cells to adhere to other cells or substrates is crucial for many yeasts. The budding yeast can switch from a unicellular lifestyle to a multicellular one. A crucial step in multicellular lifestyle adaptation is self-recognition, self-interaction, and adhesion to abiotic surfaces.

DNA-Interacting Characteristics of the Archaeal Rudiviral Protein SIRV2_Gp1.

Whereas the infection cycles of many bacterial and eukaryotic viruses have been characterized in detail, those of archaeal viruses remain largely unexplored. Recently, studies on a few model archaeal viruses such as SIRV2 (Sulfolobus islandicus rod-shaped virus) have revealed an unusual lysis mechanism that involves the formation of pyramidal egress structures on the host cell surface. To expand understanding of the infection cycle of SIRV2, we aimed to functionally characterize gp1, which is a SIRV2 gene with unknown function.

Lectin-Glycan Interaction Network-Based Identification of Host Receptors of Microbial Pathogenic Adhesins.

Unlabelled: The first step in the infection of humans by microbial pathogens is their adherence to host tissue cells, which is frequently based on the binding of carbohydrate-binding proteins (lectin-like adhesins) to human cell receptors that expose glycans. In only a few cases have the human receptors of pathogenic adhesins been described. A novel strategy-based on the construction of a lectin-glycan interaction (LGI) network-to identify the potential human binding receptors for pathogenic adhesins with lectin activity was developed.

Molecular mechanism of flocculation self-recognition in yeast and its role in mating and survival.

Unlabelled: We studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism.

Dip-pen nanolithography-assisted protein crystallization.

We demonstrate the use of dip-pen nanolithography (DPN) to crystallize proteins on surface-localized functionalized lipid layer arrays. DOPC lipid layers, containing small amounts of biotin-DOPE lipid molecules, were printed on glass substrates and evaluated in vapor diffusion and batch crystallization screening setups, where streptavidin was used as a model protein for crystallization. Independently of the crystallization system used and the geometry of the lipid layers, nucleation of streptavidin crystals occurred specifically on the DPN-printed biotinylated structures.

Engineering the carbohydrate-binding site of Epa1p from Candida glabrata: generation of adhesin mutants with different carbohydrate specificity.

The N-terminal domain of the Epa1p adhesin from Candida glabrata (N-Epa1p) is a calcium-dependent lectin, which confers the opportunistic yeast the ability to adhere to human epithelial cells. This lectin domain is able to interact with galactosides and, more precisely, with glycan molecules containing the Galβ-1,3-GalNAc disaccharide, also known as the T-antigen. Based on the crystallographic structure of the N-Epa1p domain and the role of the variable loop CBL2 in glycan binding, saturation mutagenesis on some residues of the CBL2 loop was used to increase the binding affinity of N-Epa1p for fibronectin, which was selected as a model of a human glycoprotein.

The mannose-specific lectin domains of Flo1p from Saccharomyces cerevisiae and Lg-Flo1p from S. pastorianus: crystallization and preliminary X-ray diffraction analysis of the adhesin-carbohydrate complexes.

Flo1p and Lg-Flo1p are two cell-wall adhesins belonging to the Flo (flocculation) protein family from the yeasts Saccharomyces cerevisiae and S. pastorianus. The main function of these modular proteins endowed with calcium-dependent lectin activity is to mediate cell-cell adhesion events during yeast flocculation, a process which is well known at the cellular level but still not fully characterized from a molecular perspective.

A universal fixation method based on quaternary ammonium salts (RNAlater) for omics-technologies: Saccharomyces cerevisiae as a case study.

Genomics, transcriptomics, proteomics and fluxomics are powerful omics-technologies that play a major role in today's research. For each of these techniques good sample quality is crucial. Major factors contributing to the quality of a sample is the actual sampling procedure itself and the way the sample is stored directly after sampling.

Cloning, expression, and purification of the N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae in Pichia pastoris.

Saccharomyces cerevisiae flocculation is governed by FLO genes, encoding Flo proteins (flocculins). Flo proteins are cell wall proteins consisting of three domains, sticking out of the cell wall and interacting with other yeast cells using their N-terminal mannose-binding domain. Until recently, flocculation research was focused on the genetic and cellular level.

The epithelial adhesin 1 (Epa1p) from the human-pathogenic yeast Candida glabrata: structural and functional study of the carbohydrate-binding domain.

The yeast Candida glabrata represents the second major cause of clinical candidiasis cases in the world. The ability of this opportunistic pathogen to adhere to human epithelial and endothelial cells relies on the Epa adhesins, a large set of cell-wall proteins whose N-terminal domains are endowed with a calcium-dependent lectin activity. This feature allows the yeast cells to adhere to host cells by establishing multiple interactions with the glycans expressed on their cell membrane.

The N-terminal domain of the Flo11 protein from Saccharomyces cerevisiae is an adhesin without mannose-binding activity.

The expression of the Flo11 flocculin in Saccharomyces cerevisiae offers the cell a wide range of phenotypes, depending on the strain and the environmental conditions. The most important are pseudohyphae development, invasive growth and flocculation. The mechanism of cellular adhesion mediated by Flo11p is not well understood.