Innovative solutions to sticky situations: Antiadhesive strategies for treating bacterial infections

ABSTRACT Bacterial adherence to host tissue is an essential process in pathogenesis, necessary for invasion and colonization and often required for the efficient delivery of toxins and other bacterial effectors. As existing treatment options for common bacterial infections dwindle, we find ourselves rapidly approaching a tipping point in our confrontation with antibiotic-resistant strains and in desperate need of new treatment options. Bacterial strains defective in adherence are typically avirulent and unable to cause infection in animal models. The importance of this initial binding event in the pathogenic cascade highlights its potential as a novel therapeutic target. This article seeks to highlight a variety of strategies being employed to treat and prevent infection by targeting the mechanisms of bacterial adhesion. Advancements in this area include the development of novel antivirulence therapies using small molecules, vaccines, and peptides to target a variety of bacterial infections. These therapies target bacterial adhesion through a number of mechanisms, including inhibition of pathogen receptor biogenesis, competition-based strategies with receptor and adhesin analogs, and the inhibition of binding through neutralizing antibodies. While this article is not an exhaustive description of every advancement in the field, we hope it will highlight several promising examples of the therapeutic potential of antiadhesive strategies.</jats:p

Label-free composition determination for biomolecular condensates with an arbitrarily large number of components

Biomolecular condensates are membrane-less organelles made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively, as they either require fluorescent labels, which we show can perturb composition, or can distinguish only 1-2 components. Here, we describe a label-free method based on quantitative phase microscopy to measure the composition of condensates with an arbitrarily large number of components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex aging dynamics for condensates composed of full-length proteins. In simplified multi-component protein/RNA condensates, we uncover a regime of constant condensate density and a large range of protein:RNA stoichiometry when varying average composition. The unexpected decoupling of density and composition highlights the need to determine molecular stoichiometry in multi-component condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function

Quantitative phase microscopy enables precise and efficient determination of biomolecular condensate composition

Many compartments in eukaryotic cells are protein-rich biomolecular condensates demixed from the cyto- or nucleoplasm. Although much has been learned in recent years about the integral roles condensates play in many cellular processes as well as the biophysical properties of reconstituted condensates, an understanding of their most basic feature, their composition, remains elusive. Here we combined quantitative phase microscopy (QPM) and the physics of sessile droplets to develop a precise method to measure the shape and composition of individual model condensates. This technique does not rely on fluorescent dyes or tags, which we show can significantly alter protein phase behavior, and requires 1000-fold less material than traditional label-free technologies. We further show that this QPM method measures the protein concentration in condensates to a 3-fold higher precision than the next best label-free approach, and that commonly employed strategies based on fluorescence intensity dramatically underestimate these concentrations by as much as 50-fold. Interestingly, we find that condensed-phase protein concentrations can span a broad range, with PGL3, TAF15(RBD) and FUS condensates falling between 80 and 500 mg/ml under typical in vitro conditions. This points to a natural diversity in condensate composition specified by protein sequence. We were also able to measure temperature-dependent phase equilibria with QPM, an essential step towards relating phase behavior to the underlying physics and chemistry. Finally, time-resolved QPM reveals that PGL3 condensates undergo a contraction-like process during aging which leads to doubling of the internal protein concentration coupled to condensate shrinkage. We anticipate that this new approach will enable understanding the physical properties of biomolecular condensates and their function

How to Organize a Practical Course on Light Sheet Microscopy.

Teaching the principles and practical aspects of light sheet microscopy to the new generation of scientists is paramount for realizing its potential impact on life sciences. In this chapter, we share our experiences on practical courses involving organizing EMBO-sponsored light sheet microscopy at MPI-CBG in Dresden. We describe the challenges and offer solutions for sample mounting, imaging, image analysis, and big data handling of light sheet microscopy. We also share our unique approach to teaching microscopy by performing real science on real samples in an intense course setting. We provide tips for creating an inclusive and productive atmosphere at these exhausting yet exhilarating events

The Dynamics of the Hexavalent Chromium Induced Apoptotic Patterns in vitro

Although hexavalent chromium has been shown to induce apoptosis in cells cultivated in vitro, there appear to be no studies focusing on the dynamics of this process. To find out about dynamic patterns of hexavalent chromium-induced apoptosis, we treated Hep2 cells with 150 μg/ml potassium chromate and recorded their behavior as well as appearance of some crucial organelles using different morphological and biochemical methods. We found that Hep2 cells showed the earliest observable changes at 6 hours after the treatment (blebbing, chromatin shrinkage), with the entire apoptotic process lasting up to 24 hours. While all the observed cell features clearly prove apoptosis induced by hexavalent chromium, a typical apoptotic hallmark, DNA ladder, seems not to occur in this type of cells. On the other hand, in HL-60 cells, used as a control, this ladder was observable.</jats:p

Toxic Effects of Chromium Acetate Hydroxide on Cells Cultivated <i>In Vitro</i>

Many human activities, particularly industrial ones, result in an ever-growing production of toxic waste materials. The dynamics of the toxic effects of chromium acetate hydroxide, which is found in high concentrations in a waste sediment produced in the Czech Republic, were assessed by using a battery of in vitro tests carried out on two cell lines: L-929 (mouse fibroblasts) and Hep 2 (human laryngeal cells). Various markers of cell damage were assessed by phase-contrast, video and fluorescence microscopy, fluorometry, and DNA analysis. Chromium acetate hydroxide, over a concentration range of 1–0.02mol/l induced immediate cell death by fixation, whereas, at 0.002mol/l, the treated cells died in a much slower, more discrete manner. All the detected markers of cell damage, whether immediate or slow, clearly demonstrated that the cells died by necrosis. On the other hand, test concentration of 0.001mol/l appeared to constitute a threshold at which no pathological changes of Hep 2 cells were observed over 96 hours. We conclude that chromium acetate hydroxide has a high toxic potential in vitro, which should be considered when studying the toxicity of waste materials containing it. </jats:p