Exploiting diverse chemical collections to uncover novel antifungals

The rise in drug resistance amongst pathogenic fungi, paired with the limited arsenal of antifungals available is an imminent threat to our medical system. To address this, we screened two distinct compound libraries to identify novel strategies to expand the antifungal armamentarium. The first collection wasthe RIKEN Natural Product Depository (NPDepo), which was screened for antifungal activity against four major human fungal pathogens: Candida albicans, Candida glabrata, Candida auris, and Cryptococcus neoformans. Through a prioritization pipeline, one compound, NPD6433, emerged as having broad-spectrum antifungal activity and minimal mammalian cytotoxicity. Chemical-genetic and biochemical assays demonstrated that NPD6433 inhibits the essential fungal enzyme fatty acid synthase 1 (Fas1). Treatment with NPD6433 inhibited various virulence traits in C. neoformans and C. auris, and rescued mammalian cell growth in a co-culture model with C. auris. The second compound library screened was adiversity-oriented collectionfrom Boston University. This chemical screen was focused on identifying novel molecules that enhance the activity of the widely deployed antifungal, fluconazole, against C. auris. Through this endeavour, we discovered a potent compound that enhanced fluconazole efficacy against C. auris through increasing azole intracellular accumulation. This activity was dependent on expression of the multidrug transporter geneCDR1, suggesting that this compound targets efflux mechanisms. Furthermore, this molecule significantly reduced fungal burden alone and in combination with fluconazole in a murine model of C. auris disseminated infection. Overall, this work identifies novel compounds with bioactivity against fungal pathogens, revealing important biology, and paving the way for the critical development of therapeutic strategies.

Physicochemical Investigation of Biosynthesis of a Protein Coating on Glass That Promotes Mammalian Cell Growth Using Lactobacillus rhamnosus GG Bacteria

Glass surfaces, although the first to be used for culturing ex vivo adherent cells, are not the perfect substrates for this purpose. Today, plastics dominate these applications, but in light of the global trend to reduce the use of synthetic polymers, it is reasonable to consider a return to glass vessels with coatings for these purposes. The ideal surface for cell growth is one that simulates the composition and structure of the mainly protein-based intercellular matrix. The work presented here shows a new idea of preparing porous protein coatings on glass using biosynthesis. The process utilizes the colonization of the gold nanoparticle-coated glass surface with Lactobacillus rhamnosus GG bacteria, followed by permeabilization (using ethanol) of their membrane and partial thermal degradation (at 160 °C in vacuum) of the surface-bound protein components of these microorganisms. It results in a development of coating on the glass that promotes mammalian cell growth, which has been preliminary confirmed using Vero cells. Subsequent steps in the formation of coating components were documented by reflectance ultraviolet and visible spectra and infrared spectroscopy. The presence of microorganisms and mammalian cells was confirmed using scanning electron and optical microscopy and crystalline violet staining.

Seaweed cellulose scaffolds derived from green macroalgae for tissue engineering

Extracellular matrix (ECM) provides structural support for cell growth, attachments and proliferation, which greatly impact cell fate. Marine macroalgae species Ulva sp. and Cladophora sp. were selected for their structural variations, porous and fibrous respectively, and evaluated as alternative ECM candidates. Decellularization–recellularization approach was used to fabricate seaweed cellulose-based scaffolds for in-vitro mammalian cell growth. Both scaffolds were confirmed nontoxic to fibroblasts, indicated by high viability for up to 40 days in culture. Each seaweed cellulose structure demonstrated distinct impact on cell behavior and proliferation rates. The Cladophora sp. scaffold promoted elongated cells spreading along its fibers’ axis, and a gradual linear cell growth, while the Ulva sp. porous surface, facilitated rapid cell growth in all directions, reaching saturation at week 3. As such, seaweed-cellulose is an environmentally, biocompatible novel biomaterial, with structural variations that hold a great potential for diverse biomedical applications, while promoting aquaculture and ecological agenda.

Polyamine Homeostasis in Development and Disease

Polycationic polyamines are present in nearly all living organisms and are essential for mammalian cell growth and survival, and for development. These positively charged molecules are involved in a variety of essential biological processes, yet their underlying mechanisms of action are not fully understood. Several studies have shown both beneficial and detrimental effects of polyamines on human health. In cancer, polyamine metabolism is frequently dysregulated, and elevated polyamines have been shown to promote tumor growth and progression, suggesting that targeting polyamines is an attractive strategy for therapeutic intervention. In contrast, polyamines have also been shown to play critical roles in lifespan, cardiac health and in the development and function of the brain. Accordingly, a detailed understanding of mechanisms that control polyamine homeostasis in human health and disease is needed to develop safe and effective strategies for polyamine-targeted therapy.

The silk of gorse spider mite Tetranychus lintearius represents a novel natural source of nanoparticles and biomaterials

Spider mites constitute an assemblage of well-known pests in agriculture, but are less known for their ability to spin silk of nanoscale diameters and high Young’s moduli. Here, we characterize silk of the gorse spider mite Tetranychus lintearius, which produces copious amounts of silk with nano-dimensions. We determined biophysical characteristics of the silk fibres and manufactured nanoparticles and biofilm derived from native silk. We determined silk structure using attenuated total reflectance Fourier transform infrared spectroscopy, and characterized silk nanoparticles using field emission scanning electron microscopy. Comparative studies using T. lintearius and silkworm silk nanoparticles and biofilm demonstrated that spider mite silk supports mammalian cell growth in vitro and that fluorescently labelled nanoparticles can enter cell cytoplasm. The potential for cytocompatibility demonstrated by this study, together with the prospect of recombinant silk production, opens a new avenue for biomedical application of this little-known silk.

Potential of Graphene Nanodots in Cellular Imaging and Raman Mapping

Highly-dispersed graphene nanodots (GNDs) up to 10[Formula: see text]nm are synthesized using D-galactose as a carbon precursor. The GNDs are self-passivized, emit dual-color excitation-dependent emission and highly biocompatible in cell lines. Their applicability is demonstarted for cellular bioimaging and Raman mapping of cells. Furthermore, their implications on mammalian cell growth and plant seed germination are expounded.

Cheetah: a computational toolkit for cybergenetic control

Advances in microscopy, microfluidics and optogenetics enable single-cell monitoring and environmental regulation and offer the means to control cellular phenotypes. The development of such systems is challenging and often results in bespoke setups that hinder reproducibility. To address this, we introduce Cheetah – a flexible computational toolkit that simplifies the integration of real-time microscopy analysis with algorithms for cellular control. Central to the platform is an image segmentation system based on the versatile U-Net convolutional neural network. This is supplemented with functionality to robustly count, characterise and control cells over time. We demonstrate Cheetah’s core capabilities by analysing long-term bacterial and mammalian cell growth and by dynamically controlling protein expression in mammalian cells. In all cases, Cheetah’s segmentation accuracy exceeds that of a commonly used thresholding-based method, allowing for more accurate control signals to be generated. Availability of this easy-to-use platform will make control engineering techniques more accessible and offer new ways to probe and manipulate living cells.

Non-invasive cell counting of adherent, suspended and encapsulated mammalian cells using optical density

In situ measurement to determine mammalian cell number in a non-invasive, non-destructive and reagent-free manner is needed to enable continuous cell manufacturing. An analytical method is presented for non-invasive cell counting by conducting multiwavelength spectral analysis of mammalian cells achieving a minimal detectable cell count of 62,500 at 295 nm. Light absorbance was insensitive to culture volume, giving an absolute cell count rather than a concentration. The activation state of cells was also considered. The study was extended to quantification within polymeric microcapsules as an advanced substrate for mammalian cell growth in bioreactor formats and resulted in an offset directly correlating with the absorbance maxima of the polymer. These studies provide feasibility for optical density as a simple end point to indirectly quantify mammalian cell number for continuous monitoring of cell cultures.