Electrophilic, Activation-Free Fluorogenic Reagent for Labeling Bioactive Amines.

Herein we report the preparation of BODIPY mesoionic acid fluorides through a short sequence involving an isocyanide multicomponent reaction as the key synthetic step. These novel BODIPY acid fluorides are water-stable electrophilic reagents that can be used for the fluorescent derivatization of amine-containing biomolecules using mild and activation-free reaction conditions. As a proof of principle, we have labeled the antifungal natamycin and generated a novel fluorogenic probe for imaging a variety of human and plant fungal pathogens, with excellent selectivity over bacterial cells.

Contact-induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae.

Filamentous hyphae of the human pathogen, Candida albicans, invade mucosal layers and medical silicones. In vitro, hyphal tips reorient thigmotropically on contact with small obstacles. It is not known how surface topography is sensed but hyphae lacking the cortical marker, Rsr1/Bud1, are unresponsive. We show that, on surfaces, the morphology of hyphal tips and the position of internal polarity protein complexes are asymmetrically skewed towards the substratum and biased towards the softer of two surfaces. In nano-fabricated chambers, the Spitzenkörper (Spk) responded to touch by translocating across the apex towards the point of contact, where its stable maintenance correlated with contour-following growth. In the rsr1Δ mutant, the position of the Spk meandered and these responses were attenuated. Perpendicular collision caused lateral Spk oscillation within the tip until after establishment of a new growth axis, suggesting Spk position does not predict the direction of growth in C. albicans. Acute tip reorientation occurred only in cells where forward growth was countered by hyphal friction sufficient to generate a tip force of ∼ 8.7 µN (1.2 MPa), more than that required to penetrate host cell membranes. These findings suggest mechanisms through which the organization of hyphal tip growth in C. albicans facilitates the probing, penetration and invasion of host tissue.

In Candida albicans hyphae, Sec2p is physically associated with SEC2 mRNA on secretory vesicles.

Candida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans-Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 µm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth.

The spatial distribution of the exocyst and actin cortical patches is sufficient to organize hyphal tip growth.

In the hyphal tip of Candida albicans we have made detailed quantitative measurements of (i) exocyst components, (ii) Rho1, the regulatory subunit of (1,3)-ß-glucan synthase, (iii) Rom2, the specialized guanine-nucleotide exchange factor (GEF) of Rho1, and (iv) actin cortical patches, the sites of endocytosis. We use the resulting data to construct and test a quantitative 3-dimensional model of fungal hyphal growth based on the proposition that vesicles fuse with the hyphal tip at a rate determined by the local density of exocyst components. Enzymes such as (1,3)-ß-glucan synthase thus embedded in the plasma membrane continue to synthesize the cell wall until they are removed by endocytosis. The model successfully predicts the shape and dimensions of the hyphae, provided that endocytosis acts to remove cell wall-synthesizing enzymes at the subapical bands of actin patches. Moreover, a key prediction of the model is that the distribution of the synthase is substantially broader than the area occupied by the exocyst. This prediction is borne out by our quantitative measurements. Thus, although the model highlights detailed issues that require further investigation, in general terms the pattern of tip growth of fungal hyphae can be satisfactorily explained by a simple but quantitative model rooted within the known molecular processes of polarized growth. Moreover, the methodology can be readily adapted to model other forms of polarized growth, such as that which occurs in plant pollen tubes.

Development of dynamic cell and organotypic skin models, for the investigation of a novel visco-elastic burns treatment using molecular and cellular approaches.

BACKGROUND: Burn injuries are a major cause of morbidity and mortality worldwide. Despite advances in therapeutic strategies for the management of patients with severe burns, the sequelae are pathophysiologically profound, up to the systemic and metabolic levels. Management of patients with a severe burn injury is a long-term, complex process, with treatment dependent on the degree and location of the burn and total body surface area (TBSA) affected. In adverse conditions with limited resources, efficient triage, stabilisation, and rapid transfer to a specialised intensive care burn centre is necessary to provide optimal outcomes. This initial lag time and the form of primary treatment initiated, from injury to specialist care, is crucial for the burn patient. This study aims to investigate the efficacy of a novel visco-elastic burn dressing with a proprietary bio-stimulatory marine mineral complex (MXC) as a primary care treatment to initiate a healthy healing process prior to specialist care. METHODS: A new versatile emergency burn dressing saturated in a >90% translucent water-based, sterile, oil-free gel and carrying a unique bio-stimulatory marine mineral complex (MXC) was developed. This dressing was tested using LabSkin as a burn model platform. LabSkin a novel cellular 3D-dermal organotypic full thickness human skin equivalent, incorporating fully-differentiated dermal and epidermal components that functionally models skin. Cell and molecular analysis was carried out by in vitro Real-Time Cellular Analysis (RTCA), thermal analysis, and focused transcriptomic array profiling for quantitative gene expression analysis, interrogating both wound healing and fibrosis/scarring molecular pathways. In vivo analysis was also performed to assess the bio-mechanical and physiological effects of this novel dressing on human skin. RESULTS: This hybrid emergency burn dressing (EBD) with MXC was hypoallergenic, and improved the barrier function of skin resulting in increased hydration up to 24 h. It was demonstrated to effectively initiate cooling upon application, limiting the continuous burn effect and preventing local tissue from damage and necrosis. xCELLigence RTCA® on primary human dermal cells (keratinocyte, fibroblast and micro-vascular endothelial) demonstrated improved cellular function with respect to tensegrity, migration, proliferation and cell-cell contact (barrier formation) [1]. Quantitative gene profiling supported the physiological and cellular function finding. A beneficial quid pro quo regulation of genes involved in wound healing and fibrosis formation was observed at 24 and 48 h time points. CONCLUSION: Utilisation of this EBD + MXC as a primary treatment is an effective and easily applicable treatment in cases of burn injury, proving both a cooling and hydrating environment for the wound. It regulates inflammation and promotes healing in preparation for specialised secondary burn wound management. Moreover, it promotes a healthy remodelling phenotype that may potentially mitigate scarring. Based on our findings, this EBD + MXC is ideal for use in all pre-hospital, pre-surgical and resource limited settings.

Evaluation of Corneal Cross-Linking for Treatment of Fungal Keratitis: Using Confocal Laser Scanning Microscopy on an Ex Vivo Human Corneal Model.

Purpose: Some previous reports have established the use of photoactivated chromophore-induced corneal cross-linking (PACK-CXL) in treating fungal keratitis. The results of these case reports have often been conflicting. To systematically study the effect of PACK-CXL in the management of Fusarium keratitis, we have developed an ex vivo model of human corneal infection using eye-banked human corneas. Methods: Sixteen healthy ex vivo human corneas were divided into four study groups: (1) untreated control, (2) cross-linked, (3) infected with fungal spores, and (4) infected with fungal spores and then cross-linked. All infected corneas were inoculated with Fusarium oxysporum spores. The PACK-CXL procedure was performed 24 hours post inoculation for group 4. For PACK-CXL treatment, the corneas were debrided of epithelium; then 1% (wt/vol) isotonic riboflavin was applied dropwise at 5-minute intervals for 30 minutes and during the course of UV-A cross-linking for another 30 minutes. The corneas were imaged using a confocal microscope at 48 hours post inoculation, and the Fusarium hyphal volume and spore concentration were calculated. Results: The infected and then cross-linked group had a significantly lower volume of Fusarium hyphae, compared to the infected (P = 0.001) group. In the infected and then cross-linked group there was significant inhibition of Fusarium sporulation compared with the infected (P = 0.007) group. Conclusions: A model of human corneal infection was successfully developed for investigation of the effects of PACK-CXL on fungal keratitis. A treatment regimen of combined UV-A/riboflavin-induced corneal cross-linking appears to be a valuable approach to inhibit the growth and sporulation of Fusarium and suppress the progression of fungal keratitis.

In Candida albicans, phosphorylation of Exo84 by Cdk1-Hgc1 is necessary for efficient hyphal extension.

The exocyst, a conserved multiprotein complex, tethers secretory vesicles before fusion with the plasma membrane; thus it is essential for cell surface expansion. In both Saccharomyces cerevisiae and mammalian cells, cell surface expansion is halted during mitosis. In S. cerevisiae, phosphorylation of the exocyst component Exo84 by Cdk1-Clb2 during mitosis causes the exocyst to disassemble. Here we show that the hyphae of the human fungal pathogen Candida albicans continue to extend throughout the whole of mitosis. We show that CaExo84 is phosphorylated by Cdk1, which is necessary for efficient hyphal extension. This action of Cdk1 depends on the hyphal-specific cyclin Hgc1, the homologue of G1 cyclins in budding yeast. Phosphorylation of CaExo84 does not alter its localization but does alter its affinity for phosphatidylserine, allowing it to recycle at the plasma membrane. The different action of Cdk1 on CaExo84 and ScExo84 is consistent with the different locations of the Cdk1 target sites in the two proteins. Thus this conserved component of polarized growth has evolved so that its phosphoregulation mediates the dramatically different patterns of growth shown by these two organisms.

The Cdc14p phosphatase affects late cell-cycle events and morphogenesis in Candida albicans.

We have characterized the CDC14 gene, which encodes a dual-specificity protein phosphatase in Candida albicans, and demonstrated that its deletion results in defects in cell separation, mitotic exit and morphogenesis. The C. albicans cdc14delta mutants formed large aggregates of cells that resembled those found in ace2-null strains. In cdc14delta cells, expression of Ace2p target genes was reduced and Ace2p did not accumulate specifically in daughter nuclei. Taken together, these results imply that Cdc14p is required for the activation and daughter-specific nuclear accumulation of Ace2p. Consistent with a role in cell separation, Cdc14p was targeted to the septum region during the M-G1 transition in yeast-form cells. Interestingly, hypha-inducing signals abolished the translocation of Cdc14p to the division plate, and this regulation depended on the cyclin Hgc1p, since hgc1delta mutants were able to accumulate Cdc14p in the septum region of the germ tubes. In addition to its role in cytokinesis, Cdc14p regulated mitotic exit, since synchronous cultures of cdc14delta cells exhibited a severe delay in the destruction of the mitotic cyclin Clb2p. Finally, deletion of CDC14 resulted in decreased invasion of solid agar medium and impaired true hyphal growth.