Conditional Protein Depletion in the Analysis of Antifungal Drug Resistance in Candida glabrata.

This chapter illustrates a method to generate Candida glabrata conditional depletion mutants for SNF2, an ATPase subunit of the SWI/SNF chromatin remodeling complex with potential roles in the response to azole drugs. The strategy employed utilizes a plant-specific proteolysis pathway which allows for the rapid degradation of a target protein in the presence of the phytohormone, auxin. The steps taken to generate strains expressing the auxin-inducible plant F-box protein, Tir1, and in which the auxin-binding target, IAA17, is C-terminally fused to Snf2 are described. This acute depletion strategy is suitable for studying the effects of the loss of growth-critical proteins. The rapid depletion afforded by the auxin-induced degradation avoids the potential complications of a null allele causing a severe growth defect and allows a more rapid assessment of the consequences of reduced levels of a protein of interest.

Role of the WOR1 Promoter of Candida albicans in Opaque Commitment.

During induced differentiation, the process often involves a commitment event, after which induced cells, when returned to noninducing conditions, continue to differentiate. The commitment event is rarely identified. Candida albicans differentiates from the white to opaque phenotype, a prerequisite for mating and a process accompanying colonization of the lower gastrointestinal tract and skin. In analyses of white cell populations induced to synchronously differentiate from the white to opaque phenotype, opaque commitment occurs at approximately the same time as evagination and chitin ring formation in the process of daughter cell formation, several hours after the master switch gene WOR1 is upregulated. Mutational analyses of transcription factor binding regions P1, P2, P3, P4, and P6 of the WOR1 promoter reveal that individual deletion of any of the five transcription factor binding regions does not eliminate morphological differentiation to the opaque cell phenotype under opaque-inducing conditions, but individual deletion of P2, P3, or P4, blocks opaque commitment and maintenance of the opaque phenotype after transition to noninducing conditions. These results suggest that commitment occurs at the level of the WOR1 promoter and that morphological differentiation can be dissociated from phenotypic commitment. IMPORTANCE Candida albicans, the most pervasive fungal pathogen colonizing humans, undergoes a phenotypic transition between a white and opaque phenotype. The unique opaque phenotype is necessary for mating and colonization of the lower gastrointestinal tract. Wor1, a transcription factor (TF), plays a central role in activating this transition. Under physiological conditions that induce mass conversion from white to opaque in vitro, cells commit to the opaque phenotype at the time of evagination to form a daughter cell, but several hours after upregulation of WOR1 expression. By analyzing deletion derivatives of the WOR1 promoter, we demonstrate that three of five regulatory regions of WOR1 that bind TFs involved with the regulation of the phenotypic switch are individually required for commitment to the opaque phenotype, but are not necessary for expressing the opaque phenotype. These results demonstrate that morphological differentiation can be dissociated from phenotypic commitment and that commitment occurs at the level of the WOR1 promoter.

The Use of High-Flow Nasal Cannula and the Timing of Safe Feeding in Children with Bronchiolitis.

Objective The use of high-flow nasal cannula (HFNC) as non-invasive respiratory support in children with bronchiolitis has increased over the last several years. Several studies have investigated enteral feeding safety while on HFNC. This study compares the safety of oral feeding prior to and following implementation of an HFNC feeding guideline. Patients and methods A retrospective study was designed, in children ≤2 years of age with bronchiolitis, requiring HFNC, from 2017 to 2019. We defined feeding complications on HFNC and defined safety as the absence of such complications. We gathered the following data: oral feeding timing from the HFNC initiation, duration of enteral feeding on HFNC, and HFNC flow rate at which the feeding was initiated. We compare the data prior to and post-implementation of an HFNC feeding guideline. Results Descriptive statistics were calculated separately by pre and post guideline implementation. Patients in both pre and post guideline implementation groups had no feeding complications on HFNC. Subjects in the post (n=50) vs. pre-guideline implementation (n=36) had a higher median amount of liters flow when initiating enteral feeding (8.0 vs. 6.0 respectively, p<0.024), spent fewer days in the pediatric intensive care unit (PICU) (two days vs. 0 days). Post guideline implementation, enteral feeding was initiated sooner (days nil per os [NPO] 1.0 vs 2.0). No other significant differences between the two cohorts with respect to other variables were observed.  Conclusions Our data supports that oral feeding in patients with bronchiolitis on HFNC is safe. Utilization of current guidelines allowed safe earlier feeding of children on HFNC, reducing the time spent NPO.

Reciprocal signaling and direct physical interactions between fibroblasts and breast cancer cells in a 3D environment.

Tumorigenic cells undergo cell aggregation and aggregate coalescence in a 3D Matrigel environment. Here, we expanded this 3D platform to assess the interactions of normal human dermal fibroblasts (NHDFs) and human primary mammary fibroblasts (HPMFs) with breast cancer-derived, tumorigenic cells (MDA-MB-231). Medium conditioned by MDA-MB-231 cells activates both types of fibroblasts, imbuing them with the capacity to accelerate the rate of aggregation and coalescence of MDA-MB-231 cells more than four fold. Acceleration is achieved 1) by direct physical interactions with MDA-MB-231 cells, in which activated fibroblasts penetrate the MDA-MB-231/Matrigel 3D environment and function as supporting scaffolds for MDA-MB-231 aggregation and coalescence, and 2) through the release of soluble accelerating factors, including matrix metalloproteinase (MMPs) and, in the case of activated NHDFs, SDF-1α/CXCL12. Fibroblast activation includes changes in morphology, motility, and gene expression. Podoplanin (PDPN) and fibroblast activation protein (FAP) are upregulated by more than nine-fold in activated NHDFs while activated HPMFs upregulate FAP, vimentin, desmin, platelet derived growth factor receptor A and S100A4. Overexpression of PDPN, but not FAP, in NHDF cells in the absence of MDA-MB-231-conditioned medium, activates NHDFs. These results reveal that complex reciprocal signaling between fibroblasts and cancer cells, coupled with their physical interactions, occurs in a highly coordinated fashion that orchestrates aggregation and coalescence, behaviors specific to cancer cells in a 3D environment. These in vitro interactions may reflect events involved in early tumorigenesis, particularly in cases of field cancerization, and may represent a new mechanism whereby cancer-associated fibroblasts (CAFs) promote tumor growth.

Roles of the Transcription Factors Sfl2 and Efg1 in White-Opaque Switching in a/α Strains of Candida albicans.

Candida albicans remains the most pervasive fungal pathogen colonizing humans. The majority of isolates from hosts are heterozygous at the mating type locus (MTLa/α), and a third of these have recently been shown to be capable of switching to the opaque phenotype. Here we have investigated the roles of two transcription factors (TFs) Sfl2 and Efg1, in repressing switching in a/α strains. Deleting either gene results in the capacity of a/α cells to switch to opaque en masse under facilitating environmental conditions, which include N-acetylglucosamine (GlcNAc) as the carbon source, physiological temperature (37°C), and high CO2 (5%). These conditions are similar to those in the host. Our results further reveal that while glucose is a repressor of sfl2Δ and efg1Δ switching, GlcNAc is an inducer. Finally, we show that when GlcNAc is the carbon source, and the temperature is low (25°C), the efg1Δ mutants, but not the sfl2Δ mutants, form a tiny, elongate cell, which differentiates into an opaque cell when transferred to conditions optimal for a/α switching. These results demonstrate that at least two TFs, Sfl2 and Efg1, repress switching in a/α cells and that a/α strains with either an sfl2Δ or efg1Δ mutation can switch en masse but only under physiological conditions. The role of opaque a/α cells in commensalism and pathogenesis must, therefore, be investigated.IMPORTANCE More than 95% of Candida albicans strains isolated from humans are MTLa/α, and approximately a third of these can undergo the white-to-opaque transition. Therefore, besides being a requirement for MTL-homozygous strains to mate, the opaque phenotype very likely plays a role in the commensalism and pathogenesis of nonmating, a/α populations colonizing humans.

Generating a Battery of Monoclonal Antibodies Against Firefly Luciferase for Dot Blot Analysis, Western Blot Analysis, and Immunostaining of Cells in Culture and Paraffin Sections.

Firefly luciferase (FLuc) is commonly used as a reporter gene PpyLuc1 in bioanalytical assays. We have produced five mouse-derived monoclonal antibodies (mAbs) that recognize FLuc. The mAbs, DSHB-LUC-2, DSHB-LUC-3, DSHB-LUC-9, DSHB-LUC-16, and DSHB-LUC-24, were generated by immunizing mice with purified 6xHIS-tagged FLuc (6xHis-FLuc) in suspension with an adjuvant. All five were validated by dot blots. Four of the mAbs provided strong signals in western blot analysis, and one a weak signal. All five were validated for immunostaining in fixed cell culture. Only one stained cells embedded in paraffin. The five mAbs are available at cost through the Developmental Studies Hybridoma Bank (DSHB), a nonprofit National Resource created by the National Institutes of Health.