Biology subjects

Culotta, V. C.

Publications and source records attributed to Culotta, V. C..

3 recordsLinked to original sources

bioRxiv2025

Multifunctional bending magnet beamline with a capillary optic for X-ray fluorescence studies of metals in tissue sections

Scanning fluorescence X-ray microscopy lets one non-destructively and quantitatively map the distribution of most biologically-important metals in cells and tissues. For studies on large-scale tissues and organs, a spatial resolution of several micrometers is often sufficient; in this case, bending magnets at synchrotron light sources provide abundant X-ray flux. We describe here the use of bending magnet beamline 8-BM-B at the Advanced Photon Source (APS) with two distinct microscopy stations: a pre-existing one with Kirkpatrick-Baez (KB) mirror optics for slightly higher throughput and the ability to accommodate samples tens of centimeters across, and a new prototype station with an axially-symmetric, single-bounce, capillary optic with slightly less flux, but slightly higher fluence (which affects achievable resolution at low metal concentration) and higher spatial resolution. The KB station provides{delta} res = 10.5 {micro}m spatial resolution at a per-pixel exposure time of tdwell = 100 ms and a fluence per time of 5.8x 107 photons /({micro}m2 {middle dot}s), while the prototype capillary station provides{delta} res = 6.3 {micro}m at tdwell = 50 ms and a fluence per time of 6.1x 107 photons ({micro}m2 {middle dot}s). We used image power spectral density to estimate the achieved spatial resolution{delta} res from individually acquired images, with{delta} res depending-on the optic, the fluorescence signal strength of the sample being imaged, and the method used to process raw fluorescence spectral data.

biophysics↗

bioRxiv2024

Optimized CRISPR inhibition and activation opens key avenues for systematic biological exploration in zebrafish.

The application of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) technologies in zebrafish has the potential to expand its capacity for the study of gene function significantly. We report proof-of-principle data evaluating transient expression of a codon optimized CRISPRi/a system for zebrafish across established pigmentary and growth phenotypes. A codon-optimized and catalytically inactive cas9 gene (dcas9) was cloned upstream of codon-optimized Kruppel associated box (KRAB) and methyl-CpG binding protein 2 (MeCP2) for CRISPRi, and VP64 for CRISPRa. To validate CRISPRi, we targeted key genes in melanocyte differentiation (sox10 and mitfa); and melanin production (tyrosinase; tyr). Microinjection of CRISPRi mRNA and single guide RNAs (sgRNAs) targeting the tyr promoter or 5-UTR resulted in larvae with hypopigmented epidermal melanocytes. Similarly, CRISPRi targeting of the sox10 or mitfa promoters results in hypopigmentation of epidermal melanocytes consistent with their roles upstream of tyr, and the role of sox10 in activation of mitfa. Finally, we tested both CRISPRi/a to modulate a single gene to yield hypomorphic and hypermorphic effects, selecting mrap2a as our target. This gene regulates energy homeostasis and somatic growth via inhibition of the melanocortin 4 receptor gene (mc4r). We show that inactivating or activating mrap2a with CRISPRi/a significantly decreases or increases larval body length, respectively. We demonstrate that CRISPRi/a can modulate control of zebrafish gene expression, facilitating efficient assay of candidate gene function and disease relevance.

genetics↗

bioRxiv2018

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.

microbiology↗