Uncovering the Role of OVOL1 in Placental Stem Cell Differentiation Using Saccharomyces cerevisiae

OVOL1 is a conserved transcription factor involved in regulating cytrophoblast differentiation in the placenta. Our objective for this study is to use Saccharomyces cerevisiae to uncover the role of OVOL1 in placental stem cell differentiation and proliferation. Previous research suggests that OVOL1 regulates cytotrophoblast progenitor state by regulating genome acetylation. Therefore, our study aims to determine how OVOL1 effect yeast growth and the yeast acetylome, and to use the yeast model to determine downstream targets of OVOL1. In order to understand the role of OVOL1, we will develop a yeast model and employ growth assays to assess growth defects and mass spectroscopy to assess protein acetylation. We will also identify genetic targets of OVOL1 by performing a genome-wide screen. Lastly, the interaction of OVOL1 with other proteins involved in gene repression will be analyzed using co-immunoprecipitation, and chromatin immunoprecipitation. Our results indicate that the presence of OVOL1 confers a growth defect upon yeast cells, and this defect is further exasperated when histone acetyltransferases (HAT) are deleted out of the yeast genome. However, when histone deacetylases (HDAC) are deleted in the presence of OVOL1, normal growth of yeast cells are restored. Our results indicate that there is an interaction between OVOL1 proteins and HDAC, and this could regulate growth and differentiation in the cytotrophoblasts

Fibre-based fluorescence-lifetime imaging microscopy:a real-time biopsy guidance tool for suspected lung cancer

Lung cancer is the most common cause of cancer-related deaths worldwide. Early detection improves outcomes, however, existing sampling techniques are associated with suboptimal diagnostic yield and procedure-related complications. Autofluorescence-based fluorescence-lifetime imaging microscopy (FLIM), a technique which measures endogenous fluorophore decay rates, may aid identification of optimal biopsy sites in suspected lung cancer. Our fibre-based fluorescence-lifetime imaging system, utilising 488 nm excitation, which is deliverable via existing diagnostic platforms, enables real-time visualisation and lifetime analysis of distal alveolar lung structure. We evaluated the diagnostic accuracy of the fibre-based fluorescence-lifetime imaging system to detect changes in fluorescence lifetime in freshly resected ex vivo lung cancer and adjacent healthy tissue as a first step towards future translation. The study compares paired non-small cell lung cancer (NSCLC) and non-cancerous tissues with gold standard diagnostic pathology to assess the performance of the technique. Paired NSCLC and non-cancerous lung tissues were obtained from thoracic resection patients (N=21). A clinically compatible 488 nm fluorescence-lifetime endomicroscopy platform was used to acquire simultaneous fluorescence intensity and lifetime images. Fluorescence lifetimes were calculated using a computationally-lightweight, rapid lifetime determination method. Fluorescence lifetime was significantly reduced in ex vivo lung cancer, compared with non-cancerous lung tissue [mean ± standard deviation (SD), 1.79±0.40 vs. 2.15±0.26 ns, P&lt;0.0001], and fluorescence intensity images demonstrated distortion of alveolar elastin autofluorescence structure. Fibre-based fluorescence-lifetime imaging demonstrated good performance characteristics for distinguishing lung cancer, from adjacent non-cancerous tissue, with 81.0% sensitivity and 71.4% specificity. Our novel fibre-based fluorescence-lifetime imaging system, which enables label-free imaging and quantitative lifetime analysis, discriminates ex vivo lung cancer from adjacent healthy tissue. This minimally invasive technique has potential to be translated as a real-time biopsy guidance tool, capable of optimising diagnostic accuracy in lung cancer. </p

Fibroblast Activation Protein specific optical imaging in Non-Small Cell Lung Cancer

Fibroblast activation protein (FAP) is a cell surface propyl-specific serine protease involved in the regulation of extracellular matrix. Whilst expressed at low levels in healthy tissue, upregulation of FAP on fibroblasts can be found in several solid organ malignancies, including non-small cell lung cancer, and chronic inflammatory conditions such as pulmonary fibrosis and rheumatoid arthritis. Their full role remains unclear, but FAP expressing cancer associated fibroblasts (CAFs) have been found to relate to a poor prognosis with worse survival rates in breast, colorectal, pancreatic, and non-small cell lung cancer (NSCLC). Optical imaging using a FAP specific chemical probe, when combined with clinically compatible imaging systems, can provide a readout of FAP activity which could allow disease monitoring, prognostication and potentially stratify therapy. However, to derive a specific signal for FAP any sequence must retain specificity over closely related endopeptidases, such as prolyl endopeptidase (PREP), and be resistant to degradation in areas of active inflammation. We describe the iterative development of a FAP optical reporter sequence which retains FAP specificity, confers resistance to degradation in the presence of activated neutrophil proteases and demonstrates clinical tractability ex vivo in NSCLC samples with an imaging platform

A rise in the frequency of lasR mutant Pseudomonas aeruginosa among keratitis isolates between 1993 and 2021

IntroductionPseudomonas aeruginosa causes vision threatening keratitis. The LasR transcription factor regulates virulence factors in response to the quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone. P. aeruginosa isolates with lasR mutations are characterized by an iridescent high sheen phenotype caused by a build-up of 2-heptyl-4-quinolone. A previous study demonstrated 22% (n=101) of P. aeruginosa keratitis isolates from India between 2010 and 2016 were sheen positive lasR mutants, and the sheen phenotype correlated with worse clinical outcomes for patients. In this study, a longitudinal collection of P. aeruginosa keratitis isolates from Eastern North America were screened for lasR mutations by the sheen phenotype and sequencing of the lasR gene.MethodsKeratitis isolates (n=399) were classified by sheen phenotype. The lasR gene was cloned from a subset of isolates, sequenced, and tested for loss of function or dominant-negative status based on an azocasein protease assay. A retrospective chart review compared outcomes of keratitis patients infected by sheen positive and negative isolates.ResultsA significant increase in sheen positive isolates was observed between 1993 and 2021. Extracellular protease activity was reduced among the sheen positive isolates and a defined lasR mutant. Cloned lasR alleles from the sheen positive isolates were loss of function or dominant negative and differed in sequence from previously reported ocular lasR mutant alleles. Retrospective analysis of patient information suggested significantly better visual outcomes for patients infected by sheen positive isolates.DiscussionThese results indicate an increase in lasR mutations among keratitis isolates in the United States and suggest that endemic lasR mutants can cause keratitis