Biology subjects

Phillips, E. K.

Publications and source records attributed to Phillips, E. K..

5 recordsLinked to original sources

bioRxiv2026

Mitochondrial and Cardiolipin Adaptations to Ventricular Assist Device Support in Pediatric Versus Adult Failing Myocardium

BackgroundVentricular assist devices (VADs) are used as treatment for end-stage heart failure in children and adults. We previously demonstrated decreased mitochondrial function and changes in cardiolipin, a mitochondrial phospholipid, in explanted pediatric and adult failing hearts. In this study, we tested the hypothesis that VAD unloading of failing hearts leads to positive changes in myocardial cardiolipin in both pediatric and adult hearts. MethodsVentricular tissue was collected from the same patient at time of VAD implantation and at transplant. Ejection fraction (EF), left ventricular internal diameter at end-diastole (LVIDd) and brain natriuretic peptide (BNP) were assessed pre- and post-VAD. Cardiolipin species from paired VAD core and explants were quantified using liquid chromatography mass spectrometry. Mitochondrial respiration was measured in ventricular tissue pre- and post-VAD in paired pediatric samples using the Oroboros Oxygraph-2k. ResultsVAD support led to increased EF and decreased LVIDd and BNP. The predominant cardiolipin species in cardiac mitochondria, tetralinoleoylcardiolipin, was positively remodeled in pediatric post-VAD myocardium, while adult post-VAD myocardium demonstrated significantly increased total cardiolipin and decreased oxidized cardiolipin but did not demonstrate the tetralinoleoylcardiolipin remodeling seen in pediatric hearts. In pediatric patients, VAD support resulted in significant increases in Complex I+II activity, and a trend toward increases in Complex I activity. ConclusionOur data demonstrate age-related differences in VAD-associated cardiolipin remodeling and suggest that improved mitochondrial function in pediatric VAD-supported hearts could be related to increased tetralinoleoylcardiolipin.

physiology↗

bioRxiv2025

When good guides go bad: empirical evaluation of all unique Cas9 targets in E. coli reveal widespread functionality and rules for gRNA biological design

The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease in which it can be targeted to specific DNA loci via guide RNAs (gRNAs). Reports vary widely on the actual specificity of this targeting, with some studies observing 60% of gRNAs possessing no activity against the genome, yet an assumption that inactive gRNAs are rare persists in the E. coli community. To resolve these contradictions, we evaluated the activity of nearly 500,000 unique gRNAs in the E. coli K12 MG1655 genome. We show that the overwhelming majority (at least 93%) of unique gRNAs are functional while only 0.3% are nonfunctional.These nonfunctional gRNAs exhibit strong spacer self-interaction, which can be either excluded using a simple design rule or "repaired" during library design. Finally, this work provides the greater microbial synthetic biology community both a set of nearly half a million E. coli gRNAs that have been empirically evaluated in vivo as well as a thoroughly evaluated experimental procedure, complete with appropriate controls for Cas9 activity, for conducting Cas9 assays in E. coli specifically and bacteria more generally. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/651106v3_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1038ddcorg.highwire.dtl.DTLVardef@566e37org.highwire.dtl.DTLVardef@5df6b2org.highwire.dtl.DTLVardef@ef453c_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Multilayered regulation by RNA thermometers enables precise control of Cas9 expression

Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs (gRNAs). This information should be applicable to all Cas9-based methods and technologies. NoticeThis manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (https://www.energy.gov/doe-public-access-plan). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/651296v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1cb97a4org.highwire.dtl.DTLVardef@150bf11org.highwire.dtl.DTLVardef@2ffefforg.highwire.dtl.DTLVardef@10e24c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

The small molecule CBR-5884 inhibits the Candida albicans phosphatidylserine synthase

Systemic infections by Candida spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from Candida albicans is a logical antifungal drug target due to its importance in virulence, absence in the host and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an in vitro nucleotidase-coupled malachite green-based high throughput assay for purified C. albicans Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average Z score of [~]0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited inhibitory effects against C. albicans cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting in vivo Cho1 function by inducing phenotypes consistent with the cho1{Delta}{Delta} mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a Ki of 1,550 {+/-} 245.6 nM. Thus, this compound has the potential for development into an antifungal compound.

microbiology↗

bioRxiv2018

TIM-1 SERVES AS A NONREDUNDANT RECEPTOR FOR EBOLA VIRUS, ENHANCING VIREMIA AND PATHOGENESIS

Background.T cell immunoglobulin mucin domain-1 (TIM-1) is a phosphatidylserine (PS) receptor, mediating filovirus entry into cells through interactions with PS on virions. TIM-1 expression has been implicated in Ebola virus (EBOV) pathogenesis; however, it remains unclear whether this is due to TIM-1 serving as a filovirus receptor in vivo or, as others have suggested, TIM-1 induces a cytokine storm elicited by T cell/virion interactions. Here, we use a BSL2 model virus that expresses EBOV glycoprotein and demonstrate the importance of TIM-1 as a virus receptor late during in vivo infection.\n\nMethodology/Principal findings.We used an infectious, recombinant vesicular stomatitis virus expressing EBOV glycoprotein (EBOV GP/rVSV) to assess the role of TIM-1 during in vivo infection. TIM-1-sufficient or TIM-1-deficient BALB/c interferon /{beta} receptor-/- mice were challenged with EBOV GP/rVSV-GFP or G/rVSV-GFP. While G/rVSV caused profound morbidity and mortality in both mouse strains, TIM-1-deficient mice had significantly better survival than TIM-1-expressing mice following EBOV GP/rVSV challenge. EBOV GP/rVSV load in spleen was high and unaffected by expression of TIM-1. However, infectious virus in serum, liver, kidney and adrenal gland was reduced late in infection in the TIM-1-deficient mice, suggesting that virus entry via this receptor contributes to virus load. Consistent with higher virus loads, proinflammatory chemokines trended higher in organs from infected TIM-1-sufficient mice compared to the TIM-1-deficient mice, but proinflammatory cytokines were more modestly affected. To assess the role of T cells in EBOV GP/rVSV pathogenesis, T cells were depleted in TIM-1-sufficient and -deficient mice and the mice were challenged with virus. Depletion of T cells did not alter the pathogenic consequences of virus infection.\n\nConclusions.Our studies provide evidence that at late times during EBOV GP/rVSV infection, TIM-1 increased virus load and associated mortality, consistent with an important role of this receptor in virus entry. This work suggests that inhibitors which block TIM-1/virus interaction may serve as effective antivirals, reducing virus load at late times during EBOV infection.\n\nAuthor summaryT cell immunoglobulin mucin domain-1 (TIM-1) is one of a number of phosphatidylserine (PS) receptors that mediate clearance of apoptotic bodies by binding PS on the surface of dead or dying cells. Enveloped viruses mimic apoptotic bodies by exposing PS on the outer leaflet of the viral membrane. While TIM-1 has been shown to serve as an adherence factor/receptor for filoviruses in tissue culture, limited studies have investigated the role of TIM-1 as a receptor in vivo. Here, we sought to determine if TIM-1 was critical for Ebola virus glycoprotein-mediated infection using a BSL2 model virus. We demonstrate that loss of TIM-1 expression results in decreased virus load late during infection and significantly reduced virus-elicited mortality. These findings provide evidence that TIM-1 serves as an important receptor for Ebola virus in vivo. Blocking TIM-1/EBOV interactions may be effective antiviral strategy to reduce viral load and pathogenicity at late times of EBOV infection.

microbiology↗