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Oncogenic KRAS expression generates a metabolic dependency on aerobic glycolysis, known as the Warburg effect. We report an effect of increased glycolytic flux that feeds into glycosphingolipid biosynthesis and is directly linked to KRAS oncogenic function. High resolution imaging and genetic approaches show that a defined subset of outer leaflet glycosphingolipids, including GM3 and SM4, is required to maintain KRAS plasma membrane localization, with GM3 engaging in cross-bilayer coupling to maintain inner leaflet phosphatidylserine content. Thus, glycolysis is critical for KRAS plasma membrane localization and nanoscale spatial organization. Reciprocally oncogenic KRAS selectively upregulates cellular content of these same glycosphingolipids, whose depletion in turn abrogates KRAS oncogenesis in pancreatic cancer models. Our findings expand the role of the Warburg effect beyond ATP generation and biomass building to high-level regulation of KRAS function. The positive feedforward loop between oncogenic KRAS signaling and glycosphingolipid synthesis represents a vulnerability with therapeutic potential.

AIM To investigate (1) the cytotoxic potential of the brown precipitate (BP) formed with sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX), using both a small animal model of Caenorhabditis elegans (C. elegans) and cultured human gingival fibroblasts; and (2) the chemical composition of BP using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). METHODOLOGY Brown precipitate was obtained by mixing equal volumes of 6% NaOCl and 2% CHX and separating the BP from clear supernatant by centrifugation. The brown precipitate was weighed and solubilized in dimethyl sulfoxide for cytotoxicity experiments. The cytotoxic effect of BP was assessed using C. elegans larvae and primary immortalized human gingival fibroblasts-hTERT (hTERT-hNOF) cells. Various dilutions of BP (25 ng/µL-150 ng/µL), supernatant (0.15% v/v), NaOCl (1:100-1:1000 dilutions of 6% NaOCl) or CHX (1:500-1:1000 dilutions of 2% CHX) along with vehicle control (0.5% v/v ethanol and 0.15% v/v DMSO) or untreated control (growth medium) were tested on C. elegans larvae and hTERT-hNOF cells. Viability was assessed in C. elegans larvae using stereomicroscopy and in hTERT-hNOF cells using dehydrogenase-based colorimetric assay. ToF-SIMS was used to assess the chemical composition of BP in comparison with CHX and para-chloroaniline (PCA). The C. elegans and cell line data were analysed using Log-Rank test and Student's t-test, respectively (p

KRAS is mutated in 90% of human pancreatic ductal adenocarcinomas (PDACs). To function, KRAS must localize to the plasma membrane (PM) via a C-terminal membrane anchor that specifically engages phosphatidylserine (PtdSer). This anchor-binding specificity renders KRAS–PM localization and signaling capacity critically dependent on PM PtdSer content. We now show that the PtdSer lipid transport proteins, ORP5 and ORP8, which are essential for maintaining PM PtdSer levels and hence KRAS PM localization, are required for KRAS oncogenesis. Knockdown of either protein, separately or simultaneously, abrogated growth of KRAS-mutant but not KRAS–wild-type pancreatic cancer cell xenografts. ORP5 or ORP8 knockout also abrogated tumor growth in an immune-competent orthotopic pancreatic cancer mouse model. Analysis of human datasets revealed that all components of this PtdSer transport mechanism, including the PM-localized EFR3A-PI4KIIIα complex that generates phosphatidylinositol-4-phosphate (PI4P), and endoplasmic reticulum (ER)–localized SAC1 phosphatase that hydrolyzes counter transported PI4P, are significantly up-regulated in pancreatic tumors compared to normal tissue. Taken together, these results support targeting PI4KIIIα in KRAS-mutant cancers to deplete the PM-to-ER PI4P gradient, reducing PM PtdSer content. We therefore repurposed the US Food and Drug Administration–approved hepatitis C antiviral agent, simeprevir, as a PI4KIIIα inhibitor In a PDAC setting. Simeprevir potently mislocalized KRAS from the PM, reduced the clonogenic potential of pancreatic cancer cell lines in vitro, and abrogated the growth of KRAS-dependent tumors in vivo with enhanced efficacy when combined with MAPK and PI3K inhibitors. We conclude that the cellular ER-to-PM PtdSer transport mechanism is essential for KRAS PM localization and oncogenesis and is accessible to therapeutic intervention.

Mandatory attendance, particularly in didactic settings, is a highly debated topic in higher education, including dental education. Within dental education, a large portion of education occurs in preclinical laboratories and clinical environments. There is little to no research on attendance in these settings in dental schools. This point/counterpoint paper examines the pros and cons of mandatory attendance in these highly specialized educational settings. With the backdrop of the COVID-19 pandemic that began in March 2020 and continues to impact dental education at the time of publication, this topic has become even more relevant. Viewpoint 1 claims that attendance should be mandatory because a greater exposure to preclinical and clinical environments helps foster better clinical hand skills, critical thinking, decision-making, problem-solving skills, and an overall sense of professional identity. It goes on further to suggest that there may be a link between attendance and performance in exams and that attendance is part of the dental school's responsibility. Viewpoint 2 argues that the rationale for attendance is complex, and that creating learning environments that are psychologically safe will incentivize students to attend, even without mandatory attendance policies. Furthermore, it explains that technological advances have allowed dental schools to think creatively about asynchronous learning, which by its very nature does not require attendance at a given time. The authors of both viewpoints conclude that the preclinical and clinical education and experience are critical dental education and that dental school leaders should focus on improving the quality of these experiences.

KRAS plays an essential role in regulating cell proliferation, differentiation, migration and survival. Mutated KRAS is a major driver of malignant transformation in multiple human cancers. We showed previously that fendiline (6) is an effective inhibitor of KRAS plasma membrane (PM) localization and function. In this study, we designed, synthesized and evaluated a series of new fendiline analogs to optimize its drug properties. Systemic structure-activity relationship studies by scaffold repurposing led to the discovery of several more active KRAS PM localization inhibitors such as compounds 12f (NY0244), 12h (NY0331) and 22 (NY0335) which exhibit nanomolar potencies. These compounds inhibited oncogenic KRAS-driven cancer cell proliferation at single-digit micromolar concentrations in vitro. In vivo studies in a xenograft model of pancreatic cancer revealed that 12h and 22 suppressed oncogenic KRAS-expressing MiaPaCa-2 tumor growth at a low dose range of 1–5 mg/kg with no vasodilatory effects, indicating their potential as chemical probes and anticancer therapeutics.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Introduction Understanding the physicochemical and biological properties of endodontic sealers is important for endodontic treatment planning. This study evaluated the properties of EndoSequence BC Sealer HiFlow (BCH; Brasseler USA, Savannah, GA), EndoSequence BC Sealer (BC, Brasseler USA), and AH Plus (AHP; Dentsply DeTrey, Konstanz, Germany). The effect of temperature on the setting time and flow of these sealers was also evaluated. Methods The setting time, flow, radiopacity, pH, solubility, and calcium release were investigated following ISO guidelines. The morphology and chemical composition of the sealers were evaluated by scanning electron microscopy and energy-dispersive spectroscopy. The antibacterial activity of sealers was tested against 2 strains of Enterococcus faecalis. Sealer cytotoxicity and the effects on messenger RNA expression of proinflammatory and mineralization genes were also investigated. Data analysis was performed using analysis of variance, Tukey, Kruskal-Wallis, and Dunn multiple comparison tests. P ≤ .05 was considered statistically significant. Results The setting time and flow rate of all sealers were affected by heat (P ≤ .05). The setting times and solubility of BCH and BC were significantly higher than AHP (P ≤ .0001). The radiopacity of AHP was higher than BCH and BC (P ≤ .0001). All sealers were alkaline and had antibacterial effects. Cell viability was higher for BCH and BC than AHP (P ≤ .0001). No significant differences in messenger RNA expression of proinflammatory and mineralization genes were observed. Conclusions Overall, BCH and BC had similar physicochemical and biological properties. The observed high solubility of BCH and BC as well as the high cytotoxicity of AHP might negatively impact the clinical performance of these materials. The application of heat affected the setting time and flow of all sealers.

Although dental education has traditionally been organized into basic sciences education (first and second years) and clinical education (third and fourth years), there has been growing interest in ways to better integrate the two to more effectively educate students and prepare them for practice. Since 2012, The University of Texas School of Dentistry at Houston (UTSD) has made it a priority to improve integration of basic and clinical sciences, with a focus to this point on integrating the basic sciences. The aim of this study was to determine the perspectives of basic and clinical science faculty members regarding basic and clinical sciences integration and the degree of integration currently occurring. In October 2016, all 227 faculty members (15 basic scientists and 212 clinicians) were invited to participate in an online survey. Of the 212 clinicians, 84 completed the clinician educator survey (response rate 40%). All 15 basic scientists completed the basic science educator survey (response rate 100%). The majority of basic and clinical respondents affirmed the value of integration (93.3%, 97.6%, respectively) and reported regular integration in their teaching (80%, 86.9%). There were no significant differences between basic scientists and clinicians on perceived importance (p=0.457) and comfort with integration (p=0.240), but the basic scientists were more likely to integrate (p=0.039) and collaborate (p=0.021) than the clinicians. There were no significant differences between generalist and specialist clinicians on importance (p=0.474) and degree (p=0.972) of integration in teaching and intent to collaborate (p=0.864), but the specialists reported feeling more comfortable presenting basic science information (p=0.033). Protected faculty time for collaborative efforts and a repository of integrated basic science and clinical examples for use in teaching and faculty development were recommended to improve integration. Although questions might be raised about the respondents' definition of "integration," this study provides a baseline assessment of perceptions at a dental school that is placing a priority on integration.

Human derived composite amnion-chorion membrane (ACM) has been used to facilitate wound healing due to reported anti-inflammatory properties and promotion of cell proliferation. This study aimed to assess the antimicrobial properties of the ACM using novel methods to visualize the antimicrobial efficacy of membranes in situ at different time points. Porcine Pericardium Collagen Membranes (PPCM) served as membrane controls. Circular pieces of the membranes were used in three different assays: insert, agar contact and glass-bottom well assays. Streptococcus gordonii were spotted onto the membranes and the plates were subsequently centrifuged to ensure direct bacterial contact with the membranes in the insert and agar contact assays, thus better mimicking bacterial adherence in the oral cavity. After incubation at 37 °C for 8, 24, and 48 hours, the membranes were dyed with the Live/Dead BacLight Bacterial Viability fluorescence stain and analyzed via confocal microscopy. The results demonstrated that the ACM completely inhibited bacterial growth at all time points, whereas the PPCM did not demonstrate any antimicrobial properties. Within the limits of this study, the ACM showed extremely high antimicrobial efficacy against oral streptococci. In addition, our methods may be useful in assessing antimicrobial properties for biomaterials with minimum diffusion ability, when traditional assessment methods are not applicable.

KRAS-dependent cancer cell growth is inhibited by disrupting phosphatidylserine transport to the plasma membrane by genetic knockdown of lipid exchangers ORP5 and ORP8 or by inhibition of PI4KIIIα., The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We, therefore, investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidyl-4-phosphate synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion significantly reduced proliferation and anchorage-independent growth of multiple KRAS-dependent cancer cell lines, and attenuated KRAS signaling in vivo. Similarly, functionally inhibiting ORP5 and ORP8 by inhibiting PI4KIIIα-mediated synthesis of phosphatidyl-4-phosphate at the PM selectively inhibited the growth of KRAS-dependent cancer cell lines over normal cells. Inhibiting KRAS function through regulating PM lipid PtdSer content may represent a viable strategy for KRAS-driven cancers.

K-Ras must localize to the plasma membrane (PM) for biological activity. We show here that multiple acid sphingomyelinase (ASM) inhibitors, including tricyclic antidepressants, mislocalized phosphatidylserine (PtdSer) and K-RasG12V from the PM, resulting in abrogation of K-RasG12V signaling and potent, selective growth inhibition of mutant K-Ras-transformed cancer cells. Concordantly, in nude mice, the ASM inhibitor fendiline decreased the rate of growth of oncogenic K-Ras-expressing MiaPaCa-2 tumors but had no effect on the growth of the wild-type K-Ras-expressing BxPC-3 tumors. ASM inhibitors also inhibited activated LET-60 (a K-Ras ortholog) signaling in Caenorhabditis elegans, as evidenced by suppression of the induced multivulva phenotype. Using RNA interference against C. elegans genes encoding other enzymes in the sphingomyelin (SM) biosynthetic pathway, we identified 14 enzymes whose knockdown strongly or moderately suppressed the LET-60 multivulva phenotype. In mammalian cells, pharmacological agents that target these enzymes all depleted PtdSer from the PM and caused K-RasG12V mislocalization. These effects correlated with changes in SM levels or subcellular distribution. Selected compounds, including sphingosine kinase inhibitors, potently inhibited the proliferation of oncogenic K-Ras-expressing pancreatic cancer cells. In conclusion, these results show that normal SM metabolism is critical for K-Ras function, which may present therapeutic options for the treatment of K-Ras-driven cancers.

Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems.