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Introduction: There is a growing trend of multidrug-resistant organisms (MDRO). The goal of this study was to characterize MDRO at a single center from ophthalmic cultures to better understand how treatments were tailored and to assess effect on visual acuity.Materials and Methods: The MDRO data were collected by the Multidrug-Resistant Organism Repository and Surveillance Network from the Brooke Army Medical Center clinical laboratory. Both patient- and isolate-specific data were collected and qualitatively analyzed. Primary outcome measures were organism and type of resistance, anatomic location of isolate, initial and final antibiotic choice, and visual acuity.Results: Thirty-one bacterial culture samples were analyzed from 29 patients. Twenty-two (72%) were Gram-positive and all were methicillin-resistant Staphylococcus aureus (MRSA). Nine (29%) were Gram-negative and of these five were Pseudomonas spp. Fourteen (45%) isolates were cultured from the cornea, nine (29%) from the lid, four (13%) from the conjunctiva, and four (13%) from other locations. The majority (66.6%) required adjustment of initial antibiotics following ocular culture results. Sixteen adult patients had recorded initial and final visual acuities. Fifteen of those 16 patients had stable or improved visual acuities following treatment of the infection, but five patients had a final visual acuity less than 20/200.Conclusion: This study demonstrated a high frequency of corneal MDRO infections and specifically MRSA and Pseudomonas spp. isolates. Antibiotic treatments frequently required adjustment. Further prospective study of visual outcomes from ophthalmic MDRO cultures is needed. [ABSTRACT FROM AUTHOR]

High-grade serous ovarian cancer (HGSOC) is the most common and aggressive type of ovarian cancer. Due to a lack of an early detection test and overt symptoms, many patients are diagnosed at a late stage where metastasis makes treatment very challenging. Furthermore, the current standard treatment for HGSOC patients, consisting of debulking surgery and platinum-taxane chemotherapy, reduces quality of life due to debilitating side-effects. Sadly, 80-90% of patients diagnosed with advanced stage ovarian cancer will die due to treatment resistance. As such, novel therapeutic strategies for HGSOC that are both more effective and less toxic are urgently required. Here we describe the assessment of cold atmospheric pressure (CAP) gas discharge technology as a novel treatment strategy in pre-clinical models of HGSOC. Plasma-activated media (PAM) was generated using cell growth media. HGSOC cell lines, patient ascites cells and primary tissue explants were tested for their response to PAM via analysis of cell viability, cell death and oxidative stress assays. Our data show that PAM treatment can be more effective than standard carboplatin chemotherapy at selectively targeting ovarian cancer cells in primary patient samples. Further, we also observed PAM to induce apoptosis in HGSOC cancer cell lines via induction of oxidative stress and mitochondrial-mediated apoptosis. These findings suggest that PAM is a viable therapeutic strategy to test in in vivo models of ovarian cancer, with a view to develop an intraperitoneal PAM-based therapy for HGSOC patients. Our studies validate the ability of PAM to selectively target tumour tissue and ascites cells. This work supports the development of PAM towards in vivo validation and translation into clinical practice., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Sphingosine 1-phosphate (S1P) is a signaling lipid that has broad roles, working either intracellularly through various protein targets, or extracellularly via a family of five G-protein coupled receptors . Agents that selectively and specifically target each of the S1P receptors have been sought as both biological tools and potential therapeutics. JTE-013, a small molecule antagonist of S1P receptors 2 and 4 (S1P 2 and S1P 4 ) has been widely used in defining the roles of these receptors in various biological processes. Indeed, our previous studies showed that JTE-013 had anti-acute myeloid leukaemia (AML) activity, supporting a role for S1P 2 in the biology and therapeutic targeting of AML. Here we examined this further and describe lipidomic analysis of AML cells that revealed JTE-013 caused alterations in sphingolipid metabolism, increasing cellular ceramides, dihydroceramides, sphingosine and dihydrosphingosine. Further examination of the mechanisms behind these observations showed that JTE-013, at concentrations frequently used in the literature to target S1P 2/4 , inhibits several sphingolipid metabolic enzymes, including dihydroceramide desaturase 1 and both sphingosine kinases. Collectively, these findings demonstrate that JTE-013 can have broad off-target effects on sphingolipid metabolism and highlight that caution must be employed in interpreting the use of this reagent in defining the roles of S1P 2/4 ., (© 2022. The Author(s).)

Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

There is growing evidence of the influence of sphingosine kinase (SK) enzymes on viral infection. Here, the role of sphingosine kinase 2 (SK2), an isoform of SK prominent in the brain, was defined during dengue virus (DENV) infection. Chemical inhibition of SK2 activity using two different SK2 inhibitors, ABC294640 and K145, had no effect on DENV infection in human cells in vitro. In contrast, DENV infection was restricted in SK2 -/- immortalized mouse embryonic fibroblasts (iMEFs) with reduced induction of IFN-β mRNA and protein, and mRNA for the IFN-stimulated genes (ISGs) viperin, IFIT1, IRF7 and CXCL10 in DENV-infected SK2 -/- compared to WT iMEFs. Intracranial (ic) DENV injection in C57BL/6 SK2 -/- mice induced body weight loss earlier than in WT mice but DENV RNA levels were comparable in the brain. Neither SK1 mRNA or sphingosine-1-phosphate (S1P) levels were altered following ic DENV infection in WT or SK2 -/- mice but brain S1P levels were reduced in all SK2 -/- mice, independent of DENV infection. CD8 mRNA was induced in the brains of both DENV-infected WT and SK2 -/- mice, suggesting normal CD8+ T-cell infiltration into the DENV-infected brain independent of SK2 or S1P. Thus, although SK2 may be important for replication of some viruses SK2 activity does not affect DENV infection in vitro and SK2 or S1P levels do not influence DENV infection or T-cell infiltration in the context of infection in the brain.

Sphingosine kinases (SK) are the sole enzymes responsible for the production of sphingosine 1-phosphate (S1P). S1P is a signaling molecule with a plethora of targets, acting as both a second messenger intracellularly and extracellularly via a family of cell surface G-protein-coupled S1P receptors. The two sphingosine kinases, SK1 and SK2, arise from different genes and have some distinct and overlapping cellular functions that are regulated in part by differential cellular localization, developmental expression, and catalytic properties. Here, we describe an improved method for selectively detecting SK1 activity in vitro and cell lysates via the use of the zwitterionic detergent CHAPS, which effectively inhibits SK2 activity and thus allows selective analysis of SK1 activity in a range of cell samples. The assay measures the production of 32 P-labeled S1P following the addition of exogenous sphingosine and [γ 32 P]ATP. The S1P product can be purified by Bligh-Dyer solvent extraction, separated by thin layer chromatography (TLC), and the radiolabeled S1P quantified by exposing the TLC plate to a storage phosphor screen. This sensitive, reproducible assay can be used to selectively detect SK1 activity in tissue, cell, and recombinant protein samples.

Sphingosine kinase 1 (SK1) is a key regulator of the cellular balance between proapoptotic and prosurvival sphingolipids. Oncogenic signaling by SK1 relies on its localization to the plasma membrane, which is mediated by the calcium and integrin binding protein CIB1 via its Ca 2+ -myristoyl switch function. Here we show that another member of the CIB family, CIB2, plays a surprisingly opposite role to CIB1 in the regulation of SK1 signaling. CIB2 bound SK1 on the same site as CIB1, yet it lacks the Ca 2+ -myristoyl switch function. As a result, CIB2 blocked translocation of SK1 to the plasma membrane and inhibited its subsequent signaling, which included sensitization to TNFα-induced apoptosis and inhibition of Ras-induced neoplastic transformation. CIB2 was significantly downregulated in ovarian cancer and low CIB2 expression was associated with poor prognosis in ovarian cancer patients. Notably, reintroduction of CIB2 in ovarian cancer cells blocked plasma membrane localization of endogenous SK1, reduced in vitro neoplastic growth and tumor growth in mice, and suppressed cell motility and invasiveness both in vitro and in vivo Consistent with the in vitro synergistic effects between the SK1-specific inhibitor SK1-I and standard chemotherapeutics, expression of CIB2 also sensitized ovarian cancer cells to carboplatin. Together, these findings identify CIB2 as a novel endogenous suppressor of SK1 signaling and potential prognostic marker and demonstrate the therapeutic potential of SK1 in this gynecologic malignancy. Cancer Res; 77(18); 4823-34. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Dengue virus (DENV) regulates sphingosine kinase (SK)-1 activity and chemical inhibition of SK1 reduces DENV infection. In primary murine embryonic fibroblasts (pMEFs) lacking SK1 however, DENV infection is enhanced and this is associated with induction of normal levels of interferon beta (IFN-β) but reduced levels of IFN-stimulated genes (ISGs). We have further investigated this link between SK1 and type I IFN responses. DENV infection downregulates cell-surface IFN-alpha receptor (IFNAR)1 in both wild-type (WT) and SK1 - /- pMEF, but, consistent with poor ISG responses, shows reduced induction of phosphorylated (p)-STAT1 and key IFN regulatory factors (IRF)1 and -7 in SK1 -/- pMEF. Direct IFN stimulation induced ISGs (viperin, IFIT1), CXCL10, IRF1 and -7 and p-STAT1. Responses, however, were significantly reduced in SK1 -/- pMEF, except for IFN-stimulated CXCL10 and IRF7. Poor IFN responses in SK1 -/- pMEF were associated with a small reduction in basal cell-surface IFNAR1 and IRF1 mRNA in uninfected SK1 -/- compared with WT pMEF. In contrast, treatment of cells with the SK1 inhibitor, SK1-I or expression of an inhibitory SK1 short hairpin RNA (shRNA), both of which reduce DENV infection, does not alter basal IRF1 mRNA or affect type I IFN stimulation of p-STAT1. Thus, cells genetically lacking SK1 can induce many responses normally following DENV infection, but have adaptive changes in IFNAR1 and IRF1 that compromise DENV-induced type I IFN responses. This suggests a biological link between SK1 and IFN-stimulated pathways. Other approaches to reduce SK1 activity, however, do not influence these important antiviral pathways but reduce infection and may be useful antiviral strategies., Competing Interests: The authors declare no conflict of interest.

We have previously reported that the absence of sphingosine kinase 1 (SK1) affects both dengue virus (DENV) infection and innate immune responses in vitro. Here we aimed to define SK1-dependancy of DENV-induced disease and the associated innate responses in vivo. The lack of a reliable mouse model with a fully competent interferon response for DENV infection is a challenge, and here we use an experimental model of DENV infection in the brain of immunocompetent mice. Intracranial injection of DENV-2 into C57BL/6 mice induced body weight loss and neurological symptoms which was associated with a high level of DENV RNA in the brain. Body weight loss and DENV RNA level tended to be greater in SK1-/- compared with wildtype (WT) mice. Brain infection with DENV-2 is associated with the induction of interferon-β (IFN-β) and IFN-stimulated gene (ISG) expression including viperin, Ifi27l2a, IRF7, and CXCL10 without any significant differences between WT and SK1-/- mice. The SK2 and sphingosine-1-phosphate (S1P) levels in the brain were unchanged by DENV infection or the lack of SK1. Histological analysis demonstrated the presence of a cellular infiltrate in DENV-infected brain with a significant increase in mRNA for CD8 but not CD4 suggesting this infiltrate is likely CD8+ but not CD4+ T-lymphocytes. This increase in T-cell infiltration was not affected by the lack of SK1. Overall, DENV-infection in the brain induces IFN and T-cell responses but does not influence the SK/S1P axis. In contrast to our observations in vitro, SK1 has no major influence on these responses following DENV-infection in the mouse brain., Competing Interests: The authors have declared that no competing interests exist.

Alveolar macrophages from chronic obstructive pulmonary disease patients and cigarette smokers are deficient in their ability to phagocytose apoptotic bronchial epithelial cells (efferocytosis). We hypothesized that the defect is mediated via inhibition of sphingosine kinases and/or their subcellular mislocalization in response to cigarette smoke and can be normalized with exogenous sphingosine-1-phosphate or FTY720 (fingolimod), a modulator of sphingosine-1-phosphate signaling, which has been shown to be clinically useful in multiple sclerosis. Measurement of sphingosine kinase 1/2 activities by [(32)P]-labeled sphingosine-1-phosphate revealed a 30% reduction of sphingosine kinase 1 (P < 0.05) and a nonsignificant decrease of sphingosine kinase 2 in THP-1 macrophages after 1 h cigarette smoke extract exposure. By confocal analysis macrophage sphingosine kinase 1 protein was normally localized to the plasma membrane and cytoplasm and sphingosine kinase 2 to the nucleus and cytoplasm but absent at the cell surface. Cigarette smoke extract exposure (24 h) led to a retraction of sphingosine kinase 1 from the plasma membrane and sphingosine kinase 1/2 clumping in the Golgi domain. Selective inhibition of sphingosine kinase 2 with 25 µM ABC294640 led to 36% inhibition of efferocytosis (P < 0.05); 10 µM sphingosine kinase inhibitor/5C (sphingosine kinase 1-selective inhibitor) induced a nonsignificant inhibition of efferocytosis, but its combination with ABC294640 led to 56% inhibition (P < 0.01 vs. control and < 0.05 vs. single inhibitors). Cigarette smoke-inhibited efferocytosis was significantly (P < 0.05) reversed to near-control levels in the presence of 10-100 nM exogenous sphingosine-1-phosphate or FTY720, and FTY720 reduced cigarette smoke-induced clumping of sphingosine kinase 1/2 in the Golgi domain. These data strongly support a role of sphingosine kinase 1/2 in efferocytosis and as novel therapeutic targets in chronic obstructive pulmonary disease., (© Society for Leukocyte Biology.)