Tissue-engineered vocal fold replacement in swine: Methods for functional and structural analysis.

We have developed a cell-based outer vocal fold replacement (COVR) as a potential therapy to improve voice quality after vocal fold (VF) injury, radiation, or tumor resection. The COVR consists of multipotent human adipose-derived stem cells (hASC) embedded within a three-dimensional fibrin scaffold that resembles vocal fold epithelium and lamina propria layers. Previous work has shown improved wound healing in rabbit studies. In this pilot study in pigs, we sought to develop methods for large animal implantation and phonatory assessment. Feasibility, safety, and structural and functional outcomes of the COVR implant are described. Of eight pigs studied, six animals underwent COVR implantation with harvest between 2 weeks and 6 months. Recovery of laryngeal tissue structure was assessed by vibratory and histologic analyses. Recovery of voice function was assessed by investigating acoustic parameters that were derived specifically for pigs. Results showed improved lamina propria qualities relative to an injured control animal at 6 months. Acoustic parameters reflected voice worsening immediately after surgery as expected; acoustics displayed clear voice recovery in the animal followed for 6 months after COVR. These methods form the basis for a larger-scale long-term pre-clinical safety and efficacy study.

Genistein Inhibits Clostridioides difficile Infection via Estrogen Receptors and Lysine-Deficient Protein Kinase 1.

BACKGROUND: Clostridioides difficile infection (CDI) is a debilitating nosocomial disease. Postmenopausal women may have an increased risk of CDI, suggesting estrogen influence. Soybean products contain a representative estrogenic isoflavone, genistein. METHODS: The anti-inflammatory and antiapoptotic effects of genistein were determined using primary human cells and fresh colonic tissues. The effects of oral genistein therapy among mice and hamsters were evaluated. RESULTS: Within 10 days of CDI, female c57BL/6J mice in a standard environment (regular diet) had a 50% survival rate, while those with estrogen depletion and in an isoflavone-free environment (soy-free diet) had a 25% survival rate. Oral genistein improved their 10-day survival rate to 100% on a regular diet and 75% in an isoflavone-free environment. Genistein reduced macrophage inflammatory protein-1α (MIP-1α) secretion in fresh human colonic tissues exposed to toxins. Genistein inhibited MIP-1α secretion in primary human peripheral blood mononuclear cells, abolished apoptosis and BCL-2-associated X (BAX) expression in human colonic epithelial cells, and activated lysine-deficient protein kinase 1 (WNK1) phosphorylation in both cell types. The anti-inflammatory and antiapoptotic effects of genistein were abolished by inhibiting estrogen receptors and WNK1. CONCLUSIONS: Genistein reduces CDI disease activity by inhibiting proinflammatory cytokine expression and apoptosis via the estrogen receptor/G-protein estrogen receptor/WNK1 pathways.

Low-dose exposure to PBDE disrupts genomic integrity and innate immunity in mammary tissue.

The low-dose mixture hypothesis of carcinogenesis proposes that exposure to an environmental chemical that is not individually oncogenic may nonetheless be capable of enabling carcinogenesis when it acts in concert with other factors. A class of ubiquitous environmental chemicals that are hypothesized to potentially function in this low-dose capacity are synthesized polybrominated diphenyl ethers (PBDEs). PBDEs can affect correlates of carcinogenesis that include genomic instability and inflammation. However, the effect of low-dose PBDE exposure on such correlates in mammary tissue has not been examined. In the present study, low-dose long-term (16 weeks) administration of PBDE to mice modulated transcriptomic indicators of genomic integrity and innate immunity in normal mammary tissue. PBDE increased transcriptome signatures for the Nuclear Factor Erythroid 2 Like 2 (NFE2L2) response to oxidative stress and decreased signatures for non-homologous end joining DNA repair (NHEJ). PBDE also decreased transcriptome signatures for the cyclic GMP-AMP Synthase - Stimulator of Interferon Genes (cGAS-STING) response, decreased indication of Interferon Stimulated Gene Factor 3 (ISGF3) and Nuclear Factor Kappa B (NF-κB) transcription factor activity, and increased digital cytometry estimates of immature dendritic cells (DCs) in mammary tissue. Replication of the PBDE exposure protocol in mice susceptible to mammary carcinogenesis resulted in greater tumor development. The results support the notion that ongoing exposure to low levels of PBDE can disrupt facets of genomic integrity and innate immunity in mammary tissue. Such effects affirm that synthesized PBDEs are a class of environmental chemicals that reasonably fit the low-dose mixture hypothesis.

Arachidonic acid metabolism and inflammatory biomarkers associated with exposure to polycyclic aromatic hydrocarbons.

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with systemic inflammation, yet what mechanisms regulate PAHs' inflammatory effects are less understood. This study evaluated the change of arachidonic acid (ARA) metabolites and inflammatory biomarkers in response to increased exposure to PAHs among 26 non-smoking healthy travelers from Los Angeles to Beijing. Traveling from Los Angeles to Beijing significantly increased urinary metabolites of dibenzofuran (800%), fluorene (568%), phenanthrene (277%), and pyrene (176%), accompanied with increased C-reactive protein, fibrinogen, IL-8, and IL-10, and decreased MCP-1, sCD40L, and sCD62P levels in the blood. Meanwhile, the travel increased the levels of ARA lipoxygenase metabolites that were positively associated with a panel of pro-inflammatory biomarkers. Concentrations of cytochrome P450 metabolite were also increased in Beijing and were negatively associated with sCD62P levels. In contrast, concentrations of ARA cyclooxygenase metabolites were decreased in Beijing and were negatively associated with anti-inflammatory IL-10 levels. Changes in both inflammatory biomarkers and ARA metabolites were reversed 4-7 weeks after participants returned to Los Angeles and were associated with urinary PAH metabolites, but not with other exposures such as secondhand smoke, stress, or diet. These results suggested possible roles of ARA metabolic alteration in PAHs-associated inflammatory effects.

Electronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative Biomarkers.

[Figure: see text].

Carbon Monoxide Inhibits Cytochrome P450 Enzymes CYP3A4/2C8 in Human Breast Cancer Cells, Increasing Sensitivity to Paclitaxel.

Paclitaxel (PTX) is a first-line treatment in breast cancer, though resistance develops quickly and frequently. Cytochrome P450 enzymes CYP3A4 and CYP2C8, which metabolically inactivate PTX in hepatic tissue, are overexpressed in malignant breast tissues. CYP3A4 expression correlates with PTX therapy failure and poor outcomes, though no direct evidence of CYP3A4 contributing to PTX sensitivity exists. Because CYP3A4/2C8 is susceptible to carbon monoxide (CO)-mediated inhibition and CO (a gaseous signaling molecule) has previously exhibited drug-sensitizing effects in cancer cells, we hypothesized that CO-mediated inhibition of CYP3A4/2C8 could lead to enhanced drug sensitivity. Using a photo-activated CO-releasing molecule, we have assessed the ability of CO to alter the pharmacokinetics of PTX in breast cancer cells via inhibition of CYP3A4/2C8 and determined that CO does enhance sensitivity of breast cancer cells to PTX. Inhibition of CYP3A4/2C8 by CO could therefore be a promising therapeutic strategy to enhance PTX response in breast cancer.

A sensitive LC-MS assay using derivatization with boron trifluoride to quantify curcuminoids in biological samples.

A procedure is described to measure curcumin (C), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), tetrahydrocurcumim (TC) and their glucuronidated metabolites (CG, DMCG, and BDMCG) in plasma, brain, liver and tumor samples. The procedure involves converting the analytes to their boron difluoride derivatives and analyzing them by combined liquid chromatography coupled to an ion trap mass spectrometer operating in the negative ion MSn scan mode. The method has superb limits of detection of 0.01 nM for all curcuminoids and 0.5 nM for TC and the glucuroniated metabolites, and several representative chromatograms of biological samples containing these analytes are provided. In addition, the pharmacokinetic profile of these compounds in one human who daily consumed an over-the-counter curcuminoid product shows the peak and changes in circulating concentrations achieved by this mode of administration.

Diesel Exhaust Induces Mitochondrial Dysfunction, Hyperlipidemia, and Liver Steatosis.

OBJECTIVE: Air pollution is associated with increased cardiovascular morbidity and mortality, as well as dyslipidemia and metabolic syndrome. Our goal was to dissect the mechanisms involved. Approach and Results: We assessed the effects of exposure to air pollution on lipid metabolism in mice through assessment of plasma lipids and lipoproteins, oxidized fatty acids 9-HODE (9-hydroxyoctadecadienoic) and 13-HODE (13-hydroxyoctadecadienoic), lipid, and carbohydrate metabolism. Findings were corroborated, and mechanisms were further assessed in HepG2 hepatocytes in culture. ApoE knockout mice exposed to inhaled diesel exhaust (DE, 6 h/d, 5 days/wk for 16 weeks) exhibited elevated plasma cholesterol and triglyceride levels, increased hepatic triglyceride content, and higher hepatic levels of 9-HODE and 13-HODE, as compared to control mice exposed to filtered air. A direct effect of DE exposure on hepatocytes was demonstrated by treatment of HepG2 cells with a methanol extract of DE particles followed by loading with oleic acid. As observed in vivo, this led to increased triglyceride content and significant downregulation of ACAD9 mRNA expression. Treatment of HepG2 cells with DE particles and oleic acid did not alter de novo lipogenesis but inhibited total, mitochondrial, and ATP-linked oxygen consumption rate, indicative of mitochondrial dysfunction. Treatment of isolated mitochondria, prepared from mouse liver, with DE particles and oleic acid also inhibited mitochondrial complex activity and ß-oxidation. CONCLUSIONS: DE exposure leads to dyslipidemia and liver steatosis in ApoE knockout mice, likely due to mitochondrial dysfunction and decreased lipid catabolism.

C/EBPß regulates the M2 transcriptome in ß-adrenergic-stimulated macrophages.

At the M2 terminal of the macrophage activation spectrum, expression of genes is regulated by transcription factors that include STAT6, CREB, and C/EBPß. Signaling through ß-adrenergic receptors drives M2 activation of macrophages, but little is known about the transcription factors involved. In the present study, we found that C/EBPß regulates the signaling pathway between ß-adrenergic stimulation and expression of Arg1 and several other specific genes in the greater M2 transcriptome. ß-adrenergic signaling induced Cebpb gene expression relatively early with a peak at 1 h post-stimulation, followed by peak Arg1 gene expression at 8 h. C/EBPß transcription factor activity was elevated at the enhancer region for Arg 1 at both 4 and 8 h after stimulation but not near the more proximal promoter region. Knockdown of Cebpb suppressed the ß-adrenergic-induced peak in Cebpb gene expression as well as subsequent accumulation of C/EBPß protein in the nucleus, which resulted in suppression of ß-adrenergic-induced Arg1 gene expression. Analysis of genome-wide transcriptional profiles identified 20 additional M2 genes that followed the same pattern of regulation by ß-adrenergic- and C/EBPß-signaling. Promoter-based bioinformatic analysis confirmed enrichment of binding motifs for C/EBPß transcription factor across these M2 genes. These findings pinpoint a mechanism that may be targeted to redirect the deleterious influence of ß-adrenergic signaling on macrophage involvement in M2-related diseases such as cancer.

Sperm can act as vectors for HIV-1 transmission into vaginal and cervical epithelial cells.

PROBLEM: Sperm are the major cells in semen. Human sperm possess a number of HIV-1 gp120 binding ligands including sulfogalactosylglycerolipid (SGG). However, the mechanisms of how sperm capture HIV-1 onto their surface are unclear. Furthermore, the ability of sperm to deliver HIV-1 to vaginal/cervical epithelial cells lining the lower female reproductive tract, as a first step in HIV-1 transmission, needs to be determined. METHOD OF STUDY: Sperm from healthy donors were incubated with dual-tropic HIV-1CS204 (clinical isolate), and virus capture was determined by p24 antigen ELISA. The involvement of SGG in HIV-1 capture was assessed by determining Kd values of HIV-1 gp120-SGG binding as well as computational docking of SGG to the gp120 V3 loop. The ability of sperm-associated HIV-1 to infect peripheral blood mononuclear cells (PBMCs) and TZM-bl indicator cells was determined. Lastly, infection of vaginal (Vk2/E6E7), ectocervical (Ect1/E6E7), and endocervical (End1/E6E7) epithelial cells mediated by HIV-1-associated sperm was evaluated. RESULTS: Sperm were able to capture HIV-1 in a dose-dependent manner, and the capture reached a maximum within 5 minutes. Captured HIV-1, however, could be removed from sperm by Percoll-gradient centrifugation. Affinity of gp120 for SGG was substantial, implicating sperm SGG in HIV-1 capture. Sperm-associated HIV-1 could productively infect PBMCs and TZM-bl cells, and was capable of being transmitted into vaginal/cervical epithelial cells. CONCLUSION: Sperm are able to capture HIV-1, which remains infectious and is able to be transmitted into vaginal/cervical epithelial cells, a result indicating the importance of sperm in HIV transmission.

Ethanol Induced Disordering of Pancreatic Acinar Cell Endoplasmic Reticulum: An ER Stress/Defective Unfolded Protein Response Model.

BACKGROUND & AIMS: Heavy alcohol drinking is associated with pancreatitis, whereas moderate intake lowers the risk. Mice fed ethanol long term show no pancreas damage unless adaptive/protective responses mediating proteostasis are disrupted. Pancreatic acini synthesize digestive enzymes (largely serine hydrolases) in the endoplasmic reticulum (ER), where perturbations (eg, alcohol consumption) activate adaptive unfolded protein responses orchestrated by spliced X-box binding protein 1 (XBP1). Here, we examined ethanol-induced early structural changes in pancreatic ER proteins. METHODS: Wild-type and Xbp1+/- mice were fed control and ethanol diets, then tissues were homogenized and fractionated. ER proteins were labeled with a cysteine-reactive probe, isotope-coded affinity tag to obtain a novel pancreatic redox ER proteome. Specific labeling of active serine hydrolases in ER with fluorophosphonate desthiobiotin also was characterized proteomically. Protein structural perturbation by redox changes was evaluated further in molecular dynamic simulations. RESULTS: Ethanol feeding and Xbp1 genetic inhibition altered ER redox balance and destabilized key proteins. Proteomic data and molecular dynamic simulations of Carboxyl ester lipase (Cel), a unique serine hydrolase active within ER, showed an uncoupled disulfide bond involving Cel Cys266, Cel dimerization, ER retention, and complex formation in ethanol-fed, XBP1-deficient mice. CONCLUSIONS: Results documented in ethanol-fed mice lacking sufficient spliced XBP1 illustrate consequences of ER stress extended by preventing unfolded protein response from fully restoring pancreatic acinar cell proteostasis during ethanol-induced redox challenge. In this model, orderly protein folding and transport to the secretory pathway were disrupted, and abundant molecules including Cel with perturbed structures were retained in ER, promoting ER stress-related pancreas pathology.

Focal Irradiation and Systemic TGFß Blockade in Metastatic Breast Cancer.

Purpose: This study examined the feasibility, efficacy (abscopal effect), and immune effects of TGFß blockade during radiotherapy in metastatic breast cancer patients.Experimental Design: Prospective randomized trial comparing two doses of TGFß blocking antibody fresolimumab. Metastatic breast cancer patients with at least three distinct metastatic sites whose tumor had progressed after at least one line of therapy were randomized to receive 1 or 10 mg/kg of fresolimumab, every 3 weeks for five cycles, with focal radiotherapy to a metastatic site at week 1 (three doses of 7.5 Gy), that could be repeated to a second lesion at week 7. Research bloods were drawn at baseline, week 2, 5, and 15 to isolate PBMCs, plasma, and serum.Results: Twenty-three patients were randomized, median age 57 (range 35-77). Seven grade 3/4 adverse events occurred in 5 of 11 patients in the 1 mg/kg arm and in 2 of 12 patients in the 10 mg/kg arm, respectively. Response was limited to three stable disease. At a median follow up of 12 months, 20 of 23 patients are deceased. Patients receiving the 10 mg/kg had a significantly higher median overall survival than those receiving 1 mg/kg fresolimumab dose [hazard ratio: 2.73 with 95% confidence interval (CI), 1.02-7.30; P = 0.039]. The higher dose correlated with improved peripheral blood mononuclear cell counts and a striking boost in the CD8 central memory pool.Conclusions: TGFß blockade during radiotherapy was feasible and well tolerated. Patients receiving the higher fresolimumab dose had a favorable systemic immune response and experienced longer median overall survival than the lower dose group. Clin Cancer Res; 24(11); 2493-504. ©2018 AACR.

Attenuation of Antioxidant Capacity in Human Breast Cancer Cells by Carbon Monoxide through Inhibition of Cystathionine ß-Synthase Activity: Implications in Chemotherapeutic Drug Sensitivity.

Drug resistance is a major impediment to effective treatment of breast cancer. Compared to normal cells, cancer cells have an increased antioxidant potential due to an increased ratio of reduced to oxidized glutathione (GSH/GSSG). This is known to confer therapeutic resistance. Here, we have identified a mechanism, unique to breast cancer cells, whereby cystathionine ß-synthase (CBS) promotes elevated GSH/GSSG. Lentiviral silencing of CBS in human breast cancer cells attenuated GSH/GSSG, total GSH, nuclear factor erythroid 2-related factor 2 (Nrf2), and processes downstream of Nrf2 that promote GSH synthesis and regeneration of GSH from GSSG. Carbon monoxide (CO) reduced GSH/GSSG in three breast cancer cell lines by inhibiting CBS. Furthermore, CO sensitized breast cancer cells to doxorubicin. These results provide insight into mechanism(s) by which CBS increases the antioxidant potential and the ability for CO to inhibit CBS activity to alter redox homeostasis in breast cancer, increasing sensitivity to a chemotherapeutic.

ATR inhibition facilitates targeting of leukemia dependence on convergent nucleotide biosynthetic pathways.

Leukemia cells rely on two nucleotide biosynthetic pathways, de novo and salvage, to produce dNTPs for DNA replication. Here, using metabolomic, proteomic, and phosphoproteomic approaches, we show that inhibition of the replication stress sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) reduces the output of both de novo and salvage pathways by regulating the activity of their respective rate-limiting enzymes, ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), via distinct molecular mechanisms. Quantification of nucleotide biosynthesis in ATR-inhibited acute lymphoblastic leukemia (ALL) cells reveals substantial remaining de novo and salvage activities, and could not eliminate the disease in vivo. However, targeting these remaining activities with RNR and dCK inhibitors triggers lethal replication stress in vitro and long-term disease-free survival in mice with B-ALL, without detectable toxicity. Thus the functional interplay between alternative nucleotide biosynthetic routes and ATR provides therapeutic opportunities in leukemia and potentially other cancers.Leukemic cells depend on the nucleotide synthesis pathway to proliferate. Here the authors use metabolomics and proteomics to show that inhibition of ATR reduced the activity of these pathways thus providing a valuable therapeutic target in leukemia.

Transgenic tomatoes expressing the 6F peptide and ezetimibe prevent diet-induced increases of IFN-ß and cholesterol 25-hydroxylase in jejunum.

Feeding LDL receptor (LDLR)-null mice a Western diet (WD) increased the expression of IFN-ß in jejunum as determined by quantitative RT-PCR (RT-qPCR), immunohistochemistry (IHC), and ELISA (all P < 0.0001). WD also increased the expression of cholesterol 25-hydroxylase (CH25H) as measured by RT-qPCR (P < 0.0001), IHC (P = 0.0019), and ELISA (P < 0.0001), resulting in increased levels of 25-hydroxycholesterol (25-OHC) in jejunum as determined by LC-MS/MS (P < 0.0001). Adding ezetimibe at 10 mg/kg/day or adding a concentrate of transgenic tomatoes expressing the 6F peptide (Tg6F) at 0.06% by weight of diet substantially ameliorated these changes. Adding either ezetimibe or Tg6F to WD also ameliorated WD-induced changes in plasma lipids, serum amyloid A, and HDL cholesterol. Adding the same doses of ezetimibe and Tg6F together to WD (combined formulation) was generally more efficacious compared with adding either agent alone. Surprisingly, adding ezetimibe during the preparation of Tg6F, but before addition to WD, was more effective than the combined formulation for all parameters measured in jejunum (P = 0.0329 to P < 0.0001). We conclude the following: i) WD induces IFN-ß, CH25H, and 25-OHC in jejunum; and ii) Tg6F and ezetimibe partially ameliorate WD-induced inflammation by preventing WD-induced increases in IFN-ß, CH25H, and 25-OHC.

Glutathionylation of Yersinia pestis LcrV and Its Effects on Plague Pathogenesis.

Glutathionylation, the formation of reversible mixed disulfides between glutathione and protein cysteine residues, is a posttranslational modification previously observed for intracellular proteins of bacteria. Here we show that Yersinia pestis LcrV, a secreted protein capping the type III secretion machine, is glutathionylated at Cys273 and that this modification promotes association with host ribosomal protein S3 (RPS3), moderates Y. pestis type III effector transport and killing of macrophages, and enhances bubonic plague pathogenesis in mice and rats. Secreted LcrV was purified and analyzed by mass spectrometry to reveal glutathionylation, a modification that is abolished by the codon substitution Cys273Ala in lcrV Moreover, the lcrVC273A mutation enhanced the survival of animals in models of bubonic plague. Investigating the molecular mechanism responsible for these virulence attributes, we identified macrophage RPS3 as a ligand of LcrV, an association that is perturbed by the Cys273Ala substitution. Furthermore, macrophages infected by the lcrVC273A variant displayed accelerated apoptotic death and diminished proinflammatory cytokine release. Deletion of gshB, which encodes glutathione synthetase of Y. pestis, resulted in undetectable levels of intracellular glutathione, and we used a Y. pestis ΔgshB mutant to characterize the biochemical pathway of LcrV glutathionylation, establishing that LcrV is modified after its transport to the type III needle via disulfide bond formation with extracellular oxidized glutathione.IMPORTANCEYersinia pestis, the causative agent of plague, has killed large segments of the human population; however, the molecular bases for the extraordinary virulence attributes of this pathogen are not well understood. We show here that LcrV, the cap protein of bacterial type III secretion needles, is modified by host glutathione and that this modification contributes to the high virulence of Y. pestis in mouse and rat models for bubonic plague. These data suggest that Y. pestis exploits glutathione in host tissues to activate a virulence strategy, thereby accelerating plague pathogenesis.

Cystathionine: A novel oncometabolite in human breast cancer.

In this study, we have identified cystathionine (CTH), a sulfur containing metabolite, to be selectively enriched in human breast cancer (HBC) tissues (∼50-100 pmoles/mg protein) compared with undetectable levels in normal breast tissues. The accumulation of CTH, specifically in HBC, was attributed to the overexpression of cystathionine beta synthase (CBS), its synthesizing enzyme, and the undetectable levels of its downstream metabolizing enzyme, cystathionine gamma lyase (CGL). Interestingly both CBS and CGL could not be detected in normal breast tissues. We further observed that CTH protected HBC cells against excess reactive oxygen species (ROS) and chemotherapeutic drug-induced apoptosis. Moreover, CTH promoted both mitochondrial and endoplasmic reticulum homeostasis in HBC cells. As both the mitochondria and the endoplasmic reticulum are key organelles regulating the onset of apoptosis, we reasoned that endogenous CTH could be contributing towards increasing the apoptotic threshold in HBC cells. An increased apoptotic threshold is a hallmark of all cancer types, including HBC, and is primarily responsible for drug resistance. Hence this study unravels one of the possible pathways that may contribute towards drug resistance in HBC.

[18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-ß-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-ß-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

Tg6F ameliorates the increase in oxidized phospholipids in the jejunum of mice fed unsaturated LysoPC or WD.

Mouse chow supplemented with lysophosphatidylcholine with oleic acid at sn-1 and a hydroxyl group at sn-2 (LysoPC 18:1) increased LysoPC 18:1 in tissue of the jejunum of LDL receptor (LDLR)-null mice by 8.9 ± 1.7-fold compared with chow alone. Western diet (WD) contained dramatically less phosphatidylcholine 18:1 or LysoPC 18:1 compared with chow, but feeding WD increased LysoPC 18:1 in the jejunum by 7.5 ± 1.4-fold compared with chow. Feeding LysoPC 18:1 or feeding WD increased oxidized phospholipids in the jejunum by 5.2 ± 3.0-fold or 8.6 ± 2.2-fold, respectively, in LDLR-null mice (P < 0.0004), and 2.6 ± 1.5-fold or 2.4 ± 0.92-fold, respectively, in WT C57BL/6J mice (P < 0.0001). Adding 0.06% by weight of a concentrate of transgenic tomatoes expressing the 6F peptide (Tg6F) decreased LysoPC 18:1 in the jejunum of LDLR-null mice on both diets (P < 0.0001), and prevented the increase in oxidized phospholipids in the jejunum in LDLR-null and WT mice on both diets (P < 0.008). Tg6F decreased inflammatory cells in the villi of the jejunum, decreased dyslipidemia, and decreased systemic inflammation in LDLR-null and WT mice on both diets. We conclude that Tg6F reduces diet-induced inflammation by reducing the content of unsaturated LysoPC and oxidized phospholipids in the jejunum of mice.

A Designed Inhibitor of p53 Aggregation Rescues p53 Tumor Suppression in Ovarian Carcinomas.

Half of all human cancers lose p53 function by missense mutations, with an unknown fraction of these containing p53 in a self-aggregated amyloid-like state. Here we show that a cell-penetrating peptide, ReACp53, designed to inhibit p53 amyloid formation, rescues p53 function in cancer cell lines and in organoids derived from high-grade serous ovarian carcinomas (HGSOC), an aggressive cancer characterized by ubiquitous p53 mutations. Rescued p53 behaves similarly to its wild-type counterpart in regulating target genes, reducing cell proliferation and increasing cell death. Intraperitoneal administration decreases tumor proliferation and shrinks xenografts in vivo. Our data show the effectiveness of targeting a specific aggregation defect of p53 and its potential applicability to HGSOCs.