Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase

A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium [HPLM]). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis—an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility.National Institutes of Health (U.S.) (Grant R01CA103866)National Institutes of Health (U.S.) (Grant R37AI047389

Biphasic Elimination of Tenofovir Diphosphate and Nonlinear Pharmacokinetics of Zidovudine Triphosphate in a Microdosing Study

Objective: Phase 0 studies can provide initial pharmacokinetics (PKs) data in humans and help to facilitate early drug development, but their predictive value for standard dosing is controversial. To evaluate the prediction of microdosing for active intracellular drug metabolites, we compared the PK profile of 2 antiretroviral drugs, zidovudine (ZDV) and tenofovir (TFV), in microdose and standard dosing regimens. Study Design: We administered a microdose (100 μg) of [superscript 14]C-labeled drug (ZDV or tenofovir disoproxil fumarate) with or without a standard unlabelled dose (300 mg) to healthy volunteers. Both the parent drug in plasma and the active metabolite, ZDV-triphosphate (ZDV-TP) or TFV-diphosphate (TFV-DP) in peripheral blood mononuclear cells (PBMCs) and CD4[superscript +] cells were measured by accelerator mass spectrometry. Results: The intracellular ZDV-TP concentration increased less than proportionally over the dose range studied (100 μg–300 mg), whereas the intracellular TFV-DP PKs were linear over the same dose range. ZDV-TP concentrations were lower in CD4[superscript +] cells versus total PBMCs, whereas TFV-DP concentrations were not different in CD4[superscript +] cells and PBMCs. Conclusions: Our data were consistent with a rate-limiting step in the intracellular phosphorylation of ZDV but not TFV. Accelerator mass spectrometry shows promise for predicting the PK of active intracellular metabolites of nucleosides, but nonlinearity of PK may be seen with some drugs.Johns Hopkins University (Institute for Clinical and Translational Research CTSA Grant UL1-RR025005

Enteroaggregative Escherichia coli Adherence Fimbriae Drive Inflammatory Cell Recruitment via Interactions with Epithelial MUC1

Enteroaggregative Escherichia coli (EAEC) causes diarrhea and intestinal inflammation worldwide. EAEC strains are characterized by the presence of aggregative adherence fimbriae (AAF), which play a key role in pathogenesis by mediating attachment to the intestinal mucosa and by triggering host inflammatory responses. Here, we identify the epithelial transmembrane mucin MUC1 as an intestinal host cell receptor for EAEC, demonstrating that AAF-mediated interactions between EAEC and MUC1 facilitate enhanced bacterial adhesion. We further demonstrate that EAEC infection also causes elevated expression of MUC1 in inflamed human intestinal tissues. Moreover, we find that MUC1 facilitates AAF-dependent migration of neutrophils across the epithelium in response to EAEC infection. Thus, we show for the first time a proinflammatory role for MUC1 in the host response to an intestinal pathogen. IMPORTANCE: EAEC is a clinically important intestinal pathogen that triggers intestinal inflammation and diarrheal illness via mechanisms that are not yet fully understood. Our findings provide new insight into how EAEC triggers host inflammation and underscores the pivotal role of AAFs-the principal adhesins of EAEC-in driving EAEC-associated disease. Most importantly, our findings add a new dimension to the signaling properties of the transmembrane mucin MUC1. Mostly studied for its role in various forms of cancer, MUC1 is widely regarded as playing an anti-inflammatory role in response to infection with bacterial pathogens in various tissues. However, the role of MUC1 during intestinal infections has not been previously explored, and our results describe the first report of MUC1 as a proinflammatory factor following intestinal infection

Single Dose Pharmacokinetics of Oral Tenofovir in Plasma, Peripheral Blood Mononuclear Cells, Colonic Tissue, and Vaginal Tissue

HIV seroconversion outcomes in preexposure prophylaxis (PrEP) trials of oral tenofovir (TFV)-containing regimens are highly sensitive to drug concentration, yet less-than-daily dosing regimens are under study. Description of TFV and its active moiety, TFV diphosphate (TFV-DP), in blood, vaginal tissue, and colon tissue may guide the design and interpretation of PrEP clinical trials. Six healthy women were administered a single oral dose of 300 mg tenofovir disoproxil fumarate (TDF) and 4.3 mg (12.31 MBq, 333 μCi) [superscript 14]C-TDF slurry. Blood was collected every 4 h for the first 24 h, then at 4, 8, 11, and 15 days postdosing. Colonic and vaginal samples (tissue, total and CD4+ cells, luminal fluid and cells) were collected 1, 8 and 15 days postdose. Samples were analyzed for TFV and TFV-DP. Plasma TFV demonstrated triphasic decay with terminal elimination half-life median [interquartile range (IQR)] 69 h (58–77). Peripheral blood mononuclear cell (PBMC) TFV-DP demonstrated biphasic peaks (median 12 h and 96 h) followed by a terminal 48 h (38–76) half-life; C[subscript max] was 20 fmol/million cells (2–63). One day postdose, the TFV-DP paired colon:vaginal tissue concentration ratio was 1 or greater in all subjects' tissue homogenates, median 124 (range 1–281), but was not sustained. The ratio was lower and more variable in cells extracted from tissue. Among all sample types, TFV and TFV-DP half-life ranged from 23 to 139 h. PBMC TFV-DP rose slowly in the hours after dosing indicating that success with exposure-driven dosing regimens may be sensitive to timing of the dose prior to exposure. Colonic tissue homogenate TFV-DP concentrations were greater than in vaginal homogenate at 24 h, but not in cells extracted from tissue. These and the other pharmacokinetic findings will guide the interpretation and design of future PrEP trials.National Center for Advancing Translational Sciences (U.S.) (Grant UL1RR025005)National Institutes of Health (U.S.)National Institutes of Health (U.S.) (Roadmap for Medical Research

Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis

Neutrophil influx into the intestinal lumen is a critical response to infectious agents, but is also associated with severe intestinal damage observed in idiopathic inflammatory bowel disease. The chemoattractant hepoxilin A3, an eicosanoid secreted from intestinal epithelial cells by the apically restricted efflux pump multidrug resistance protein 2 (MRP2), mediates this neutrophil influx. Information about a possible counterbalance pathway that could signal the lack of or resolution of an apical inflammatory signal, however, has yet to be described. We now report a system with such hallmarks. Specifically, we identify endocannabinoids as the first known endogenous substrates of the apically restricted multidrug resistance transporter P-glycoprotein (P-gp) and reveal a mechanism, which we believe is novel, for endocannabinoid secretion into the intestinal lumen. Knockdown or inhibition of P-gp reduced luminal secretion levels of N-acyl ethanolamine-type endocannabinoids, which correlated with increased neutrophil transmigration in vitro and in vivo. Additionally, loss of CB2, the peripheral cannabinoid receptor, led to increased pathology and neutrophil influx in models of acute intestinal inflammation. These results define a key role for epithelial cells in balancing the constitutive secretion of antiinflammatory lipids with the stimulated secretion of proinflammatory lipids via surface efflux pumps in order to control neutrophil infiltration into the intestinal lumen and maintain homeostasis in the healthy intestine

Seasonal changes in patterns of gene expression in avian song control brain regions.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by altering the hormonal and photoperiodic conditions in seasonally-breeding Gambel's white-crowned sparrows and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. We found that 363 genes varied by more than 1.5 fold (>log(2) 0.585) in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity

Inhibitory Activity of Bevacizumab to Differentiation of Retinoblastoma Cells

Vascular endothelial growth factor (VEGF) is a major regulator in retinal and choroidal angiogenesis, which are common causes of blindness in all age groups. Recently anti-VEGF treatment using anti-VEGF antibody has revolutionarily improved the visual outcome in patients with vaso-proliferative retinopathies. Herein, we demonstrated that bevacizumab as an anti-VEGF antibody could inhibit differentiation of retinoblastoma cells without affection to cellular viability, which would be mediated via blockade of extracellular signal-regulated kinase (ERK) 1/2 activation. The retinoblastoma cells expressed VEGFR-2 as well as TrkA which is a neurotrophin receptor associated with differentiation of retinoblastoma cells. TrkA in retinoblastoma cells was activated with VEGF treatment. Interestingly even in the concentration of no cellular death, bevascizumab significantly attenuated the neurite formation of differentiated retinoblastoma cells, which was accompanied by inhibition of neurofilament and shank2 expression. Furthermore, bevacizumab inhibited differentiation of retinoblastoma cells by blockade of ERK 1/2 activation. Therefore, based on that the differentiated retinoblastoma cells are mostly photoreceptors, our results suggest that anti-VEGF therapies would affect to the maintenance or function of photoreceptors in mature retina

Bi-Directional Sexual Dimorphisms of the Song Control Nucleus HVC in a Songbird with Unison Song

Sexually dimorphic anatomy of brain areas is thought to be causally linked to sex differences in behaviour and cognitive functions. The sex with the regional size advantage (male or female) differs between brain areas and species. Among adult songbirds, males have larger brain areas such as the HVC (proper name) and RA (robust nucleus of the arcopallium) that control the production of learned songs. Forest weavers (Ploceus bicolor) mated pairs sing a unison duet in which male and female mates learn to produce identical songs. We show with histological techniques that the volume and neuron numbers of HVC and RA were ≥1.5 times larger in males than in females despite their identical songs. In contrast, using in-situ hybridizations, females have much higher (30–70%) expression levels of mRNA of a number of synapse-related proteins in HVC and/or RA than their male counterparts. Male-typical and female-typical sexual differentiation appears to act on different aspects of the phenotypes within the same brain areas, leading females and males to produce the same behaviour using different cellular mechanisms

A Randomized Trial to Assess Anti-HIV Activity in Female Genital Tract Secretions and Soluble Mucosal Immunity Following Application of 1% Tenofovir Gel

Preclinical and early phase clinical microbicide studies have not consistently predicted the outcome of efficacy trials. To address this gap, candidate biomarkers of microbicide pharmacodynamics and safety were evaluated in a double-blind, placebo-controlled trial of tenofovir gel, the first microbicide to demonstrate significant protection against HIV acquisition.30 women were randomized to apply a single daily dose of tenofovir or placebo gel for 14 consecutive days. Anti-HIV activity was measured in cervicovaginal lavage (CVL) on Days 0, 3, 7, 14 and 21 by luciferase assay as a surrogate marker of pharmacodynamics. Endogenous activity against E. coli and HSV-2 and concentrations of immune mediators were quantified in CVL as candidate biomarkers of safety. Tenofovir levels were measured in CVL and blood.A significant increase in anti-HIV activity was detected in CVL from women who applied tenofovir gel compared to their endogenous anti-HIV activity in genital tract secretions on Day 0 and compared to activity in CVL from women in the placebo group. The activity correlated significantly with CVL concentration of tenofovir (r = 0.6, p<0.001) and fit a sigmoid E(max) pharmacodynamic model. Anti-HIV activity in CVL from women who applied tenofovir persisted when virus was introduced in semen, whereas endogenous anti-HIV activity decreased. Tenofovir did not trigger an inflammatory response or induce sustained loss in endogenous antimicrobial activity or immune mediators.Tenofovir gel had no deleterious impact on soluble mucosal immunity. The increased anti-HIV activity in CVL, which persisted in the presence of semen and correlated with tenofovir concentration, is consistent with the efficacy observed in a recent clinical trial. These results promote quantified CVL anti-HIV activity as a surrogate of tissue pharmacodynamics and as a potential biomarker of adherence to product. This simple, feasible and inexpensive bioassay may promote the development of models more predictive of microbicide efficacy.ClinicalTrials.gov NCT00594373