Primate fetal hepatic responses to maternal obesity: epigenetic signalling pathways and lipid accumulation.

KEY POINTS: Maternal obesity (MO) and exposure to a high-fat, high-simple-carbohydrate diet during pregnancy predisposes offspring to obesity, metabolic and cardiovascular disorders in later life. Underlying molecular pathways and potential epigenetic factors that are dysregulated in MO were identified using unbiased transcriptomic methods. There was increased lipid accumulation and severe steatosis in the MO baboon fetal liver suggesting that these offspring are on an early trajectory of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. ABSTRACT: Maternal obesity (MO) increases offspring cardiometabolic disease risk. Altered fetal liver development in response to the challenge of MO has metabolic consequences underlying adverse offspring life-course health outcomes. Little is known about the molecular pathways and potential epigenetic changes regulating primate fetal liver responses to MO. We hypothesized that MO would induce fetal baboon liver epigenetic changes resulting in dysregulation of key metabolic pathways that impact lipid metabolism. MO was induced prior to pregnancy by a high-fat, high-fructose diet. Unbiased gene and microRNA (small RNA Seq) abundance analyses were performed on fetal baboon livers at 0.9 gestation and subjected to pathway analyses to identify fetal liver molecular responses to MO. Fetal baboon liver lipid and glycogen content were quantified by the Computer Assisted Stereology Toolbox. In response to MO, fetal livers revealed dysregulation of TCA cycle, proteasome, oxidative phosphorylation, glycolysis and Wnt/ß-catenin signalling pathways together with marked lipid accumulation supporting our hypothesis that multiple pathway dysregulation detrimentally impacts lipid management. This is the first study of MO programming of the non-human primate fetal liver using unbiased transcriptome analysis to detect changes in hepatic gene expression levels and identify potential microRNA epigenetic regulators of metabolic disruption.

Prematurity disrupts glomeruli development, whereas prematurity and hyperglycemia lead to altered nephron maturation and increased oxidative stress in newborn baboons.

BackgroundPremature birth occurs when nephrogenesis is incomplete and has been linked to increased renal pathologies in the adult. Metabolic factors complicating preterm birth may have additional consequences for kidney development. Here, we evaluated the effects of prematurity and hyperglycemia on nephrogenesis in premature baboons when compared with those in term animals.MethodsBaboons were delivered prematurely (67% gestation; n=9) or at term (n=7) and survived for 2-4 weeks. Preterm animals were classified by glucose control during the first 5 days of life: normoglycemic (PtN; serum glucose 50-100 mg/dl, n=6) and hyperglycemic (PtH; serum glucose 150-250 mg/dl, n=3). Kidneys were assessed histologically for glomeruli relative area, maturity, size, and overall morphology. Kidney lysates were evaluated for oxidative damage with 4-hydroxynonenal (4-HNE) antibody.ResultsHistological examination revealed decreased glomeruli relative area (P<0.05), fewer glomerular generations (P<0.01), and increased renal corpuscle area (P<0.001) in preterm compared with those in term animals. Numbers of apoptotic glomeruli were similar between groups. PtH kidneys exhibited reduced nephrogenic zone width (P<0.0001), increased numbers of mature glomeruli (P<0.05), and increased 4-HNE staining compared with those in PtN kidneys.ConclusionPrematurity interrupts normal kidney development, independent of glomerular cell apoptosis. When prematurity is complicated by hyperglycemia; kidney development shifts toward accelerated maturation and increased oxidative stress.

Favorable Outcomes of Preterm Infants With Parenteral Nutrition-associated Liver Disease Treated With Intravenous Fish Oil-based Lipid Emulsion.

OBJECTIVES: The aim of the study was to determine the acute and long-term outcomes of preterm infants treated with an intravenous fish oil-based lipid emulsion (FishLE) for parenteral nutrition-associated liver disease (PNALD). METHODS: Preterm infants 14 days to 24 months of age with anatomic short gut or severe intestinal dysmotility, serum direct bilirubin ≥4 mg/dL, and requiring >60% calories from parenteral nutrition were eligible. Enrolled infants received 1 g ·â€Škg ·â€Šday of FishLE until resolution of direct hyperbilirubinemia or return of enteral nutrition. Acute clinical effects and biochemical markers of liver function were monitored. Growth and developmental scores at 6 and 12 months postmenstrual age (PMA) were assessed and compared with controls matched by gestational age (GA). RESULTS: Thirteen patients with mean GA of 28 ±â€Š4 weeks were treated and compared with 119 GA-matched controls. Their mean direct bilirubin was 9.8 ±â€Š6.4 mg/dL at enrollment. All infants had resolution of cholestasis after study completion. There were no acute adverse events, deaths, or liver/intestinal transplants. Weight and head circumference were similar between FishLE-treated patients and controls at 6- and 12-month PMA. Cognitive and motor scores were decreased at 6 and 12 months PMA in FishLE-treated infants. Logistic regression analysis showed that prolonged hospitalization was detrimental to cognitive and motor development, whereas treatment was not. CONCLUSIONS: The use of intravenous FishLEs in premature infants appears to be safe and reverses PNALD despite significant liver disease and intestinal failure. This therapy should be used in preterm infants with PNALD and followed long term to evaluate development.

Variability in the Treatment of Prehospital Hypoglycemia: A Structured Review of EMS Protocols in the United States.

BACKGROUND: In many industries, limiting variability in process has been associated with a reduction in errors. Hypoglycemia is a common prehospital diabetic emergency for which most EMS systems have a treatment protocol. OBJECTIVE: To examine the treatment variability for prehospital hypoglycemia within EMS protocols in the U.S. METHODS: EMS protocols were reviewed in a structured fashion from 2 sources: the website www.emsprotocols.org and through manual identification from the 50 largest populated cities in the U.S. Data was abstracted by trained investigators regarding the concentration of glucose recommended for the parenteral reversal of hypoglycemia, clinical treatment thresholds, dose recommendations, follow-up care, and non-transport policies. Descriptive statistics were used to summarize the findings. We also reviewed these EMS protocols for the protocol's effective date, the presence of a specific hypoglycemia patient non-transport policy, the use of dilutions of hypertonic dextrose for pediatric patients, glucagon use, and CBG or GCS for patient follow-up. RESULTS: Protocols were retrieved from 185 EMS agencies of a variety of sizes across the U.S. Seventy percent specified only D50 for the treatment of hypoglycemia in adult patients, 8% only D10, and 22% either D10 or D50. Most protocols (85%), which used D50, specified concentration dilutions for pediatric patients. The most frequently specified initial dose of glucose was 25 g of glucose for adults (73-78%), 0.5 g/kg for pediatric (70%), and 0.5 g/kg for neonates (45%). The median blood glucose level threshold for treatment was 60 mg/dl for patients of all ages, but the mean treatment threshold levels for adults, pediatric patients and neonates were statistically different (p < 0.0001). Nearly all protocols (97%) allowed for the use of glucagon in the absence of vascular access. Patient follow up with a repeat CBG was recommended in 32%, both CBG and GCS in 31%, GCS only in 4%, and no follow-up was specified in 33% of the protocols. A specific policy permitting the non-transport of select patients whose hypoglycemia had been corrected was noted in slightly less than half (49%) of the protocols. CONCLUSIONS: In the U.S., EMS protocols for the treatment of hypoglycemia vary significantly. Further studies are warranted to determine the factors underlying this variability and effects on patient outcomes.

Thankful for the little things: A meta-analysis of gratitude interventions.

A recent qualitative review by Wood, Froh, and Geraghty (2010) cast doubt on the efficacy of gratitude interventions, suggesting the need to carefully attend to the quality of comparison groups. Accordingly, in a series of meta-analyses, we evaluate the efficacy of gratitude interventions (ks = 4-18; Ns = 395-1,755) relative to a measurement-only control or an alternative-activity condition across 3 outcomes (i.e., gratitude, anxiety, psychological well-being). Gratitude interventions outperformed a measurement-only control on measures of psychological well-being (d = .31, 95% confidence interval [CI = .04, .58]; k = 5) but not gratitude (d = .20; 95% CI [-.04, .44]; k = 4). Gratitude interventions outperformed an alternative-activity condition on measures of gratitude (d = .46, 95% CI [.27, .64]; k = 15) and psychological well-being (d = .17, 95% CI [.09, .24]; k = 20) but not anxiety (d = .11, 95% CI [-.08, .31]; k = 5). More-detailed subdivision was possible on studies with outcomes assessing psychological well-being. Among these, gratitude interventions outperformed an activity-matched comparison (d = .14; 95% CI [.01, .27]; k = 18). Gratitude interventions performed as well as, but not better than, a psychologically active comparison (d = -.03, 95% CI [-.13, .07]; k = 9). On the basis of these findings, we summarize the current state of the literature and make suggestions for future applied research on gratitude. (PsycINFO Database Record

Review of the demographic factors and toxicology related to deaths due to pregabalin in Northern Ireland.

The potential for recreational abuse of the analgesic and antiepileptic drug pregabalin is now well established in the literature. The potential minimum lethal dose in post mortem cases is however less defined. All post mortem examinations in Northern Ireland where the cause of death was found to be due to pregabalin were examined for demographic and toxicological analysis. Deaths are generally seen in young men, especially 30-39-year-olds, many of whom have a history of substance misuse and are often prescribed pregabalin. Until recently, prescription rates have been on the rise regionally. The overall median post mortem peripheral blood concentration of pregabalin found in this study is 10.6 mg/L, however this rises when concurrent drugs and alcohol are considered and in cases where pregabalin is considered responsible for death alone (i.e. outside of multidrug toxicity). Pregabalin peripheral blood concentrations returned in this study suggest previously offered minimum lethal dosages may need to be revised downward.

Hyperglycemia increases the risk of death in extremely preterm baboons.

BACKGROUND: Transient neonatal hyperglycemia (HG) has been reported in up to 80% of extremely preterm human infants. We hypothesize that severe HG is associated with increased morbidity and mortality in preterm baboons. METHODS: Sixty-six baboons born at 67% of gestation were studied. HG was defined as serum glucose level ≥150 mg/dl during the first week of life. Animals were stratified into two groups: severe HG (≥8 events) and nonsevere HG (<8 events). RESULTS: HG developed in 65 of the 66 (98%) baboons that were included. A total of 3,386 glucose measurements were obtained. The mean serum glucose level was 159 ± 69 mg/dl for the severe HG group and 130 ± 48 mg/dl for the nonsevere HG group during the first week of life. No differences were found in gender, birth weight, sepsis, patent ductus arteriosus, or oxygenation/ventilation indexes between groups. Severe HG was associated with early death even after controlling for sepsis, postnatal steroid exposure, and catecholamine utilization. CONCLUSION: HG is common in preterm baboons and is not associated with short-term morbidity. Severe HG occurring in the first week of life is associated with early death in preterm baboons.

A homogeneous method for investigation of methylation-dependent protein-protein interactions in epigenetics.

Methylation of lysine residues on the tails of histone proteins is a major determinant of the transcription state of associated DNA coding regions. The interplay among methylation states and other histone modifications to direct transcriptional outcome is referred to as the histone code. In addition to histone methyltransferases and demethylases which function to modify the methylation state of lysine sidechains, other proteins recognize specific histone methylation marks essentially serving as code readers. While these interactions are highly specific with respect to site and methylation state of particular lysine residues, they are generally weak and therefore difficult to monitor by traditional assay techniques. Herein, we present the design and implementation of a homogeneous, miniaturizable, and sensitive assay for histone methylation-dependent interactions. We use AlphaScreen, a chemiluminescence-based technique, to monitor the interactions of chromodomains (MPP8, HP1beta and CHD1), tudor domains (JMJD2A) and plant homeodomains (RAG2) with their cognate trimethyllysine histone partners. The utility of the method was demonstrated by profiling the binding specificities of chromo- and tudor domains toward several histone marks. The simplicity of design and the sensitive and robust nature of this assay should make it applicable to a range of epigenetic studies, including the search for novel inhibitors of methylation-dependent interactions.

Specificity of human aldo-keto reductases, NAD(P)H:quinone oxidoreductase, and carbonyl reductases to redox-cycle polycyclic aromatic hydrocarbon diones and 4-hydroxyequilenin-o-quinone.

Polycyclic aromatic hydrocarbons (PAHs) are suspect human lung carcinogens and can be metabolically activated to remote quinones, for example, benzo[a]pyrene-1,6-dione (B[a]P-1,6-dione) and B[a]P-3,6-dione by the action of either P450 monooxygenase or peroxidases, and to non-K region o-quinones, for example B[a]P-7,8-dione, by the action of aldo keto reductases (AKRs). B[a]P-7,8-dione also structurally resembles 4-hydroxyequilenin o-quinone. These three classes of quinones can redox cycle, generate reactive oxygen species (ROS), and produce the mutagenic lesion 8-oxo-dGuo and may contribute to PAH- and estrogen-induced carcinogenesis. We compared the ability of a complete panel of human recombinant AKRs to catalyze the reduction of PAH o-quinones in the phenanthrene, chrysene, pyrene, and anthracene series. The specific activities for NADPH-dependent quinone reduction were often 100-1000 times greater than the ability of the same AKR isoform to oxidize the cognate PAH-trans-dihydrodiol. However, the AKR with the highest quinone reductase activity for a particular PAH o-quinone was not always identical to the AKR isoform with the highest dihydrodiol dehydrogenase activity for the respective PAH-trans-dihydrodiol. Discrete AKRs also catalyzed the reduction of B[a]P-1,6-dione, B[a]P-3,6-dione, and 4-hydroxyequilenin o-quinone. Concurrent measurements of oxygen consumption, superoxide anion, and hydrogen peroxide formation established that ROS were produced as a result of the redox cycling. When compared with human recombinant NAD(P)H:quinone oxidoreductase (NQO1) and carbonyl reductases (CBR1 and CBR3), NQO1 was a superior catalyst of these reactions followed by AKRs and last CBR1 and CBR3. In A549 cells, two-electron reduction of PAH o-quinones causes intracellular ROS formation. ROS formation was unaffected by the addition of dicumarol, suggesting that NQO1 is not responsible for the two-electron reduction observed and does not offer protection against ROS formation from PAH o-quinones.

Evidence for the aldo-keto reductase pathway of polycyclic aromatic trans-dihydrodiol activation in human lung A549 cells.

Polycyclic aromatic hydrocarbons (PAHs) are tobacco carcinogens implicated in the causation of human lung cancer. Metabolic activation is a key prerequisite for PAHs to cause their deleterious effects. Using human lung adenocarcinoma (A549) cells, we provide evidence for the metabolic activation of (+/-)-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-trans-dihydrodiol) by aldo-keto reductases (AKRs) to yield benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione), a redox-active o-quinone. We show that B[a]P-7,8-trans-dihydrodiol (AKR substrate) and B[a]P-7,8-dione (AKR product) lead to the production of intracellular reactive oxygen species (ROS) (measured as an increase in dichlorofluorescin diacetate fluores-cence) and that similar changes were not observed with the regioisomer (+/-)-trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene or the diol-epoxide, (+/-)-anti-7,8-dihydroxy-9alpha,10beta-epoxy-7,8,9,10-tetrahydro-B[a]P. B[a]P-7,8-trans-dihydrodiol and B[a]P-7,8-dione also caused a decrease in glutathione levels and an increase in NADP(+)/NADPH ratios, with a concomitant increase in single-strand breaks (as measured by the comet assay) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo). The specificity of the comet assay was validated by coupling it to human 8-oxo-guanine glycosylase (hOGG1), which excises 8-oxo-Gua to yield single-strand breaks. The levels of 8-oxo-dGuo observed were confirmed by an immunoaffinity purification stable isotope dilution ([(15)N(5)]-8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry (LC-ESI/MRM/MS) assay. B[a]P-7,8-trans-dihydrodiol produced DNA strand breaks in the hOGG1-coupled comet assay as well as 8-oxo-dGuo (as measured by LC-ESI/MRM/MS) and was enhanced by a catechol O-methyl transferase (COMT) inhibitor, suggesting that COMT protects against o-quinone-mediated redox cycling. We conclude that activation of PAH-trans-dihydrodiols by AKRs in lung cells leads to ROS-mediated genotoxicity and contributes to lung carcinogenesis.

Effect of environmental enrichment and group size on the water use and waste in grower-finisher pigs.

The grower-finisher stage accounts for 64% of the total on-farm herd water use. Part of this is consumed by the pigs, but a part is also wasted. Drinking water usage and wastage is affected by different factors. We investigated how different group sizes and different levels of enrichment affect water usage (ingested plus wasted), water wastage, behavior and performance in grower-finisher pigs. Pigs (n = 672), 11 weeks of age (77 ± 2 days) were used for the experiment. The effect of group size: SMALL (12 pigs), MEDIUM (24 pigs), and LARGE (48 pigs) was assessed across two levels of enrichment (LOW-wooden post, hanging rubber toy, HIGH-Same as LOW + fresh grass). There was no effect of group size on water use or wastage. Pigs with HIGH enrichment (10.4 ± 0.4 L/pig/day) used less water than LOW enrichment (11.0 ± 0.4 L/pig/day; p < 0.001). The water wastage/drinker/hour was lower in pens with HIGH enrichment than LOW (p = 0.003). The drinking bout number (p = 0.037) and total occupancy/hour (p = 0.048) was also higher for pens with LOW than HIGH enrichment. Aggressive and harmful behaviour were performed less in LARGE groups and pens with HIGH enrichment. Thus, HIGH enrichment allowance reduced water usage and wastage so may have benefits for the environment, as well as animal welfare.

80 Years of vision: preventing blindness from retinopathy of prematurity.

Retinopathy of prematurity (ROP) is one of the leading yet preventable causes of childhood blindness worldwide. The purpose of this review is to provide a practical template for observational and treatment methods in order to reduce the overall incidence of any ROP and to improve both short-term and long-term outcomes once Type 1 ROP (treatable ROP) develops.

Malassezia Hepatic Abscess in a Neonate.

Malassezia sp. require exogenous lipid for growth and can cause disseminated infection in neonates requiring intravenous lipid infusions. Usually, Malassezia infection in neonates presents as fungemia or hematogenous dissemination into bone or lungs. We present a presumed case of Malassezia liver abscess associated with lipid infusion via a mispositioned umbilical venous catheter.

Effect of different cleaning procedures on water use and bacterial levels in weaner pig pens.

Pork is one of the most globally eaten meats and the pig production chain contributes significantly to the water footprint of livestock production. However, very little knowledge is available about the on-farm factors that influence freshwater use in the pig production chain. An experiment was conducted to quantify the effect of three different washing treatments on freshwater use, bacterial levels [(total bacterial counts; TBC), Enterobacteriaceae and Staphylococcus] and cleaning time in washing of pens for weaning pigs. Three weaner rooms were selected with each room having 10 pens and a capacity to hold up to 14 pigs each. Pigs were weaned and kept in the pens for 7 weeks. Finally, the pens were cleaned before the next batch of pigs moved in. The washing treatments used were power washing and disinfection (WASH); presoaking followed by power washing and disinfection (SOAK), and presoaking followed by detergent, power washing and disinfection (SOAK + DETER). A water meter was used to collect water use data and swab samples were taken to determine the bacterial levels. The results showed that there was no overall effect of washing treatments on water use. However, there was an effect of treatment on the washing time (p<0.01) with SOAK and SOAK+DETER reducing the washing time per pen by 2.3 minutes (14%) and 4.2 minutes (27%) compared to WASH. Nonetheless, there was an effect of sampling time (before or after washing) (p<0.001) on the levels of TBC and Staphylococcus, but no effect was seen on Enterobacteriaceae levels. Thus, the washing treatments used in this study had no effect on the water use of the pork production chain. Although there was no difference in both water use and bacterial load, from a producer perspective, presoaking and detergent use can save time and labour costs, so this would be the preferred option.

Phospho-STAT5 and phospho-Akt expression in chronic myeloproliferative neoplasms.

The majority of Myeloproliferative Neoplasms (MPNs) are characterised by mutations in genes encoding molecules or receptors involved in cell signalling, the most common being the JAK2 V617F mutation. This mutation leads to ligand-independent activation of downstream signalling pathways by constitutive phosphorylation. The signalling pathways affected include the Janus kinase-signal transducers and activators of transcription (JAK-STAT) and phosphotidylinositide-3 kinase (PI3K) pathways, which regulate cell survival and apoptosis respectively. Monoclonal antibodies to phospho-STAT5 and phospho-Akt were generated and assessed by immunocytochemistry on bone marrow biopsies of MPN patients with JAK2 V617F, JAK2 exon 12, MPL exon 10 and KIT D816V mutations. JAK2 V617F mutation was associated with significantly increased levels of phosphorylated STAT5 and Akt in haemopoietic cells, most marked in megakaryocytes. In contrast, JAK2 exon 12 and MPL exon 10 mutations did not affect the level of phosphorylation. In systemic mastocytosis with KIT D618V mutation there was significantly increased expression of phosphorylated STAT5 and Akt in neoplastic mast cells although there was no change in the expression in other haemopoietic cells. JAK2 V617F is associated with upregulated phosphorylation of STAT5 and Akt in megakaryocytes, and to a lesser extent in other haemopoietic cells. Immunocytochemistry of bone marrow trephines for these phospho-proteins can be used as a supplementary diagnostic test with a high negative predictive value.

Air Leak Syndrome: Pneumoperitoneum in a Ventilated Neonate.

Air leak syndrome has several manifestations and is common in neonates with meconium aspiration syndrome (MAS) due to air trapping. While pneumoperitoneum is classically a result of intestinal perforation, intra-abdominal free air may be a less common presentation of air leak syndrome. In the ventilated neonate, there is insufficient clinical evidence outlining management of pneumoperitoneum in this situation. We report a case of a term neonate with MAS and air leak syndrome who developed benign pneumoperitoneum (BPPT).

Comparisons of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol activation by human cytochrome P450 and aldo-keto reductase enzymes: effect of redox state and expression levels.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are metabolically activated to proximate carcinogenic trans-dihydrodiols. PAH trans-dihydrodiols are further activated in humans by cytochrome P450 (P450) 1A1 and 1B1 to yield diol-epoxides or by aldo-keto reductases (AKR) 1A1 and 1C1-1C4 to yield reactive and redox-active o-quinones. Reconstituted in vitro systems were used to compare the steady-state kinetic constants for human P450 (P450 1A1 and 1B1) and AKR (AKR1A1, AKR1C1-1C4) mediated metabolism of (+/-)- trans-7,8-dihydroxy-7,8-dihydrobenzo[ a]pyrene ((+/-)-B[ a]P-7,8-diol) at physiological pH. It was found that P450 isoforms yielded much greater k cat/ K m values than AKR enzymes. Initial rates of (+/-)-B[ a]P-7,8-diol oxidation were measured for AKR1A1, AKR1C2, P450 1A1, and P450 1B1 as the ratio of NADPH/NAD (+) cofactors was varied to determine the redox state necessary for AKRs to successfully compete for trans-dihydrodiols. P450 and AKR enzymes equally competed for (+/-)-B[ a]P-7,8-diol substrate at an NADPH/NAD (+) ratio equal to 0.001. The resting NADPH/NAD (+) ratio was determined in A549 human lung adenocarcinoma cells to be 0.28. These data suggest that the P450 pathway would be favored over the AKR pathway if the enzymes were equally expressed. Basal mRNA transcript levels of AKR1C1-1C3 exceed those of both basal and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced P450 1A1 and 1B1 by up to 90-fold in A549 cells as measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods. AKR expression levels were comparable to TCDD-induced P450 1A1 and 1B1 in HBEC-KT immortalized normal human bronchial epithelial cells. Functional assays of both A549 and HBEC-KT cell lysates demonstrated a lack of TCDD-inducible P450 1A1/1B1 activity but robust basal expression of AKR1A1 and AKR1C activities, where the functional assay for P450 detection is 300-fold more sensitive than the functional assay for AKR isoforms. These data suggest that AKR enzymes may effectively compete with P450 1A1/1B1 for PAH trans-dihydrodiol activation in human lung cells.

Oxidation of PAH trans-dihydrodiols by human aldo-keto reductase AKR1B10.

AKR1B10 has been identified as a potential biomarker for human nonsmall cell lung carcinoma and as a tobacco exposure and response gene. AKR1B10 functions as an efficient retinal reductase in vitro and may regulate retinoic acid homeostasis. However, the possibility that this enzyme is able to activate polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols to form reactive and redox-active o-quinones has not been investigated to date. AKR1B10 was found to oxidize a wide range of PAH trans-dihydrodiol substrates in vitro to yield PAH o-quinones. Reactions of AKR1B10 proceeded with improper stereochemistry, since it was specific for the minor (+)-benzo[a]pyrene-7S,8S-dihydrodiol diastereomer formed in vivo. However, AKR1B10 displayed reasonable activity in the oxidation of both the (-)-R,R and (+)-S,S stereoisomers of benzo[g]chrysene-11,12-dihydrodiol and oxidized the potentially relevant, albeit minor, (+)-benz[a]anthracene-3S,4S-dihydrodiol metabolite. We find that AKR1B10 is therefore likely to play a contributing role in the activation of PAH trans-dihydrodiols in human lung. AKR1B10 retinal reductase activity was confirmed in vitro and found to be 5- to 150-fold greater than the oxidation of PAH trans-dihydrodiols examined. AKR1B10 was highly expressed at the mRNA and protein levels in human lung adenocarcinoma A549 cells, and robust retinal reductase activity was measured in lysates of these cells. The much greater catalytic efficiency of retinal reduction compared to PAH trans-dihydrodiol metabolism suggests AKR1B10 may play a greater role in lung carcinogenesis through dysregulation of retinoic acid homeostasis than through oxidation of PAH trans-dihydrodiols.

Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic-Industry Partnership.

The RecA/LexA axis of the bacterial DNA damage (SOS) response is a promising, yet nontraditional, drug target. The SOS response is initiated upon genotoxic stress, when RecA, a DNA damage sensor, induces LexA, the SOS repressor, to undergo autoproteolysis, thereby derepressing downstream genes that can mediate DNA repair and accelerate mutagenesis. As genetic inhibition of the SOS response sensitizes bacteria to DNA damaging antibiotics and decreases acquired resistance, inhibitors of the RecA/LexA axis could potentiate our current antibiotic arsenal. Compounds targeting RecA, which has many mammalian homologues, have been reported; however, small-molecules targeting LexA autoproteolysis, a reaction unique to the prokaryotic SOS response, have remained elusive. Here, we describe the logistics and accomplishments of an academic-industry partnership formed to pursue inhibitors against the RecA/LexA axis. A novel fluorescence polarization assay reporting on RecA-induced self-cleavage of LexA enabled the screening of 1.8 million compounds. Follow-up studies on select leads show distinct activity patterns in orthogonal assays, including several with activity in cell-based assays reporting on SOS activation. Mechanistic assays demonstrate that we have identified first-in-class small molecules that specifically target the LexA autoproteolysis step in SOS activation. Our efforts establish a realistic example for navigating academic-industry partnerships in pursuit of anti-infective drugs and offer starting points for dedicated lead optimization of SOS inhibitors that could act as adjuvants for current antibiotics.

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1.

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation-based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.