Covalent fragment-based drug discovery for target tractability.

An important consideration in drug discovery is the prioritization of tractable protein targets that are not only amenable to binding small molecules, but also alter disease biology in response to small molecule binding. Covalent fragment-based drug discovery has emerged as a powerful approach to aid in the identification of such protein targets. The application of irreversible binding mechanisms enables the identification of fragment hits for challenging-to-target proteins, allows proteome-wide screening in a cellular context, and makes it possible to determine functional effects with modestly potent ligands without the requirement for extensive compound optimization. Here, we provide an overview of recent approaches to covalent fragment-based screening and discuss how these have been applied to establish the tractability of unexplored binding sites on protein targets.

Implications of neonatal absence of innate immune mediated NFκB/AP1 signaling in the murine liver.

BACKGROUND: The developmental immaturity of the innate immune system helps explains the increased risk of infection in the neonatal period. Importantly, innate immune signaling pathways such as p65/NFκB and c-Jun/AP1 are responsible for the prevention of hepatocyte apoptosis in adult animals, yet whether developmental immaturity of these pathways increases the risk of hepatic injury in the neonatal period is unknown. METHODS: Using a murine model of endotoxemia (LPS 5 mg/kg IP x 1) in neonatal (P3) and adult mice, we evaluated histologic evidence of hepatic injury and apoptosis, presence of p65/NFκB and c-Jun/AP1 activation and associated transcriptional regulation of apoptotic genes. RESULTS: We demonstrate that in contrast to adults, endotoxemic neonatal (P3) mice exhibit a significant increase in hepatic apoptosis. This is associated with absent hepatic p65/NFκB signaling and impaired expression of anti-apoptotic target genes. Hepatic c-Jun/AP1 activity was attenuated in endotoxemic P3 mice, with resulting upregulation of pro-apoptotic factors. CONCLUSIONS: These results demonstrate that developmental absence of innate immune p65/NFκB and c-Jun/AP1 signaling, and target gene expression is associated with apoptotic injury in neonatal mice. More work is needed to determine if this contributes to long-term hepatic dysfunction, and whether immunomodulatory approaches can prevent this injury. IMPACT: Various aspects of developmental immaturity of the innate immune system may help explain the increased risk of infection in the neonatal period. In adult models of inflammation and infection, innate immune signaling pathways such as p65/NFκB and c-Jun/AP1 are responsible for a protective, pro-inflammatory transcriptome and regulation of apoptosis. We demonstrate that in contrast to adults, endotoxemic neonatal (P3) mice exhibit a significant increase in hepatic apoptosis associated with absent hepatic p65/NFκB signaling and c-Jun/AP1 activity. We believe that these results may explain in part hepatic dysfunction with neonatal sepsis, and that there may be unrecognized developmental and long-term hepatic implications of early life exposure to systemic inflammatory stress.

GSK3ß/NF-κB -dependent transcriptional regulation of homeostatic hepatocyte Tnf production.

Maintenance of hepatocyte homeostasis plays an important role in mediating the pathogenesis of many diseases. A growing body of literature has established a critical role played by tumor necrosis factor-α (TNFα) in maintaining hepatocyte homeostasis; however, the transcriptional mechanisms underlying constitutive Tnf expression are unknown. Whole liver fractions and primary hepatocytes from adult control C57BL/6 mice and the murine hepatocyte cell line AML12 were assessed for constitutive Tnf expression. Impacts of glycogen synthase kinase-3 ß (GSK3ß) and nuclear factor κB (NF-κB) inhibition on constitutive Tnf expression were assessed in AML12 cells. Finally, AML12 cell proliferation following GSK3ß and NF-κB inhibition was evaluated. Constitutive Tnf gene expression is present in whole liver, primary hepatocytes, and cultured AML12 hepatocytes. Cytokine-induced Tnf gene expression is regulated by NF-κB activation. Pharmacological inhibition of GSK3ß resulted in a time- and dose-dependent inhibition of Tnf gene expression. GSK3ß inhibition decreased nuclear levels of the NF-κB subunits p65 and p50. We determined that NF-κB transcription factor subunit p65 binds to consensus sequence elements present in the murine TNFα promoter and inhibition of GSK3ß decreases binding and subsequent Tnf expression. Finally, AML12 cell growth was significantly reduced following GSK3ß and NF-κB inhibition. These results demonstrate that GSK3ß and NF-κB are essential for mediating Tnf expression and constitutive hepatocyte cell growth. These findings add to a growing body of literature on TNFα mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in mediating response to various disease states in the liver.NEW & NOTEWORTHY Maintenance of hepatocyte homeostasis plays an important role in controlling the pathogenesis of many diseases. Our findings add to a growing body of literature on tumor necrosis factor-α (TNFα)-mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in regulating this response.

Consistency and Variation in Reasoning About Physical Assembly.

The ability to reason about how things were made is a pervasive aspect of how humans make sense of physical objects. Such reasoning is useful for a range of everyday tasks, from assembling a piece of furniture to making a sandwich and knitting a sweater. What enables people to reason in this way even about novel objects, and how do people draw upon prior experience with an object to continually refine their understanding of how to create it? To explore these questions, we developed a virtual task environment to investigate how people come up with step-by-step procedures for recreating block towers whose composition was not readily apparent, and analyzed how the procedures they used to build them changed across repeated attempts. Specifically, participants (N = 105) viewed 2D silhouettes of eight unique block towers in a virtual environment simulating rigid-body physics, and aimed to reconstruct each one in less than 60 s. We found that people built each tower more accurately and quickly across repeated attempts, and that this improvement reflected both group-level convergence upon a tiny fraction of all possible viable procedures, as well as error-dependent updating across successive attempts by the same individual. Taken together, our study presents a scalable approach to measuring consistency and variation in how people infer solutions to physical assembly problems.

EEG microstate quantifiers and state space descriptors during anaesthesia in patients with postoperative delirium: a descriptive analysis.

Postoperative delirium is a serious sequela of surgery and surgery-related anaesthesia. One recommended method to prevent postoperative delirium is using bi-frontal EEG recording. The single, processed index of depth of anaesthesia allows the anaesthetist to avoid episodes of suppression EEG and excessively deep anaesthesia. The study data presented here were based on multichannel (19 channels) EEG recordings during anaesthesia. This enabled the analysis of various parameters of global electrical brain activity. These parameters were used to compare microstate topographies under anaesthesia with those in healthy volunteers and to analyse changes in microstate quantifiers and EEG global state space descriptors with increasing exposure to anaesthesia. Seventy-three patients from the Surgery Depth of Anaesthesia and Cognitive Outcome study (SRCTN 36437985) received intraoperative multichannel EEG recordings. Altogether, 720 min of artefact-free EEG data, including 210 min (29.2%) of suppression EEG, were analysed. EEG microstate topographies, microstate quantifiers (duration, frequency of occurrence and global field power) and the state space descriptors sigma (overall EEG power), phi (generalized frequency) and omega (number of uncorrelated brain processes) were evaluated as a function of duration of exposure to anaesthesia, suppression EEG and subsequent development of postoperative delirium. The major analyses involved covariate-adjusted linear mixed-effects models. The older (71 ± 7 years), predominantly male (60%) patients received a median exposure of 210 (range: 75-675) min of anaesthesia. During seven postoperative days, 21 patients (29%) developed postoperative delirium. Microstate topographies under anaesthesia resembled topographies from healthy and much younger awake persons. With increasing duration of exposure to anaesthesia, single microstate quantifiers progressed differently in suppression or non-suppression EEG and in patients with or without subsequent postoperative delirium. The most pronounced changes occurred during enduring suppression EEG in patients with subsequent postoperative delirium: duration and frequency of occurrence of microstates C and D progressed in opposite directions, and the state space descriptors showed a pattern of declining uncorrelated brain processes (omega) combined with increasing EEG variance (sigma). With increasing exposure to general anaesthesia, multiple changes in the dynamics of microstates and global EEG parameters occurred. These changes varied partly between suppression and non-suppression EEG and between patients with or without subsequent postoperative delirium. Ongoing suppression EEG in patients with subsequent postoperative delirium was associated with reduced network complexity in combination with increased overall EEG power. Additionally, marked changes in quantifiers in microstate C and in microstate D occurred. These putatively adverse intraoperative trajectories in global electrical brain activity may be seen as preceding and ultimately predicting postoperative delirium.

Detecting emerald green in 19thC book bindings using vis-NIR spectroscopy.

Detection and identification of heavy metal-based pigments in 19th-century bookbindings is crucial to avoid human user exposure to toxic substances. Vibrant green bookbindings with arsenical emerald green are particularly problematic due to their friability. A pilot study at St Andrews University tested 800 green bookbindings for arsenic presence using visible near-infrared spectrometry, a technique not previously applied to the detection of heavy metals in bindings. The ASD TerraSpec Halo portable spectrometer that is normally used in geology to identify minerals in rocks, is used here to collect hyperspectral reflectance data between 350 and 2500 nm. Raman spectroscopy and Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM-EDS) are used here to validate hyperspectral test results. The study finds that bookbindings containing emerald green have a distinctive pattern in the visible part of the spectrum that is distinguishable from other green pigments. This finding opens up the possibility for all collecting institutions to test bindings for this toxic compound in a non-destructive, cost-effective and efficient manner.

A statewide study of disparities in local policies and tobacco, vape, and cannabis retail environments.

The current study: (1) assesses sociodemographic disparities in local policies related to tobacco and cannabis retail, and (2) examines the cross-sectional association between policy strength and retailer densities of tobacco, e-cigarette (vape), and cannabis retailers within California cities and county unincorporated areas (N = 539). We combined (a) American Community Survey data (2019 5-year estimates), (b) 2018 tobacco, vape, and cannabis retailer locations from a commercial data provider, (c) 2017 tobacco and vape retail environment policy data from American Lung Association, and (d) 2018 cannabis policy data from California Cannabis Local Laws Database. Conditional autoregressive models examined policy strength associations with sociodemographic composition and retailer density in California jurisdictions. Jurisdictions with larger percentages of Black and foreign-born residents had stronger tobacco and vape policies. For cannabis policy, only income had a small, significant positive association with policy strength. Contrary to hypothesis, tobacco/vape policies were not significantly associated with retailer density, but cannabis policy strength was associated with lower cannabis retailer density (relative rate = 0.58, 95% Uncertainty Interval 0.47-0.70)-this effect was completely driven by storefront bans. Thus, storefront cannabis bans were the only policy studied that was associated with lower cannabis retailer density. Further research is needed to understand policies and disparities in retail environments for tobacco, vape, and cannabis, including data on the prospective association between policy implementation and subsequent retailer density, and the role of enforcement.

Isolation and identification of chlorate-reducing Hafnia sp. from milk.

Chlorate has become a concern in the food and beverage sector, related to chlorine sanitizers in industrial food production and water treatment. It is of particular concern to regulatory bodies due to the negative health effects of chlorate exposure. This study investigated the fate of chlorate in raw milk and isolated bacterial strains of interest responsible for chlorate breakdown. Unpasteurized milk was demonstrated to have a chlorate-reducing capacity, breaking down enriched chlorate to undetectable levels in 11 days. Further enrichment and isolation using conditions specific to chlorate-reducing bacteria successfully isolated three distinct strains of Hafnia paralvei. Chlorate-reducing bacteria were observed to grow in a chlorate-enriched medium with lactate as an electron donor. All isolated strains were demonstrated to reduce chlorate in liquid medium; however, the exact mechanism of chlorate degradation was not definitively identified in this study.

Randomized Comparative Effectiveness Trial of 2 Federally Recommended Strategies to Reduce Excess Body Fat in Overweight, Low-Income Patients: MyPlate.gov vs Calorie Counting.

PURPOSE: Since 2011, US authorities have supported the following 2 approaches to healthier body fat composition: the Centers for Disease Control and Prevention National Diabetes Prevention Program's calorie counting (CC) approach and the US Department of Agriculture's MyPlate (adherence to federal nutrition guidelines). The purpose of this study was to compare the effect of CC vs MyPlate approaches on satiety/satiation and on achieving healthier body fat composition among primary care patients. METHODS: We conducted a randomized controlled trial comparing the CC and MyPlate approaches from 2015 to 2017. The adult participants were overweight, of low income, and were mostly Latine (n = 261). For both approaches, community health workers conducted 2 home education visits, 2 group education sessions, and 7 telephone coaching calls over a period of 6 months. Satiation and satiety were the primary patient-centered outcome measures. Waist circumference and body weight were the primary anthropometric measures. Measures were assessed at baseline, 6 months, and 12 months. RESULTS: Satiation and satiety scores increased for both groups. Waist circumference was significantly decreased in both groups. MyPlate, but not CC, resulted in lower systolic blood pressure at 6 months but not at 12 months. Participants for both MyPlate and CC reported greater quality of life and emotional well-being and high satisfaction with their assigned weight-loss program. The most acculturated participants showed the greatest decreases in waist circumference. CONCLUSIONS: A MyPlate-based intervention might be a practical alternative to the more traditional CC approach to promoting satiety and facilitating decreases in central adiposity among low-income, mostly Latine primary care patients.

Innate Immune Zonation in the Liver: NF-κB (p50) Activation and C-Reactive Protein Expression in Response to Endotoxemia Are Zone Specific.

Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial heterogeneity of hepatocyte metabolic function; however, whether innate immune function is zonated remains unknown. To test this question, we exposed adult C57BL/6 mice to endotoxemia, and hepatic tissue was assessed for the acute phase response (APR). The zone-specific APR was evaluated in periportal and pericentral/centrilobular hepatocytes isolated using digitonin perfusion and on hepatic tissue using RNAscope and immunohistochemistry. Western blot, EMSA, chromatin immunoprecipitation, and immunohistochemistry were used to determine the role of the transcription factor NF-κB in mediating hepatic C-reactive protein (CRP) expression. Finally, the ability of mice lacking the NF-κB subunit p50 (p50-/-) to raise a hepatic APR was evaluated. We found that endotoxemia induces a hepatocyte transcriptional APR in both male and female mice, with Crp, Apcs, Fga, Hp, and Lbp expression being enriched in pericentral/centrilobular hepatocytes. Focusing our work on CRP expression, we determined that NF-κB transcription factor subunit p50 binds to consensus sequence elements present in the murine CRP promoter. Furthermore, pericentral/centrilobular hepatocyte p50 nuclear translocation is temporally associated with zone-specific APR during endotoxemia. Lastly, the APR and CRP expression is blunted in endotoxemic p50-/- mice. These results demonstrate that the murine hepatocyte innate immune response to endotoxemia includes zone-specific activation of transcription factors and target gene expression. These results support further study of zone-specific hepatocyte innate immunity and its role in the development of various disease states.

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD <20 µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.

"Ancestral recipes": a mixed-methods analysis of MyPlate-based recipe dissemination for Latinos in rural communities.

BACKGROUND: The Latinx population experiences some of the highest rates of chronic disease, including obesity and type II diabetes. Such conditions may be especially burdensome in rural Latinx communities that often face barriers to accessing disease prevention resources and public health programs. METHODS: Diverse stakeholders (i.e., patients, community members, system of healthcare clinics, community food bank) tailored an existing cookbook, based on the U.S. Department of Agriculture MyPlate healthy eating and dietary guidelines, for local ingredients, health literacy, and language for rural Latinx and Indigenous Latin Americans. The cookbook recipes were disseminated widely via virtual cooking demonstrations, food distribution events, and social media. Pre- and posttest surveys were used to assess changes in diabetes knowledge measured by the 24-item American Diabetes Association Diabetic Knowledge Questionnaire and confidence in dietary behavior change over time measured by 4 questions of the 17-item Mediterranean Diet Index. A mixed effects, repeated measures analysis was conducted with gender ID, age range and educational attainment included as covariates and assessment interval as the predictor (pretest vs posttest) and change in confidence about adhering to four specific components of the Mediterranean diet. Focus groups elicited information on participants' motivation and ability to use the recipes and eat healthy foods following the virtual cooking demonstration participation. RESULTS: A total of 20 virtual cooking demonstrations were conducted and 60 participants completed a pretest survey and 54 a posttest survey, a subsample (n = 19) participated in one of three focus groups. Most participants were female, identified as Latinx/Hispanic, were between the ages of 40-49, and spoke Spanish. 17% identified as Indigenous Latin American specifically as Purépecha, an indigenous group from Michoacán, Mexico. Survey and focus group findings indicated at posttest an increase in diabetes knowledge among participants with no prior diagnosis of chronic health conditions and more confidence in limiting sugary beverages and refined wheat pasta/white rice among indigenous participants. Focus group discussions explicated the quantitative findings. CONCLUSION: This study brought together patients and key stakeholders committed to addressing the social determinants of health and it mobilized the community to develop culturally vetted health education materials. The findings indicate the need for increased access to evidence-based nutrition education and to culturally appropriate food products that can be easily incorporated into daily food preparation.

Family functioning within the context of families with adolescent children in urban India.

Research on family functioning within given cultural contexts is needed. This study aims to describe salient dimensions of family functioning in two urban contexts in India and to examine differences in family functioning by sociodemographic groups. We measured differences in family functioning using cross-sectional survey questionnaire data collected from 13 to 15-year-old adolescents and one of their parents/primary caregivers in Mumbai (n = 843) and Kolkata (n = 913) during 2019-2020. We drew a multi-stage sample representative of neighborhoods and households in both cities. We assessed a multi-dimensional family functioning latent factor that included parent-reported measures (parent-adolescent communication, family cohesion, and parent monitoring of peers) and adolescent-reported measures (parent support, family cohesion, and parent supervision). Our results support an overall measure of family functioning manifested by multiple dimensions for parent- and adolescent-reported data. Families with male adolescents had worse adolescent-reported family functioning in Mumbai and parent-reported family functioning in Kolkata. Higher socioeconomic status was associated with better parent-reported family functioning in both cities and better adolescent-reported family functioning in Kolkata. Muslim religious identification in Kolkata and the Hindi native language in both cities were associated with better adolescent-reported family functioning. Our findings indicate heterogeneity in family functioning across demographic and social-cultural groups within the two urban contexts of India. This study may inform the development of culturally congruent prevention interventions for families with adolescents in India.

Comparative Effectiveness RCT of Two Weight Loss Strategies in Primary Care Patients: MyPlate.gov vs Calorie Counting.

Context: Since 2011, the U.S. government has supported two approaches to achieve healthier body fat composition: the Diabetes Prevention Program calorie counting (CC) approach, and adherence to federal nutrition guidelines at www.choosemyplate.gov (MyPlate). Objective: Compare the effect of the CC versus MyPlate approach on satiety/satiation and on achieving healthier body fat composition in the primary care setting. Study Design: Randomized, controlled trial comparing the MyPlate and CC approaches from 2015 to 2017. Setting: A federally qualified health center in Long Beach, California. Population: Adult, low-income, mostly Latina patients (N=261) with a BMI between 27 and 40.4 were randomized to condition and followed for twelve months (76.6% retention). Interventions: Eleven health education sessions featuring MyPlate versus CC messages. Community health workers conducted two home visits, two group education sessions and 7 telephone coaching calls over six months. Outcome Measures: Satiation and satiety were primary patient-centered outcomes. Waist circumference and body weight were primary anthropometric measures. These were assessed at baseline, 6- and 12-months follow-up. Results: Satiation and satiety scores increased for both groups; neither group lost significant body weight, and only the MyPlate condition reduced waist circumference by 2 cm at 12 months. Both conditions reported consuming proportionately more fruits and vegetables and fewer sugary beverages at 12 months. MyPlate but not CC participants experienced lower systolic blood pressure at 6 months follow-up; neither group had lower blood pressure at 12 months. Both MyPlate and CC participants reported higher quality of life and emotional well-being at 12 months and high satisfaction with their assigned weight loss program. At 12 months follow-up, the most acculturated participants experienced the greatest reduction in waist circumference. Conclusions: A MyPlate-based intervention may be a practical alternative to the more traditional CC approach to promoting satiety and facilitating reduction in central adiposity among low-income mostly Latina overweight primary care patients. Our results align with recommendations favoring a diet rich in diverse, fiber-rich foods. More research is warranted to investigate satiety-enhancing approaches to desirable weight control in diverse populations and the use of community health workers as change agents.

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a K D <20 µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.

Neonatal Selenium Deficiency Decreases Selenoproteins in the Lung and Impairs Pulmonary Alveolar Development.

Decreased selenium (Se) levels during childhood and infancy are associated with worse respiratory health. Se is biologically active after incorporation into Se-containing antioxidant enzymes (AOE) and proteins. It is unknown how decreased maternal Se during pregnancy and lactation impacts neonatal pulmonary selenoproteins, growth, and lung development. Using a model of neonatal Se deficiency that limits Se intake to the dam during pregnancy and lactation, we evaluated which neonatal pulmonary selenoproteins are decreased in both the saccular (postnatal day 0, P0) and early alveolar (postnatal day 7, P7) stages of lung development. We found that Se deficient (SeD) pups weigh less and exhibit impaired alveolar development compared to Se sufficient (SeS) pups at P7. The activity levels of glutathione peroxidase (GPx) and thioredoxin reductase (Txnrd) were decreased at P0 and P7 in SeD lungs compared to SeS lungs. Protein content of GPx1, GPx3 and Txnrd1 were decreased in SeD lungs at P0 and P7, whereas Txnrd2 content was unaltered compared to SeS controls. The expression of NRF-2 dependent genes and several non-Se containing AOE were similar between SeS and SeD lungs. SeD lungs exhibited a decrease in selenoprotein N, an endoplasmic reticulum protein implicated in alveolar development, at both time points. We conclude that exposure to Se deficiency during pregnancy and lactation impairs weight gain and lung growth in offspring. Our data identify multiple selenoproteins in the neonatal lung that are vulnerable to decreased Se intake, which may impact oxidative stress and cell signaling under physiologic conditions as well as after oxidative stressors.

Absence of IκBß/NFκB signaling does not attenuate acetaminophen-induced hepatic injury.

Acetaminophen (N-acetyl-p-aminophenol [APAP]) toxicity is a common cause of acute liver failure. Innate immune signaling and specifically NFκB activation play a complex role in mediating the hepatic response to toxic APAP exposures. While inflammatory innate immune responses contribute to APAP-induced injury, these same pathways play a role in regeneration and repair. Previous studies have shown that attenuating IκBß/NFκB signaling downstream of TLR4 activation can limit injury, but whether this pathway contributes to APAP-induced hepatic injury is unknown. We hypothesized that the absence of IκBß/NFκB signaling in the setting of toxic APAP exposure would attenuate APAP-induced hepatic injury. To test this, we exposed adult male WT and IκBß-/- mice to APAP (280 mg/kg, IP) and evaluated liver histology at early (2-24 hr) and late (48-72 hr) time points. Furthermore, we interrogated the hepatic expression of NFκB inflammatory (Cxcl1, Tnf, Il1b, Il6, Ptgs2, and Ccl2), anti-inflammatory (Il10, Tnfaip3, and Nfkbia), and Nrf2/antioxidant (Gclc, Hmox, and Nqo1) target genes previously demonstrated to play a role in APAP-induced injury. Conflicting with our hypothesis, we found that hepatic injury was similar in WT and IκBß-/- mice. Acutely, the induced expression of some target genes was similar in WT and IκBß-/- mice (Tnfaip3, Nfkbia, and Gclc), while others were either not induced (Cxcl1, Tnf, Ptgs2, and Il10) or significantly attenuated (Ccl2) in IκBß-/- mice. At later time points, APAP-induced hepatic expression of Il1b, Il6, and Gclc was significantly attenuated in IκBß-/- mice. Based on these findings, the therapeutic potential of targeting IκBß/NFκB signaling to treat toxic APAP-induced hepatic injury is likely limited.

Chronic, but not sub-chronic, stress increases binge-like alcohol consumption in male and female c57BL6 mice.

Stress is known to contribute to mental illness and alcohol use disorders, which are highly prevalent and lead to considerable disability. These stress-related disorders are characterized by significant sex differences, which remain poorly understood. Preclinical research comparing the effects of stress in males and females has the potential to provide new insights into the neurobiology of these conditions. The current study compared the effects of chronic and sub-chronic exposure to variable environmental stressors on binge-like alcohol consumption using the drinking-in-the-dark model in male and female c57BL6 mice. The results reveal that chronic, but not sub-chronic, exposure to variable stress increases alcohol intake in both sexes. Stress-induced alterations in gene expression were also compared in the nucleus accumbens, a brain region widely known to play a key role in stress susceptibility and reward processing. Real-time PCR data indicate that chronic, but not sub-chronic, environmental stress leads to downregulation of adenosine 2A (A2A) receptor mRNA. By contrast, sub-chronic stress increased CREB expression, while chronic stress did not. Several sex differences in the effects of stress on gene expression were also noted. Our results demonstrate that reductions in A2A receptor mRNA in the nucleus accumbens are associated with the increased binge drinking of chronically stressed animals, but future work will be required to determine the functional importance of this gene expression change. Continuing to define the molecular alterations associated with stress-induced increases in alcohol intake has the potential to provide insights into the development and progression of stress-related disorders.

Development of potent inhibitors by fragment-linking strategies.

Fragment-based drug discovery (FBDD) is a method of identifying small molecule hits that can be elaborated rationally through fragment growing, merging and linking, to afford high-affinity ligands for biological targets. Despite the promised theoretical potential of fragment linking, examples are still surprisingly sparse and remain overshadowed by the successes of fragment growing. The aim of this review was to outline a number of key examples of fragment-linking strategies and discuss their strengths and limitations. Structure-based approaches including X-ray crystallography and in silico methods of fragment optimization are discussed, as well as fragment linking guided by NMR experiments. Target-guided approaches, exploiting the biological target to assemble its own inhibitors through dynamic combinatorial chemistry (DCC) and kinetic target-guided synthesis (KTGS), are identified as alternative efficient methods for fragment linking.