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Contains fulltext : 282509.pdf (Publisher’s version ) (Open Access) Although the genomic and immune microenvironmental landscape of follicular lymphoma (FL) has been extensively investigated, little is known about the potential biological differences between stage I and stage III/IV disease. Using next-generation sequencing and immunohistochemistry, 82 FL nodal stage I cases were analyzed and compared with 139 FL stage III/IV nodal cases. Many similarities in mutations, chromosomal copy number aberrations, and microenvironmental cell populations were detected. However, there were also significant differences in microenvironmental and genomic features. CD8+ T cells (P = .02) and STAT6 mutations (false discovery rate [FDR]

Naphthoquinone (1,4-NQ) and its derivatives (NQs, juglone, plumbagin, 2-methoxy-1,4-NQ, and menadione) have a variety of therapeutic applications, many of which are attributed to redox cycling and the production of reactive oxygen species (ROS). We previously demonstrated that NQs also oxidize hydrogen sulfide (H2S) to reactive sulfur species (RSS), potentially conveying identical benefits. Here we use RSS-specific fluorophores, mass spectroscopy, EPR and UV-Vis spectrometry, and oxygen-sensitive optodes to examine the effects of thiols and thiol-NQ adducts on H2S-NQ reactions. In the presence of glutathione (GSH) and cysteine (Cys), 1,4-NQ oxidizes H2S to both inorganic and organic hydroper-/hydropolysulfides (R2Sn, R=H, Cys, GSH; n = 2–4) and organic sulfoxides (GSnOH, n = 1, 2). These reactions reduce NQs and consume oxygen via a semiquinone intermediate. NQs are also reduced as they form adducts with GSH, Cys, protein thiols, and amines. Thiol, but not amine, adducts may increase or decrease H2S oxidation in reactions that are both NQ- and thiol-specific. Amine adducts also inhibit the formation of thiol adducts. These results suggest that NQs may react with endogenous thiols, including GSH, Cys, and protein Cys, and that these adducts may affect both thiol reactions as well as RSS production from H2S.

1,4-Napththoquinones (NQs) are clinically relevant therapeutics that affect cell function through production of reactive oxygen species (ROS) and formation of adducts with regulatory protein thiols. Reactive sulfur species (RSS) are chemically and biologically similar to ROS and here we examine RSS production by NQ oxidation of hydrogen sulfide (H

In utero dietary exposures are linked to the development of metabolic syndrome in adult offspring. These dietary exposures can potentially impact gut microbial composition and offspring metabolic health. Female BALB/c mice were administered a lard, lard + flaxseed oil, high sugar, or control diet 4 wk before mating, throughout mating, pregnancy, and lactation. Female offspring were offered low-fat control diet at weaning. Fecal 16S sequencing was performed. Untargeted metabolomics was performed on visceral adipose tissue (VAT) of adult female offspring. Immunohistochemistry was used to determine adipocyte size, VAT collagen deposition, and macrophage content. Hippurate was administered via weekly intraperitoneal injections to low-fat and high-fat diet-fed female mice and VAT fibrosis and collagen 1A (COL1A) were assessed by immunohistochemistry. Lard diet exposure was associated with elevated body and VAT weight and dysregulated glucose metabolism. Lard + flaxseed oil attenuated these effects. Lard diet exposures were associated with increased adipocyte diameter and VAT macrophage count. Lard + flaxseed oil reduced adipocyte diameter and fibrosis compared with the lard diet. Hippurate-associated bacteria were influenced by lard versus lard + flax exposures that persisted to adulthood. VAT hippurate was increased in lard + flaxseed oil compared with lard diet. Hippurate supplementation mitigated VAT fibrosis pathology. Maternal high-fat lard diet consumption resulted in long-term metabolic and gut microbiome programming in offspring, impacting VAT inflammation and fibrosis, and was associated with reduced VAT hippurate content. These traits were not observed in maternal high-fat lard + flaxseed oil diet-exposed offspring. Hippurate supplementation reduced VAT fibrosis. These data suggest that detrimental effects of early-life high-fat lard diet exposure can be attenuated by dietary omega-3 polyunsaturated fatty acid supplementation.

Abstract Background The objective of this study was to increase understanding of the complex interactions between diet, obesity, and the gut microbiome of adult female non-human primates (NHPs). Subjects consumed either a Western (n=15) or Mediterranean (n=14) diet designed to represent human dietary patterns for 31 months. Body composition was determined using CT, fecal samples were collected, and shotgun metagenomic sequencing was performed. Gut microbiome results were grouped by diet and adiposity. Results Diet was the main contributor to gut microbiome bacterial diversity. Adiposity within each diet was associated with subtle shifts in the proportional abundance of several taxa. Mediterranean diet-fed NHPs with lower body fat had a greater proportion of Lactobacillus animalis than their higher body fat counterparts. Higher body fat Western diet-fed NHPs had more Ruminococcus champaneliensis and less Bacteroides uniformis than their low body fat counterparts. Western diet-fed NHPs had significantly higher levels of Prevotella copri than Mediterranean diet NHPs. Western diet-fed subjects were stratified by P. copri abundance (P. copriHIGH versus P. copriLOW), which was not associated with adiposity. Overall, Western diet-fed animals in the P. copriHIGH group showed greater proportional abundance of B. ovatus, B. faecis, P. stercorea, P. brevis, and Faecalibacterium prausnitzii than those in the Western P. copriLOW group. Western diet P. copriLOW subjects had a greater proportion of Eubacterium siraeum. E. siraeum negatively correlated with P. copri proportional abundance regardless of dietary consumption. In the Western diet group, Shannon diversity was significantly higher in P. copriLOW when compared to P. copriHIGH subjects. Furthermore, gut E. siraeum abundance positively correlated with HDL plasma cholesterol indicating that those in the P. copriLOW population may represent a more metabolically healthy population. Untargeted metabolomics on urine and plasma from Western diet-fed P. copriHIGH and P. copriLOW subjects suggest early kidney dysfunction in Western diet-fed P. copriHIGH subjects. Conclusions In summary, the data indicate diet to be the major influencer of gut bacterial diversity. However, diet and adiposity must be considered together when analyzing changes in abundance of specific bacterial taxa. Interestingly, P. copri appears to mediate metabolic dysfunction in Western diet-fed NHPs.

Background: The unfolded protein response (UPR) is an endoplasmic reticulum pathway stimulated when the protein folding demand of a cell surpasses the bio-energetic capacity. UPR master regulator and protein chaperone, GRP78, binds to the three signaling arms (PERK, ATF6, and IRE1) rendering them inactive. When unfolded protein accumulate, GRP78 unbinds from these components enabling UPR activation. We, among other, previously showed elevated UPR in breast tumors promoted therapeutic resistance. However, studies exploring whether combinatoral therapies targeting UPR coupled with standard of care chemotherapeutics modulates the development of co-morbidities remain under-explored. Methods: 4T1-luciferase triple negative breast cancer cells were injected into the mammary fat pad of BALBc mice. Tumor-bearing mice were treated with 1 x weekly 30 µM GRP78, PERK, or IRE1 morpholino +/- four 1 x weekly 2.5 mg/kg doxorubicin (cumulative dose 10 mg/kg). Tumor volume was measured by calipers and IVIS. Cardiac function was determined by VEVO ultrasound in morpholino + DOX treated BALBc and WT/GRP78 heterozygous mice. In vitro and ex-vivo viability experiments were performed using ACEA xCELLigence RTCA system. Results: Targeting GRP78 or IRE1 potentiated doxorubicin-mediated tumor killing while targeting PERK enhanced 4T1 tumor growth and prevented chemotherapy efficacy. Targeting GRP78 or PERK reduced cardiac function in response to doxorubicin resulting in poor overall survival. However, targeting XBP1 either through upstream IRE1 protein reduction or inhibition of IRE1 activity protected in vivo heart function. In vitro pre-treatment of cardiomyoblast H9C2 cells with KIRA8 (a XBP1 inhibitor) reduced doxorubicin-mediated cell death and reduced chemotherapy-induced apoptosis as measured by cleaved caspase-3 and cleaved PARP. Ex-vivo treatment of primary cardiac cells isolated from wild-type and GRP78 heterozygous mice indicate inhibition of XBP1 ameliorated GRP78 heterozygous sensitization to doxorubicin, suggesting the elevated cardiotoxicities observed by systemic GRP78 targeting is resulting from elevated IRE1 activation. Conclusions: We now show that while targeting GRP78 and IRE1 in vivo enhances tumor doxorubicin responsiveness, targeting these proteins results in differential effects on chemotherapy-mediated cardiotoxicities. Targeting PERK enhanced tumor growth and reduced chemotherapy responsiveness. Our study highlights the importance of testing these pre-clinical drugs on cardiac function to reduce future clinical trial side effects. Citation Format: David Soto-Pantoja, Kenysha Clear, Katherine Loree Cook. Targeting the unfolded protein response components differentially sensitizes breast tumors to doxorubicin while modulating chemotherapy-induced cardiotoxicities [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-14-19.

Breast tumors have their own specific microbiota, distinct from normal mammary gland tissue. Patients with breast cancer that present with locally advanced disease often undergo neoadjuvant chemotherapy to reduce tumor size prior to surgery to allow breast conservation or limit axillary lymph node dissection. The purpose of our study was to evaluate whether neoadjuvant chemotherapy modulates the tumor microbiome and the potential impact of microbes on breast cancer signaling. Using snap-frozen aseptically collected breast tumor tissue from women who underwent neoadjuvant chemotherapy (n = 15) or women with no prior therapy at time of surgery (n = 18), we performed 16S rRNA-sequencing to identify tumoral bacterial populations. We also stained breast tumor microarrays to confirm presence of identified microbiota. Using bacteria-conditioned media, we determined the effect of bacterial metabolites on breast cancer cell proliferation and doxorubicin therapy responsiveness. We show chemotherapy administration significantly increased breast tumor Pseudomonas spp. Primary breast tumors from patients who developed distant metastases displayed increased tumoral abundance of Brevundimonas and Staphylococcus. We confirmed presence of Pseudomonas in breast tumor tissue by IHC staining. Treatment of breast cancer cells with Pseudomonas aeruginosa conditioned media differentially effected proliferation in a dose-dependent manner and modulated doxorubicin-mediated cell death. Our results indicate chemotherapy shifts the breast tumor microbiome and specific microbes correlate with tumor recurrence. Further studies with a larger patient cohort may provide greater insights into the role of microbiota in therapeutic outcome and develop novel bacterial biomarkers that could predict distant metastases. Implications: Breast tumor microbiota are modified by therapy and affects molecular signaling.

In the canonical pathway for mitochondrial H

1,4-Napththoquinones (NQs) are clinically relevant therapeutics that affect cell function through production of reactive oxygen species (ROS) and formation of adducts with regulatory protein thiols. Reactive sulfur species (RSS) are chemically and biologically similar to ROS and here we examine RSS production by NQ oxidation of hydrogen sulfide (H2S) using RSS-specific fluorophores, liquid chromatography-mass spectrometry, UV-Vis absorption spectrometry, oxygen-sensitive optodes, thiosulfate-specific nanoparticles, HPLC-monobromobimane derivatization, and ion chromatographic assays. We show that NQs, catalytically oxidize H2S to per- and polysulfides (H2Sn, n = 2–6), thiosulfate, sulfite and sulfate in reactions that consume oxygen and are accelerated by superoxide dismutase (SOD) and inhibited by catalase. The approximate efficacy of NQs (in decreasing order) is, 1,4-NQ ≈ juglone ≈ plumbagin > 2-methoxy-1,4-NQ ≈ menadione >> phylloquinone ≈ anthraquinone ≈ menaquinone ≈ lawsone. We propose that the most probable reactions are an initial two-electron oxidation of H2S to S0 and reduction of NQ to NQH2. S0 may react with H2S or elongate H2Sn in variety of reactions. Reoxidation of NQH2 likely involves a semiquinone radical (NQ·−) intermediate via several mechanisms involving oxygen and comproportionation to produce NQ and superoxide. Dismutation of the latter forms hydrogen peroxide which then further oxidizes RSS to sulfoxides. These findings provide the chemical background for novel sulfur-based approaches to naphthoquinone-directed therapies.

It has been unclear what role metabolism is playing in the pathophysiology of chronic lymphocytic leukemia (CLL). One reason is that the study of CLL metabolism is challenging due to the resting nature of circulating CLL cells. Also, it is not clear if any of the genomic aberrations observed in this disease have any impact on metabolism. Here, we demonstrate that CLL cells in proliferation centers exhibit upregulation of several molecules involved in glycolysis and mitochondrial metabolism. Comparison of CXCR4/CD5 intraclonal cell subpopulations showed that these changes are paralleled by increases in the metabolic activity of the CXCR4

Drawing from dispositional mindfulness research and stress and coping theories, we tested whether adolescents’ dispositional mindfulness was associated with perceptions of peer victimization and exclusion and internalizing symptoms. We further explored the role of dispositional mindfulness as a protective factor buffering the impact of peer victimization and exclusion (PVE) on internalizing symptoms. Participants were 361 (40% boys) adolescents aged between 11 and 18 years ( M = 14.9, SD = 1.4) who completed a questionnaire to assess dispositional mindfulness, perceptions of PVE, social anxiety and depressive symptoms, and loneliness. As expected, more frequent experience of PVE was associated with reporting more symptoms of social anxiety, depression, and loneliness. Further, adolescents who reported higher dispositional mindfulness also reported fewer symptoms of social anxiety, depression, and loneliness, even after controlling for gender and experiences of PVE. Dispositional mindfulness was not protective against (i.e., did not buffer) the effects of PVE on internalizing symptoms. Instead, we found that PVE had a stronger association with symptoms of social anxiety, depression, and loneliness when mindfulness was high relative to when it was medium or low. Yet, victimization was associated with greater social anxiety, depressive symptoms, and loneliness at all levels of mindfulness.

Drawing from attachment and emotion theories, we tested a model whereby emerging adults’ anxious and avoidant attachment would have specific associations with dysregulation and suppression of sadness and anger and would be unique correlates of emotional and behavioral problems. Participants were 383 (47% men) students between 16 and 23 years (M = 19.6, SD = 1.58) who completed a questionnaire to assess attachment, emotion dysregulation and suppression, depressive and social anxiety symptoms, and aggressive behavior. In a path model, greater anxious attachment was associated with more emotion dysregulation, whereas greater avoidant attachment was associated with greater emotion suppression. Greater sadness dysregulation was uniquely and significantly associated with depression and social anxiety but not aggression, whereas greater anger dysregulation was associated with aggressive behavior but not depression and anxiety. Also, participants with elevated attachment insecurities reported heightened emotional and behavioral problems. Anxious attachment had the most pervasive impact on all forms of symptoms, either directly or indirectly via emotion dysregulation. Yet, there was also evidence that a focus on regulation of sadness, relative to anger, identified unique links with depression and social anxiety, relative to aggressive behavior.

Introduction Adolescents higher in the trait of dispositional mindfulness report fewer socioemotional problems. Focusing on the domain of peer stressors, we tested a model of adolescents’ mindfulness as a resource that undergirds more constructive stress coping responses, in turn resulting in fewer socioemotional problems. Method The participants were 361 Australian secondary school students (40% boys; ages 11 to 18; M = 14.9 years; SD = 1.4). Each completed a questionnaire to report four facets of dispositional mindfulness; engagement and disengagement coping and involuntary stress responses to recent peer interpersonal stressful events; and socioemotional problems of loneliness, social anxiety, and depression. Results Adolescents who reported more dispositional mindfulness, including facets of awareness, describing, non-judgement and non-reactivity, were lower in involuntary peer stress responses, disengagement coping, loneliness, social anxiety, and depression; associations of mindfulness facets with engagement coping were mixed. Mediational path models showed that almost all the significant negative associations of dispositional mindfulness with loneliness, social anxiety and depression were fully indirect via peer stress coping responses. Further, an alternative model, which tested whether loneliness, social anxiety and depression were the instigators of stress coping responses via mindfulness, had an adequate but poorer fit. Conclusion As hypothesized, the benefit of adolescents' dispositional mindfulness for reducing loneliness, social anxiety and depression seems to be indirect, with positive peer stress coping responses key mediators in these indirect pathways. It is less likely that the alternative occurs, whereby adolescents’ socioemotional problems are the foundation for mindfulness and peer stress coping responses.

Experiencing stressful events that threaten feelings of social belonging can have far-reaching negative impacts on well-being, but there are individual differences in sensitivity to threat that might be explained by dispositional traits. In particular, naturally occurring dispositional mindfulness may be one trait that can explain such differences. To test this possibility, a pool of 495 young adults completed a measure of dispositional mindfulness and a subset of 90 (M = 19 years, SD = 1.3), selected to represent the full range of mindfulness scores, participated in an induced social rejection task (Cyberball). Threat appraisal was collected by asking about perceived exclusion and rejection post-Cyberball, and participants reported their mood and friendliness before, after, and at 3-mins of recovery, and their self-esteem and life meaning after Cyberball and at recovery. Participants higher in mindfulness reported better mood and less unfriendliness prior to Cyberball. Directly after playing Cyberball, a more heightened appraisal of threat, but not mindfulness, was associated with worse mood, less friendliness, lower self-esteem, and less life meaning. Mindfulness directly mitigated the negative effects of rejection on feelings of friendliness post rejection. When mindfulness and threat appraisal were considered in interaction, the association of perceived threat with pre- to post- changes in positive mood and friendliness was strongly negative when mindfulness was high relative to low. Further, mindfulness was associated with better recovery of mood and life meaning by 3-min after Cyberball, and these effects were additive rather than interactive. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Additional file 1: Supplemental Table 1. Non-human primate diets replicating human Western and Mediterranean dietary patterns. Supplemental Figure S1. Individual read statistics on each sample. Samples ranged from 13,694,588 to 28,756,486 reads with no significant differences in reads between dietary patterns. Supplemental Figure S2. Diet, not pen effects, drive microbiome populations. A. PCoA of bacterial beta diversity based on the Bray-Curtis dissimilarity; different solid color spheres indicates subjects housed in same pen. B. Shannon diversity of each subject; different solid color spheres indicates subjects housed in same pen. Supplemental Figure S3. Diet shifts viral and protozoa populations in the gut microbiome. A. Relative abundance of viral species in different fecal samples is visualized by bar plots. Samples are aggregated by diet cohort. Each colored box represents a viral species. The height of a color box represents the relative abundance of that organism within the sample. “Other” represents lower abundance taxa (

Obesity and poor diet often go hand-in-hand, altering metabolic signaling and thereby impacting breast cancer risk and outcomes. We have recently demonstrated that dietary patterns modulate mammary microbiota populations. An important and largely open question is whether the microbiome of the gut and mammary gland mediates the dietary effects on breast cancer. To address this, we performed fecal transplants between mice on control or high-fat diets (HFD) and recorded mammary tumor outcomes in a chemical carcinogenesis model. HFD induced protumorigenic effects, which could be mimicked in animals fed a control diet by transplanting HFD-derived microbiota. Fecal transplants altered both the gut and mammary tumor microbiota populations, suggesting a link between the gut and breast microbiomes. HFD increased serum levels of bacterial lipopolysaccharide (LPS), and control diet–derived fecal transplant reduced LPS bioavailability in HFD-fed animals. In vitro models of the normal breast epithelium showed that LPS disrupts tight junctions (TJ) and compromises epithelial permeability. In mice, HFD or fecal transplant from animals on HFD reduced expression of TJ-associated genes in the gut and mammary gland. Furthermore, infecting breast cancer cells with an HFD-derived microbiome increased proliferation, implicating tumor-associated bacteria in cancer signaling. In a double-blind placebo-controlled clinical trial of patients with breast cancer administered fish oil supplements before primary tumor resection, dietary intervention modulated the microbiota in tumors and normal breast tissue. This study demonstrates a link between the gut and breast that mediates the effect of diet on cancer. Significance: This study demonstrates that diet shifts the microbiome in the gut and the breast tumor microenvironment to affect tumorigenesis, and oral dietary interventions can modulate the tumor microbiota in patients with breast cancer.

OBJECTIVES: Poor diet and obesity often go hand-in-hand and are difficult to discern which variable is the major driver of the gut microbiome. The objective of this study was to determine the impact of obesity within dietary exposures on the gut microbiome and metabolic parameters using a non-human primate model. METHODS: Female M. fasicularis monkeys were fed a Western or Mediterranean diet for 2.5 years. We performed metagenomics sequencing on fecal samples obtained at 26 months. DNA was isolated from feces using Qiagen PowerSoil DNA extraction kit and metagenomics sequencing was performed for multikingdom microbiome analysis. DEXA scans for body adiposity and metabolic profiling were measured in each subject before the end of the study. Subjects were grouped by body fat composition (Lean (≤10% body fat) or Overweight/Obese (≥20% body fat)) and the impact of diet and adiposity was determine on the gut microbiome. Gut microbiota populations were correlated with metabolic parameters. RESULTS: Diet is the main determinant on gut microbiome α-diversity. Obesity had no significant outcome on Shannon diversity. Obesity within each dietary pattern can influence certain gut microbes. Lean Mediterranean diet-fed animals had significantly higher L. animals and C. comes that overweight animals fed the same diet. Obese Western diet-fed animals displayed elevated proportional abundance of S. infantarius and R. chanpaneliensis that lean Western diet-fed animals. Independent of adiposity, Western diet consumption lead to two distinct microbiome populations; P. copri high and P. copri low. P. copri(HIGH) displayed reduced α-diversity, increased abundance of other Prevotella species (P. stercorea, P. brevis, and P. bryantii), and increased F. prausnitzii. P. copri negatively correlated with α-diversity. P. copri(LOW) displayed increased proportional abundance of E. siraeum. Gut E. siraeum populations positively correlated with plasma HDL cholesterol levels. CONCLUSIONS: Our data indicates that diet is a potent regulator of the gut microbiome, while body adiposity can subtly shift specific gut microbiota taxa within subjects fed a specific dietary pattern. Moreover, our data indicates at a sub-group of metabolically healthier subjects on a Western diet characterized by low P. copri microbiota abundance. FUNDING SOURCES: NIH and DOD BCRP.

Dysregulation of emotions is a risk for social anxiety symptoms, whereas dispositional mindfulness has been proposed as assisting with emotion regulation. The aim of the current study was to examine the unique associations of dysregulation and mindfulness with adolescents' social anxiety, while focusing on the conceptual overlap and the empirical connection between dysregulation and mindfulness. Participants were 336 Australian adolescents (53% girls; 12–15 years) who completed questionnaires. Dysregulation and mindfulness were moderately correlated. Factor analysis revealed two factors accounting for 59% of the variance. The first factor, active dysregulation, had high positive loadings for five dysregulation and high negative loadings for two mindfulness subscales. The second factor, passive regulation, had a high negative loading for one dysregulation and high positive loadings for three mindfulness subscales. Both active and passive composite scores had unique associations with heightened anxiety symptoms. Regression analyses of the original subscales indicated that the dysregulation subscale limited strategies, and the mindfulness subscales observing and describing were uniquely associated with anxiety; strategies and observing were associated with more symptoms, whereas describing was associated with fewer. Interventions that address dysregulation and low capacity for mindfulness may be beneficial for adolescents with heightened social anxiety symptoms.

Summary: Recent identification of a mammary gland-specific microbiome led to studies investigating bacteria populations in breast cancer. Malignant breast tumors have lower Lactobacillus abundance compared with benign lesions, implicating Lactobacillus as a negative regulator of breast cancer. Diet is a main determinant of gut microbial diversity. Whether diet affects breast microbiome populations is unknown. In a non-human primate model, we found that consumption of a Western or Mediterranean diet modulated mammary gland microbiota and metabolite profiles. Mediterranean diet consumption led to increased mammary gland Lactobacillus abundance compared with Western diet-fed monkeys. Moreover, mammary glands from Mediterranean diet-fed monkeys had higher levels of bile acid metabolites and increased bacterial-processed bioactive compounds. These data suggest that diet directly influences microbiome populations outside the intestinal tract in distal sites such as the mammary gland. Our study demonstrates that diet affects the mammary gland microbiome, establishing an alternative mechanistic pathway for breast cancer prevention. : Using a non-human primate model of women’s health, Shively et al. demonstrate that diet plays a critical role in determining microbiota populations in tissues outside the gut, such as the mammary gland. These microbial populations modulate localized bile acid and bacterial-modified metabolites to potentially influence anticancer signaling pathways. Keywords: breast, mammary gland, diet, microbiome, bile acid, hippurate, oxidative stress

// David R. Soto-Pantoja 1, 3 , Adam S. Wilson 1 , Kenysha YJ. Clear 1 , Brian Westwood 1 , Pierre L. Triozzi 2, 3 and Katherine L. Cook 1, 3 1 Department of Surgery, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 2 Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 3 Comprehensive Cancer Center of Wake Forest Baptist Medical Center, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA Correspondence to: Katherine L. Cook, email: klcook@wakehealth.edu Keywords: breast cancer, melanoma, immune therapy, ipilimumab, CTLA4 Received: March 22, 2017 Accepted: July 23, 2017 Published: August 03, 2017 ABSTRACT The unfolded protein response (UPR) is a stress pathway controlled by GRP78 to mediate IRE1, PERK, and ATF6 signaling. We show that targeting GRP78, IRE1, and PERK differentially regulates macrophage polarization. Specifically, PERK targeting enhanced macrophage proliferation and macrophage-mediated killing but not GRP78 or IRE1. Targeting UPR in cancer cells also differentially affected macrophage cytolytic capacity. Tumoral IRE1 or GRP78 inhibition enhanced macrophage-mediated cancer cell clearance. Conditioned media from GRP78-silenced cancer cells caused reciprocal regulation of CD80 and CD206, suggesting control of plasticity by secreted factors. GRP78 targeting in mice resulted in a cytokine shift and increased tumoral CD80+/CD68+ cells, suggesting an M1-like profile. Targeting UPR in both macrophage and cancer cells indicates that PERK or GRP78 reduction enhances macrophage clearance of cancer cells. Recent evidence suggests that macrophage polarization influences immune checkpoint therapy resistance. To determine whether UPR effects immunotherapy resistance, analysis of matched melanoma patient PBMC before/after developing ipilimumab resistance demonstrated increased UPR signaling and an M2-like macrophage population, supporting a novel role of UPR signaling and innate immune regulation in anti-CTLA-4 therapy resistance. These data suggest that targeting GRP78 or PERK promotes an anti-tumor immune response by either directly promoting macrophage cytolytic activity or indirectly by shifting tumoral cytokine secretion.

Administration of exogenous 5-aminolevulinic acid (5-ALA) to cancerous tissue leads to intracellular production of photoactive protoporphyrin IX, a biosynthetic process that enables photodynamic therapy and fluorescence-guided surgery of cancer. Cell uptake of 5-ALA is limited by its polar structure and there is a need for non-toxic chemical additives that can enhance its cell permeation. Two zinc-bis(dipicolylamine) (ZnBDPA) compounds were evaluated for their ability to promote uptake of 5-ALA into Chinese Hamster Ovary (CHO-K1) cells and produce protoporphyrin IX. One of the ZnBDPA compounds was found to be quite effective, and a systematic comparison of cells incubated with 5-ALA (100 μM) for 6 hours showed that the presence of this ZnBDPA compound (10 μM) produced 3-fold more protoporphyrin IX than cells treated with 5-ALA alone. The results of mechanistic studies suggest that the ZnBDPA compound does not interact strongly with the 5-ALA. Rather, the additive is membrane active and transiently disrupts the cell membrane, permitting 5-ALA permeation. The membrane disruption is not severe enough to induce cell toxicity or allow passage of larger macromolecules like plasmid DNA.

Attachment theory and previous research on emotion regulation (ER) suggest that ER will be associated with adult attachment orientation, with the expectation of different associations of attachment avoidance, anxiety, and security with specific ER patterns. In addition, research has shown that the emotion under consideration and the context may matter to patterns of ER and associations between attachment and ER. In the present study, we examined associations between attachment representations, and emotion specific (sadness, worry, and anger) ER among late adolescents and young adults aged 16 to 23 years ( M = 19.6, SD = 1.58). In addition, to consider context, participants were randomly assigned to report ER following insecurity priming or no priming. Participants were 383 (181 male, 202 female) students who completed a self-report questionnaire. As expected, multivariate regression results examining all attachment orientations simultaneously showed that attachment anxiety was associated with greater dysregulation (sadness, worry, and anger), but also more anger suppression. In contrast, attachment avoidance was associated with greater suppression (sadness and worry), but also more anger dysregulation. Attachment security was associated with less dysregulation (sadness, worry, and anger), and less sadness and worry suppression. Finally, sadness and anger dysregulation were higher when reported after insecurity priming compared to the standard no prime condition, but few associations between attachment orientations and ER were moderated by condition. The results suggest that individuals’ attachment representations are associated with ER, with security a benefit to adaptive ER, and anxiety and avoidance playing different roles in maladaptive ER for different emotions.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype with a 5-year survival rate significantly worse when compared to other breast cancer types. There are no targeted therapy options, limiting these patients to cytotoxic chemotherapy regimens. Our group demonstrated increased tumoral Pseudomonas abundance in breast cancer patients receiving neoadjuvant chemotherapy, suggesting that systemic anticancer therapy administration may modulate microbiota populations. The purpose of our study was to determine whether gut microbiota populations correlate with chemotherapeutic responsiveness and may be a predictive biomarker outcome. We wanted to determine the impact of shifting the microbiome on chemotherapy efficacy. To do so, 8-week old female BALB/c mice were injected with 4T1-luciferase cells in the mammary fat pad. Once tumors reached 100 mm3, mice were either untreated (control group), treated with 1x weekly 2.5 mg/kg doxorubicin (DOX) for 4 weeks, or treated with doxorubicin +antibiotics (mixture of streptomycin, ampicillin, and colistin in the drinking water to ablate the microbiome). Tumor size was monitored. Tumors and lungs were collected after the study. Fecal samples were collected at T0 (before treatment) and T4 (after treatment). Mice receiving DOX were stratified into DOX-responders or DOX-nonresponders based upon tumor size. Mice from DOX-responders and DOX +antibiotics groups displayed reduced tumor weight and decreased lung metastatic burden. 16S-bacterial sequencing indicates elevated fecal Ruminococcus correlates with DOX-nonresponsiveness and increased abundance of Oscillospira and Bacteroidales is associated with better therapeutic outcome. Protein analysis of tumor tissue indicates a significant increase in apoptosis in DOX-responders and DOX +antibiotic groups. To determine whether modulating the gut microbiome influences drug responsiveness, BALB/c mice were stratified into a control group or group receiving lard diet-derived fecal transplant (LDFT) by oral gavage. Mice were injected with 4T1-luciferase cells in the mammary fat pad. Once tumors reached 100 mm3, mice were treated with 1x weekly 2.5 mg/kg DOX for 4 weeks. Tumors and lungs were collected at the end of the study. Fecal samples were collected at T0 (before gavage), T3 (after 3 weeks of LDFT and before DOX-treatment), and T7 (at end of study). All tumors from LDFT-group were larger and displayed reduced chemotherapy responsiveness when compared with control animals, suggesting gut microbiome populations can modulate chemotherapy resistance. Taken together, our data demonstrate that chemotherapy efficacy is modulated by gut microbiome and suggest that modulation of the gut microbiome through dietary or probiotic interventions may affect therapeutic outcomes. Moreover, fecal microbiota populations could be used as a predictive biomarker of chemotherapeutic responsiveness. Citation Format: Kenysha Clear, Adam Wilson, Akiko Chiba, Katherine L. Cook, Alaa Bawaneh. Gut microbiome populations modulate neoadjuvant chemotherapy responsiveness in preclinical triple-negative breast cancer murine model [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B15.

Cell death is involved in many pathological conditions, and there is a need for clinical and preclinical imaging agents that can target and report cell death. One of the best known biomarkers of cell death is exposure of the anionic phospholipid phosphatidylserine (PS) on the surface of dead and dying cells. Synthetic zinc(II)-bis(dipicolylamine) (Zn2BDPA) coordination complexes are known to selectively recognize PS-rich membranes and act as cell death molecular imaging agents. However, there is a need to improve in vivo imaging performance by selectively increasing target affinity and decreasing off-target accumulation. This present study compared the cell death targeting ability of two new deep-red fluorescent probes containing phenoxide-bridged Zn2BDPA complexes. One probe was a bivalent version of the other and associated more strongly with PS-rich liposome membranes. However, the bivalent probe exhibited self-quenching on the membrane surface, so the monovalent version produced brighter micrographs of dead and dying cells in cell culture and also better fluorescence imaging contrast in two living animal models of cell death (rat implanted tumor with necrotic core and mouse thymus atrophy). An (111)In-labeled radiotracer version of the monovalent probe also exhibited selective cell death targeting ability in the mouse thymus atrophy model, with relatively high amounts detected in dead and dying tissue and low off-target accumulation in nonclearance organs. The in vivo biodistribution profile is the most favorable yet reported for a Zn2BDPA complex; thus, the monovalent phenoxide-bridged Zn2BDPA scaffold is a promising candidate for further development as a cell death imaging agent in living subjects.

Diverse qualities of parent–adolescent relationships, such as warmth and communication, explain some of the variability in the quality and satisfaction of offspring’s later romantic relationships. In this chapter, we briefly summarize theories and research linking qualities of the parent–adolescent relationship to offspring’s romantic relationship outcomes (e.g., relationship satisfaction). After this section, we focus more precisely on one aspect of parenting, autonomy support, which has been infrequently studied but may be critical to offspring’s positive development of intimacy and romantic relationships. Based on Self-Determination Theory and past research, we define parental autonomy support as parents’ support for volition, open communication, and guidance in coping and decision-making, and identify five intrapersonal and five interpersonal competencies that should develop out of autonomy-supportive parenting. We end by outlining a model of how and why parental autonomy support may foster the development of optimal romantic relationships via offspring’s developing intrapersonal and interpersonal competencies.

Triple negative breast cancer (TNBC) accounts for 15-20% of all breast cancers and predominately affects young and minority women. TNBC is characterized by a high recurrence rate, with about 34% of TNBC patients relapsing around 2.6 years. This type of breast cancer lacks targeted therapy options and is limited to chemotherapy treatment options. The purpose of our study was to determine whether gut microbiota populations correlate with chemotherapeutic responsiveness and may be a predictive biomarker of outcome. Moreover, we want to determine the impact of targeting the microbiome on chemotherapy efficacy. In this study, 8-week old female BALB/c mice were injected with TNBC 4T1-luciferase cells into their L4/5 mammary fat pad. Once tumors developed, mice were either untreated (control group), treated with 1 x weekly 2.5 mg/kg IV doxorubicin (DOX), or treated with doxorubicin + antibiotics (mixture of streptomycin, ampicillin, and colistin in the drinking water to ablate host bacterial populations). Tumors were collected at the end of the study. Fecal samples were collected at time point 0 weeks (T0; before therapy when tumors were approximately 100 mm3) and time point 4 weeks (T4; after treatment). We subdivided the doxorubicin treated group into DOX-responders (tumors stopped growing or shrank) or DOX-nonresponders (tumors continued to grow on treatment). Tumors from the DOX-responders and DOX + antibiotics groups were significantly smaller and mice from these groups displayed reduced lung weight, suggesting decreased lung metastatic burden. 16S-bacterial sequencing analysis was performed on breast tumor tissue and feces. We demonstrate that at T0, elevated fecal Ruminococcus correlates with DOX-nonresponsiveness. Furthermore, at T4, we show increased fecal abundance of Oscillospira and Bacteroidales are associated with better therapeutic outcome. Protein analysis of primary 4T1 breast tumor tissue indicate a significant elevation of apoptosis (cleaved caspase-3 protein levels) in DOX-responders and DOX + antibiotic treated mice. We also stained breast tumor tissue and lung tissue for bacteria to confirm presence of microbiota by immunohistochemistry. Taken together our data demonstrates that chemotherapy efficacy is modulated by the microbiome. Moreover, fecal microbiota populations could be used as a predictive biomarker of chemotherapeutic responsiveness and suggests that modulation of the gut microbiome through dietary or probiotic interventions may affect therapeutic outcomes. Citation Format: Alaa Bawaneh, Adam S. Wilson, Kenysha YJ Clear, Akiko Chiba, Katherine L. Cook. Gut microbiota population may be used to predict chemotherapeutic responsiveness in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 650.

This feature article describes the development of synthetic zinc(ii)-dipicolylamine (ZnDPA) receptors as selective targeting agents for anionic membranes in cell culture and living subjects. There is a strong connection between anionic cell surface charge and disease, and ZnDPA probes have been employed extensively for molecular imaging and targeted therapeutics. Fluorescence and nuclear imaging applications include detection of diseases such as cancer, neurodegeneration, arthritis, and microbial infection, and also quantification of cell death caused by therapy. Therapeutic applications include selective targeting of cytotoxic agents and drug delivery systems, photodynamic inactivation, and modulation of the immune system. The article concludes with a summary of expected future directions.

This feature article describes the development of synthetic zinc(ii)-dipicolylamine (ZnDPA) receptors as selective targeting agents for anionic membranes in cell culture and living subjects. There is a strong connection between anionic cell surface charge and disease, and ZnDPA probes have been employed extensively for molecular imaging and targeted therapeutics. Fluorescence and nuclear imaging applications include detection of diseases such as cancer, neurodegeneration, arthritis, and microbial infection, and also quantification of cell death caused by therapy. Therapeutic applications include selective targeting of cytotoxic agents and drug delivery systems, photodynamic inactivation, and modulation of the immune system. The article concludes with a summary of expected future directions.

Liposomes containing membrane-anchored pH-sensitive optical probes are valuable sensors for monitoring pH in various biomedical samples. The dynamic range of the sensor is maximized when the probe pKa is close to the expected sample pH. While some biomedical samples are close to neutral pH there are several circumstances where the pH is 1 or 2 units lower. Thus, there is a need to fine-tune the probe pKa in a predictable way. This investigation examined two lipid-conjugated optical probes, each with appended deep-red cyanine dyes containing indoline nitrogen atoms that are protonated in acid. The presence of anionic phospholipids in the liposomes stabilized the protonated probes and increased the probe pKa values by < 1 unit. The results show that rational modification of the membrane composition is a general non-covalent way to fine-tune the pKa of an optical liposome sensor for optimal pH sensing performance.

An admixture of zinc(II)-bis(dipicolylamine) receptor with covalently attached paramagnetic relaxation agent and fluorine-labeled phosphate indicator enables (19)F NMR detection of phosphorylated analytes with amplified switched-on signal intensity.

The development of immunotherapy is a major recent advance in clinical oncology. However the majority of patients do not respond and those who do, experience resistance and relapse. Therefore, understanding the molecular mechanisms of immune checkpoint inhibitor resistance is critical to develop combinatorial drug strategies to potentiate therapeutic responsiveness to reduce mortality. Highly secretory cells, such as T-cells, have a larger endoplasmic reticulum (ER) to handle the increased protein translation capacity required by the cell. Therefore, T-cells may be highly sensitive to ER stress. The high nutrient needs of the tumor deplete resources in the microenvironment subjugating infiltrating T-cells to reduced nutrient availability resulting in stress. Our data indicate that co-culturing melanoma cells with T-cells increase T-cell specific unfolded protein response (UPR) signaling. Stimulating ER stress decreased cytolytic T-cell function in TALL-104 T-cells, enabling Mun2b melanoma survival. Knockdown of PERK restored T-cell killing capacity in ER stress induced cells. In an ex-vivo model of antigen-specific mediated T-cell death, Pmel-1 T-cells stimulated with gp100 displayed increased B16 melanoma killing when treated with GSK2606414 (a small molecule PERK inhibitor). PERK inhibition also elevated glycolysis proteins and mitochondrial bioenergetics in T-cells, suggesting PERK inhibition increases T-cell metabolism. As proof-of-concept, we determined the effect of PERK inhibition in a syngeneic B16 melanoma model. Male C57/Bl6 mice were inoculated with B16 melanoma cells. At 5 days post injection, mice were treated with a control IgG, antisense morpholino targeting PERK, PD1 antibody, or a combination of PERK morpholino and PD1 antibody. PERK morpholino treatment alone was sufficient to reduce tumor volume by 46.5%. PD1 antibody therapy alone reduced tumor volume by 47.4%. The combination of PD1 antibody and PERK targeting therapy significantly reduced B16 melanoma tumor volume by 64.5% demonstrating that targeting PERK in vivo enhanced immunocheckpoint therapy efficacy. Treated B16 tumors were homogenized and infiltrating T-cells isolated by flow cytometry. Gated CD3+ infiltrating leukocytes were then counted for CD8 and PD1 expression. Tumor infiltrating CD8+ T-cells doubled with PERK inhibition alone, giving further proof indicating that PERK may be a novel immune checkpoint target. Small molecule PERK inhibitors were previously shown to cause β-islet damage and insulin resistance; therefore we tested the effect of our PERK morpholino on glucose tolerance in the B16 melanoma model. PERK morpholino treatment had no overall impact on glucose tolerance and therefore may be a novel therapeutic to induce T-cell metabolism and potentiate immune checkpoint therapy efficacy with reduced off-target toxicities. Citation Format: David R. Soto-Pantoja, Yismeilin R. Feliz-Mosquea, Kenysha YJ Clear, Adam S. Wilson, Katherine L. Cook. Targeting PKR-like endoplasmic reticulum kinase modulates metabolism to promote T-cell effector function and PD1 immunotherapy responsiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 449.

Polar lipids are a broad class of amphiphilic biomolecules that serve diverse biological roles encompassing energy storage, cell compartmentalization, and cell signaling. The first part of the chapter focuses on the structures of polar lipids and their self-assembly properties. The next section summarizes biological strategies for polar lipid recognition, and finally synthetic receptors are described. The supramolecular receptors operate by targeting the hydrophobic tail, the polar headgroup, or simultaneously both the headgroup and tail. The synthetic receptors include container molecules that accommodate the hydrophobic tail, or small molecules that use a combination of polar non-covalent, metal coordination, or reversible covalent bonds to associate with the polar headgroup. Some of the synthetic receptors have been converted into molecular imaging agents and others exhibit antimicrobial activity. The chapter concludes with a summary of the challenges in polar lipid recognition and future directions.

BACKGROUND AND OBJECTIVES Autoimmune hemolytic anemiA (AIHA) is rarely caused by IgM warm autoantibodies, and is sometimes difficult to diagnose. We describe three patients with severe AIHA caused by IgM warm autoantibodies; in two of the three, the hemolysis was fatal. MATERIALS AND METHODS Conventional hematologic and serologic procedures were used. RESULTS The IgM autoantibodies in all three cases were directed against determinants on or associated with glycophorin A (Ena, Wrb, and Pr). The IgM autoantibodies and unusual serological characteristics, in that the agglutinins were detected or greatly enhanced only by the presence of albumin or a low pH, and/or the agglutinins reacted optimally at 20-30 degrees C. CONCLUSIONS IgM warm autoantibodies directed at determinants on or associated with glycophorin A appear to cause severe (often fatal) hemolytic anemia.

A binary mixture of Tb(3+) and pyrocatechol violet (PV) forms a 1 : 1 Tb(3+)/PV complex that can be used in a dye displacement assay. Addition of dipicolinate (DPA) to the Tb(3+)/DPA complex simultaneously produces a PV color change from blue to yellow and luminescence emission from the newly formed Tb(3+)/DPA complex.

Although researchers have begun to examine the issue of ethnic and cultural factors in childhood sexual abuse (CSA), relatively little has been done to look at possible ethnic and cultural differences in psychological symptoms related to CSA. This study investigated the relationship between ethnicity and symptom presentation among Hispanic, African American, and Caucasian sexually abused girls. The study examined the relationship between ethnicity and depression, ethnicity and post-trauma intrusive symptoms, and ethnicity and post-trauma avoidance symptoms. Results indicated that African American girls had significantly higher levels of post-trauma avoidance symptoms than Hispanic girls, but not Caucasian girls. No significant differences were found between ethnic groups for depression or intrusive symptoms.

The synthesis, antibacterial activity and stability to human dehydropeptidase-1 (DHP-1) of three small series of carbapenems carrying carbon-linked substituents at C-2 are described. C-2 Ethenyl carbapenems showed moderate antibacterial activity but poor stability to DHP-1, C-2 Oxyiminomethyl carbapenems demonstrated variable activity and stability C-2 alpha-(Hydroxy)benzyl carbapenems were the most promising and showed good potency and DHP-1 stability.