Mechanistic Data Research Articles

Bacterial multispecies interaction mechanisms dictate biogeographic arrangement between the oral commensals Corynebacterium matruchotii and Streptococcus mitis.

Polymicrobial biofilms are present in many environments particularly in the human oral cavity where they can prevent or facilitate the onset of disease. While recent advances have provided a clear picture of both the constituents and their biogeographic arrangement, it is still unclear what mechanisms of interaction occur between individual species in close proximity within these communities. In this study, we investigated two mechanisms of interaction between the highly abundant supragingival plaque (SUPP) commensal Corynebacterium matruchotii and Streptococcus mitis which are directly adjacent/attached in vivo. We discovered that C. matruchotii enhanced the fitness of streptococci dependent on its ability to detoxify streptococcal-produced hydrogen peroxide and its ability to oxidize lactate also produced by streptococci. We demonstrate that the fitness of adjacent streptococci was linked to that of C. matruchotii and that these mechanisms support the previously described "corncob" arrangement between these species but that this is favorable only in aerobic conditions. Furthermore, we utilized scanning electrochemical microscopy to quantify lactate production and consumption between individual bacterial cells for the first time, revealing that lactate oxidation provides a fitness benefit to S. mitis not due to pH mitigation. This study describes mechanistic interactions between two highly abundant human commensals that can explain their observed in vivo spatial arrangements and suggest a way by which they may help preserve a healthy oral bacterial community. IMPORTANCE As the microbiome era matures, the need for mechanistic interaction data between species is crucial to understand how stable microbiomes are preserved, especially in healthy conditions where the microbiota could help resist opportunistic or exogenous pathogens. Here we reveal multiple mechanisms of interaction between two commensals that dictate their biogeographic relationship to each other in previously described structures in human supragingival plaque. Using a novel variation for chemical detection, we observed metabolite exchange between individual bacterial cells in real time validating the ability of these organisms to carry out metabolic crossfeeding at distal and temporal scales observed in vivo. These findings reveal one way by which these interactions are both favorable to the interacting commensals and potentially the host.

Refine and Strengthen SAR-Based Read-Across by Considering Bioactivation and Modes of Action.

Structure-activity relationship (SAR)-based read-across is an important and effective method to establish the safety of a data-poor target chemical (structure of interest (SOI)) using hazard data from structurally similar source chemicals (analogues). Many methods use quantitative similarity scores to evaluate the structural similarity for searching and selecting analogues as well as for evaluating analogue suitability. However, studies suggest that read-across based purely on structural similarity cannot accurately predict the toxicity of an SOI. As mechanistic data become available, we gain a greater understanding of the mode of action (MOA), the relationship between structures and metabolism/bioactivation pathways, and the existence of "activity cliffs" in chemical chain length, which can improve the analogue rating process. For this purpose, the current work identifies a series of classes of chemicals where a small change at a key position can result in a significant change in metabolism and bioactivation pathways and may eventually result in significant changes in chemical toxicity that have a big impact on the suitability of analogues for read-across. Additionally, a series of SAR-based read-across case studies are presented, which cover a variety of chemical classes that commonly link to different toxic endpoints. The case study results indicate that SAR-based read-across can be refined and strengthened by considering MOAs or proposed reactive metabolite formation pathways, which can improve the overall accuracy, consistency, transparency, and confidence in evaluating analogue suitability.

Unnatural Amino Acid Crosslinking for Increased Spatiotemporal Resolution of Chromatin Dynamics.

The utilization of an expanded genetic code and in vivo unnatural amino acid crosslinking has grown significantly in the past decade, proving to be a reliable system for the examination of protein-protein interactions. Perhaps the most utilized amino acid crosslinker, p-benzoyl-(l)-phenylalanine (pBPA), has delivered a vast compendium of structural and mechanistic data, placing it firmly in the upper echelons of protein analytical techniques. pBPA contains a benzophenone group that is activated with low energy radiation (~365 nm), initiating a diradical state that can lead to hydrogen abstraction and radical recombination in the form of a covalent bond to a neighboring protein. Importantly, the expanded genetic code system provides for site-specific encoding of the crosslinker, yielding spatial control for protein surface mapping capabilities. Paired with UV-activation, this process offers a practical means for spatiotemporal understanding of protein-protein dynamics in the living cell. The chromatin field has benefitted particularly well from this technique, providing detailed mapping and mechanistic insight for numerous chromatin-related pathways. We provide here a brief history of unnatural amino acid crosslinking in chromatin studies and outlooks into future applications of the system for increased spatiotemporal resolution in chromatin related research.

Synergistic Binding of the Halide and Cationic Prime Substrate of l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States.

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl- or Br-), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three nongaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2 all coordinate directly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H•) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the l-lysine 4-chlorinase, BesD. After addition of 2OG, subsequent coordination of the halide to the cofactor and binding of cationic l-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon the addition of O2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and l-Lys. The surprising lability of the BesD•[Fe(IV)=O]•Cl•succinate•l-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in l-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply (i) that BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between l-Lys binding and chloride coordination following the loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

A blend of medium-chain fatty acids, butyrate, organic acids, and a phenolic compound accelerates microbial maturation in newly weaned piglets.

Inclusion of additive blends is a common dietary strategy to manage post-weaning diarrhea and performance in piglets. However, there is limited mechanistic data on how these additives improve outcomes during this period. To evaluate the effects of Presan FX (MCOA) on the intestinal microbiota and metabolome, diets with or without 0.2% MCOA were compared. Pigs fed MCOA showed improved whole-body metabolism 7 days post-weaning, with decreased (P < 0.05) creatine, creatinine and β-hydroxybutyrate. Alterations in bile-associated metabolites and cholic acid were also observed at the same time-point (P < 0.05), suggesting MCOA increased bile acid production and secretion. Increased cholic acid was accompanied by increased tryptophan metabolites including indole-3-propionic acid (IPA) in systemic circulation (P = 0.004). An accompanying tendency toward increased Lactobacillus sp. in the small intestine was observed (P = 0.05). Many lactobacilli have bile acid tolerance mechanisms and contribute to production of IPA, suggesting increased bile acid production resulted in increased abundance of lactobacilli capable of tryptophan fermentation. Tryptophan metabolism is associated with the mature pig microbiota and many tryptophan metabolites such as IPA are considered beneficial to gut barrier function. In conclusion, MCOA may help maintain tissue metabolism and aid in microbiota re-assembly through bile acid production and secretion.

Phytotoxic Effects of Al on Root Growth Are Confounded in the Presence of Fulvic and Humic Acids

Background and Aims: In acidic soils, aluminum (Al) toxicity remains a critical crop limitation that can be ameliorated by organic amendments through Al complexation with high-molecular-weight carbon compounds, particularly fulvic and humic acids (FA and HA) However, no research discriminates between the direct effects of FA and HA on plant growth and the indirect effect that occurs through ameliorating Al toxicity. This study delineates the direct and indirect effects of FA and HA on plant growth. Methods: Eucalyptus and Hay FA and HA, and Al effects on maize (Zea mays) root growth were investigated using dilute nutrient solution. Five Al concentrations (0–270 µM) were combined with four organic acid (OA) treatments, including Nil-OA, FA40, and HA40 (each at 40 mg C L−1) and a combined treatment FA40HA40 (80 mg C L−1). Results: Eucalyptus FA and HA stimulated root growth by ~20% compared with root growth in the Nil-OA (17.4 cm). In the absence of Al, Hay FA and HA inhibited root growth (by ~20%) compared with the Nil-OA but the addition of Al resulted in stimulation of root growth. In the presence of FA and HA, root growth was not inhibited by nominally toxic monomeric Al (Al3+) concentrations (~20 µM Al). However, when expressed on a relative basis to remove the direct effect of the ligand, the response was consistent with Al toxicity. Conclusions: The effects of FA and HA were either inhibitory or stimulatory depending on the source while both sources of FA and HA mitigated Al toxicity through complexation. The study provides mechanistic data that highlights limitations of soil bioassays where the direct effects of organic ligands on root growth are confounded with the indirect effect of their reduction of Al toxicity. These two independent processes must be considered in evaluating the amelioration of Al by organic amendments.

The potential role of the microbiota in prostate cancer pathogenesis and treatment.

The human body hosts a complex and dynamic population of trillions of microorganisms - the microbiota - which influences the body in homeostasis and disease, including cancer. Several epidemiological studies have associated specific urinary and gut microbial species with increased risk of prostate cancer; however, causal mechanistic data remain elusive. Studies have associated bacterial generation of genotoxins with the occurrence of TMPRSS2-ERG gene fusions, a common, early oncogenic event during prostate carcinogenesis. A subsequent study demonstrated the role of the gut microbiota in prostate cancer endocrine resistance, which occurs, at least partially, through the generation of androgenic steroids fuelling oncogenic signalling via the androgen receptor. These studies present mechanistic evidence of how the host microbiota might be implicated in prostate carcinogenesis and tumour progression. Importantly, these findings also reveal potential avenues for the detection and treatment of prostate cancer through the profiling and modulation of the host microbiota. The latter could involve approaches such as the use of faecal microbiota transplantation,prebiotics, probiotics, postbiotics or antibiotics, which can be used independently or combined with existing treatments to reverse therapeutic resistance and improve clinical outcomes in patients with prostate cancer.

Conjugation of Vancomycin with a Single Arginine Improves Efficacy against Mycobacteria by More Effective Peptidoglycan Targeting.

Drug resistant bacterial infections have emerged as one of the greatest threats to public health. The discovery and development of new antimicrobials and anti-infective strategies are urgently needed to address this challenge. Vancomycin is one of the most important antibiotics for the treatment of Gram-positive infections. Here, we introduce the vancomycin-arginine conjugate (V-R) as a highly effective antimicrobial against actively growing mycobacteria and difficult-to-treat mycobacterial biofilm populations. Further improvement in efficacy through combination treatment of V-R to inhibit peptidoglycan synthesis and ethambutol to inhibit arabinogalactan synthesis underscores the ability to identify compound synergies to more effectively target the Achilles heel of the cell-wall assembly. Moreover, we introduce mechanistic activity data and a molecular model derived from a d-Ala-d-Ala-bound vancomycin structure that we hypothesize underlies the molecular basis for the antibacterial improvement attributed to the arginine modification that is specific to peptidoglycan chemistry employed by mycobacteria and distinct from Gram-positive pathogens.

Geniposide alleviated bile acid-associated NLRP3 inflammasome activation by regulating SIRT1/FXR signaling in bile duct ligation-induced liver fibrosis

BackgroundGeniposide (GE), the active compound derived from Gardeniae Fructus, possesses valuable bioactivity for liver diseases, but GE effects on bile duct ligation (BDL)-induced cholestasis remain unclear. This study aimed to elucidate the influence of GE on BDL-induced liver fibrosis and to investigate the underlying mechanisms. MethodsGE (25 or 50 mg/kg) were intragastrical administered to C57BL/6 J mice for two weeks to characterize the hepatoprotective effect of GE on BDL-induced liver fibrosis. NLRP3 inflammasome activation was detected in vivo, and BMDMs were isolated to explore whether GE directly inhibited NLRP3 inflammasome activation. Serum bile acid (BA) profiles were assessed utilizing UPLC-MS/MS, and the involvement of SIRT1/FXR pathways was identified to elucidate the role of SIRT1/FXR in the hepaprotective effect of GE. The veritable impact of SIRT1/FXR signaling was further confirmed by administering the SIRT1 inhibitor EX527 (10 mg/kg) to BDL mice treated with GE. ResultsGE treatment protected mice from BDL-induced liver fibrosis, with NLRP3 inflammasome inhibition. However, development in vitro experiments revealed that GE could not directly inhibit NLRP3 activation under ATP, monosodium urate, and nigericin stimulation. Further mechanistic data showed that GE activated SIRT1, which subsequently deacetylated FXR and restored CDCA, TUDCA, and TCDCA levels, thereby contributing to the observed hepaprotective effect of GE. Notably, EX527 treatment diminished the hepaprotective effect of GE on BDL-induced liver fibrosis. ConclusionThis study first proved the hepaprotective effect of GE on liver fibrosis in BDL mice, which was closely associated with the restoration of BA homeostasis and NLRP3 inflammasome inhibition. The activation of SIRT1 and the subsequent FXR deacetylation restored the BA profiles, especially CDCA, TUDCA, and TCDCA contents, which was the main contributor to NLRP3 inhibition and the hepaprotective effect of GE. Overall, our work provides novel insights that GE as well as Gardeniae Fructus might be the potential attractive candidate for ameliorating BDL-induced liver fibrosis.

RWTC-MBTA: autologous vaccine prevents metastases via antitumor immune responses

BackgroundAutologous tumor cell-based vaccines (ATVs) aim to prevent and treat tumor metastasis by activating patient-specific tumor antigens to induce immune memory. However, their clinical efficacy is limited. Mannan-BAM (MB), a pathogen-associated molecular pattern (PAMP), can coordinate an innate immune response that recognizes and eliminates mannan-BAM-labeled tumor cells. TLR agonists and anti-CD40 antibodies (TA) can enhance the immune response by activating antigen-presenting cells (APCs) to present tumor antigens to the adaptive immune system. In this study, we investigated the efficacy and mechanism of action of rWTC-MBTA, an autologous whole tumor cell vaccine consisting of irradiated tumor cells (rWTC) pulsed with mannan-BAM, TLR agonists, and anti-CD40 antibody (MBTA), in preventing tumor metastasis in multiple animal models.MethodsThe efficacy of the rWTC-MBTA vaccine was evaluated in mice using breast (4T1) and melanoma (B16-F10) tumor models via subcutaneous and intravenous injection of tumor cells to induce metastasis. The vaccine’s effect was also assessed in a postoperative breast tumor model (4T1) and tested in autologous and allogeneic syngeneic breast tumor models (4T1 and EMT6). Mechanistic investigations included immunohistochemistry, immunophenotyping analysis, ELISA, tumor-specific cytotoxicity testing, and T-cell depletion experiments. Biochemistry testing and histopathology of major tissues in vaccinated mice were also evaluated for potential systemic toxicity of the vaccine.ResultsThe rWTC-MBTA vaccine effectively prevented metastasis and inhibited tumor growth in breast tumor and melanoma metastatic animal models. It also prevented tumor metastasis and prolonged survival in the postoperative breast tumor animal model. Cross-vaccination experiments revealed that the rWTC-MBTA vaccine prevented autologous tumor growth, but not allogeneic tumor growth. Mechanistic data demonstrated that the vaccine increased the percentage of antigen-presenting cells, induced effector and central memory cells, and enhanced CD4+ and CD8+ T-cell responses. T-cells obtained from mice that were vaccinated displayed tumor-specific cytotoxicity, as shown by enhanced tumor cell killing in co-culture experiments, accompanied by increased levels of Granzyme B, TNF-α, IFN-γ, and CD107a in T-cells. T-cell depletion experiments showed that the vaccine’s antitumor efficacy depended on T-cells, especially CD4+ T-cells. Biochemistry testing and histopathology of major tissues in vaccinated mice revealed negligible systemic toxicity of the vaccine.ConclusionThe rWTC-MBTA vaccine demonstrated efficacy in multiple animal models through T-cell mediated cytotoxicity and has potential as a therapeutic option for preventing and treating tumor metastasis with minimal systemic toxicity.

Circular RNA circSLC7A11 contributes to progression and stemness of laryngeal squamous cell carcinoma via sponging miR-877-5p from LASP1

BackgroundLaryngeal squamous cell carcinoma (LSCC) belongs to tumors of head and neck. Circular RNA circSLC7A11 functions as oncogenes in various tumors. However, the role of circSLC7A11 in LSCC remains largely unknown. Here, we aimed to clarify the circSLC7A11 function in LSCC. MethodsRelevance between circSLC7A11 expressions and LSCC clinicopathological was checked using chi-square. Relevance between circSLC7A11 expressions and LSCC patients' survival time was validated using Kaplan-Meier analysis. CircSLC7A11 expressions in LSCC tissues and cells were determined using quantitative real-time PCR. CircSLC7A11 functions in LSCC were examined by Cell Counting Kit-8, EdU analysis, Western blot, flow cytometry, sphere formation assay, and Transwell analysis. Meanwhile, circSLC7A11 mechanism in LSCC was determined using dual-luciferase reporter analysis, RNA pull-down, RNA Immunoprecipitation. ResultsCircSLC7A11 was highly expressed in LSCC, and high circSLC7A11 expressions were interrelated to the TNM stage. Also, LSCC patients with high circSLC7A11 owned shorter overall survival. Functional studies revealed that circSLC7A11 knockdown reduced LSCC cell proliferation, migration, invasion, stemness characteristics, and enhanced cell apoptosis. Mechanistic study data corroborated that circSLC7A11 targeted miR-877-5p, miR-877-5p targeted LASP1. LASP1 was negatively interrelated to miR-877-5p and was positively interrelated to circSLC7A11 in LSCC tissues. Also, circSLC7A11 knockdown reduced the LASP1 levels, and miR-877-5p inhibitor co-transfection reversed this reduction. Rescue assays further demonstrated that circSLC7A11 accelerated LSCC through miR-877-5p/LASP1. ConclusionCircSLC7A11 exerted oncogenic functions in LSCC by miR-877-5p/LASP1, hinting that circSLC7A11 was a novel biomarker for LSCC.

Highly Selective Fe-Catalyzed Nitrogen Fixation to Hydrazine Enabled by Sm(II) Reagents with Tailored Redox Potential and pKa.

Controlling product selectivity in multiproton, multielectron reductions of unsaturated small molecules is of fundamental interest in catalysis. For the N2 reduction reaction (N2RR) in particular, parameters that dictate selectivity for either the 6H+/6e- product ammonia (NH3) or the 4H+/4e- product hydrazine (N2H4) are poorly understood. To probe this issue, we have developed conditions to invert the selectivity of a tris(phosphino)borane iron catalyst (Fe), with which NH3 is typically the major product of N2R, to instead favor N2H4 as the sole observed fixed-N product (>99:1). This dramatic shift is achieved by replacing moderate reductants and strong acids with a very strongly reducing but weakly acidic SmII-(2-pyrrolidone) core supported by a hexadentate dianionic macrocyclic ligand (SmII-PH) as the net hydrogen-atom donor. The activity and efficiency of the catalyst with this reagent remain high (up to 69 equiv of N2H4 per Fe and 67% fixed-N yield per H+). However, by generating N2H4 as the kinetic product, the overpotential of this Sm-driven reaction is 700 mV lower than that of the mildest reported set of NH3-selective conditions with Fe. Mechanistic data support assignment of iron hydrazido(2-) species FeNNH2 as selectivity-determining: we infer that protonation of FeNNH2 at Nβ, favored by strong acids, releases NH3, whereas one-electron reduction to FeNNH2-, favored by strong reductants such as SmII-PH, produces N2H4 via reactivity initiated at Nα. Spectroscopic data also implicate a role for SmIII-binding to anionic FeN2- (via an Fe-N2- -SmIII species) with respect to catalytic efficacy.

Research advances in identification procedures of endocrine disrupting chemicals.

Endocrine disrupting chemicals (EDCs) are increasingly concerned substance endangering human health and environment. However, there is no unified standard for identifying chemicals as EDCs, which is also controversial internationally. In this review, the procedures for EDC identification in different organizations/countries were described. Importantly, three aspects to be considered in identifying chemical substances as EDCs were summarized, which were mechanistic data, animal experiments, and epidemiological information. The relationships between them were also discussed. To elaborate more clearly on these three aspects of evidence, scientific data on some chemicals including bisphenol A, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane and perchlorate were collected and evaluated. Altogether, the above three chemicals were assessed for interfering with hormones and elaborated their health hazards from macroscopic to microscopic. This review is helpful for standardizing the identification procedure of EDCs.

P-503 Immunological therapies in women undergoing assisted conception: a systematic review and meta-analysis

Abstract Study question What is the effectiveness and safety of immunological therapies in women undergoing assisted conception? Summary answer The evidence on the overall effectiveness and safety of immunomodulators was scarce and uncertain, although some drugs may lead to improved outcomes in selected women. What is known already Embryo implantation is a pre-requisite for pregnancy success. It requires a state of immune tolerance within the maternal endometrium permitting the attachment and subsequent invasion into the decidua of the semi-allogenic blastocyst. Mechanistic data suggesting that heightened immune responses may impair implantation have led to the widespread use of immunomodulatory therapies in clinical practice. These include drugs such as aspirin, heparin, corticosteroids, intralipid and intravenous immunoglobulin (IVIG). However, despite the common use of immunomodulatory drugs in women having in vitro fertilisation (IVF), there is no consensus on their effectiveness and safety, owing to a paucity of well-designed interventional studies. Study design, size, duration We searched MEDLINE, EMBASE, PubMed and CENTRAL until September 2022. We selected randomised controlled trials (RCTs) investigating immunological therapies in women undergoing IVF, including aspirin, heparin, corticosteroids, granulocyte-colony stimulating factor (G-CSF), intralipid, intravenous immunoglobulin (IVIG), tumour necrosis factor-alpha inhibitors and peripheral blood mononuclear cells (PBMCs), evaluated alone or in combination, versus no intervention, placebo or any other immunomodulator(s). The primary outcomes were the rates of live birth or ongoing pregnancy (LBR/OPR) and miscarriage per participant. Participants/materials, setting, methods Two reviewers independently selected studies and extracted data. We conducted pairwise meta-analyses according to participants’ phenotypes as reported by the trialists, including: good prognosis, previous implantation failure, autoimmunity, high inflammation, thin endometrium, and low ovarian reserve. Using the Cochrane-RoB-1 tool, we restricted our primary analyses to RCTs at low risk of selection and other biases. We presented effect estimates as risk ratio (RR) with 95% confidence interval (CI) and considered I2&amp;gt;50% as representing substantial heterogeneity. Main results and the role of chance Our searches identified 14,893 records. We included a total of 84 studies in the qualitative synthesis, of which 43 contributed to the meta-analyses, evaluating in total 7,301 participants and 12 treatment comparisons. The primary analysis showed that aspirin resulted in little to no difference in LBR (RR 0.97, 95% CI 0.94-1.23; n = 688 women; moderate-certainty evidence) and MR (RR 1.20, 95% CI 0.73-1.99; n = 988 women; low-certainty evidence) compared with placebo. The data did not identify a difference in LBR/OPR in women using subcutaneous heparin (RR 1.30, 95% CI 0.80-2.12; n = 150 women; very low-certainty evidence) or IVIG (RR 1.28, 95% CI 0.32-5.16; n = 51 women; very low-certainty evidence) versus placebo. For the remaining interventions, the primary analysis was uncertain of an effect or insufficient for quantitative synthesis. However, the sensitivity analysis including all studies, irrespective of risk of bias, showed that corticosteroids may improve LBR in women with thyroid autoimmunity (RR 2.33, 95% CI 1.04-5.25; n = 60 women; very low-certainty evidence); intrauterine G-CSF may improve LBR in women with a thin endometrium (RR 2.57, 95% CI 1.24-5.29; n = 304 women; very low-certainty evidence); and intrauterine PBMCs may improve LBR in women with previous implantation failure (RR 2.03, 95% CI 1.33-3.10; n = 312 women; very low-certainty evidence). Limitations, reasons for caution For most of the interventions under study, the evidence was of low or very-low certainty, owing to limitations in study design, imprecision, indirectness and inconsistency. Further, very few studies reported on adverse events, highlighting a worrying lack of data on the safety of immunological therapies for women undergoing assisted conception. Wider implications of the findings A scarcity of well-designed RCTs limits the evidence on immunomodulators in women undergoing IVF. However, we identified positive signals for some therapies in specific conditions (e.g. corticosteroids for thyroid autoimmunity, intrauterine G-CSF for thin endometrium, and PBMCs in women with previous implantation failure). This merits further investigation in high-quality trials. Trial registration number PROSPERO registration number CRD42021294031

Genetic deletion of hepatic NCOR1 protects from atherosclerosis by promoting alternative bile acid-metabolism and sterol excretion

BackgroundThe nuclear receptor corepressor 1 (NCOR1) plays an important role in the regulation of gene expression in immunometabolic conditions by connecting chromatin-modifying enzymes, coregulators and transcription factors. NCOR1 has been shown to be involved in cardiometabolic diseases. Recently, we demonstrated that the deletion of macrophage NCOR1 aggravates atherosclerosis by promoting CD36-triggered foam cell formation via PPARG derepression.PurposeSince NCOR1 modulates the function of several key regulators involved in hepatic lipid and bile acid metabolism, we hypothesized that its deletion in hepatocytes alters lipid metabolism and atherogenesis.MethodsTo test this hypothesis, we generated hepatocyte-specific Ncor1 knockout mice on a Ldlr-/- background. Besides assessing the progression of the disease in thoracoabdominal aortae en face, we analyzed hepatic cholesterol and bile acid metabolism at expression and functional levels.ResultsOur data demonstrate that liver-specific Ncor1 knockout mice on an atherosclerosis-prone background develop less atherosclerotic lesions than controls. Interestingly, under chow diet, plasma cholesterol levels of liver-specific Ncor1 knockout mice were slightly higher compared to control, but strongly reduced compared to control mice after feeding them an atherogenic diet for 12 weeks. Moreover, the hepatic cholesterol content was decreased in liver-specific Ncor1 knockout compared to control mice. Our mechanistic data revealed that NCOR1 reprograms the synthesis of bile acids towards the alternative pathway, which in turn reduce bile hydrophobicity and enhances fecal cholesterol excretion.ConclusionsOur data suggest that hepatic Ncor1 deletion in mice decreases atherosclerosis development by reprograming bile acid metabolism and enhancing fecal cholesterol excretion.

Perspectives on Mycophenolate Mofetil in the Management of Autoimmunity.

Before becoming a cornerstone in the treatment of numerous immune-mediated diseases, mycophenolate mofetil (MMF) was first introduced as an immunosuppressive agent in transplant immunology and later received the attention of rheumatologists and clinicians involved in the management of autoimmune diseases. MMF is now a widespread immunosuppressive drug for the treatment of several conditions, including lupus nephritis, interstitial lung disease associated with systemic sclerosis, and anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis while being efficacious also as rescue therapy in various orphan diseases, including dermatomyositis and IgA-associated nephropathy. Similarly, case reports or series support a possible use of MMF in other rare autoimmune diseases. Beyond modulating lymphocyte activation, MMF acts on other immune and non-immune cells and these effects may explain the therapeutic profile of this medication. The effects of MMF are broadly characterized by the impact on the immune system and the antiproliferative and antifibrotic changes induced. In this latter case, mechanistic data on fibroblasts may in the future allow to reevaluate the use of MMF in selected patients with inflammatory arthritis or systemic sclerosis. Attention must be paid towards the possible occurrence of adverse events, such as gastrointestinal complaints and teratogenicity, while the risk of infections and cancer related to MMF needs to be further investigated.

EPEN-03. ZFTA-RELA LOCALIZATION DYNAMICS REGULATE TRANSCRIPTIONAL CONTROL

Abstract ZFTA gene fusions are the most frequent driver events in supratentorial ependymoma (EPN). ZFTA-fusion proteins act as oncogenic transcription factors (TFs) to aberrantly activate oncogenic transcriptional programs. Our data indicates that ZFTA fusion proteins form distinct punctate structures with heterogeneous patterns of liquid-phase-like characteristics. These condensates are closely associated with transcription as seen by nascent RNA FISH and recruitment of various members of the transcription machinery, such as BRD4, MED1, and RNA POLII. Our genomic footprinting data indicates that many key transcription factors, such as Sox9, share similar genomic occupancy patterns with ZFTA fusions suggesting that these transcription factors are potentially recruited to these condensates, establishing transcriptional hubs. Our mutagenesis studies reveal that the ZFTA zinc finger domain is indispensable for condensate formation, and may bridge ZFTA fusion proteins to DNA at unique genomic loci. Condensate-forming deficient mutants fail to establish the same transcriptional program and are unable to drive tumor formation. This mechanistic data has led to our efforts to establish drug-chemical screen approaches against ZFTA-RELA localization/dynamics to identify novel therapeutic targets.

Bromo- and Extra-Terminal Domain Inhibitors Induce Mitochondrial Stress in Pancreatic Ductal Adenocarcinoma.

Identifying novel, unique, and personalized molecular targets for patients with pancreatic ductal adenocarcinoma (PDAC) remains the greatest challenge in altering the biology of fatal tumors. Bromo- and extra-terminal domain (BET) proteins are activated in a noncanonical fashion by TGFβ, a ubiquitous cytokine in the PDAC tumor microenvironment (TME). We hypothesized that BET inhibitors (BETi) represent a new class of drugs that attack PDAC tumors via a novel mechanism. Using a combination of patient and syngeneic murine models, we investigated the effects of the BETi drug BMS-986158 on cellular proliferation, organoid growth, cell-cycle progression, and mitochondrial metabolic disruption. These were investigated independently and in combination with standard cytotoxic chemotherapy (gemcitabine + paclitaxel [GemPTX]). BMS-986158 reduced cell viability and proliferation across multiple PDAC cell lines in a dose-dependent manner, even more so in combination with cytotoxic chemotherapy (P < 0.0001). We found that BMS-986158 reduced both human and murine PDAC organoid growth (P < 0.001), with associated perturbations in the cell cycle leading to cell-cycle arrest. BMS-986158 disrupts normal cancer-dependent mitochondrial function, leading to aberrant mitochondrial metabolism and stress via dysfunctional cellular respiration, proton leakage, and ATP production. We demonstrated mechanistic and functional data that BETi induces metabolic mitochondrial dysfunction, abrogating PDAC progression and proliferation, alone and in combination with systemic cytotoxic chemotherapies. This novel approach improves the therapeutic window in patients with PDAC and offers another treatment approach distinct from cytotoxic chemotherapy that targets cancer cell bioenergetics.

The Combination of a Human Biomimetic Liver Microphysiology System with BIOLOGXsym, a Quantitative Systems Toxicology (QST) Modeling Platform for Macromolecules, Provides Mechanistic Understanding of Tocilizumab- and GGF2-Induced Liver Injury.

Biologics address a range of unmet clinical needs, but the occurrence of biologics-induced liver injury remains a major challenge. Development of cimaglermin alfa (GGF2) was terminated due to transient elevations in serum aminotransferases and total bilirubin. Tocilizumab has been reported to induce transient aminotransferase elevations, requiring frequent monitoring. To evaluate the clinical risk of biologics-induced liver injury, a novel quantitative systems toxicology modeling platform, BIOLOGXsym™, representing relevant liver biochemistry and the mechanistic effects of biologics on liver pathophysiology, was developed in conjunction with clinically relevant data from a human biomimetic liver microphysiology system. Phenotypic and mechanistic toxicity data and metabolomics analysis from the Liver Acinus Microphysiology System showed that tocilizumab and GGF2 increased high mobility group box 1, indicating hepatic injury and stress. Tocilizumab exposure was associated with increased oxidative stress and extracellular/tissue remodeling, and GGF2 decreased bile acid secretion. BIOLOGXsym simulations, leveraging the in vivo exposure predicted by physiologically-based pharmacokinetic modeling and mechanistic toxicity data from the Liver Acinus Microphysiology System, reproduced the clinically observed liver signals of tocilizumab and GGF2, demonstrating that mechanistic toxicity data from microphysiology systems can be successfully integrated into a quantitative systems toxicology model to identify liabilities of biologics-induced liver injury and provide mechanistic insights into observed liver safety signals.

Abstract B058: Exploring the oncogenic role of six-transmembrane epithelial antigen of the prostate in the context of androgen receptor signaling

Abstract Prostate cancer is the second leading cause of death among American men, where pathogenesis is predominantly driven by dysregulation of the Androgen Receptor (AR). The current standard of care for metastatic disease includes a regime of androgen deprivation therapy (ADT) and AR antagonists, which increases survival to 24-48 months. Selective pressure of the therapy often leads to resurgence of AR activity resulting in castrate resistant disease, for which there are no curative treatment options. Moreover, once the disease has progressed to castrate resistance, AR directed therapies are no longer viable as a sole treatment option. Six-Transmembrane Epithelial Antigen of the Prostate 1-4 (STEAP) proteins are a family of ferric metalloreductases involved in maintaining iron homeostasis. STEAP expression is enriched in androgen sensitive and hormone refractory disease states, where increased expression correlates with high Gleason Score. STEAP proteins display greater intratumoral homogeneity at both primary and metastatic sites than the canonically used Prostate Specific Membrane Antigen (PSMA), indicating attractive diagnostic and therapeutic potential. Differential expression profiles have been observed among the STEAP family members depending on AR status in-vitro, suggesting a dependence on the AR signaling network. Previous literature indicates silencing of STEAPs in-vitro decreased cell viability and reduced proliferative, invasive, and migratory phenotypes; moreover in-vivo xenografts in which STEAPs are knocked down exhibited a significant reduction in tumor growth. Preliminary data suggests that treatment with Enzalutamide reduces the expression of STEAP proteins, implying a mechanistic link to the AR signaling axis. AMG509, a bispecific STEAP1-CD3 antibody, is the only STEAP directed therapeutic actively in clinical trials for prostate cancer (NCT04221542). There is limited mechanistic data regarding how STEAP directed therapies exert their anti-tumor function. Despite sharing similar architecture, localization, and biological implications, each STEAP protein retains unique functional roles within the cell. We plan to perform a systematic investigation on the oncogenic roles of individual STEAP protein in the context of AR dependent and independent prostate cancer models. In-vitro pharmacological AR manipulations will be used to evaluate changes in STEAP expression in multiple disease models. Genetic manipulations of STEAPs will be carried out under normal growth and ADT conditions to investigate the effects on growth, migration, oxidative stress, and cell cycle regulation. Given their principal role as ferric reductases, iron supplementation and iron drop-out will be examined in parallel. Novel treatment regimens will be explored using a combination of AR and STEAP directed therapeutics. Collectively, this study aims to uncover both the overlapping and distinct functions of STEAP proteins within the AR signaling axis and to illuminate their usefulness as a therapeutic target. Citation Format: Candice Bizzaro, Latese Evens, Moriah Cunningham, Jasibel Vasquez Gonzalez, Matthew Schiewer. Exploring the oncogenic role of six-transmembrane epithelial antigen of the prostate in the context of androgen receptor signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B058.