Requires Protein Synthesis Research Articles

A DAP5/eIF3d alternate mRNA translation mechanism promotes differentiation and immune suppression by human regulatory T cells

Regulatory T cells (Treg cells) inhibit effector T cells and maintain immune system homeostasis. Treg cell maturation in peripheral sites requires inhibition of protein kinase mTORC1 and TGF-beta-1 (TGF-beta). While Treg cell maturation requires protein synthesis, mTORC1 inhibition downregulates it, leaving unanswered how Treg cells achieve essential mRNA translation for development and immune suppression activity. Using human CD4+ T cells differentiated in culture and genome-wide transcription and translation profiling, here we report that TGF-beta transcriptionally reprograms naive T cells to express Treg cell differentiation and immune suppression mRNAs, while mTORC1 inhibition impairs translation of T cell mRNAs but not those induced by TGF-beta. Rather than canonical mTORC1/eIF4E/eIF4G translation, Treg cell mRNAs utilize the eIF4G homolog DAP5 and initiation factor eIF3d in a non-canonical translation mechanism that requires cap-dependent binding by eIF3d directed by Treg cell mRNA 5’ noncoding regions. Silencing DAP5 in isolated human naive CD4+ T cells impairs their differentiation into Treg cells. Treg cell differentiation is mediated by mTORC1 downregulation and TGF-beta transcriptional reprogramming that establishes a DAP5/eIF3d-selective mechanism of mRNA translation.

Mitochondria-More than just ATP for CTLs.

Cytotoxic T cells require protein synthesis in mitochondria for prolonged killing.

P–424 Evaluating the relationship between endoplasmic reticulum stress and recurrent pregnancy loss in a young population cohort

Abstract Study question Does endoplasmic reticulum (ER) stress evaluated by X-box binding protein 1 (XBP–1) among patients aged 18–30 years play a role in recurrent pregnancy loss (RPL)? Summary answer High levels of XBP–1 observed in patients with known RPL indicate that ER stress plays an important role in RPL. What is known already Female reproductive tract undergoes dynamic changes during oogenesis which require protein synthesis, folding, maturation, which take place in the ER. ER is also responsible for destruction of unfolded/misfolded proteins. Excess accumulation of these faulty proteins leads to ER stress, which actives unfolded protein response (UPR). XBP–1 is a transcription factor involved in UPR and regulates ER stress-mediated apoptosis. Regulation of ER homeostasis is important in folliculogenesis, oocyte maturation and embryogenesis. It is also known that ER stress has a positive correlation with age and it is associated with age-related diseases. Study design, size, duration This prospective case-controlled study was conducted at University of Health Sciences Turkey, Istanbul Kanuni Sultan Suleyman Training and Research Hospital Department of Obstetrics and Gynecology between March 2020 – September 2020. A total of 70 subjects were included in the study. All patients gave their written informed consent before their enrollment in the study. Participants/materials, setting, methods 38 patients aged 18–30 years with a history of RPL were included in the study. Patients who had miscarriages due to fetal abnormalities, patients with infections, endocrine or genetic disorders, smokers, alcohol and/or drug abusers, with acute/chronic inflammatory diseases, patients using steroids, anti-inflammatory and antioxidant medications were excluded from the study. Age-matched 32 healthy subjects without RPL were included in the control group. XBP–1 levels were determined using Human XBP–1 ELISA Kit (Elabscience Co., USA). Main results and the role of chance The mean age in the control group and in the study group were 25.21±3.3 and 25.26±2.6, respectively and they were statistically similar (p = 0.324). When groups were compared according to thyroid stimulating hormone (TSH) levels and body mass index (BMI), which are additional risk factors of RPL both groups were statistically similar (p = 0.642 and 0.942, respectively). As expected gravidity and abortus numbers were significantly higher in the study group (p < 0.001). A mean XBP–1 level of 1233.41± 3902.97 was determined in the control group. The mean value of the study group was calculated to be 2251.49± 9621.12. Mean XBP–1 level in the study group was significantly high (p < 0.001). A receiver operating curve (ROC) analysis was conducted in the study group. The area under the curve was found to be 87% (95% CI: 79% - 95%). The specificity was 75%, sensitivity was 89%, positive LR was 3.5, negative LR was 0.15, positive predictive value was 80% and negative predictive value was 87% for the cut-off XBP–1 level at 1364.68 pg/mL. Limitations, reasons for caution Small sample size is an important limitation of this study. In addition, evaluating XBP–1 only in serum samples does not let us drive any conclusions on the local changes of ER stress. Studies with larger samples sizes and studying XBP–1 levels in tissue samples of endometrial material is needed. Wider implications of the findings: The significantly high levels of XBP–1 in RPL patients younger than 30 years, indicate higher ER stress in this group even when age dependent increase in ER stress is calculated out of the equation. XBP–1 can be a promising marker in evaluating patients with a fertility wish for RPL risk. Trial registration number NCT04455256

The hydrophobicity of mutations targeting D1:Val219 modifies formate and diuron binding in the quinone-Fe-acceptor complex of Photosystem II.

The D1:Val219 residue of Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 was mutated to alanine or isoleucine, creating the V219A and V219I mutants, respectively. Oxygen evolution was slowed in these mutants, while chlorophyll a fluorescence induction assays indicated slowed electron transfer. As previously observed [Erickson J.M., Rahire, M., Rochaix, J.-D. and Mets. L. (1985) Science, 228, 204-207], the V219I mutant was resistant to 3,4-dichloro-1,1-dimethyl urea (DCMU); however, the V219A strain displayed no DCMU resistance. Additionally, the V219A strain was less sensitive to the addition of formate than the control, while the V219I strain was more sensitive to formate. Both mutant strains were susceptible to photodamage and required protein synthesis for recovery. We hypothesize that the sensitivity to DCMU and the extent of bicarbonate-reversible formate-induced inhibition, as well as the capacity for recovery in cells following photodamage, are influenced by the hydrophobicity of the environment associated with the Val219 residue in D1.

Exoproteome Perspective on the Bile Stress Response of Lactobacillus johnsonii.

Probiotics must not only exert a health-promoting effect but also be capable of adapting to the harsh environment of the gastrointestinal (GI) tract. Probiotics in the GI tract must survive the cell wall-disrupting effect of bile acids. We investigated the exoproteome of Lactobacillus johnsonii PF01 and C1-10 under bile stress. A comparative analysis revealed the similarities between the two L. johnsonii exoproteomes, as well as their different responses to bile. The large number of metabolic proteins in L. johnsonii revealed its metabolic adaptation to meet protein synthesis requirements under bile stress. In addition, cell wall modifications occurred in response to bile. Furthermore, some extracellular proteins of L. johnsonii may have moonlighting function in the presence of bile. Enolase, L-lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, 50s ribosomal protein L7/L12, and cellobiose-specific phosphotransferase system (PTS) sugar transporter were significantly upregulated under bile stress, suggesting a leading role in the collective bile stress response of L. johnsonii from its exoproteome perspective.

Energy partitioning for growth by rabbits fed groundnut and stylo santhes forages supplemented with concentrate

Forty eight crossbred (California X New Zealand White) rabbits were used to evaluate energy partitioning of rabbits fed forages supplemented with concentrate. The rabbits were randomly allocated to three treatments consisting of sole Stylosanthes hamata (stylo),sole Arachis hypogea (groundnut) haulms and 50:50 mixture of both forages (mixed), and four class groups: weaners, 6-8 weeks old (0.79kg), growers, 11-12 weeks old (1.15kg), pubertal, 15-16 weeks old (1.36kg) and adult, >16 weeks old (1.55kg) in a completely randomized design (3 x 4 factorial experiment). The rabbits were individually housed and offered 150g forage and 50g concentrate diet in separate feeders at 08.00hr. Feeding sole or mixed forages did not significantly affect daily gain or body composition of rabbits. DE intake, DE retention as protein, fat and growth, DE requirement for maintenance, protein synthesis, fat synthesis and for growth were similar for sole and mixed forages. Efficiency of utilization of DE for growth was significantly higher for mixed forage than sole stylo and ranged between 0.41-0.43. DE intake was similar for all classes of rabbits. Weaners had significantly higher retained energy as protein, fat and growth, DE requirement for protein and fat synthesis and for growth and efficiency of DE utilization for growth than growing, pubertal and adult rabbits. Efficiency of DE utilization for growth ranged between 0.39-0.47. Retained energy as protein, fat and for growth were similar for weaner and grower rabbits on mixed forage but higher than for pubertal and adult rabbits. For sole stylo, weaner rabbits retained more energy as protein, fat and growth than grower, pubertal and adult rabbits while for sole groundnut haulms, weaner and pubertal rabbits retained more energy as protein, fat and growth than grower and adult rabbits. DE utilization was better for mixed than stylo. It is concluded that feeding a mixture of groundnut and stylo forages slightly increased gain and improved efficiency of DE utilization resulting in better performance of the rabbits.

Small Nucleolar RNA Expression Profiling in Cartilage.

Osteoarthritis (OA) presents as a change in the articular chondrocyte phenotype. The origin of the phenotype change is poorly understood. Small nucleolar RNAs (snoRNAs) direct chemical modification of other RNA substrates and are involved in endoribonucleolytic pre-rRNA processing. They have thereby a role by fine-tuning spliceosome and ribosome function and can thus accommodate changing requirements for protein synthesis in OA. Here we describe both targeted and global methods for snoRNA isolation and quantification from whole cartilage.

Abstract IA24: Imaging cancer metabolism in patients: Beyond FDG PET

Abstract Molecular imaging is a tool that can visualize and quantify biological processes at the molecular and cellular levels in living systems1, and it is therefore ideally suited to the study of cancer metabolism in patients2. In the clinic, molecular imaging of cancer metabolism has been most widely exploited using the elevated rate of glycolysis observed in most tumors as a target for cancer detection and staging using positron emission tomography (PET) and the glucose analog 18F-fluordeoxyglucose (FDG)3. FDG PET, now combined with anatomic imaging in the form of PET/CT, has become an important tool for cancer detection, staging, and response assessment, widely used in oncologic clinical practices around the world. Clinical use of FDG PET has increased our appreciation of aberrant glycolysis as a feature of more aggressive, less differentiated tumors4. At the same time, the variability of tumor FDG uptake seen in clinical imaging has highlighted some of the limitations of imaging tumor glycolysis and has spurred the development of new imaging probes and novel imaging modalities designed to image other facets of cancer energy metabolism. In addition, the use of therapeutics that are targeted to and/or influenced by cancer metabolic phenotype5 drive applications of cancer metabolic imaging to clinical challenges beyond detection that include predicting and monitoring response to targeted therapy6. This talk will briefly review the underpinnings of the current use of FDG PET/CT for cancer staging and response evaluation. The talk will beyond FDG PET and discuss alternative PET probes targeted to other metabolic substrates such as amino acids, lipids, and TCA intermediates7, as well as new instrumentation that offers unique capabilities for imaging whole-body cancer metabolism imaging8. The talk will also highlight other methods for imaging cancer metabolism in patients, especially hyperpolarized 13C-labeled metabolic substrates such as pyruvate9. Unique features and advantages of each modality will be highlighted, as will approaches to image analysis common across modalities, including quantitative imaging and kinetic analysis. Finally, the talk will emphasize benefit of multi-modality imaging as an approach to fully characterize cancer metabolism, using glutaminolysis imaging as an example of active investigation. Supported in part by Komen SAC130060, NIH P30-CA016520, and NIH R01-CA211337.

Sulfur-Containing Amino Acids and Lipid Metabolism

The metabolism of methionine and cysteine in the body tissues determines the concentrations of several metabolites with various biologic activities, including homocysteine, hydrogen sulfide (H2S), taurine, and glutathione. Hyperhomocysteinemia, which is correlated with lower HDL cholesterol in blood in volunteers and animal models, has been associated with an increased risk for cardiovascular diseases. In humans, the relation between methionine intake and hyperhomocysteinemia is dependent on vitamin status (vitamins B-6 and B-12 and folic acid) and on the supply of other amino acids. However, lowering homocysteinemia by itself is not sufficient for decreasing the risk of cardiovascular disease progression. Other compounds related to methionine metabolism have recently been identified as being involved in the risk of atherosclerosis and steatohepatitis. Indeed, the metabolism of sulfur amino acids has an impact on phosphatidylcholine (PC) metabolism, and anomalies in PC synthesis due to global hypomethylation have been associated with disturbances of lipid metabolism. In addition, impairment of H2S synthesis from cysteine favors atherosclerosis and steatosis in animal models. The effects of taurine on lipid metabolism appear heterogeneous depending on the populations of volunteers studied. A decrease in the concentration of intracellular glutathione, a tripeptide involved in redox homeostasis, is implicated in the etiology of cardiovascular diseases and steatosis. Last, supplementation with betaine, a compound that allows remethylation of homocysteine to methionine, decreases basal and methionine-stimulated homocysteinemia; however, it adversely increases plasma total and LDL cholesterol. The study of these metabolites may help determine the range of optimal and safe intakes of methionine and cysteine in dietary proteins and supplements. The amino acid requirement for protein synthesis in different situations and for optimal production of intracellular compounds involved in the regulation of lipid metabolism also needs to be considered for dietary attenuation of atherosclerosis and steatosis risk.

The role of the mTOR pathway in models of drug-induced reward and the behavioural constituents of addiction.

Exposure to drugs of abuse induces neuroadaptations in critical nodes of the so-called reward systems that are thought to mediate the transition from controlled drug use to the compulsive drug-seeking that characterizes addictive disorders. These neural adaptations are likely to require protein synthesis, which is regulated, among others, by the mechanistic target of the rapamycin kinase (mTOR) signalling cascade. We have performed a narrative review of the literature available in PubMed about the involvement of the mTOR pathway in drug-reward and addiction-related phenomena. The aim of this study was to review the underlying architecture of this complex intracellular network and to discuss the alterations of its components that are evident after exposure to drugs of abuse. The aim was also to delineate the effects that manipulations of the mTOR network have on models of drug reward and on paradigms that recapitulate some of the psychological components of addiction. There is evidence for the involvement of the mTOR pathway in the acute and rewarding effects of drugs of abuse, especially psychostimulants. However, the data regarding opiates are scarce. There is a need to use sophisticated animal models of addiction to ascertain the real role of the mTOR pathway in this pathology and not just in drug-mediated reward. The involvement of this pathway in behavioural addictions and impulsivity should also be studied in detail in the future. Although there is a plethora of data about the modulation of mTOR by drugs of abuse, the involvement of this signalling pathway in addictive disorders requires further research.

Mechanisms associated with the antidepressant-like effects of L-655,708.

Previous research has demonstrated that selective modulation of hippocampal transmission by systemic administration of an α5-GABAA receptor negative allosteric modulator, L-655,708, reproduces the sustained antidepressant-like (AD-like) effect of R,S-ketamine in the absence of any psychotomimetic or abuse-related effects. Pharmacological, electrophysiological (whole-cell patch clamp), and behavioral approaches were used to examine the mechanisms by which L-655,708 produces plasticity within the hippocampus that accounts for its sustained AD-like effect in rats. Inhibitors of either transcription or translation prevented the sustained AD-like effect of L-655,708. Unlike R,S-ketamine, L-655,708 did not cause an increase in the phosphorylation of the receptor for BDNF, TrkB, in the ventral hippocampus (vHipp) 30 or 60 min after its administration nor did administration of the TrkB inhibitor, K252a, directly into the vHipp, block the sustained AD-like effect of L-655,708. Similar to previous results with R,S-ketamine, administration of L-655,709 increased levels of GluA1 in the mPFC and, blockade of such receptors by direct administration of NBQX into the mPFC blocked the sustained AD-like effect of L-655,708. Patch-clamp recordings of ventral CA1 pyramidal cells 24 h after a single systemic administration of L-655,708 revealed a significant increase in input resistance, which resulted in an approximately two-fold increase in action potential frequency. These experiments indicate that the sustained AD-like effects of L-655,708 require protein synthesis and plasticity of GluA1 glutamate receptors in the mPFC. The drug also caused changes in GABAA receptor gating properties in the vHipp with resultant changes in ventral CA1 that indirectly increases neuronal excitability. Such effects likely contribute to its sustained AD-like activity.

Neur1 and Neur2 are required for hippocampus-dependent spatial memory and synaptic plasticity.

Neur1 and Neur2, mouse homologs of the Drosophila neur gene, consist of two neuralized homology repeat domains and a RING domain. Both Neur1 and Neur2 are expressed in the whole adult brain and encode E3 ubiquitin ligases, which play a crucial role in the Notch signaling pathways. A previous study reported that overexpression of Neur1 enhances hippocampus-dependent memory, whereas the role of Neur2 remains largely unknown. Here, we aimed to elucidate the respective roles of Neur1 and Neur2 in hippocampus-dependent memory using three lines of genetically modified mice: Neur1 knock-out, Neur2 knock-out, and Neur1 and Neur2 double knock-out (D-KO). Our results showed that spatial memory was impaired when both Neur1 and Neur2 were deleted, but not in the individual knock-out of either Neur1 or Neur2. In addition, basal synaptic properties estimated by input-output relationships and paired-pulse facilitation did not change, but a form of long-term potentiation that requires protein synthesis was specifically impaired in the D-KO mice. These results collectively suggest that Neur1 and Neur2 are crucially involved in hippocampus-dependent spatial memory and synaptic plasticity.

The Apparent Requirement for Protein Synthesis during G2 Phase Is due to Checkpoint Activation

SUMMARYProtein synthesis inhibitors (e.g., cycloheximide) block mitotic entry, suggesting that cell cycle progression requires protein synthesis until right before mitosis. However, cycloheximide is also known to activate p38 mitogen-activated protein kinase (MAPK), which can delay mitotic entry through a G2/M checkpoint. Here, we ask whether checkpoint activation or a requirement for protein synthesis is responsible for the cycloheximide effect. We find that p38 inhibitors prevent cycloheximide-treated cells from arresting in G2 phase and that G2 duration is normal in approximately half of these cells. The Wee1 inhibitor MK-1775 and Wee1/Myt1 inhibitor PD0166285 also prevent cycloheximide from blocking mitotic entry, raising the possibility that Wee1 and/or Myt1 mediate the cycloheximide-induced G2 arrest. Thus, protein synthesis during G2 phase is not required for mitotic entry, at least when the p38 checkpoint pathway is abrogated. However, M phase progression is delayed in cycloheximide-plus-kinase-inhibitor-treated cells, emphasizing the different requirements of protein synthesis for timely entry and completion of mitosis.

SnoRNA signatures in cartilage ageing and osteoarthritis

Osteoarthritis presents as a change in the chondrocyte phenotype and an imbalance between anabolic and catabolic processes. Age affects its onset and progression. Small nucleolar RNAs (SnoRNAs) direct chemical modification of RNA substrates to fine-tune spliceosomal and rRNA function, accommodating changing requirements for splicing and protein synthesis during health and disease. Articular cartilage from young, old and OA knees was used in a microarray study to identify alterations in snoRNA expression. Changes in snoRNAs in osteoarthritis-like conditions were studied in chondrocytes using interleukin-1 and osteoarthritic synovial fluid. SNORD26 and SNORD96A knockdown and overexpression were undertaken using antisense oligonucleotides and overexpression plasmids. We identified panels of snoRNAs differentially expressed due to ageing (including SNORD96A, SNORD44) and osteoarthritis (including SNORD26 and SNORD116). In vitro experiments using osteoarthritis-like conditions affected snoRNA expression. Knockdown or overexpression of SNORD26 or SNORD96A resulted in changes in chondrogenic, hypertrophic, rRNA and osteoarthritis related gene expression. We demonstrate that snoRNA expression changes in cartilage ageing, and osteoarthritis and in osteoarthritis-like conditions, and when the expression of these snoRNAs is altered this affects chondrogenic and hypertrophic gene expression. Thus, we propose an additional dimension in the molecular mechanisms underlying cartilage ageing and osteoarthritis through the dysregulation of snoRNAs.

Norepinephrine stabilizes translation-dependent, homosynaptic long-term potentiation through mechanisms requiring the cAMP sensor Epac, mTOR and MAPK.

Neuromodulators regulate higher-order cognitive processes including learning and memory through modulation of synaptic transmission and plasticity. Norepinephrine is a neuromodulator that is secreted throughout the brain in response to novelty or increased arousal, which alters neural circuits by increasing the modifiability of CNS synapses. Norepinephrine activates metabotropic receptors, initiating complex intracellular signalling cascades that can promote enduring changes in synaptic strength including long-term potentiation (LTP). In particular, activation of beta-adrenergic receptors (β-ARs) by norepinephrine enhances LTP through downstream engagement of signalling cascades which upregulate protein synthesis at synapses. Here, we sought to determine the select signalling pathways recruited by norepinephrine to promote homosynaptic LTP at hippocampal synapses in mice. Application of norepinephrine initiated a long-lasting form of homosynaptic LTP that requires protein synthesis. Norepinephrine-mediated enhancement of LTP was reduced by inhibition of mammalian target of rapamycin and exchange protein directly activated by cAMP (Epac) but not cAMP-dependent protein kinase A, suggesting that the endogenous β-AR ligand norepinephrine may preferentially recruit Epac signalling to promote enduring changes in synaptic strength. These findings advance our understanding of the mechanisms through which norepinephrine regulates synaptic plasticity associated with formation of new memories.

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage.

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.

Induction of Samhd1 by interferon gamma and lipopolysaccharide in murine macrophages requiresIRF1.

SAMHD1 is an enzyme with phosphohydrolase activity. Mutations in SAMHD1 have been linked to the development of Aicardi-Goutières syndrome in humans. This enzyme also has the capacity to restrict HIV virus replication in macrophages. Here, we report that Samhd1 is highly expressed in murine macrophages and is regulated by proinflammatory (IFN-γ and LPS) but not by anti-inflammatory (IL-4 or IL-10) activators. The induction of Samhd1 follows the pattern of an intermediate gene that requires protein synthesis. In transient transfection experiments using the Samhd1 promoter, we found that a fragment of 27 bps of this gene, falling between -937 and -910 bps relative to the transcription start site, is required for IFN-γ-dependent activation. Using EMSAs, we determined that IFN-γ treatment led to the elimination of a protein complex. Chromatin immunoprecipitation assays and siRNA experiments revealed that IRF1 is required for IFN-γ- or LPS-induced Samhd1 expression. Therefore, our results indicate that Samhd1 is stimulated by proinflammatory agents IFN-γ and LPS. Moreover, they reveal that these two agents, via IRF1, eliminate a protein complex that may be related to a repressor, thereby, triggering Samhd1 expression.

O2-Tuned Protein Synthesis Machinery in Escherichia coli-Based Cell-Free System.

Involved in most aerobic biochemical processes, oxygen affects cellular functions, and organism behaviors. Protein synthesis, as the underlying biological process, is unavoidably affected by the regulation of oxygen delivery and utilization. Bypassing the cell wall, cell-free protein synthesis (CFPS) systems are well adopted for the precise oxygen regulation analysis of bioprocesses. Here a reliable flow platform was developed for measuring and analyzing the oxygen regulation on the protein synthesis processes by combining Escherichia coli-based CFPS systems and a tube-in-tube reactor. This platform allows protein synthesis reactions conducted in precisely controlled oxygen concentrations. For analysis of the intrinsic role of oxygen in protein synthesis, O2-tuned CFPS systems were explored with transcription-translation related parameters (transcripts, energy, reactive oxygen species, and proteomic pathway analysis). It was found that 2% of oxygen was the minimum requirement for protein synthesis. There was translation-related protein degradation in the high oxygen condition leading to a reduction. By combining the precise gas level controlling and open biosystems, this platform is also potential for fundamental understanding and clinical applications by diverse gas regulation in biological processes.

Evaluation of brain angiotensin and plasticity related genes following fear memory retrieval

BackgroundFear learning is an adaptive response to traumatic events and gradual decline of fear response on repeated exposure to stimuli can lead to fear extinction. However, a single presentation of a cue threat can reactive the fear memory through an alternative process called reconsolidation. Here we sought to investigate the role of the renin angiotensin systems (RAS) in reconsolidation to determine whether recent findings from RAS extinction studies in rodents and humans might also be explained by interference with reconsolidation.MethodsUsing Pavlovian auditory fear conditioning, a single retrieval tone was presented to initiate memory recall. It has been previously shown that fear memories, after retrieval, are in a labile state for a time‐limited window of six hours and require protein synthesis for consolidation. Without any pharmacological intervention, mRNA levels at early (40mins) and late (4hr) phases of reconsolidation were compared to examine gene expression pattern changes in common RAS and plasticity genes in different brain regions during memory reconsolidation.ResultsTo validate our behavior model of single retrieval presentation, we compared the expression of immediate early genes (IEGs) ‐ cfos, Egr‐1 and Arc in the basolateral amygdala (BLA) – critical for fear learning and memory. These IEG’s were induced rapidly at 40mins with 2–3‐fold (p<0.001; n=12) increases in mRNA expression and returned to the baseline within 4hrs comparable to animals which did not receive the retrieval tone. The RAS distribution in the brain follows a distinct pattern which we confirmed in our lab by measuring the expression level of angiotensinogen (Agt) in telencephalon (forebrain) and diencephalon (mid and hindbrain) relative to liver. There is a 3‐fold increase in Agt (p=0.025, n=6) mRNA levels at 40mins. Angiotensin converting enzyme (Ace) mRNA levels decreased by half (p = 0.0169, n=6) and consequently Ace2 levels increased 2‐fold at early reconsolidation phase in BLA. The angiotensin type1 receptor (Agtr1) showed increased expression (p=0.0213, n=10) at 4hrs whereas angiotensin type 2 receptor (Agtr2) had steady rise at both 40mins and 4hrs (p=0.0016, n=10). The BLA mRNA levels for tachykinin receptor 3 (Tac3r), previously shown to modulate fear memory and angiotensin II, was elevated by 3‐fold at both 40mins and 4hrs. We also examined the CA1 hippocampal region, but unlike the BLA, there was no significant change in the expression levels of these.ConclusionHere we identified two distinct pathways for brain RAS in the BLA during a time course of memory reconsolidation, including early phase activation of Ace2 and a later phase increase in angiotensin receptors (Agtr1 and Agtr2) mRNA levels. These data suggest that, similar to transcriptional regulation of IEGs in the BLA, angiotensin‐related genes undergo dynamic regulation during reconsolidation following fear memory retrieval.Support or Funding InformationNIH1R01HL137103‐01, AHA15CSA24340001 Evaluation of brain angiotensin and plasticity related genes following fear memory retrieval in BLA (− decrease; + increase) Gene Gene Symbol Changes Peak Time 1. Angiotensin Type 1 Receptor AT1R + 4 hour 2. Angiotensin Type 2 Receptor AT2R + 40 mins and 4hrs 3. Angiotensin Converting Enzyme Ace − 40 mins 4. Angiotensin Converting Enzyme 2 Ace 2 + 40 mins 5. Angiotensinogen Agt + 40 mins 6. Tachykinin Receptor 3 Tac3R ++ 40mins and 4 hrs 7. IEG Fos ++ 40mins 8. IEG Arc ++ 40mins 9. IEG Egr‐1 ++ 40mins

Tissue Responses to Shiga Toxin in Human Intestinal Organoids.

Background & AimsShiga toxin (Stx)-producing Escherichia coli (eg, O157:H7) infection produces bloody diarrhea, while Stx inhibits protein synthesis and causes the life-threatening systemic complication of hemolytic uremic syndrome. The murine intestinal tract is resistant to O157:H7 and Stx, and human cells in culture fail to model the complex tissue responses to intestinal injury. We used genetically identical, human stem cell–derived intestinal tissues of varying complexity to study Stx toxicity in vitro and in vivo.MethodsIn vitro susceptibility to apical or basolateral exposure to Stx was assessed using human intestinal organoids (HIOs) derived from embryonic stem cells, or enteroids derived from multipotent intestinal stem cells. HIOs contain a lumen, with a single layer of differentiated epithelium surrounded by mesenchymal cells. Enteroids only contain epithelium. In vivo susceptibility was assessed using HIOs, with or without an enteric nervous system, transplanted into mice.ResultsStx induced necrosis and apoptotic death in both epithelial and mesenchymal cells. Responses that require protein synthesis (cellular proliferation and wound repair) also were observed. Epithelial barrier function was maintained even after epithelial cell death was seen, and apical to basolateral translocation of Stx was seen. Tissue cross-talk, in which mesenchymal cell damage caused epithelial cell damage, was observed. Stx induced mesenchymal expression of the epithelial marker E-cadherin, the initial step in mesenchymal–epithelial transition. In vivo responses of HIO transplants injected with Stx mirrored those seen in vitro.ConclusionsIntestinal tissue responses to protein synthesis inhibition by Stx are complex. Organoid models allow for an unprecedented examination of human tissue responses to a deadly toxin.