Formation of memory assemblies through the DNA-sensing TLR9 pathway.

As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3-5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.

Immune and Neural Response to Acute Social Stress in Adolescent Humans and Rodents.

Studies in adults have linked stress-related activation of the immune system to the manifestation of psychiatric conditions. Using a translational design, this study aimed to examine the impact of social stress on immune activity in adolescents and on neuronal activity in a preclinical mouse model. Participants were 31 adolescents (ages 12-19), including 25 with mood and anxiety symptoms. Whole-blood samples were collected before and after the Trier Social Stress Test (TSST), a stress-inducing public speaking task, then cultured for 6 hours in the presence and absence of the inflammatory endotoxin lipopolysaccharide (LPS). Effects of TSST and LPS on 41 immune biomarkers were examined using repeated-measures analysis of variance. Separately, juvenile (8-week-old) male mice were non-stressed or exposed to reminder social defeat then intraperitoneally injected with saline or LPS (n = 6/group). Brains were perfused and collected for immunohistochemistry and confocal microscopy at 0, 1, 6, and 24 hours post-injection. Activity was determined by the density of cFos-positive neurons in the paraventricular hypothalamus, paraventricular thalamus, and basolateral amygdala, regions known to show sustained activation to immunological challenge. Analyses in the adolescent study indicated a strong effect of LPS but no effects of TSST or TSST×LPS interaction on immune biomarkers. Similarly, reminder social defeat did not induce sustained neuronal activity changes comparable to LPS immunological challenge in juvenile mice. Our convergent findings across species suggest that the acute immune response to stress documented in adults is not present in youth. Thus, aging and chronicity effects may play an important role in the inflammatory response to acute psychosocial stress.

Pyrazolone-type compounds (part II): in vitro and in silico evaluation of antioxidant potential; structure-activity relationship.

The pyrazolone class comprises a variety of hybrid compounds displaying diverse biological actions. Although studied for decades, these compounds are still of interest due to their facile chemical transformations. In our previous work, we presented the synthetic route of functionalised pyrazolone derivatives. The presence of pyrazolone structural motif in many drugs, such as edaravone, prompted us to investigate the antioxidant features of the selected compounds. In this paper, we provide an extensive in vitro and in silico description of the antioxidant properties of selected pyrazolone analogues. The obtained in vitro results revealed their great antiradical potency against the DPPH radical (IC50 values in the 2.6-7.8 µM range), where the best results were obtained for analogues bearing a catechol moiety. Density functional theory (DFT) was used to assess their antioxidant capacity from the thermodynamic aspect. Here, good agreement with in vitro results was achieved. DFT was employed for the prediction of the most preferable radical scavenging pathway, also. In polar solvents, the SPLET mechanism is a favourable scavenging route, whereas in nonpolar solvents the HAT is slightly predominant. Furthermore, antioxidant mechanisms were studied in the presence of relevant reactive oxygen species. The obtained values of the reaction enthalpies with the selected radicals revealed that HAT is slightly prevailing in polar solvents, while the SPLET mechanism is dominant in nonpolar solvents. Regarding the well-known antioxidant features of the drug edaravone, these findings represent valuable data for this pyrazolone class and could be used as the basis for further investigations.

Sex-specific role of hippocampal NMDA-Erk-mTOR signaling in fear extinction of adolescent mice.

Adolescence is a key phase of development for perturbations in fear extinction, with inability to adequately manage fear a potent factor for developing psychiatric disorders in adulthood. However, while behavioral correlates of adolescent fear regulation are established to a degree, molecular mediators of extinction learning in adolescence remain largely unknown. In this study, we observed fear acquisition and fear extinction (across 4 and 7 days) of adolescent and adult mice of both sexes and investigated how hippocampal levels of different plasticity markers relate to extinction learning. While fear was acquired evenly in males and females of both ages, fear extinction was found to be impaired in adolescent males. We also observed lower levels of GluA1, GLUN2A and GLUN2B subunits in male adolescents following fear acquisition, with an increase in their expression, as well as the activity of Erk-mTOR pathway over subsequent extinction sessions, which was paralleled with improved extinction learning. On the other hand, we detected no changes in plasticity-related proteins after fear acquisition in females, with alterations in GluA1, GluA4 and GLUN2B levels across fear extinction sessions. Additionally, we did not discern any pattern regarding the Erk-mTOR activity in female mice associated with their extinction performance. Overall, our research identifies sex-specific synaptic properties in the hippocampus that underlie developmentally regulated differences in fear extinction learning. We also point out hippocampal NMDA-Erk-mTOR signaling as the driving force behind successful fear extinction in male adolescents, highlighting this pathway as a potential therapeutic target for fear-related disorders in the adolescent population.

Pyrazolone-type compounds: synthesis and in silico assessment of antiviral potential against key viral proteins of SARS-CoV-2.

Coronavirus outbreak is still a major public health concern. The high mutation ability of SARS-CoV-2 periodically delivers more transmissible and dangerous variants. Hence, the necessity for an efficient and inexpensive antiviral agent is urgent. In this work, pyrazolone-type compounds were synthesised, characterised using spectroscopic methods and theoretical tools, and evaluated in silico against proteins of SARS-CoV-2 responsible for host cell entry and reproduction processes, i.e., spike protein (S), Mpro, and PLpro. Five of twenty compounds are newly synthesised. In addition, the crystal structure of a pyrazolone derivative bearing a vanillin moiety is determined. The obtained in silico results indicate a more favourable binding affinity of pyrazolone analogues towards Mpro, and PLpro in comparison to drugs lopinavir, remdesivir, chloroquine, and favipiravir, while in the case of S protein only lopinavir exerted higher binding affinity. Also, the investigations were performed on ACE2 and the spike RBD-ACE2 complex. The obtained results for these proteins suggest that selected compounds could express antiviral properties by blocking the binding to the host cell and viral spreading, also. Moreover, several derivatives expressed multitarget antiviral action, blocking both binding and reproduction processes. Additionally, in silico ADME/T calculations predicted favourable features of the synthesised compounds, i.e., drug-likeness, oral bioavailability, as well as good pharmacokinetic parameters related to absorption, metabolism, and toxicity. The obtained results imply the great potential of synthesised pyrazolones as multitarget agents against SARS-CoV-2 and represent a valuable background for further in vitro investigations.

Evaluation of antioxidant and cytotoxic properties of phenolic N-acylhydrazones: structure-activity relationship.

Cancer is still a relentless public health issue. Particularly, colorectal cancer is the third most prevalent cancer in men and the second in women. Moreover, cancer development and growth are associated with various cell disorders, such as oxidative stress and inflammation. The quest for efficient therapeutics is a challenging task, especially when it comes to achieving both cytotoxicity and selectivity. Herein, five series of phenolic N-acylhydrazones were synthesized and evaluated for their antioxidant potency, as well as their influence on HCT-116 and MRC-5 cells viability. Among 40 examined analogues, 20 of them expressed antioxidant activity against the DPPH radical. Furthermore, density functional theory was employed to estimate the antioxidant potency of the selected analogues from the thermodynamical aspect, as well as the preferable free-radical scavenging pathway. Cytotoxicity assay exposed enhanced selectivity of a number of analogues toward cancer cells. The structure-activity analysis revealed the impact of the type and position of the functional groups on both cell viability and selectivity toward cancer cells.

Stress-induced changes of the cholinergic circuitry promote retrieval-based generalization of aversive memories.

Generalization, the process of applying knowledge acquired in one context to other contexts, often drives the expression of similar behaviors in related situations. At the cellular level, generalization is thought to depend on the activity of overlapping neurons that represent shared features between contexts (general representations). Using contextual fear conditioning in mice, we demonstrate that generalization can also occur in response to stress and result from reactivation of specific, rather than general context representations. We found that generalization emerges during memory retrieval, along with stress-induced abnormalities of septohippocampal oscillatory activity and acetylcholine release, which are typically found in negative affective states. In hippocampal neurons that represent aversive memories and drive generalization, cholinergic septohippocampal afferents contributed to a unique reactivation pattern of cFos, Npas4, and repressor element-1 silencing transcription factor (REST). Together, these findings suggest that generalization can be triggered by perceptually dissimilar but valence-congruent memories of specific aversive experiences. Through promoting the reactivation of such memories and their interference with ongoing behavior, abnormal cholinergic signaling could underlie maladaptive cognitive and behavioral generalization linked to negative affective states.

Sex-specific contribution of glucocorticoid receptor alpha isoforms to anxiety and depressive-like behavior in mice.

Adolescent stress predisposes individuals to increased risk for anxiety and depression in adulthood. The stress response is mediated by the glucocorticoid receptor (GR) via regulation of GR-responsive genes involved in brain reaction to stress. Although dysregulation of GR in depression is well documented, this is the first study investigating the role of GRα isoforms in pathogenesis of depression. We exposed adolescent male and female C57BL/6J mice to chronic unpredictable stress (CUS) for 12 days starting at postnatal day 28 (PND28). Tests evaluating anxiety and depressive-like behaviors were performed at PND70. We analyzed corticosterone concentrations in serum, levels of GRα isoforms (95, 67, 50, 40, and 25 kDa), and mRNA levels of GR-responsive genes (GR, FKBP5, BDNF, and IL-1ß) in the hippocampus and the prefrontal cortex (PFC). CUS increased anxiety and depressive-like behavior in adult animals of both sexes, but did not affect corticosterone serum levels, 95 and 67 kDa GR isoforms. However, the levels of shorter GRα isoforms (50, 40, and 25 kDa) were altered in adult mice underwent CUS, in sex- and brain structure-specific way. Changes in gene expression revealed that female depressive-like behavior could be related to increased levels of IL-1ß in hippocampus and reduced BDNF levels in both hippocampus and PFC. However, in males, adolescent CUS increased expression of GR in adult hippocampus and BDNF in PFC. These findings suggest that adolescent stress altered levels of GRα isoforms, especially those with lower molecular weight, in sex- and tissue-specific ways, contributing to anxiety and depression in adult mice.

Antioxidant and cytotoxic activities of selected salicylidene imines: experimental and computational study.

Selected salicylidene imines were evaluated for their antioxidant and cytotoxic potentials. Several of them exerted potent scavenging capacity towards ABTS radical and hydrogen peroxide. The insight into the preferable antioxidative mechanism was reached employing density functional theory. In the absence of free radicals, the SPLET mechanism is dominant in polar surroundings, while HAT is prevailing in a non-polar environment. The results obtained for the reactions of the most active compounds with some medically relevant radicals pointed out competition between HAT and SPLET mechanisms. The assessment of their cytotoxic properties revealed inhibition of ER-a human breast adenocarcinoma cells or estrogen-independent prostate cancer cells. Molecular docking study with the cyclooxygenase (COX) 2 enzyme was performed to examine the most probable bioactive conformations and possible interactions between the tested derivatives and COX-2 binding pocket.

GluN2A-ERK-mTOR pathway confers a vulnerability to LPS-induced depressive-like behaviour.

Inflammation plays a key role in the pathogenesis of the major depressive disorder. Namely, neuroinflammation can induce the production of neuroactive metabolites that interfere with N-methyl-D-aspartate receptors (NMDAR)-mediated glutamatergic neurotransmission and contribute to depressive-like behaviour. On the other hand, mammalian target of rapamycin (mTOR) activity with synaptogenic effects is the main mediator of antidepressant effects of several potent NMDAR antagonists. In this study, we investigated the specific role of GluN2A subunits of NMDAR on the activity of mTOR signaling and behaviour in lipopolysaccharide (LPS)-induces model of depression. The results showed that mice lacking GluN2A subunit did not display depressive-like behavior after the immune challenge, opposite to LPS-treated wild-type mice. Specifically, in GluN2A knockout mice, we estimated the activity of the mTOR pathway in the hippocampus and prefrontal cortex (PFC) by measuring synaptic levels of upstream regulators (p-Akt, p-ERK, and p-GSK3ß) and downstream effectors (p-mTOR, and p-p70S6K) of mTOR activity. In addition, we assessed the changes in the levels of two important synaptic markers, GluA1 and PSD-95. Contrary to downregulated mTOR signaling and decreased synaptic markers in LPS-treated wild-type animals, the resilience of GluN2A KO mice to depressive-like behaviour was paralleled with sustained mTOR signaling activity synaptic stability in hippocampus and PFC. Finally, we disclosed that resistance of GluN2A knockouts to LPS-induced depressive-like behavior was ERK-dependent. These findings demonstrate that GluN2A-ERK-mTOR signaling is a vulnerability factor of inflammation-related depressive behaviour, making this signaling pathway the promising target for developing novel antidepressants.

Protocol for assessing the role of hippocampal perineuronal nets in aversive memories.

Perineuronal nets (PNNs) are emerging as critical regulators of memory-related neuronal processes. However, their exact contribution depends on type of memory, consolidation stage, or brain region, and remains to be fully investigated. We describe here a protocol to evaluate the importance of PNNs in the dorsal hippocampus in different stages of aversive memories using a mouse model. The protocol provides detailed instructions for surgical implantation of hippocampal cannulas, drug infusion, contextual fear conditioning procedures, and immunohistochemistry for PNN visualization. For complete details on the use and execution of this protocol, please refer to Jovasevic et al. (2021).

Green One-Pot Synthesis of Coumarin-Hydroxybenzohydrazide Hybrids and Their Antioxidant Potency.

Compounds from the plant world that possess antioxidant abilities are of special importance for the food and pharmaceutical industry. Coumarins are a large, widely distributed group of natural compounds, usually found in plants, often with good antioxidant capacity. The coumarin-hydroxybenzohydrazide derivatives were synthesized using a green, one-pot protocol. This procedure includes the use of an environmentally benign mixture (vinegar and ethanol) as a catalyst and solvent, as well as very easy isolation of the desired products. The obtained compounds were structurally characterized by IR and NMR spectroscopy. The purity of all compounds was determined by HPLC and by elemental microanalysis. In addition, these compounds were evaluated for their in vitro antioxidant activity. Mechanisms of antioxidative activity were theoretically investigated by the density functional theory approach and the calculated values of various thermodynamic parameters, such as bond dissociation enthalpy, proton affinity, frontier molecular orbitals, and ionization potential. In silico calculations indicated that hydrogen atom transfer and sequential proton loss-electron transfer reaction mechanisms are probable, in non-polar and polar solvents respectively. Additionally, it was found that the single-electron transfer followed by proton transfer was not an operative mechanism in either solvent. The conducted tests indicate the excellent antioxidant activity, as well as the low potential toxicity, of the investigated compounds, which makes them good candidates for potential use in food chemistry.

Pyrazole Derivatives of Medically Relevant Phenolic Acids: Insight into Antioxidative and Anti-LOX Activity.

BACKGROUND: From the point of view of medicinal chemistry, compounds containing phenolic and pyrazolic moiety are significant since they are often constituents of bioactive compounds. OBJECTIVE: The aims of this study were to synthesize pyrazole derivatives of medically relevant phenolic acids, confirm their structure, and evaluate their antioxidative and anti-LOX activities. METHODS: Phenolic pyrazole derivatives were obtained, starting from esters of medically relevant phenolic acids. The structures of all obtained compounds were determined by NMR and IR spectroscopy, and UV-Vis spectrophotometry. In addition, the single-crystal X-ray diffraction was used. Pyrazole derivatives were tested for their in vitro antioxidative (DPPH assay), and lipoxygenase (LOX) inhibitory activities. Radical quenching mechanism was estimated using DFT and thermodynamic approach, while molecular docking was used to estimate the binding mode within the enzyme. RESULTS: Pyrazole derivatives were obtained in high yields. The crystal structure of a new compound 3e was determined. Pyrazole derivative with catechol moiety 3d exhibited excellent radical scavenging activity, while compound 3b exhibited the best anti-LOX activity. Molecular docking study revealed that there is no direct interaction of any ligand with the active site of LOX-Ib, but pyrazoles 3a-e behave as inhibitors blocking the approach of linoleic acid to the active site. CONCLUSION: In this research, protocatechuic and vanillic acid pyrazole derivatives have been obtained for the first time. In vitro antioxidative assay suggests that pyrazole derivate of protocatechuic acid is a powerful radical scavenger, while anti-LOX assay indicates a pyrazole derivative with 4-hydroxyphenyl moiety.

Disruption of the NMDA receptor GluN2A subunit abolishes inflammation-induced depression.

Recent reports have demonstrated that lipopolysaccharide (LPS)-induced depressive-like behaviour is mediated via NMDA receptor. In this study, we further investigated the role of GluN2 A subunit of NMDA receptor in synaptic processes in the prefrontal cortex (PFC) and hippocampus of GluN2 A knockout (KO) mice in LPS-induced depressive-like behavior. Our data suggest that LPS-treated mice, lacking GluN2 A subunit, did not exhibit depressive-like behaviour. This was accompanied by unaltered levels of IL-6 and significant changes in neuroplasticity markers and glutamate receptor subunits composition in PFC and hippocampus. In particular, an immune challenge in GluN2 A KO mice resulted in unchanged PSA-NCAM levels and proBDNF increase in both brain structures as well as in increase in BDNF levels in hippocampus. Furthermore, the absence of GluN2 A resulted in increased levels of all NCAM isoforms in PFC upon LPS which was followed with a decrease in GluN1 and GluN2B subunits. The levels of AMPA receptor subunits (GluA1, GluA3, and GluA4) in the hippocampus of GluN2 A mice were unaltered upon the treatment and abundantly present in the PFC of KO mice. These results indicate that the GluN2 A subunit is critical in neuroinflammation-related depression, that its absence abolishes LPS-induced depressive phenotype, sustains PSA-NCAM levels, increases proBDNF signalling in the PFC and hippocampus and potentiates synaptic stabilization through NCAM in the PFC upon an immune challenge.

Glucocorticoid receptor alpha translational isoforms as mediators of early adversities and negative emotional states.

Childhood trauma (CT) increases the risk for psychopathology through disturbed acquisition and extinction of fear. The effects of CT are mediated by abnormalities of the hypothalamic-pituitary-adrenal axis and glucocorticoid receptor (GR). Since, the alterations in GRα translational isoforms have been documented in psychiatric disorders we sought to: 1) explore whether multiple GRα isoforms in the human peripheral blood mononuclear cells of two independent cohorts (whole cell n = 40; and nuclear extracts n = 43, adult subjects) mediate the effect of CT on negative affectivity (NA) measured by Depression, Anxiety and Stress Scales (DASS), and 2) examine their role/function during fear extinction in the animal model. In multiple regression analysis, CT, nuclear 40-kDa GRα, their interactions and FKBP5 explained 22%-35% of variance in DASS scores. Structural equation modeling showed that CT had a significant direct effect on 40-kDa and DASS in both cohorts, and on the nuclear 25-kDa GRα. The association between 40-kDa and total DASS was significantly mediated by nuclear FKBP5, whereas on DASS anxiety, over FKBP5 in both cohorts and nuclear full length GRα. Nuclear 40-kDa GRα and its interaction with CT had a significant direct effect on DASS anxiety. In mice, the successful extinction learning was followed by nuclear translocation of 40-kDa GRα and induction of BDNF exon IV expression. Our data revealed that the association between CT and adult NA in non-clinical subjects is mediated by the GRα translational isoforms, in particular 40-kDa GRα, and emphasized its role in fear extinction and neural plasticity.

Acetophenone Mannich bases: study of ionic liquid catalysed synthesis and antioxidative potential of products.

Three-component Mannich reaction of acetophenone or 4-iodoacetophenone with a variety of substituted anilines and benzaldehyde, catalysed with diethanolammonium chloroacetate, was performed under mild conditions. Mannich bases (MBs), of which five are new, were obtained in good to excellent yields. All compounds were characterized using elemental analysis, NMR and IR. In addition, detailed experimental and simulated UV-Vis spectral characterization of these compounds is presented here for the first time. In vitro antioxidative potential of synthetized MBs was evaluated using 2,2-diphenyl-1-picryl-hydrazyl radical and density functional theory (DFT) thermodynamical study. It was shown that compounds with anisidine moiety express moderate antioxidative activity. Mechanism of the organocatalysed Mannich reaction was thoroughly inspected by means of DFT. The reaction undergoes the hydrogen bonding-assisted mechanism. Moreover, the proposed rate determining step of the overall reaction is water elimination in the process of iminium ion formation. To the extent of our knowledge, this is the first detailed report on the influence of this type of catalyst on the formation of iminium ion, as a crucial intermediate for the whole reaction.

Dicoumarol derivatives: Green synthesis and molecular modelling studies of their anti-LOX activity.

Dicoumarol derivatives were synthesized in the InCl3 catalyzed pseudo three-component reactions of 4-hydroxycoumarin with aromatic aldehydes in excellent yields. The reactions were performed in water under microwave irradiation. All synthesized compounds were characterized using NMR, IR, and UV-Vis spectroscopy, as well as with TD-DFT. Obtained dicoumarols were subjected to evaluation of their in vitro lipid peroxidation and soybean lipoxygenase inhibition activities. It was shown that five of ten examined compounds (3e, 3h, 3b, 3d, 3f) possess significant potential of antilipid peroxidation (84-97%), and that compounds 3b, 3e, 3h provided the highest soybean lipoxygenase (LOX-Ib) inhibition (IC50 = 52.5 µM) and 3i somewhat lower activity (IC50 = 55.5 µM). The bioactive conformations of the best LOX-Ib inhibitors were obtained by means of molecular docking and molecular dynamics. It was shown that, within the bioactive conformations interior to LOX-Ib active site, the most active compounds form the pyramidal structure made of two 4-hydroxycoumarin cores and a central phenyl substituent. This form serves as a spatial barrier which prevents LOX-Ib Fe2+/Fe3+ ion activity to generate the coordinative bond with the C13 hydroxyl group of the α-linoleate. It is worth pointing out that the most active compounds 3b, 3e, 3h and 3i can be candidates for further examination of their in vitro and in vivo anti-inflammatory activity and that molecular modeling study results provide possibility to screen bioactive conformations and elucidate the mechanism of dicoumarols anti-LOX activity.

A new efficient domino approach for the synthesis of pyrazolyl-phthalazine-diones. Antiradical activity of novel phenolic products.

Pyrazolyl-phthalazine-dione derivatives (PPDs) were synthetized in the ionic liquid catalyzed one-pot multicomponent reaction of acetylacetone, 2,3-dihydrophthalazine-1,4-dione, and different aldehydes in moderate to good yields. Six new PPDs were obtained, and the crystal structure of 2-acetyl-1-(4-fluorophenyl)-3-methyl-1H-pyrazolo[1,2-b]phthalazine-5,10-dione (PPD-4) was determined. The most interesting structural features of the novel PPD-4 is the formation of a rather short intermolecular distance between the F atom of one molecule and the midpoint of the neighbouring six-membered heterocyclic ring. This interaction arranges all molecules into parallel supramolecular chains. UV-Vis spectra of all PPDs were acquired and compared to the simulated ones obtained with TD-DFT. All synthetized compounds were subjected to evaluation of their in vitro antioxidative activity using a stable DPPH radical. It was shown that PPD-7, with a catechol motive, is the most active antioxidant, while PPD-9, with two neighbouring methoxy groups to the phenolic OH, exerted a somewhat lower, but significant antioxidative potential. The results of DFT thermodynamical study are in agreement with experimental findings that PPD-7 and PPD-9 should be considered as powerful radical scavengers. In addition, the obtained theoretical results (bond dissociation and proton abstraction energies) specify SPLET as a prevailing radical scavenging mechanism in polar solvents, and HAT in solvents with lower polarity. On the other hand, the obtained reaction enthalpies for inactivation of free radicals suggest competition between HAT and SPLET mechanisms, except in the case of the ˙OH radical in polar solvents, where HAT is labeled as prefered.