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OBJECTIVES: To observe the effect of electroacupuncture (EA) of "Zusanli" (ST36) and "Sanyinjiao" (SP6) on cancer pain and concomitant negative emotion in cancer pain model mice, and to explore its molecular mechanisms in the basolateral amygdala (BLA) by using transcriptomics techniques. METHODS: C57BL/6 mice were randomized into sham operation, model and EA groups, with 10 mice in each group. The cancer pain model was established by injecting PBS suspension containing Lewis lung cancer cells into the femur. The mice in the EA group received EA stimulation(1 mA, 2 Hz) on ST36 and SP6 from the 10th day after modeling, 20 min per day for 12 successive days. The bone damage of the distal femur was observed with X-ray and H.E. staining, respectively. The mechanical pain threshold (MPT) was detected by using von Frey. The depression-like behavior was detected by using sucrose-preference test (sucrose preference index in 12 h), and the immobility (feeling of despair) duration of forced swimming within 4 min. The BLA tissue was extracted for RNA sequencing (RNA library construction, and screening differential gene profiling by transcriptomic sequencing) and bioinformatics analysis. The real-time PCR was used to validate the mRNA expression of differentially expressed genes：tumor necrosis factor superfamily 8 (Tnfsf8), bone marrow stromal cell antigen 1 (Bst1), prodynorphin (Pdyn) and voltage-gated sodium channelß4 (Scn4b). RESULTS: H.E. staining and X-ray showed significant bone damage in the distal femur in cancer pain mice. In contrast to the sham operation group, the MPT on the 1st , 4th, 7th , 10th, 14th and 21st day after modeling and sucrose preference index were significantly decreased (P<0.001, P<0.000 1), and the immobility time of the forced swimming was considerably increased in the model group (P<0.001). In contrast to the model group, the MPT values on the 14th and 21st day and sucrose preference index were obviously increased (P<0.000 1, P<0.05), and the immobility time was strikingly decreased in the EA group (P<0.01). RNA sequencing showed that a total of 404 differentially expressed genes (205 up-regulated, 199 down-regulated) were screened in the model group compared with the sham operation group, and a total of 329 differentially expressed genes (206 up-regulated and 123 down-regulated) were screened in the EA group compared with the model group. Venn diagram analysis of the differentially expressed genes showed that 45 up-regulated and 28 down-regulated genes in the model group were completely reversed by EA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the screened differentially expressed genes revealed that the above differential genes were mainly enriched in the ligand receptor activity, cytokine receptor binding, and cytokine activity related to neuro-inflammation, as well as in neuropeptide signaling pathways related to neuronal excitability, and calcium ion mediated signal transduction. The analysis of KEGG pathway showed that the differentially expressed genes were mainly enriched in the inflammation-related pathways, such as interleukin-17 pathway. Validation analysis of the differentially expressed genes showed that the expression levels of Tnfsf8 and Bst1 were significantly up-regulated in the model group compared with the sham operation group (P<0.01, P<0.05), and down-regulated by EA (P<0.01, P<0.05), while the expression levels of Pdyn and Scn4b were down-regulated in the model group in comparison with the sham operation group (P<0.01), and up-regulated by EA (P<0.05, P<0.01), which was consistent with the changing trend of the gene sequencing results. CONCLUSIONS: Acupuncture of ST36 and SP6 can significantly relieve cancer pain and concomitant negative emotion in cancer pain mice, which may be related to its functions in alleviating neuro-inflammation and relieving the abnormal activities of specific neurons in the BLA.

Enkephalin Rescues Temporomandibular Joint Pain-Related Behavior in Rats.

Temporomandibular joint disorders include a variety of clinical syndromes that are difficult to manage if associated with debilitating severe jaw pain. Thus, seeking additional experimental therapies for temporomandibular joint pain reduction is warranted. Targeted enkephalin gene therapy approaches provide clear promise for pain control. The studies detailed here indicate significant analgesia and protection of joint tissue are provided after injection of an overexpression viral vector gene therapy near the joint. The viral vector gene therapy described provides overexpression of naturally occurring opioid peptides after its uptake by trigeminal nerve endings. The viral vectors act as independent "minipump" sources for the opioid peptide synthesis in the neuronal cytoplasm producing the intended biological function, reduction of pain, and tissue repair. The antinociceptive effects provided with this delivery method of opioid expression persist for over 4 weeks. This is coincident with the expected time frame for the duration of the transgene overproduction of the endogenous opioid peptide before its diminution due to dormancy of the virus. These experimental studies establish a basis for the use of replication-defective herpes simplex type 1-based gene therapy for severe chronic inflammatory temporomandibular joint destruction and pain. As innovative means of significantly reducing joint inflammation and preserving tissue architecture, gene therapies may extend their clinical usefulness for patients with temporomandibular joint disorders.

Social Isolation Induces Changes in the Monoaminergic Signalling in the Rat Medial Prefrontal Cortex.

(1) Background: The effects of short-term social isolation during adulthood have not yet been fully established in rats behaviourally, and not at all transcriptomically in the medial prefrontal cortex (mPFC). (2) Methods: We measured the behavioural effects of housing adult male rats in pairs or alone for 10 days. We also used RNA sequencing to measure the accompanying gene expression alterations in the mPFC of male rats. (3) Results: The isolated animals exhibited reduced sociability and social novelty preference, but increased social interaction. There was no change in their aggression, anxiety, or depression-like activity. Transcriptomic analysis revealed a differential expression of 46 genes between the groups. The KEGG pathway analysis showed that differentially expressed genes are involved in neuroactive ligand-receptor interactions, particularly in the dopaminergic and peptidergic systems, and addiction. Subsequent validation confirmed the decreased level of three altered genes: regulator of G protein signalling 9 (Rgs9), serotonin receptor 2c (Htr2c), and Prodynorphin (Pdyn), which are involved in dopaminergic, serotonergic, and peptidergic function, respectively. Antagonizing Htr2c confirmed its role in social novelty discrimination. (4) Conclusions: Social homeostatic regulations include monoaminergic and peptidergic systems of the mPFC.

Genetic Variants in KNDy Pathway Lack Association with Premature Ovarian Insufficiency in Mexican Women: A Sequencing-Based Cohort Study.

Previous studies have demonstrated the essential role of the Kisspeptin/Neurokinin B/Dynorphin A (KNDy) pathway in female reproductive biology by regulating the activity of the hypothalamic-pituitary-gonadal axis. Identified loss-of-function mutations in these genes are linked to various reproductive disorders. This study investigated genetic disorders linked to mutations in the KNDy genes related to premature ovarian insufficiency (POI). A cohort of 14 Mexican POI patients underwent genetic screening using PCR-SSCP and Sanger sequencing, assessing the genetic variations' impact on protein function thereafter using multiple in silico tools. The PCR excluded extensive deletions, insertions, and duplications, while SSCP detected five genetic variants. Variations occurred in the KISS1 (c.58G>A and c.242C>G), KISS1R (c.1091A>T), PDYN (c.600C>T), and OPRK1 (c.36G>T) genes, whereas no genetic anomalies were found in NK3/NK3R genes. Each single-nucleotide variant underwent genotyping using PCR-SSCP in 100 POI-free subjects. Their allelic frequencies paralleled the patient group. These observations indicate that allelic variations in the KNDy genes may not contribute to POI etiology. Hence, screening for mutations in KNDy genes should not be a part of the diagnostic protocol for POI.

Modulation of endogenous opioid signaling by inhibitors of puromycin-sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin-sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

The Foundational Science of Endogenous Opioids and Their Receptors.

The function of endogenous opioids spans from initiating behaviors that are critical for survival, to responding to rapidly changing environmental conditions. A network of interconnected systems throughout the body characterizes the endogenous opioid system (EOS). EOS receptors for beta-endorphin, enkephalin, dynorphin, and endomorphin underpin the diverse functions of the EOS across biological systems. This chapter presents a succinct yet comprehensive summary of the structure of the EOS, EOS receptors, and their relationship to other biological systems.

Endogenous Opioids and Exercise-Related Hypoalgesia: Modern Models, Measurement, and Mechanisms of Action.

This chapter will focus on the role exercise appears to have on activation and modulating factors within the central nervous system related to endogenous like opioids and its possible contribution to exercise-induced hypoalgesia. The implications for the exercise-mediated alterations of CNS activation factors related to opioids, specifically endorphins and enkephalins, will be presented. In this update, we discuss utilization of new technology and methods to monitor mechanisms of opioid involvement to suggest their contribution with exercise mediated hypoalgesia as well as their relationships to alterations of perceptions of pain and mood. Several special populations were included to suggest that not all individuals will respond to the exercise by mediating hypoalgesia. Factors that may confound the current understanding and suggestions from the recent literature will be presented as well as suggestions for future investigations.

Amadori and Heyns rearrangement products of bioactive peptides as potential new ligands of galectin-3.

Non-enzymatic cascade reactions between amines and reducing sugars are known as Maillard reaction. The late phase of these reactions consists of advanced glycation end products (AGEs), which have been implicated in the pathogenesis of numerous human diseases. Recent evidence suggests that galectin-3 acts as a receptor for AGEs and some early products of the Maillard reaction. The early phase of the Maillard reaction, which consists of 1-amino-1-deoxyketoses (Amadori compounds) and 2-amino-2-deoxyaldoses (Heyns compounds), was the subject of our study. The binding interactions between galectin-3 and the Amadori and Heyns compounds of leucine-enkephalin (YGGFL), leucine-enkephalin methyl ester (YGGFL-OMe), truncated enkephalin (YGG and Y) and tetrapeptide (LSKL) were measured using the AlphaScreen competitive binding assay. The affinity of galectin-3 for Amadori and Heyns compounds depends on both the sugar moiety and the amino acid sequence of the model compounds. The best results were obtained with Leu-enkephalin derivatives of Amadori (IC50 = 6.06 µm) and Heyns (IC50 = 8.6 µm) compound, respectively.

A single-vector intersectional AAV strategy for interrogating cellular diversity and brain function.

As discovery of cellular diversity in the brain accelerates, so does the need for tools that target cells based on multiple features. Here we developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), an adeno-associated virus-based, single-construct, intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches to deliver molecular cargo to specific neuronal subtypes. ConVERGD offers benefits over existing intersectional expression platforms, such as expanded intersectional targeting with up to five recombinase-based features, accommodation of larger and more complex payloads and a vector that is easy to modify for rapid toolkit expansion. In the present report we employed ConVERGD to characterize an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus that co-express the endogenous opioid gene prodynorphin (Pdyn). These studies showcase ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn-expressing NE+ locus coeruleus neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents.

Organization of enkephalinergic neuronal system in the central nervous system of the gecko Hemidactylus frenatus.

Enkephalins are endogenous opioid pentapeptides that play a role in neurotransmission and pain modulation in vertebrates. However, the distribution pattern of enkephalinergic neurons in the brains of reptiles has been understudied. This study reports the organization of the methionine-enkephalin (M-ENK) and leucine-enkephalin (L-ENK) neuronal systems in the central nervous system of the gecko Hemidactylus frenatus using an immunofluorescence labeling method. Although M-ENK and L-ENK-immunoreactive (ir) fibers extended throughout the pallial and subpallial subdivisions, including the olfactory bulbs, M-ENK and L-ENK-ir cells were found only in the dorsal septal nucleus. Enkephalinergic perikarya and fibers were highly concentrated in the periventricular and lateral preoptic areas, as well as in the anterior and lateral subdivisions of the hypothalamus, while enkephalinergic innervation was observed in the hypothalamic periventricular nucleus, infundibular recess nucleus and median eminence. The dense accumulation of enkephalinergic content was noticed in the pars distalis of the hypophysis. In the thalamus, the nucleus rotundus and the dorsolateral, medial, and medial posterior thalamic nuclei contained M-ENK and L-ENK-ir fibers, whereas clusters of M-ENK and L-ENK-ir neurons were observed in the pretectum, mesencephalon, and rhombencephalon. The enkephalinergic fibers were also seen in the area X around the central canal, as well as the dorsal and ventral horns. The widespread distribution of enkephalin-containing neurons within the central nervous system implies that enkephalins regulate a variety of functions in the gecko, including sensory, behavioral, hypophysiotropic, and neuroendocrine functions.

Estrogen promotes gonadotropin-releasing hormone expression by regulating tachykinin 3 and prodynorphin systems in chicken.

The "KNDy neurons" located in the hypothalamic arcuate nucleus (ARC) of mammals are known to co-express kisspeptin, neurokinin B (NKB), and dynorphin (DYN), and have been identified as key mediators of the feedback regulation of steroid hormones on gonadotropin-releasing hormone (GnRH). However, in birds, the genes encoding kisspeptin and its receptor GPR54 are genomic lost, leaving unclear mechanisms for feedback regulation of GnRH by steroid hormones. Here, the genes tachykinin 3 (TAC3) and prodynorphin (PDYN) encoding chicken NKB and DYN neuropeptides were successfully cloned. Temporal expression profiling indicated that TAC3, PDYN and their receptor genes (TACR3, OPRK1) were mainly expressed in the hypothalamus, with significantly higher expression at 30W than at 15W. Furthermore, overexpression or interference of TAC3 and PDYN can regulate the GnRH mRNA expression. In addition, in vivo and in vitro assays showed that estrogen (E2) could promote the mRNA expression of TAC3, PDYN, and GnRH, as well as the secretion of GnRH/LH. Mechanistically, E2 could dimerize the nuclear estrogen receptor 1 (ESR1) to regulate the expression of TAC3 and PDYN, which promoted the mRNA and protein expression of GnRH gene as well as the secretion of GnRH. In conclusion, these results revealed that E2 could regulate the GnRH expression through TAC3 and PDYN systems, providing novel insights for reproductive regulation in chickens.

Plasma proenkephalin and neutrophil gelatinase-associated lipocalin predict mortality in ICU patients with acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) is a common complication in patients admitted to intensive care unit (ICU) and mortality rates for this condition are high. To reduce the high incidence of short-term mortality, reliable prognostic indicators are required to facilitate early diagnosis and treatment of AKI. We assessed the ability of plasma proenkephalin (p­PENK) and plasma neutrophil gelatinase-associated lipocalin (p­NGAL) to predict 28-day mortality in AKI patients in intensive care. METHODS: This prospective study, carried out between January 2019 and December 2019, comprised 150 patients (100 male) diagnosed with AKI after excluding 20 patients discharged within 24 h and those with missing hospitalization data. Blood samples were collected to determine admission p-PENK and p-NGAL levels. The study outcome was 28­day mortality. RESULTS: The mean patient age was 68 years (female, 33%). The average P­PENK and p­NGAL levels were 0.24 ng/µL and 223.70 ng/mL, respectively. P­PENK levels >0.36 ng/µL and p­NGAL levels >230.30 ng/mL were used as critical values to reliably indicate 28­day mortality for patients with AKI (adjusted hazard ratios 0.785 [95% confidence interval 0.706-0.865, P<0.001] and 0.700 [95% confidence interval 0.611-0.789, P<0.001], respectively). This association was significant for mortality in patients in intensive care with AKI. Baseline p-PENK (0.36 ng/µL) and p-NGAL (230.30 ng/mL) levels and their respective cut-off values showed clinical value in predicting 28-day mortality. CONCLUSION: Serum PENK and NGAL levels, when used in conjunction, improved the accuracy of predicting 28-day mortality in patients with AKI while retaining sensitivity and specificity.

Research Note: Morphine influences circulating and tissue concentrations of met-enkephalin and proenkephalin (PENK) expression and plasma concentrations of corticosterone in chickens.

The effects of the administration of the opioid agonist, morphine, on plasma and tissue concentrations of Met-enkephalin were determined in 14 wk old female chickens. In addition, effects of morphine on proenkephalin (PENK) expression were examined. Plasma concentrations of Met-enkephalin were reduced 10 minutes after morphine administration. Plasma concentrations of peptides that contain Met-enkephalin motifs were decreased 30 minutes after morphine administration. Tissue concentrations of Met-enkephalin tended to be depressed following morphine administration. Adrenal concentrations of PENK peptides containing Met-enkephalin motifs were decreased in chickens challenged with morphine. Expression of PENK in the anterior pituitary gland and adrenal glands were decreased in morphine treated compared to control pullets. In contrast, plasma concentrations of corticosterone were elevated 10 min after morphine treatment. Morphine also induced changes in mu (µ) opioid receptors and delta (δ) opioid receptors in both anterior pituitary tissue and adrenal tissues.

Biomarkers Improve Diagnostics of Sepsis in Adult Patients With Suspected Organ Dysfunction Based on the Quick Sepsis-Related Organ Failure Assessment (qSOFA) Score in the Emergency Department.

OBJECTIVES: Consensus regarding biomarkers for detection of infection-related organ dysfunction in the emergency department is lacking. We aimed to identify and validate biomarkers that could improve risk prediction for overt or incipient organ dysfunction when added to quick Sepsis-related Organ Failure Assessment (qSOFA) as a screening tool. DESIGN: In a large prospective multicenter cohort of adult patients presenting to the emergency department with a qSOFA score greater than or equal to 1, admission plasma levels of C-reactive protein, procalcitonin, adrenomedullin (either bioavailable adrenomedullin or midregional fragment of proadrenomedullin), proenkephalin, and dipeptidyl peptidase 3 were assessed. Least absolute shrinkage and selection operator regression was applied to assess the impact of these biomarkers alone or in combination to detect the primary endpoint of prediction of sepsis within 96 hours of admission. SETTING: Three tertiary emergency departments at German University Hospitals (Jena University Hospital and two sites of the Charité University Hospital, Berlin). PATIENTS: One thousand four hundred seventy-seven adult patients presenting with suspected organ dysfunction based on qSOFA score greater than or equal to 1. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The cohort was of moderate severity with 81% presenting with qSOFA = 1; 29.2% of these patients developed sepsis. Procalcitonin outperformed all other biomarkers regarding the primary endpoint (area under the curve for receiver operating characteristic [AUC-ROC], 0.86 [0.79-0.93]). Adding other biomarkers failed to further improve the AUC-ROC for the primary endpoint; however, they improved the model regarding several secondary endpoints, such as mortality, need for vasopressors, or dialysis. Addition of procalcitonin with a cutoff level of 0.25 ng/mL improved net (re)classification by 35.2% compared with qSOFA alone, with positive and negative predictive values of 60.7% and 88.7%, respectively. CONCLUSIONS: Biomarkers of infection and organ dysfunction, most notably procalcitonin, substantially improve early prediction of sepsis with added value to qSOFA alone as a simple screening tool on emergency department admission.

Pre-proenkephalin 1 is Downregulated Under Unloading and is Involved in Osteoblast Biology.

Pre-proenkephalin 1 (Penk1) is a pro-neuropeptide that belongs to the typical opioid peptide's family, having analgesic properties. We previously found Penk1 to be the most downregulated gene in a whole gene profiling analysis performed in osteoblasts subjected to microgravity as a model of mechanical unloading. In this work, Penk1 downregulation was confirmed in the bones of two in vivo models of mechanical unloading: tail-suspended and botulinum toxin A (botox)-injected mice. Consistently, in the sera from healthy volunteers subjected to bed rest, we observed an inverse correlation between PENK1 and bed rest duration. These results prompted us to investigate a role for this factor in bone. Penk1 was highly expressed in mouse bone, but its global deletion failed to impact bone metabolism in vivo. Indeed, Penk1 knock out (Penk1-/-) mice did not show an overt bone phenotype compared to the WT littermates. Conversely, in vitro Penk1 gene expression progressively increased during osteoblast differentiation and its transient silencing in mature osteoblasts by siRNAs upregulated the transcription of the Sost1 gene encoding sclerostin, and decreased Wnt3a and Col1a1 mRNAs, suggesting an altered osteoblast activity due to an impairment of the Wnt pathway. In line with this, osteoblasts treated with the Penk1 encoded peptide, Met-enkephalin, showed an increase of Osx and Col1a1 mRNAs and enhanced nodule mineralization. Interestingly, primary osteoblasts isolated from Penk1-/- mice showed lower metabolic activity, ALP activity, and nodule mineralization, as well as a lower number of CFU-F compared to osteoblasts isolated from WT mice, suggesting that, unlike the transient inhibition, the chronic Penk1 deletion affects both osteoblast differentiation and activity. Taken together, these results highlight a role for Penk1 in the regulation of the response of the bone to mechanical unloading, potentially acting on osteoblast differentiation and activity in a cell-autonomous manner.

Applications of biaryl cyclization in the synthesis of cyclic enkephalin analogs with a highly restricted flexibility.

A series of 10 cyclic, biaryl analogs of enkephalin, with Tyr or Phe residues at positions 1 and 4, were synthesized according to the Miyaura borylation and Suzuki coupling methodology. Biaryl bridges formed by side chains of the two aromatic amino acid residues are of the meta-meta, meta-para, para-meta, and para-para configuration. Conformational properties of the peptides were studied by CD and NMR. CD studies allowed only to compare conformations of individual peptides while NMR investigations followed by XPLOR calculations provided detailed information on their conformation. Reliability of the XPLOR calculations was confirmed by quantum chemical ones performed for one of the analogs. No intramolecular hydrogen bonds were found in all the peptides. They are folded and adopt the type IV ß-turn conformation. Due to a large steric strain, the aromatic carbon atoms forming the biaryl bond are distinctly pyramidalized. Seven of the peptides were tested in vitro for their affinity for the µ-opioid receptor.

In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the µ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as µ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and ß-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and ß-arrestin2 recruitment pathways. Compared to the reference agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting ß-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and ß-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. In addition, the extremely high potency of many NSOs now infiltrating illicit drug markets further contributes to the danger posed to public health.

The impact of multifunctional enkephalin analogs and morphine on the protein changes in crude membrane fractions isolated from the rat brain cortex and hippocampus.

Endogenous opioid peptides serve as potent analgesics through the opioid receptor (OR) activation. However, they often suffer from poor metabolic stability, low lipophilicity, and low blood-brain barrier permeability. Researchers have developed many strategies to overcome the drawbacks of current pain medications and unwanted biological effects produced by the interaction with opioid receptors. Here, we tested multifunctional enkephalin analogs LYS739 (MOR/DOR agonist and KOR partial antagonist) and LYS744 (MOR/DOR agonist and KOR full antagonist) under in vivo conditions in comparison with MOR agonist, morphine. We applied 2D electrophoretic resolution to investigate differences in proteome profiles of crude membrane (CM) fractions isolated from the rat brain cortex and hippocampus exposed to the drugs (10 mg/kg, seven days). Our results have shown that treatment with analog LYS739 induced the most protein changes in cortical and hippocampal samples. The identified proteins were mainly associated with energy metabolism, cell shape and movement, apoptosis, protein folding, regulation of redox homeostasis, and signal transduction. Among these, the isoform of mitochondrial ATP synthase subunit beta (ATP5F1B) was the only protein upregulation in the hippocampus but not in the brain cortex. Contrarily, the administration of analog LYS744 caused a small number of protein alterations in both brain parts. Our results indicate that the KOR full antagonism, together with MOR/DOR agonism of multifunctional opioid ligands, can be beneficial in treating chronic pain states by reducing changes in protein expression levels but retaining analgesic efficacy.

Impact of angiotensin receptor-neprilysin inhibition (ARNI) in improving ejection fraction and left and right ventricular remodeling in heart failure.

Angiotensin receptor neprilysin inhibitors (ARNI), a new therapeutic class of agents acting on the renin angiotensin aldosterone system (RAAS) and neutral endopeptidase system has been developed in treatment of ventricular remodeling and has attracted considerable attention. The first in class is LCZ696, which is a molecule that combines Valsartan (ARB) and Sacubitril (neprilysin inhibitor) within a single substance. Sacubitril-Valsartan is the first angiotensin receptor enkephalin inhibitors (ARNI), which can block angiotensin II type 1 receptor (AT1R) while inhibiting enkephalin (NEP) and effectively reverse ventricular remodeling in heart failure patients. It has been recommended by the European and American authoritative guidelines on heart failure as Class I for the treatment of chronic heart failure particularly as intensive care medicine. Sacubitril-Valsartan demonstrated significant effects in improving left ventricular performance and remodeling in patients with heart failure with reduced ejection fraction. Sacubitril acts on increased levels of circulating natriuretic peptides by preventing their enzymatic breakdown and Valsartan, which acts to lessen the effects of the RAAS. However, not more research has been done on its effects on the right ventricle remodeling. This review aimed to assess the impact of angiotensin receptor neprilysin inhibitors on left and right ventricular remodeling in heart failure patients.