DUSP22 Ameliorates Endothelial-to-Mesenchymal Transition in HUVECs through Smad2/3 and MAPK Signaling Pathways.

Endothelial-to-mesenchymal transition (EndMT) is the process by which endothelial cells lose their endothelial properties and acquire mesenchymal characteristics. Dual-specific protein phosphatase 22 (DUSP22) inactivates various protein kinases and transcription factors by dephosphorylating serine/threonine residues: hence, it plays a key role in many diseases. The aim of this study was to explore the functional role of DUSP22 in EndMT. In the transforming growth factor-ß-induced EndMT model in human umbilical vein endothelial cells (HUVECs), we observed a downregulation of DUSP22 expression. This DUSP22 deficiency could aggravate EndMT. Conversely, the overexpression of DUSP22 could ameliorate EndMT. We used signaling pathway inhibitors to verify our results and found that DUSP22 could regulate EndMT through the smad2/3 and the mitogen-activated protein kinase (MAPK) signaling pathways. In summary, DUSP22 ameliorates EndMT in HUVECs in vitro through the smad2/3 and MAPK signaling pathways.

Endothelial POFUT1 controls injury-induced liver fibrosis by repressing fibrinogen synthesis.

BACKGROUND & AIMS: NOTCH signaling in liver sinusoidal endothelial cells (LSECs) regulates liver fibrosis, a pathological feature of chronic liver diseases. POFUT1 is an essential regulator of NOTCH signaling. Here, we investigated the role of LSEC-expressed POFUT1 in liver fibrosis. METHODS: Endothelial-specific Pofut1 knockout mice were generated and experimental liver fibrosis was induced by chronic carbon tetrachloride exposure or common bile duct ligation. Liver samples were assessed by ELISA, histology, electron microscopy, immunostaining and RNA in situ hybridization. LSECs and hepatic stellate cells (HSCs) were isolated for gene expression analysis by RNA sequencing, qPCR, and western blotting. Signaling crosstalk between LSECs and HSCs was investigated by treating HSCs with supernatant from LSEC cultures. Liver single-cell RNA sequencing datasets from patients with cirrhosis and healthy individuals were analyzed to evaluate the clinical relevance of gene expression changes observed in mouse studies. RESULTS: POFUT1 loss promoted injury-induced LSEC capillarization and HSC activation, leading to aggravated liver fibrosis. RNA sequencing analysis revealed that POFUT1 deficiency upregulated fibrinogen expression in LSECs. Consistently, fibrinogen was elevated in LSECs of patients with cirrhosis. HSCs treated with supernatant from LSECs of Pofut1 null mice showed exacerbated activation compared to those treated with supernatant from control LSECs, and this effect was attenuated by knockdown of fibrinogen or by pharmacological inhibition of fibrinogen receptor signaling, altogether suggesting that LSEC-derived fibrinogen induced the activation of HSCs. Mechanistically, POFUT1 loss augmented fibrinogen expression by enhancing NOTCH/HES1/STAT3 signaling. CONCLUSIONS: Endothelial POFUT1 prevents injury-induced liver fibrosis by repressing the expression of fibrinogen, which functions as a profibrotic paracrine signal to activate HSCs. Therapies targeting the POFUT1/fibrinogen axis offer a promising strategy for the prevention and treatment of fibrotic liver diseases. IMPACT AND IMPLICATIONS: Paracrine signals produced by liver vasculature play a major role in the development of liver fibrosis, which is a pathological hallmark of most liver diseases. Identifying those paracrine signals is clinically relevant in that they may serve as therapeutic targets. In this study, we discovered that genetic deletion of Pofut1 aggravated experimental liver fibrosis in mouse models. Moreover, fibrinogen was identified as a downstream target repressed by Pofut1 in liver endothelial cells and functioned as a novel paracrine signal that drove liver fibrosis. In addition, fibrinogen was found to be relevant to cirrhosis and may serve as a potential therapeutic target for this devastating human disease.

Paternal preconception alcohol consumption increased Angiotensin II-mediated vasoconstriction in male offspring cerebral arteries via oxidative stress-AT1R pathway.

Alcohol consumption is popular worldwidely and closely associated with cardiovascular diseases. Influences of paternal preconception alcohol consumption on offspring cerebral arteries are largely unknown. Male rats were randomly given alcohol or water before being mated with alcohol-naive females to produce alcohol- and control-sired offspring. Middle cerebral artery (MCA) was tested with a Danish Myo Technology wire myograph, patch-clamp, IONOPTIX, immunofluorescence and quantitative PCR. Alcohol consumption enhanced angiotensin II (AngII)-mediated constriction in male offspring MCA mainly via AT1R. PD123,319 only augmented AngII-induced constriction in control offspring. AngII and Bay K8644 induced stronger intracellular calcium transient in vascular smooth muscle cells (VSMCs) from MCA of alcohol offspring. L-type voltage-dependent calcium channel (L-Ca2+ ) current at baseline and after AngII-stimulation was higher in VSMCs. Influence of large-conductance calcium-activated potassium channel (BKC a ) was lower. Caffeine induced stronger constriction and intracellular calcium release in alcohol offspring. Superoxide anion was higher in alcohol MCA than control. Tempol and thenoyltrifluoroacetone alleviated AngII-mediated contractions, while inhibition was significantly higher in alcohol group. The mitochondria were swollen in alcohol MCA. Despite lower Kcnma1 and Prkce expression, many genes expressions were higher in alcohol group. Hypoxia induced reactive oxygen species production and increased AT1R expression in control MCA and rat aorta smooth muscle cell line. In conclusion, this study firstly demonstrated paternal preconception alcohol potentiated AngII-mediated vasoconstriction in offspring MCA via ROS-AT1R. Alcohol consumption increased intracellular calcium via L-Ca2+ channel and endoplasmic reticulum and decreased BKCa function. The present study provided new information for male reproductive health and developmental origin of cerebrovascular diseases.

Epigenetic repression of Cend1 by lysine-specific demethylase 1 is essential for murine heart development.

Epigenetic regulation of heart development remains incompletely understood. Here we show that LSD1, a histone demethylase, plays a crucial role in regulating cardiomyocyte proliferation during heart development. Cardiomyocyte-specific deletion of Lsd1 in mice inhibited cardiomyocyte proliferation, causing severe growth defect of embryonic and neonatal heart. In vivo RNA-seq and in vitro functional studies identified Cend1 as a target suppressed by LSD1. Lsd1 loss resulted in elevated Cend1 transcription associated with increased active histone mark H3K4me2 at Cend1 promoter. Cend1 knockdown relieved the cell-cycle arrest and proliferation defect caused by LSD1 inhibition in primary rat cardiomyocytes. Moreover, genetic deletion of Cend1 rescued cardiomyocyte proliferation defect and embryonic lethality in Lsd1 null embryos. Consistently, LSD1 promoted the cell cycle of cardiomyocytes derived from human-induced pluripotent stem cells by repressing CEND1. Together, these findings reveal an epigenetic regulatory mechanism involving the LSD1-CEND1 axis that controls cardiomyocyte proliferation essential for murine heart development.

Applied anatomy of female pelvic plexus for nerve-sparing radical hysterectomy(NSRH).

BACKGROUND: Nerve-sparing radical hysterectomy(NSRH)has the advantage of reducing postoperative complications and improving postoperative quality of life. The separation and protection of the pelvic plexus in NSRH is extremely important and challenging. METHODS: 24 female cadaveric hemipelves were dissected. Morphologic patterns and compositions of pelvic plexus as well as relationship of pelvic plexus to the surrounding structures were observed and documented. RESULTS: Two patterns of superior hypogastric plexus were observed, including fenestrated and cord-like shape. The origin of bilateral hypogastric nerves were inferiorly to upper margin of promontory about 1.6 ± 0.1 cm and parallel to the ureter in front of the sacrum. Pelvic splanchnic nerves(PSN)from the second sacral nerve, the third sacral nerve and the forth sacral nerve were observed combing with the hypogastric nerves within the lateral rectal ligament. The sacral sympathetic trunk can be identified anteriorly or medially to the anterior sacral foramen. We identified the boundaries of pelvic plexus as following: the upper margin is formed by the PSNs from the third sacral nerve, posterior margin by inferior rectal artery, and anteriorly by vesical venous plexus. The uterine branches from pelvic plexus were observed accompanying with uterine artery, while other branches were inferiorly to the artery. The PSNs were located beneath the deep uterine veins within the cardinal ligament. The upper margin of pelvic plexus was observed directly approach to urinary bladder within the vesico-vaginal ligament as a single trunk accompanying with ureter, between the middle and inferior vesical veins. CONCLUSIONS: Our study clarified the intricate arrangement, distribution and relationship of female pelvic plexus and the related structures to provide reference index for NSRH application. The innervation patterns of bladder and uterine were clarified, and by tracing these visceral branches of pelvic plexus, we suggest several new important land markers for NSRH.

S1P-S1PR3-RAS promotes the progression of S1PR3hi TAL1+ T-cell acute lymphoblastic leukemia that can be effectively inhibited by an S1PR3 antagonist.

TAL1+ T-cell acute lymphoblastic leukemia (T-ALL) is a distinct subtype of leukemia with poor outcomes. Through the cooperation of co-activators, including RUNX1, GATA3, and MYB, the TAL1 oncoprotein extends the immature thymocytes with autonomy and plays an important role in the development of T-ALL. However, this process is not yet well understood. Here, by investigating the transcriptome and prognosis of T-ALL from multiple cohorts, we found that S1PR3 was highly expressed in a subset of TAL1+ T-ALL (S1PR3hi TAL1+ T-ALL), which showed poor outcomes. Through pharmacological and genetic methods, we identified a specific survival-supporting role of S1P-S1PR3 in TAL1+ T-ALL cells. In T-ALL cells, TAL1-RUNX1 up-regulated the expression of S1PR3 by binding to the enhancer region of S1PR3 gene. With hyperactivated S1P-S1PR3, T-ALL cells grew rapidly, partly by activating the KRAS signal. Finally, we assessed S1PR3 inhibitor TY-52156 in T-ALL patient-derived xenografts (PDXs) mouse model. We found that TY-52156 attenuated leukemia progression efficiently and extended the lifespan of S1PR3hi TAL1+ T-ALL xenografts. Our findings demonstrate that S1PR3 plays an important oncogenic role in S1PR3hi TAL1+ T-ALL and may serve as a promising therapeutic target.

Comparative study of pancreatic vessels and mesopancreas of rhesus monkeys and humans.

Introduction: With the introduction of the concept of mesopancreas defining the perineural structures that includes neurovascular bundle and lymph nodes extending from the posterior surface of the pancreatic head to behind the mesenteric vessels,Total Mesopancreas Excision (TMpE) based on this theory has facilitated the development of pancreatic cancer surgery in clinical practice in recent years. However, the existence of so called mesopancreas in the human body is still in debate and the comparative study of mesopancreas of rhesus monkey and human have not been well investigated. Purpose: The aim of our study is to compare the pancreatic vessels and fascia of human and rhesus monkeys in anatomical and embryological perspectives and to support the utilization of rhesus monkey as animal model. Methods: In this study, 20 rhesus monkey cadavers were dissected and their mesopancreas location, relationships and arterial distribution were analyzed. We compared the location and developmental patterns of mesopancreas in macaques and humans. Results: The results showed that the distribution of pancreatic arteries in rhesus monkeys was the same as that in humans, which is consistent with phylogenetic similarities. However, the morphological features of the mesopancreas and greater omentum is anatomically different from that of humans, including (1) the greater omentum is not connected to the transverse colon in monkeys. (2) The presence of the dorsal mesopancreas of the rhesus monkey suggests that it be an intraperitoneal organ. Comparative anatomical studies of mesopancreas and arteries in macaques and humans showed characteristic patterns of mesopancreas and similarities in pancreatic artery development in nonhuman primates, consistent with phylogenetic differentiation.

In vitro fertilization with frozen embryo transfer increased histamine-mediated contractile sensitivity via PKCß in human umbilical vein.

OBJECTIVE: In vitro fertilization-embryo transfer (IVF-ET) technologies (especially frozen ET) have been widely used, which might affect maternal and fetal health. Information regarding influence of IVF-ET on the vasoconstriction of human umbilical vein (HUV) is limited. This study determined effects of frozen ET on histamine-mediated vascular responses in HUV and related mechanisms. METHODS AND RESULTS: HUVs were collected from frozen ET conceived pregnancy and spontaneously conceived pregnancy (control). Histamine concentration in umbilical plasma was higher in frozen ET group than the control. Histamine-mediated contractile response curve was left-shifted in the frozen ET group when comparing with the control. In isolated HUV rings, H1R showed a critical role in regulating vascular constriction, while H2R played little roles in regulating vessel tone. Iberiotoxin and 4-aminopyridine didn't significantly change histamine-mediated constriction in HUVs. Histamine-induced vasoconstrictions were significantly decreased by nifedipine, KN93, or GF109203X, while the inhibitory effects were significantly greater in the frozen ET group in comparison to the control. The constrictions by Bay K8644, phenylephrine, or PDBu were stronger in frozen ET, respectively. There was a decrease in the protein expressions of H1R and H2R, an increase in protein expressions of BKCaα and PKCß. CONCLUSIONS: Histamine-induced constriction in HUV was mainly via H1R. The increased sensitivity to histamine in HUV following frozen ET cycles were linked to the enhanced PKCß protein expression and function. The new data and findings in this study provide important insight into influences of frozen ET on fetal vessel development and potential influence in long-term.

Controllable Preparation of Eucommia Wood-Derived Mesoporous Activated Carbon as Electrode Materials for Supercapacitors.

Activated carbons (ACs) for supercapacitors were synthesized from Eucommia ulmoides Oliver (EUO) wood by H3PO4 with systemic activation processes. The target structure of ACs could be prepared by adjusting the technological parameters. As the H3PO4 concentration was 25%, the mass ratio of feedstocks to activator was 1:4, the activation time was 6 h, and the activation temperature was 400 °C, the obtained AC revealed a high specific surface area (2033.87 m2·g-1) and well-developed mesoporous (the rate of mesoporous was 96.4%) with the best economic feasibility. Besides, it possessed excellent electrochemical performance: the maximum specific capacitance reached up to 252 F·g-1, the charging and discharging period was 3098.2 s at 0.2 A·g-1, and the retention rate of specific capacitance reached 92.3% after 10,000 cycles. This low temperature and convenience technology provide a valuable reference for synthesizing the EUO-based ACs, making high-value utilization on the EUO branches, and owning a broad application prospect in supercapacitors.

[The Effect of KRAS on Proliferation and Apoptosis of T-ALL Cell Lines].

OBJECTIVE: To investigate the function of RAS protein on the progression of the T-ALL cell lines in vitro. METHODS: The DNA of the T-ALL cells was purified then amplified the coding regions of three RAS genes (KRAS, NRAS, HRAS) by PCR reaction. After T-A cloning, the coding regions of KRAS, NRAS and HRAS were sequenced by Sanger Sequencing. The siRNA oligonucleotides were cloned into the pSEH-361 vector, which were then packaged into retroviral together with pAMPHO and pVSVG in the HEK-293T cells. The T-ALL cells were infected with the retrovirus. The gene expressions were detected by qRT-PCR and Western blot. The T-ALL cells were stained with Annexin V-PE/7-AAD and the apoptotic cells were detected by flow cytometry. The T-ALL cells were stained with Hoechst 33258, and the cell cycle distribution was determined by flow cytometry. The expression of cleaved-Caspase 3 was stained with antibody and observed with fluorescence microscope. RESULTS: For RAS genes, beside the Loucy and the P12-ICH cells harbored KRAS c.6187G>A (p.KRASG12D) homozygous mutant, no missense mutation of RAS was found in other T-ALL cells genome. The pan RAS inhibitor compound 3144 showed toxicity to all tested T-ALL cells, except PEER (IC50=47.916 µmol/L). Similarly, Tipifarnib induced apoptosis of multiple T-ALL cell lines except for the PEER cells (IC50=94.2265 µmol/L). After KRAS knock-down, the T-ALL cells showed significant apoptosis and an arrested cell cycle. CONCLUSION: The KRAS protein is vital for the progression of the T-ALL cells in vitro, it is a potential therapeutic target for T-ALL patients.

Sex Differences at Early Old Stage in Glycolipid Metabolism and Fatty Liver in Offspring Prenatally Exposed to Chinese Great Famine.

Background: About 50 years ago, Chinese Great Famine (CGF) affected the entire population in China, and its long-term influence on the offspring has attracted significant attention for research. However, information on possible metabolic differences between sexes is limited. This study explored whether there might be sex differences in the risks of development of glucolipid metabolic dysfunction and fatty liver following prenatal exposure to CGF. Materials and Methods: There were 11,417 subjects around 55 years of age (6,661 women and 4,756 men). They were divided as the exposed group in which the fetal stage was in CGF, and the unexposed group included those born after CGF. Analysis focused on comparisons between sexes. Results: Compared to the unexposed group, the BMI and triglyceride (P < 0.05) in men were higher in exposed group, while waist circumference and blood sugar (P < 0.05) in the exposed women were significantly higher. With the ages being properly balanced, the risks of glycolipid metabolic dysfunction were significantly higher in both men and women in the exposed than in the unexposed group (P < 0.001). Prenatal exposure to CGF significantly increased risks of abnormal BMI (P < 0.001, 95% CI: 2.305-2.93), blood sugar (P < 0.05, 95% CI: 1.050-1.401), triglycerides (P < 0.05, 95% CI: 1.006-1.245), and fatty liver (P < 0.001, 95% CI: 1.121-1.390) in men, and increased risks of abnormal blood sugar (P < 0.05, 95% CI: 1.024-1.689) and positive urine sugar (P < 0.05, 95% CI: 1.062-6.211) in women. Height and body weight were either the same or higher in the exposed subjects compared with the unexposed ones, regardless of sexes. Conclusion: This study is the first to identify sex differences in the long-term effects of CGF on metabolism and fatty liver. Importance of the findings include the benefits of prescribing medicine for the early prevention of certain diseases for each sex before aging based on the differences revealed. This study also shows "catch-up growth" in the offspring prenatally exposed to CGF as possible mechanisms underlying the long-term effects.

[Application of Flow Cytometry Combined Fluorescence in Situ Hybridization to Indentify the Lymphocyte Subtypies with Epstein-Barr Virus Infection].

OBJECTIVE: To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization (Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients' peripheral blood sample. METHODS: Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children's Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope. RESULTS: It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV+ cells from cell lines and blood samples of patients were identified successfully. CONCLUSION: A Flow-FISH technology is established, which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV+ related proliferative diseases.

In utero hypoxia attenuated acetylcholine-mediated vasodilatation via CHRM3/p-NOS3 in fetal sheep MCA: role of ROS/ERK1/2.

Hypoxia can lead to adult middle cerebral artery (MCA) dysfunction and increase the risk of cerebrovascular diseases. It is largely unknown whether intrauterine hypoxia affects fetal MCA vasodilatation. This study investigated the effects and mechanisms of intrauterine hypoxia on fetal MCA vasodilatation. Near-term fetal sheep were exposed to intrauterine hypoxia. Human umbilical vein endothelial cells (HUVECs) were exposed to hypoxia in cellular experiments. Vascular tone measurement, molecular analysis, and transmission electron microscope (TEM) were utilized to determine vascular functions, tissue anatomy, and molecular pathways in fetal MCA. In fetal MCA, acetylcholine (ACh) induced reliable relaxation, which was markedly attenuated by intrauterine hypoxia. Atropine, P-F-HHSiD, L-NAME, and u0126 blocked most ACh-mediated dilation, while AF-DX 116 and tropicamide partially inhibited the dilation. Indomethacin and SB203580 did not significantly change ACh-mediated dilation. Tempol and PS-341 could restore the attenuated ACh-mediated vasodilatation following intrauterine hypoxia. The mRNA expression levels of CHRM2 and CHRM3 and the protein levels of CHRM3, p-NOS3, SOD2, ERK1/2, p-ERK1/2, MAPK14, and p-MAPK14 were significantly reduced by intrauterine hypoxia. The dihydroethidium assay showed that the production of ROS was increased under intrauterine hypoxia. TEM analysis revealed endothelial cells damaged by intrauterine hypoxia. In HUVECs, hypoxia increased ROS formation and decreased the expression of CHRM3, p-NOS3, SOD1, SOD2, SOD3, ERK1/2, p-ERK1/2, and p-MAPK14, while tempol and PS-341 potentiated p-NOS3 protein expression. In conclusion, in utero hypoxia reduced ACh-mediated vasodilatation in ovine MCA predominantly via decreased CHRM3 and p-NOS3, and the decreased NOS3 bioactivities might be attributed to ROS and ERK1/2.

The tethered peptide activation mechanism of adhesion GPCRs.

Adhesion G-protein-coupled receptors (aGPCRs) are characterized by the presence of auto-proteolysing extracellular regions that are involved in cell-cell and cell-extracellular matrix interactions1. Self cleavage within the aGPCR auto-proteolysis-inducing (GAIN) domain produces two protomers-N-terminal and C-terminal fragments-that remain non-covalently attached after receptors reach the cell surface1. Upon dissociation of the N-terminal fragment, the C-terminus of the GAIN domain acts as a tethered agonist (TA) peptide to activate the seven-transmembrane domain with a mechanism that has been poorly understood2-5. Here we provide cryo-electron microscopy snapshots of two distinct members of the aGPCR family, GPR56 (also known as ADGRG1) and latrophilin 3 (LPHN3 (also known as ADGRL3)). Low-resolution maps of the receptors in their N-terminal fragment-bound state indicate that the GAIN domain projects flexibly towards the extracellular space, keeping the encrypted TA peptide away from the seven-transmembrane domain. High-resolution structures of GPR56 and LPHN3 in their active, G-protein-coupled states, reveal that after dissociation of the extracellular region, the decrypted TA peptides engage the seven-transmembrane domain core with a notable conservation of interactions that also involve extracellular loop 2. TA binding stabilizes breaks in the middle of transmembrane helices 6 and 7 that facilitate aGPCR coupling and activation of heterotrimeric G proteins. Collectively, these results enable us to propose a general model for aGPCR activation.

MiR-652-5p elevated glycolysis level by targeting TIGAR in T-cell acute lymphoblastic leukemia.

The effect of glycolysis remains largely elusive in acute T lymphoblastic leukemia (T-ALL). Increasing evidence has indicated that the dysregulation of miRNAs is involved in glycolysis, by targeting the genes coding glycolysis rate-limiting enzymes. In our previous studies, we found that overexpression of the ARRB1-derived miR-223 sponge repressed T-ALL progress and reduced the expression of miR-652-5p. However, little is known about miR-652-5p on T-ALL. Here, we showed that impaired miR-652-5p expression inhibited growth, promoted apoptosis of T-ALL cells in vitro and prolonged overall survival (OS) in vivo. Based on the GO enrichment of miR-652-5p target genes, we uncovered that impaired miR-652-5p decreased glycolysis, including reduced the lactate, pyruvate, ATP level and the total extracellular acidification rate (ECAR), elevated oxygen consumption rate (OCR) in T-ALL cell lines. Mechanically, miR-652-5p targeted the 3'UTR of Tigar mRNA and inhibited its expression. Furthermore, the alteration of glycosis level was attributed to Tigar overexpression, consistent with the effect of impaired miR-652-5p. Additionally, Tigar suppressed the expression of PFKFB3, a glycolysis rate-limiting enzyme, in vivo and in vitro. Taken together, our results demonstrate that impaired miR-652-5p/Tigar axis could repress glycolysis, thus to slow growth of T-ALL cells, which support miR-652-5p as a novel potential drug target for T-ALL therapeutics.

Luteolin increases susceptibility to macrolides by inhibiting MsrA efflux pump in Trueperella pyogenes.

Trueperella pyogenes (T. pyogenes) is an opportunistic pathogen associated with a variety of diseases in many domestic animals. Therapeutic treatment options for T. pyogenes infections are becoming limited due to antimicrobial resistance, in which efflux pumps play an important role. This study aims to evaluate the inhibitory activity of luteolin, a natural flavonoid, on the MsrA efflux pump and investigate its mechanism. The results of antimicrobial susceptibility testing indicated that the susceptibility of msrA-positive T. pyogenes isolates to six macrolides increased after luteolin treatment, while the susceptibility of msrA-negative isolates showed no change after luteolin treatment. It is suspected that luteolin may increase the susceptibility of T. pyogenes isolates by inhibiting MsrA activity. After 1/2 MIC luteolin treatment for 36 h, the transcription level of the msrA gene and the expression level of the MsrA protein decreased by 55.0-97.7% and 36.5-71.5%, respectively. The results of an affinity test showed that the equilibrium dissociation constant (KD) of luteolin and MsrA was 6.462 × 10-5 M, and hydrogen bonding was predominant in the interaction of luteolin and MsrA. Luteolin may inhibit the ATPase activity of the MsrA protein, resulting in its lack of an energy source. The current study illustrates the effect of luteolin on MsrA in T. pyogenes isolates and provides insight into the development of luteolin as an innovative agent in combating infections caused by antimicrobial-resistant bacteria.

Identification of a Novel Delta Opioid Receptor Agonist Chemotype with Potential Negative Allosteric Modulator Capabilities.

The δ-opioid receptor (δOR) holds great potential as a therapeutic target. Yet, clinical drug development, which has focused on δOR agonists that mimic the potent and selective tool compound SNC80 have largely failed. It has increasingly become apparent that the SNC80 scaffold carries with it potent and efficacious ß-arrestin recruitment. Here, we screened a relatively small (5120 molecules) physical drug library to identify δOR agonists that underrecruit ß-arrestin, as it has been suggested that compounds that efficaciously recruit ß-arrestin are proconvulsant. The screen identified a hit compound and further characterization using cellular binding and signaling assays revealed that this molecule (R995045, compound 1) exhibited ten-fold selectivity over µ- and κ-opioid receptors. Compound 1 represents a novel chemotype at the δOR. A subsequent characterization of fourteen analogs of compound 1, however did not identify a more potent δOR agonist. Computational modeling and in vitro characterization of compound 1 in the presence of the endogenous agonist leu-enkephalin suggest compound 1 may also bind allosterically and negatively modulate the potency of Leu-enkephalin to inhibit cAMP, acting as a 'NAM-agonist' in this assay. The potential physiological utility of such a class of compounds will need to be assessed in future in vivo assays.

Modulating ß-arrestin 2 recruitment at the δ- and µ-opioid receptors using peptidomimetic ligands.

µ-Opioid receptor agonists provide potent and effective acute analgesia; however, their therapeutic window narrows considerably upon repeated administration, such as required for treating chronic pain. In contrast, bifunctional µ/δ opioid agonists, such as the endogenous enkephalins, have potential for treating both acute and chronic pain. However, enkephalins recruit ß-arrestins, which correlate with certain adverse effects at µ- and δ-opioid receptors. Herein, we identify the C-terminus of Tyr-ψ[(Z)CF[double bond, length as m-dash]CH]-Gly-Leu-enkephalin, a stable enkephalin derivative, as a key site to regulate bias of both δ- and µ-opioid receptors. Using in vitro assays, substitution of the Leu5 carboxylate with amides (NHEt, NMe2, NCyPr) reduced ß-arrestin recruitment efficacy through both the δ-opioid and µ-opioid, while retaining affinity and cAMP potency. For this series, computational studies suggest key ligand-receptor interactions that might influence bias. These findings should enable the discovery of a range of tool compounds with previously unexplored biased µ/δ opioid agonist pharmacological profiles.

Prenatal hypoxia induced ETBR activation and abnormal ROS signalling in pulmonary artery cells of rat offspring.

Pulmonary arterial hypertension is a progressive disorder characterized by remodeling and increased small pulmonary arteries resistance. Endothelin-1 (ET-1) was related to PAH and ET-1 receptors were up-regulated selectively in the lung when exposed to toxic factor hypoxia. However, the role of ET-1 signaling in the pathogenesis of prenatal hypoxia-induced pulmonary abnormalities remains to be elucidated. Pregnant rats were divided into prenatal hypoxia (10.5 % O2 from gestational day 4-21) and control group. Their three-month-old offspring male rats were tested for vascular functions and molecular analysis, DNA methylation was assessed for cellular hypoxia. Functional testing showed that ET-1-mediated vasoconstriction was enhanced, and the expressions of endothelin A receptor/B receptor (ETAR/ETBR), inositol 1,4,5-trisphosphate receptor, type 1, and the sensitivity of calcium channels were increased in the small pulmonary arteries following prenatal hypoxia. q-PCR and DHE staining showed that the expressions of NADPH oxidase 1/4 (Nox1/4) were up-regulated, along with the increased production of superoxide anion. Furthermore, superoxide anion promoted ET-1-mediated pulmonary artery contraction. In the pulmonary artery smooth muscle cell experiments, q-PCR, Western Blot, CCK8 and DHE staining showed that the expressions of ETBR, Nox1/4, and superoxide anion were increased by hypoxia, along with promoted cell proliferation. 2,2,6,6-Tetramethyl-1-piperidinyloxy reversed hypoxia-induced cell proliferation. ETBR antagonist BQ788 inhibited hypoxia-increased expressions of Nox1/4, superoxide anion production, and proliferation of cells. Moreover, methylation analysis indicated that hypoxia decreased the methylation levels of the ETBR promoter in the pulmonary artery smooth muscle cells. The results indicated that prenatal toxic factor hypoxia resulted in abnormal ETBR activation, which enhanced ET-1-mediated vasoconstriction of pulmonary arteries and pulmonary artery smooth muscle cell proliferation through ETBR/Nox1/4-derived ROS pathway.

[Induction of mouse T lymphocyte differentiation in vitro by thymic organoids].

Objective To induce the differentiation of hematopoietic stem progenitor cells (HSPCs) into T cell by creating thymic organoids and simulating the three-dimensional structure of thymus tissue in vitro. Methods The retroviral vector expressing the DLL1 and Green fluorescent protein (GFP) was constructed, and the OP9-DLL1 cell line was established in OP9 cells with the aid of retroviral infection. The mRNA and protein level of DLL1 in OP9-DLL1 cells was detected by quantitative real-time PCR and Western blot respectively. Immunofluorescence assay was used to detect the DLL1 protein expression and distribution in OP9-DLL1 cells. HSPCs were extracted from E13.5 fetal liver and bone marrow of C57BL/6 mouse, and mixed with OP9-DLL1 cells in an appropriate ratio respectively, then compacted by centrifuging and cultured at the air-liquid interface in medium. Fluorescence microscope was used to observe the growth of thymic organoids. Flow cytometry was used to detect the expression of T cell surface markers, including CD3, CD4, CD8, CD25, CD44, CD45, CD117 and TCRß. Immunofluorescence cytochemical staining was used to observe the distribution of hematopoietic cells in thymic organoids. Results The retroviral vector expressing DLL1 and GFP was successfully constructed. The OP9 cells were infected with the retrovirus constructed, and OP9-DLL1 cells were obtained by GFP screening. The mRNA and protein level of DLL1 in OP9-DLL1 cells significantly increased, and DLL1 was expressed in the membrane OP9-DLL1 cells. During the 40 days of culture, the thymic organoids remained in good condition and increased gradually in volume. The thymic organoids induced programmed differentiation of T cells, and differentiation of HSPCs into CD3+ T cells. Conclusion OP9-DLL1 cells can be used to construct thymic organoids and to induce differentiation of HSPCs into T cells in vitro.