O-Fucose and Fringe-modified NOTCH1 extracellular domain fragments as decoys to release niche-lodged hematopoietic progenitor cells.

Successful hematopoietic progenitor cell (HPC) transplant therapy is improved by mobilizing HPCs from the bone marrow niche in donors. Notch receptor-ligand interactions are known to retain HPCs in the bone marrow, and neutralizing antibodies against Notch ligands, Jagged-1 or Delta-like ligand (DLL4), or NOTCH2 receptor potentiates HPC mobilization. Notch-ligand interactions are dependent on posttranslational modification of Notch receptors with O-fucose and are modulated by Fringe-mediated extension of O-fucose moieties. We previously reported that O-fucosylglycans on Notch are required for Notch receptor-ligand engagement controlling hematopoietic stem cell quiescence and retention in the marrow niche. Here, we generated recombinant fragments of NOTCH1 or NOTCH2 extracellular domain carrying the core ligand-binding regions (EGF11-13) either as unmodified forms or as O-fucosylglycan-modified forms. We found that the addition of O-fucose monosaccharide or the Fringe-extended forms of O-fucose to EGF11-13 showed substantial increases in binding to DLL4. Furthermore, the O-fucose and Fringe-extended NOTCH1 EGF11-13 protein displayed much stronger binding to DLL4 than the NOTCH2 counterpart. When assessed in an in vitro 3D osteoblastic niche model, we showed that the Fringe-extended NOTCH1 EGF11-13 fragment effectively released lodged HPC cells with a higher potency than the NOTCH2 blocking antibody. We concluded that O-fucose and Fringe-modified NOTCH1 EGF11-13 protein can be utilized as effective decoys for stem cell niche localized ligands to potentiate HPC egress and improve HPC collection for hematopoietic cell therapy.

Dipeptidyl peptidase-4 inhibitor MK-626 restores insulin secretion through enhancing autophagy in high fat diet-induced mice.

Autophagy is cellular machinery for maintenance of ß-cell function and mass. The current study aimed to investigate the regulatory effects of MK-626, a dipeptidyl peptidase-4 inhibitor, on insulin secretion through the activation of autophagy in high fat diet-induced obese mice. C57BL/6 mice were fed with a rodent diet containing 45 kcal% fat for 16 weeks to induce obesity and then were received either vehicle or MK-626 (3 mg/kg/day) orally during the final 4 weeks. Mouse islets were isolated. Phosphorylation of serine/threonine-protein kinase mTOR and levels of light chain 3B I (LC3B I), LC3B II, sequestosome-1 (SQSTM1/p62) and autophagy-related protein-7 (Atg7) were examined by Western blotting. Glucagon like-peptide-1 (GLP-1) level and insulin secretion were measured by ELISA. GLP-1 level in plasma was decreased in obese mice, which was elevated by dipeptidyl peptidase-4 inhibitor MK-626. In the islets of obese mice, phosphorylation of mTOR, ratio of LC3B I and LC3B II, and level of p62 were elevated and the expression of Atg7 and insulin secretion were reduced compared to those of C57BL/6 mice. However, such effects were reversed by MK-626. Autophagy activator rapamycin stimulated insulin secretion in obese mice but autophagy inhibitor chloroquine treatment inhibited insulin secretion in obese mice administrated by MK-626. Furthermore, the beneficial effects of MK-626 were inhibited by GLP-1 receptor antagonist exendin 9-39. The present study reveals the activation of autophagy to mediate the anti-diabetic effect of GLP-1.

Berberine improves endothelial function by inhibiting endoplasmic reticulum stress in the carotid arteries of spontaneously hypertensive rats.

Activation of endoplasmic reticulum (ER) stress in endothelial cells leads to increased oxidative stress and often results in cell death, which has been implicated in hypertension. The present study investigated the effects of berberine, a botanical alkaloid purified from Coptidis rhizoma, on ER stress in spontaneously hypertensive rats (SHRs) and the underling mechanism. Isolated carotid arteries from normotensive WKYs and SHRs were suspended in myograph for isometric force measurement. Protein phosphorylations and expressions were determined by Western blotting. Reactive oxygen species (ROS) level was measured by DHE staining. SHR carotid arteries exhibited exaggerated acetylcholine-triggered endothelium-dependent contractions (EDCs) and elevated ROS accumulation compared with WKY arteries. Moreover, Western blot analysis revealed the reduced AMPK phosphorylation, increased eIF2α phosphorylation, and elevated levels of ATF3, ATF6, XBP1 and COX-2 in SHR carotid arteries while these pathological alterations were reversed by 12 h-incubation with berberine. Furthermore, AMPK inhibitor compound C or dominant negative AMPK adenovirus inhibited the effects of berberine on above-mentioned marker proteins and EDCs. More importantly, ROS scavengers, tempol and tiron plus DETCA, or ER stress inhibitors, 4-PBA and TUCDA normalized the elevated levels of ROS and COX-2 expression, and attenuated EDCs in SHR arteries. Taken together, the present results suggest that berberine reduces EDCs likely through activating AMPK, thus inhibiting ER stress and subsequently scavenging ROS leading to COX-2 down-regulation in SHR carotid arteries. The present study thus provides additional insights into the vascular beneficial effects of berberine in hypertension.

Preservation of nitric oxide-induced relaxation of porcine coronary artery: roles of the dimers of soluble guanylyl cyclase, phosphodiesterase type 5, and cGMP-dependent protein kinase.

Soluble guanylyl cyclase (sGC), phosphodiesterase type 5 (PDE5), and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) are all dimeric. The present study was to determine the role of their dimeric status in nitric oxide-induced vasodilatation. In isolated porcine coronary arteries, after 20 h incubation with serum-free medium, serum-containing medium, or phosphate-buffered saline solution, the protein levels of the dimers of sGC, PDE5, and PKG were diminished while the monomer levels remained unchanged, associated with reduced cGMP elevation in response to DETA NONOate and decreased PDE5 activity; the activity of PKG was not significantly altered. DETA NONOate caused a greater relaxation in arteries incubated for 20 vs. 2 h. The relaxant response was largely abolished by 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, an sGC inhibitor. Zaprinast, a PDE5 inhibitor, had no effect on relaxation caused by DETA NONOate of arteries incubated for 20 h but augmented the response incubated for 2 h. A greater relaxation to 8-bromo-guanosine 3'5'-cyclic monophosphate occurred in arteries incubated for 20 than for 2 h. The protein level of the dimers but not monomers of PDE5 was reduced by dithiothreitol and unaffected by hydrogen peroxide, accompanied with decreased PDE5 activity and reduced response to DETA NONOate. These results demonstrate that the dimeric but not monomeric status of sGC and PDE5 of coronary arteries are closely related to their activities. The preserved vasodilator response after 20 h incubation may result in part from a synchronous reduction of the dimer levels of sGC and PDE5 as well as an augmented response to cGMP.

cIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries.

cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3',5'-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (Po2: 25-30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5'-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr(853), which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK.

Protein kinase Cδ contributes to phenylephrine-mediated contraction in the aortae of high fat diet-induced obese mice.

The down-regulation of α-adrenoceptor-mediated signaling casacade has been implicated in obesity but the underlying mechanism remains largely unknown. The present study investigated whether inositol 1,4,5-trisphosphate (IP3) receptor and protein kinase C (PKC) were involved in the reduction of α1-adrenoceptor agonist phenylephrine-evoked contraction in aortae of high fat diet-induced obese (DIO) mice. C57BL/6 mice were fed with a rodent diet containing 45 kcal% fat for 16 weeks to induce obesity. Isolated mouse aortae were suspended in myograph for isometric force measurement. Protein phosphorylations and expressions were determined by Western blotting. In C57BL/6 mouse aortae, phenylephrine-induced contraction was partially inhibited by either IP3 receptor antagonist heparin or PKC inhibitor GFX, and the combined treatment with heparin and GFX abolished the contraction. Phenylephrine-induced contraction was significantly less in the aortae of DIO mice than those of control mice; only GFX but not heparin attenuated the contraction, indicating a diminishing role of IP3 receptor in DIO mice. Western blotting showed the reduced expression and phosphorylation of IP3 receptor and the down-regulated expression of PKC, PKCß, PKCδ, and PKCζ in DIO mouse aortae. Importantly, PKCδ was more likely to maintain phenylephrine-mediated contraction in DIO mouse aortae because that (1) PKCδ inhibitor rottlerin but not PKCα and PKCß inhibitor Gö6976, PKCß inhibitor hispidin, or PKCζ pseudosubstrate inhibitor attenuated the contraction; and (2) PKCδ phosphorylation was increased but phosphorylations of PKCα, PKCß, and PKCζ were reduced in DIO mouse aortae. The present study thus provides additional insights into the cellular mechanisms responsible for vascular dysfunction in obesity.

Cross regulation between cGMP-dependent protein kinase and Akt in vasodilatation of porcine pulmonary artery.

cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation.

Pd(II) coordination molecule modified g-C3N4 for boosting photocatalytic hydrogen production.

The photocatalytic H2 production activity of polymer carbon nitride (g-C3N4) is limited by the rapid recombination of photoelectron-hole pairs and slow surface reduction dynamic process. Here, a supramolecular complex (named R-TAP-Pd(II)) was fabricated via self-assembly of (R)-N-(1-phenylethyl)-4-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)benzamide (R-TAP) with Pd(II) and used to modify g-C3N4. In the R-TAP-Pd(II)@g-C3N4 composite photocatalyst, the spin polarization of R-TAP-Pd(II) can promote charge transfer and inhibit photogenerated carrier recombination, as confirmed by spectral tests and photoelectrochemical performance tests. Electrochemical tests and in situ X-ray photoelectron spectroscopy (XPS) tests proved that the Pd(II) ion in the R-TAP-Pd(II) molecule can serve as active sites to accelerate H2 production. The R-TAP-Pd(II)@g-C3N4 presented a photocatalytic H2 generation rate of 1085 µmol g-1 h-1 when exposed to visible light, which was a about 278-fold increase compared with g-C3N4. This work finds a new approach to boost the photocatalytic efficiency of g-C3N4 via supramolecular self-assembly.

PANX1-mediated ATP release confers FAM3A's suppression effects on hepatic gluconeogenesis and lipogenesis.

BACKGROUND: Extracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet ß cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed. METHODS: db/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein-protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays. RESULTS: In livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers. CONCLUSIONS: PANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A's suppressive effects on hepatic gluconeogenesis and lipogenesis.

FAM3A maintains metabolic homeostasis by interacting with F1-ATP synthase to regulate the activity and assembly of ATP synthase.

Reduced mitochondrial ATP synthase (ATPS) capacity plays crucial roles in the pathogenesis of metabolic disorders. However, there is currently no effective strategy for synchronously stimulating the expressions of ATPS key subunits to restore its assembly. This study determined the roles of mitochondrial protein FAM3A in regulating the activity and assembly of ATPS in hepatocytes. FAM3A is localized in mitochondrial matrix, where it interacts with F1-ATPS to initially activate ATP synthesis and release, and released ATP further activates P2 receptor-Akt-CREB pathway to induce FOXD3 expression. FOXD3 synchronously stimulates the transcriptions of ATPS key subunits and assembly genes to increase its assembly and capacity, augmenting ATP synthesis and inhibiting ROS production. FAM3A, FOXD3 and ATPS expressions were reduced in livers of diabetic mice and NAFLD patients. FOXD3 expression, ATPS capacity and ATP content were reduced in various tissues of FAM3A-deficient mice with dysregulated glucose and lipid metabolism. Hepatic FOXD3 activation increased ATPS assembly to ameliorate dysregulated glucose and lipid metabolism in obese mice. Hepatic FOXD3 inhibition or knockout reduced ATPS capacity to aggravate HFD-induced hyperglycemia and steatosis. In conclusion, FAM3A is an active ATPS component, and regulates its activity and assembly by activating FOXD3. Activating FAM3A-FOXD3 axis represents a viable strategy for restoring ATPS assembly to treat metabolic disorders.

Hepatocyte-secreted FAM3D ameliorates hepatic steatosis by activating FPR1-hnRNP U-GR-SCAD pathway to enhance lipid oxidation.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide; however, the underlying mechanisms remain poorly understood. FAM3D is a member of the FAM3 family; however, its role in hepatic glycolipid metabolism remains unknown. Serum FAM3D levels are positively correlated with fasting blood glucose levels in patients with diabetes. Hepatocytes express and secrete FAM3D, and its expression is increased in steatotic human and mouse livers. Hepatic FAM3D overexpression ameliorated hyperglycemia and steatosis in obese mice, whereas FAM3D-deficient mice exhibited exaggerated hyperglycemia and steatosis after high-fat diet (HFD)-feeding. In cultured hepatocytes, FAM3D overexpression or recombinant FAM3D protein (rFAM3D) treatment reduced gluconeogenesis and lipid deposition, which were blocked by anti-FAM3D antibodies or inhibition of its receptor, formyl peptide receptor 1 (FPR1). FPR1 overexpression suppressed gluconeogenesis and reduced lipid deposition in wild hepatocytes but not in FAM3D-deficient hepatocytes. The addition of rFAM3D restored FPR1's inhibitory effects on gluconeogenesis and lipid deposition in FAM3D-deficient hepatocytes. Hepatic FPR1 overexpression ameliorated hyperglycemia and steatosis in obese mice. RNA sequencing and DNA pull-down revealed that the FAM3D-FPR1 axis upregulated the expression of heterogeneous nuclear ribonucleoprotein U (hnRNP U), which recruits the glucocorticoid receptor (GR) to the promoter region of the short-chain acyl-CoA dehydrogenase (SCAD) gene, promoting its transcription to enhance lipid oxidation. Moreover, FAM3D-FPR1 axis also activates calmodulin-Akt pathway to suppress gluconeogenesis in hepatocytes. In conclusion, hepatocyte-secreted FAM3D activated the FPR1-hnRNP U-GR-SCAD pathway to enhance lipid oxidation in hepatocytes. Under obesity conditions, increased hepatic FAM3D expression is a compensatory mechanism against dysregulated glucose and lipid metabolism.

IL6 Mediates Suppression of T- and NK-cell Function in EMT-associated TKI-resistant EGFR-mutant NSCLC.

PURPOSE: Patients with advanced non-small cell lung cancer (NSCLC) harboring activating EGFR mutations are initially responsive to tyrosine kinase inhibitors (TKI). However, therapeutic resistance eventually emerges, often via secondary EGFR mutations or EGFR-independent mechanisms such as epithelial-to-mesenchymal transition. Treatment options after EGFR-TKI resistance are limited as anti-PD-1/PD-L1 inhibitors typically display minimal benefit. Given that IL6 is associated with worse outcomes in patients with NSCLC, we investigate whether IL6 in part contributes to this immunosuppressed phenotype. EXPERIMENTAL DESIGN: We utilized a syngeneic genetically engineered mouse model (GEMM) of EGFR-mutant NSCLC to investigate the effects of IL6 on the tumor microenvironment and the combined efficacy of IL6 inhibition and anti-PD-1 therapy. Corresponding in vitro studies used EGFR-mutant human cell lines and clinical specimens. RESULTS: We identified that EGFR-mutant tumors which have oncogene-independent acquired resistance to EGFR-TKIs were more mesenchymal and had markedly enhanced IL6 secretion. In EGFR-mutant GEMMs, IL6 depletion enhanced activation of infiltrating natural killer (NK)- and T-cell subpopulations and decreased immunosuppressive regulatory T and Th17 cell populations. Inhibition of IL6 increased NK- and T cell-mediated killing of human osimertinib-resistant EGFR-mutant NSCLC tumor cells in cell culture. IL6 blockade sensitized EGFR-mutant GEMM tumors to PD-1 inhibitors through an increase in tumor-infiltrating IFNγ+ CD8+ T cells. CONCLUSIONS: These data indicate that IL6 is upregulated in EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance and suppressed T- and NK-cell function. IL6 blockade enhanced antitumor immunity and efficacy of anti-PD-1 therapy warranting future clinical combinatorial investigations.

CD70 is a therapeutic target upregulated in EMT-associated EGFR tyrosine kinase inhibitor resistance.

Effective therapeutic strategies are needed for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations that acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) mediated by epithelial-to-mesenchymal transition (EMT). We investigate cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches and identify CD70 as being highly upregulated in EMT-associated resistance. Moreover, CD70 upregulation is an early event in the evolution of resistance and occurs in drug-tolerant persister cells (DTPCs). CD70 promotes cell survival and invasiveness, and stimulation of CD70 triggers signal transduction pathways known to be re-activated with acquired TKI resistance. Anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting chimeric antigen receptor (CAR) T cell and CAR NK cells show potent activity against EGFR TKI-resistant cells and DTPCs. These results identify CD70 as a therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits clinical investigation.

Heterogeneity in relaxation of different sized porcine coronary arteries to nitrovasodilators: role of PKG and MYPT1.

The present study was to determine the role of the type I isoform of cGMP-dependent protein kinase (PKG I) and its downstream effector myosin phosphatase target subunit 1 (MYPT1) in the responses of different sized coronary arteries to nitrovasodilators. Relaxations of isolated porcine coronary arteries were determined by isometric tension recording technique. Protein levels of PKG I and its effectors were analyzed by Western blotting. The activities of PKG I and MYPT1 were studied by analyzing phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and MYPT1, respectively. Nitroglycerin, DETA NONOate, and 8-Br-cGMP caused greater relaxations in large than in small coronary arteries. Relaxations were attenuated to a greater extent by Rp-8-Br-PET-cGMPS (a PKG inhibitor) in large vs. small arteries. The expressions of PKG I and MYPT1 in large arteries were more abundant than in small arteries. DETA NONOate stimulated phosphorylation of VASP at Ser239 and inhibited phosphorylation of MYPT1 at Thr853 to a greater extent in large than in small arteries. A suppressed phosphorylation of MYPT1 at Thr853 was caused by 8-Br-cGMP in large but not small arteries, which was inhibited by Rp-8-Br-PET-cGMPS. These results suggest that the greater responsiveness of large coronary arteries to nitrovasodilators result in part from greater activities of PKG I and MYPT1. Dysfunction in nitric oxide signaling is implicated in the vulnerability of large coronary arteries to certain disorders such as atherosclerosis and spasm. Augmentation of PKG I-MYPT1 signaling may be of therapeutic benefit for combating these events.

A Gaijin-like miniature inverted repeat transposable element is mobilized in rice during cell differentiation.

BACKGROUND: Miniature inverted repeat transposable element (MITE) is one type of transposable element (TE), which is largely found in eukaryotic genomes and involved in a wide variety of biological events. However, only few MITEs were proved to be currently active and their physiological function remains largely unknown. RESULTS: We found that the amplicon discrepancy of a gene locus LOC_Os01g0420 in different rice cultivar genomes was resulted from the existence of a member of Gaijin-like MITEs (mGing). This result indicated that mGing transposition was occurred at this gene locus. By using a modified transposon display (TD) analysis, the active transpositions of mGing were detected in rice Jiahua No. 1 genome under three conditions: in seedlings germinated from the seeds received a high dose γ-ray irradiation, in plantlets regenerated from anther-derived calli and from scutellum-derived calli, and were confirmed by PCR validation and sequencing. Sequence analysis revealed that single nucleotide polymorphisms (SNPs) or short additional DNA sequences at transposition sites post mGing transposition. It suggested that sequence modification was possibly taken place during mGing transposition. Furthermore, cell re-differentiation experiment showed that active transpositions of both mGing and mPing (another well studied MITE) were identified only in regenerated plantlets. CONCLUSIONS: It is for the first time that mGing active transposition was demonstrated under γ-ray irradiation or in cell re-differentiation process in rice. This newly identified active MITE will provide a foundation for further analysis of the roles of MITEs in biological process.

Inhibition of phosphoinositide 3-kinase potentiates relaxation of porcine coronary arteries induced by nitroglycerin by decreasing phosphodiesterase type 5 activity.

BACKGROUND: Vessel tension can be modulated by phosphoinositide 3-kinase (PI3K) acting on l-type calcium channel, rho kinase and phosphodiesterase (PDE) type 3 in smooth muscle cells. Inhibition of PI3K could increase the relaxation of porcine coronary arteries to nitroglycerin independent of this pathway, and the aim of the present study was therefore to determine the underlying mechanisms. METHODS AND RESULTS: Isolated porcine coronary arteries were dissected from the heart and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The response of these vessels was studied by using the organ chamber technique; the content of cyclic guanosine monophosphate (cGMP) was determined by using enzyme-linked immunosorbent assay kit; and PI3K and Akt activity were determined by measuring the phosphorylation level of their downstream signaling molecule on Western blot. Inhibition of PI3K with 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) potentiated the relaxation of porcine coronary arteries to nitroglycerin and nitric oxide (NO), but not to 8-bromo-guanosine 3'5'-cyclic monophosphate, isoproterenol or (R)-(+)-trans-4-(1-Aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y27632). Increased relaxation induced by LY294002 was eliminated by Akt1/2 kinase inhibitor (Akt-I: 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate) or zaprinast, but was not affected by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, nifedipine or milrinone. Inhibition of Akt caused similar effects as LY294002. Incubation with LY294002 or Akt-I decreased the activity of PI3K and Akt but augmented the elevation of cGMP caused by NO. Enhanced cGMP elevation induced by LY294002 or Akt-I was also eliminated by zaprinast. CONCLUSIONS: PI3K-Akt signaling may affect vascular tone through a stimulatory effect on PDE type 5.

Expression of a Cytochrome P450 Gene from Bermuda Grass Cynodon dactylon in Soybean Confers Tolerance to Multiple Herbicides.

Bermuda grass (Cynodon dactylon) is notoriously difficult to control with some commonly used herbicides. We cloned a cytochrome P450 gene from Bermuda grass, named P450-N-Z1, which was found to confer tolerance to multiple herbicides in transgenic Arabidopsis. These herbicides include: (1) acetolactate synthase (ALS) inhibitor herbicides nicosulfuron and penoxsulam; (2) p-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide mesotrione; (3) synthetic auxin herbicide dicamba; (4) photosynthesis inhibitor bentazon. We further generated transgenic soybean plants expressing P450-N-Z1, and found that these transgenic soybean plants gained robust tolerance to nicosulfuron, flazasulfuron, and 2,4-dichlorophenoxyacetic acid (2,4-D) in greenhouse assays. A field trial demonstrated that transgenic soybean is tolerant to flazasulfuron and 2,4-D at 4-fold and 2-fold the recommended rates, respectively. Furthermore, we also demonstrated that flazasulfuron and dicamba are much more rapidly degraded in vivo in the transgenic soybean than in non-transgenic soybean. Therefore, P450-N-Z1 may be utilized for engineering transgenic crops for herbicide tolerance.

Imipramine activates FAM3A-FOXA2-CPT2 pathway to ameliorate hepatic steatosis.

Mitochondrial FAM3A has been revealed to be a viable target for treating diabetes and nonalcoholic fatty liver disease (NAFLD). However, its distinct mechanism in ameliorating hepatic steatosis remained unrevealed. High-throughput RNA sequencing revealed that carnitine palmityl transferase 2 (CPT2), one of the key enzymes for lipid oxidation, is the downstream molecule of FAM3A signaling pathway in hepatocytes. Intensive study demonstrated that FAM3A-induced ATP release activated P2 receptor to promote the translocation of calmodulin (CaM) from cytoplasm into nucleus, where it functioned as a co-activator of forkhead box protein A2 (FOXA2) to promote the transcription of CPT2, increasing free fatty acid oxidation and reducing lipid deposition in hepatocytes. Furthermore, antidepressant imipramine activated FAM3A-ATP-P2 receptor-CaM-FOXA2-CPT2 pathway to reduce lipid deposition in hepatocytes. In FAM3A-deficient hepatocytes, imipramine failed to activate CaM-FOXA2-CPT2 axis to increase lipid oxidation. Imipramine administration significantly ameliorated hepatic steatosis, hyperglycemia and obesity of obese mice mainly by activating FAM3A-ATP-CaM-FOXA2-CPT2 pathway in liver and thermogenesis in brown adipose tissue (BAT). In FAM3A-deficient mice fed on high-fat-diet, imipramine treatment failed to correct the dysregulated lipid and glucose metabolism, and activate thermogenesis in BAT. In conclusion, imipramine activates FAM3A-ATP-CaM-FOXA2-CPT2 pathway to ameliorate steatosis. For depressive patients complicated with metabolic disorders, imipramine may be recommended in priority as antidepressive drug.

Multi-modal molecular programs regulate melanoma cell state.

Melanoma cells display distinct intrinsic phenotypic states. Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines. We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly conserved within melanoma tumors, are associated with distinct immune features within tumors, and differentially correlate with checkpoint inhibitor and adoptive T cell therapy efficacy. Through integrative analyses we demonstrate highly complex multi-omic regulation of melanoma cell intrinsic programs that provide key insights into the molecular maintenance of phenotypic states. These findings have implications for cancer biology and the identification of new therapeutic strategies. Further, these deeply characterized cell lines will serve as an invaluable resource for future research in the field.

Altered Regulation of HIF-1α in Naive- and Drug-Resistant EGFR-Mutant NSCLC: Implications for a Vascular Endothelial Growth Factor-Dependent Phenotype.

INTRODUCTION: The treatment of patients with EGFR-mutant NSCLC with vascular endothelial growth factor (VEGF) inhibitors in combination with EGFR inhibitors provides a greater benefit than EGFR inhibition alone, suggesting that EGFR mutation status may define a patient subgroup with greater benefit from VEGF blockade. The mechanisms driving this potentially enhanced VEGF dependence are unknown. METHODS: We analyzed the effect of EGFR inhibition on VEGF and HIF-1α in NSCLC models in vitro and in vivo. We determined the efficacy of VEGF inhibition in xenografts and analyzed the impact of acquired EGFR inhibitor resistance on VEGF and HIF-1α. RESULTS: NSCLC cells with EGFR-activating mutations exhibited altered regulation of VEGF compared with EGFR wild-type cells. In EGFR-mutant cells, EGFR, not hypoxia, was the dominant regulator of HIF-1α and VEGF. NSCLC tumor models bearing classical or exon 20 EGFR mutations were more sensitive to VEGF inhibition than EGFR wild-type tumors, and a combination of VEGF and EGFR inhibition delayed tumor progression. In models of acquired EGFR inhibitor resistance, whereas VEGF remained overexpressed, the hypoxia-independent expression of HIF-1α was delinked from EGFR signaling, and EGFR inhibition no longer diminished HIF-1α or VEGF expression. CONCLUSIONS: In EGFR-mutant NSCLC, EGFR signaling is the dominant regulator of HIF-1α and VEGF in a hypoxia-independent manner, hijacking an important cellular response regulating tumor aggressiveness. Cells with acquired EGFR inhibitor resistance retained elevated expression of HIF-1α and VEGF, and the pathways were no longer EGFR-regulated. This supports VEGF targeting in EGFR-mutant tumors in the EGFR inhibitor-naive and refractory settings.