Targeted deletion of RIC8A in mouse neural precursor cells interferes with the development of the brain,eyes, and muscles

Autosomal recessive disorders such as Fukuyama congenital muscular dystrophy, Walker–Warburg syndrome, and the muscle–eye–brain disease are characterized by defects in the development of patient's brain, eyes, and skeletal muscles. These syndromes are accompanied by brain malformations like type II lissencephaly in the cerebral cortex with characteristic overmigrations of neurons through the breaches of the pial basement membrane. The signaling pathways activated by laminin receptors, dystroglycan and integrins, control the integrity of the basement membrane, and their malfunctioning may underlie the pathologies found in the rise of defects reminiscent of these syndromes. Similar defects in corticogenesis and neuromuscular disorders were found in mice when RIC8A was specifically removed from neural precursor cells. RIC8A regulates a subset of G‐protein α subunits and in several model organisms, it has been reported to participate in the control of cell division, signaling, and migration. Here, we studied the role of RIC8A in the development of the brain, muscles, and eyes of the neural precursor‐specific conditional Ric8a knockout mice. The absence of RIC8A severely affected the attachment and positioning of radial glial processes, Cajal‐Retzius’ cells, and the arachnoid trabeculae, and these mice displayed additional defects in the lens, skeletal muscles, and heart development. All the discovered defects might be linked to aberrancies in cell adhesion and migration, suggesting that RIC8A has a crucial role in the regulation of cell–extracellular matrix interactions and that its removal leads to the phenotype characteristic to type II lissencephaly‐associated diseases. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 374–390, 2018     

Hypoxia-Targeting Drug Evofosfamide (TH-302) Enhances Sunitinib Activity in Neuroblastoma Xenograft Models

Antiangiogenic therapy has shown promising results in preclinical and clinical trials. However, tumor cells acquire resistance to this therapy by gaining ability to survive and proliferate under hypoxia induced by antiangiogenic therapy. Combining antiangiogenic therapy with hypoxia-activated prodrugs can overcome this limitation. Here, we have tested the combination of antiangiogenic drug sunitinib in combination with hypoxia-activated prodrug evofosfamide in neuroblastoma. In vitro, neuroblastoma cell line SK-N-BE(2) was 40-folds sensitive to evofosfamide under hypoxia compared to normoxia. In IV metastatic model, evofosfamide significantly increased mice survival compared to the vehicle (P=.02). In SK-N-BE(2) subcutaneous xenograft model, we tested two different treatment regimens using 30 mg/kg sunitinib and 50 mg/kg evofosfamide. Here, sunitinib therapy when started along with evofosfamide treatment showed higher efficacy compared to single agents in subcutaneous SK-N-BE(2) xenograft model, whereas sunitinib when started 7 days after evofosfamide treatment did not have any advantage compared to treatment with either single agent. Immunofluorescence of tumor sections revealed higher number of apoptotic cells and hypoxic areas compared to either single agent when both treatments were started together. Treatment with 80 mg/kg sunitinib with 50 mg/kg evofosfamide was significantly superior to single agents in both xenograft and metastatic models. This study confirms the preclinical efficacy of sunitinib and evofosfamide in murine models of aggressive neuroblastoma. Sunitinib enhances the efficacy of evofosfamide by increasing hypoxic areas, and evofosfamide targets hypoxic tumor cells. Consequently, each drug enhances the activity of the other.     

Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo.In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.     

Redox-active trace metal-induced release of high mobility group box 1(HMGB1) and inflammatory cytokines in fibroblast-like synovial cells is Toll-like receptor 4 (TLR4) dependent

Rheumatoid arthritis (RA) is a chronic autoimmune systemic inflammatory disease that is characterized by synovial inflammation and bone erosion. We have investigated the mechanism(s) by which essential trace metals may initiate and propagate inflammatory phenotypes in synovial fibroblasts. We used HIG-82, rabbit fibroblast-like synovial cells (FLS), as a model system for potentially initiating RA through oxidative stress. We used potassium peroxychromate (PPC, Cr⁺⁵), ferrous chloride (FeCl_2, Fe⁺²), and cuprous chloride (CuCl, Cu⁺) trace metal agents as exogenous pro-oxidants. Intracellular ROS was quantified by fluorescence microscopy and confirmed by flow cytometry (FC). Protein expression levels were measured by western blot and FC, while ELISA was used to quantify the levels of cytokines. Trace metal agents in different valence states acted as exogenous pro-oxidants that generate reactive oxygen species (ROS), which signal through TLR4 stimulation. ROS/TLR4- coupled activation resulted in the release of HMGB1, TNF-α, IL-1β, and IL-10 in conjunction with upregulation of myeloid-related protein (MRP8/14) inflammatory markers that may contribute to the RA pathophysiology. Our results indicate that oxidant-induced TLR4 activation can release HMGB1 in combination with other inflammatory cytokines to mediate pro-inflammatory actions that contribute to RA pathogenesis. The pathway by which inflammatory and tissue erosive changes may occur in this model system possibly underlies the need for functioning anti-HMGB1-releasing agents and antioxidants that possess both dual trace metal chelating and oxidant scavenging properties in a directed combinatorial therapy for RA.     

Novel urushiol derivatives as HDAC8 inhibitors: rational design,virtual screening,molecular docking and molecular dynamics studies

Three series of novel urushiol derivatives were designed by introducing a hydroxamic acid moiety into the tail of an alkyl side chain and substituents with differing electronic properties or steric bulk onto the benzene ring and alkyl side chain. The compounds’ binding affinity toward HDAC8 was screened by Glide docking. The highest-scoring compounds were processed further with molecular docking, MD simulations, and binding free energy studies to analyze the binding modes and mechanisms. Ten compounds had Glide scores of ?8.2 to ?10.2, which revealed that introducing hydroxy, carbonyl, amino, or methyl ether groups into the alkyl side chain or addition of –F, –Cl, sulfonamide, benzamido, amino, or hydroxy substituents on the benzene ring could significantly increase binding affinity. Molecular docking studies revealed that zinc ion coordination, hydrogen bonding, and hydrophobic interactions contributed to the high calculated binding affinities of these compounds toward HDAC8. MD simulations and binding free energy studies showed that all complexes possessed good stability, as characterized by low RMSDs, low RMSFs of residues, moderate hydrogen bonding and zinc ion coordination and low values of binding free energies. Hie147, Tyr121, Phe175, Hip110, Phe119, Tyr273, Lys21, Gly118, Gln230, Leu122, Gly269, and Gly107 contributed favorably to the binding; and Van der Waals and electrostatic interactions provided major contributions to the stability of these complexes. These results show the potential of urushiol derivatives as HDAC8 binding lead compounds, which have great therapeutic potential in the treatment of various malignancies, neurological disorders, and human parasitic diseases.     

Abscisic acid catabolism enhances dormancy release of grapevine buds

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It was formerly proposed that dormancy is maintained by abscisic acid (ABA)‐mediated repression of bud–meristem activity and that removal of this repression triggers dormancy release. It was also proposed that such removal of repression may be achieved via natural or artificial up‐regulation of VvA8H‐CYP707A4, which encodes ABA 8′‐hydroxylase, and is the most highly expressed paralog in grapevine buds. The current study further examines these assumptions, and its experiments reveal that (a) hypoxia and ethylene, stimuli of bud dormancy release, enhance expression of VvA8H‐CYP707A4 within grape buds, (b) the VvA8H‐CYP707A4 protein accumulates during the natural transition to the dormancy release stage, and (c) transgenic vines overexpressing VvA8H‐CYP707A4 exhibit increased ABA catabolism and significant enhancement of bud break in controlled and natural environments and longer basal summer laterals. The results suggest that VvA8H‐CYP707A4 functions as an ABA degrading enzyme, and are consistent with a model in which the VvA8H‐CYP707A4 level in the bud is up‐regulated by natural and artificial bud break stimuli, which leads to increased ABA degradation capacity, removal of endogenous ABA‐mediated repression, and enhanced regrowth. Interestingly, it also hints at sharing of regulatory steps between latent and lateral bud outgrowth.     

LXR/ApoE Activation Restricts Innate Immune Suppression in Cancer

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Gluconeogenesis and the peroxisome

The interaction between hydroperoxides, cytochrome P450 and 8-anilino-1-naphthalenesulfonic acid (ANS) has been investigated. The addition of ANS to the cytochrome P450 solution did not effect the P450 Soret absorption peak or the reduced CO difference spectrum, suggesting that ANS may not bind to P450 heme directly. H2O2 or CuOOH alone did not effect ANS fluorescence and absorption spectra indicating that no detectable reaction occurs between hydroperoxide and ANS in the absence of P450. The reconstituted system of cytochrome P450, P450 reductase, lipid and NADPH did not mediate ANS metabolism. In the presence of P450, the addition of either H2O2 or CuOOH, however, leads to a decrease in ANS absorption around 258 nm and 350 nm indicating possible destruction of ANS. ANS destruction was confirmed with the disappearance of the ANS elution peak in the reverse phase HPLC profiles and with the changes in P450-bound ANS fluorescence intensity and the shift of max of ANS. Moreover , a very sensitive method to detect trace fluorescent products of ANS by thin layer chromatography has been developed based on the fact that ANS fluorescence is enhanced more than 1000-fold by the organic solvent butanol. A UV-sensitive fluorescent product was detected on thin layer chromatography profiles of the reaction mixtures. P450 was also observed to be modified by a fluorescent derivative of ANS, when the fluorescence was enhanced by butanol. These results also show that an organic compound which can not be metabolized by the reconstituted system of cytochrome P450 and NADPH-P450 reductase is metabolized by the reconstituted system of P450 and hydroperoxide, suggesting the activities of these two systems may not be completely comparable. (Mol Cell Biochem 167: 159-168, 1997)     

Cloning and sequencing of complement component C9 and its linkage to DOC-2 in the pufferfish Fugu rubripes

The Japanese pufferfish Fugu rubripes has a 400 Mb genome with high gene density and minimal non-coding complexity, and is therefore an ideal vertebrate model for sequence comparison. The identification of regions of conserved synteny between Fugu and humans would greatly accelerate the mapping and ordering of genes. Fugu C9 was cloned and sequenced as a first step in an attempt to characterize the region in Fugu homologous to human chromosome 5p13. The 11 exons of the Fugu C9 gene share 33% identity with human C9 and span 2.9 kb of genomic DNA. By comparison, human C9 spans 90 kb, representing a 30-fold difference in size. We have also determined by cosmid sequence scanning that DOC-2, a tumour suppresser gene which also maps to human 5p13, lies 6–7 kb from C9 in a head-to-head or 5′ to 5′ orientation. These results demonstrate that the Fugu C9/DOC-2 locus is a region of conserved synteny. Sequence scanning of overlapping cosmids has identified two other genes, GAS-1 and FBP, both of which map to human chromosome 9q22, and lie adjacent to the Fugu C9/DOC-2 locus, indicating the boundary between two syntenic regions.