We aimed at characterizing microRNAs activated downstream of MUC4-associated signaling in pancreatic adenocarcinoma. We investigated the MUC4-miR-210-3p reciprocal regulation and deciphered miR-210-3p biological roles in vitro and in vivo. We showed a MUC4-miR-210-3p negative feedback loop that involves NF-κB in PDAC-derived cells and the miR-210-3p anti-tumoral functions, suggesting a complex balance between antagonistic pro-oncogenic functions of the oncomucin MUC4 and anti-tumoral roles of the miR-210-3p.Background: Pancreatic adenocarcinoma (PDAC) is a deadly cancer with an extremely poor prognosis. MUC4 membrane-bound mucin is neoexpressed in early pancreatic neoplastic lesions and is associated with PDAC progression and chemoresistance. In cancers, microRNAs  are crucial regulators of carcinogenesis, chemotherapy response and even metastatic processes. In this study, we aimed at identifying and characterizing miRNAs activated downstream of MUC4-associated signaling in pancreatic adenocarcinoma. MiRnome analysis comparing MUC4-KD versus Mock cancer cells showed that MUC4 inhibition impaired miR-210-3p expression. Therefore, we aimed to better understand the miR-210-3p biological roles. Methods: miR-210-3p expression level was analyzed by RT-qPCR in PDAC-derived cell lines , as well as in mice and patients tissues. The MUC4-miR-210-3p regulation was investigated using luciferase reporter construct and chromatin immunoprecipitation experiments. Stable cell lines expressing miR-210-3p or anti-miR-210-3p were established using CRISPR/Cas9 technology or lentiviral transduction. We evaluated the biological activity of miR-210-3p in vitro by measuring cell proliferation and migration and in vivo using a model of subcutaneous xenograft. Results: miR-210-3p expression is correlated with MUC4 expression in PDAC-derived cells and human samples, and in pancreatic PanIN lesions of Pdx1-Cre; LstopL-KrasG12D mice. MUC4 enhances miR-210-3p expression levels via alteration of the NF-κB signaling pathway. Chromatin immunoprecipitation experiments showed p50 NF-κB subunit binding on miR-210-3p promoter regions. We established a reciprocal regulation since miR-210-3p repressed MUC4 expression via its 3′-UTR. MiR-210-3p transient transfection of PANC89, PANC-1 and MiaPACA-2 cells led to a decrease in cell proliferation and migration. These biological effects were validated in cells overexpressing or knocked-down for miR-210-3p. Finally, we showed that miR-210-3p inhibits pancreatic tumor growth and proliferation in vivo. Conclusion: We identified a MUC4-miR-210-3p negative feedback loop in early-onset PDAC, but also revealed new functions of miR-210-3p in both in vitro and in vivo proliferation and migration of pancreatic cancer cells, suggesting a complex balance between MUC4 pro-oncogenic roles and miR-210-3p anti-tumoral effects. Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive and highly drug-resistant cancer. At the time of diagnosis, more than 80% of PDACs are already locally advanced or metastatic. In addition, the 5-year survival rate of patients with local or metastatic PDAC treated with surgery or palliative chemotherapy, respectively, remains very low <10%) ..2]. OwinO-glycoprotein (up to 930 kDa). This heterodimeric membrane-bound protein is composed of two subunits: MUC4α and MUC4β. The extracellular mucin-type subunit, MUC4α, represents a typical hyper-glycosylated region. The membrane-bound EGF-like subunit, MUC4β, contains EGF-like domains and is implicated in receptor–ligand interactions with the transmembrane growth factor receptor ErbB2/HER2  and  (once), and TE supplemented with 50 mM NaCl) (×2). Elution of cross-linking complexes was performed at 65°C using elution buffer  and RecoverAll™ Total Nucleic Acid Isolation Kit . MiRNA was measured by RT-qPCR using a TaqMan® MicroRNA Assays protocol  and CFX96 ™ Real-Time System . Briefly, 5 ng of total RNA was reverse transcribed using a TaqMan MicroRNA Reverse Transcription kit, including Taqman microRNA primers specific for miR-210-3p , RNU48 (001006) and snoRNA202 (001232) from Thermo Fisher Scientific, following manufacturer’s instructions. The qPCR was carried out using TaqMan Gene Expression Master Mix II with Taqman microRNA specific primers using recommended PCR cycling conditions. Expression levels were normalized using RNU48 (human cells and tissues), or snoRNA202 (mice samples) and were measured using the 2−ΔΔCt method.Formalin-fixed paraffin-embedded (FFPE) sample RNAs (from mice and human cells) were purified, respectively, using NucleoSpinw/v) non-fat dry milk in Tris-buffered saline (Tween-20). MUC4 and β-actin antibodies were diluted in Tris-buffered saline Tween-20 and incubated overnight at 4 °C. Peroxidase-conjugated secondary antibodies were used . Immunoreactive bands were visualized with the LAS4000 device  using the Super Signal® West Pico chemoluminescent substrate . Band density was quantified with the image analysis software ImageJ and represented as histograms.Total protein extraction and Western blotting analysis were carried out as described before ,46. Afte4 cells were cultured over 96 h. MTT  was then added to the medium for 1 h. Formazan crystals were dissolved using 100 µL of dimethyl sulfoxide . Optical density was evaluated at 570 nm using the Multiskan™ FC microplate photometer .MTT assay: Cell viability was measured using MTT assay. Briefly, 10® . Cell confluency was measured and analyzed every 12 h over 96 h using the IncuCyte software. Experiments were performed in triplicate.Cell proliferation: Cells were seeded in 96-well plates and incubated in the live cell imaging system IncuCyte5 in 96-well plates  and incubated until confluence. Wounds were homogenously generated manually using the 96-well Wound Maker . Pictures were collected every 2 h during 24 h using IncuCyte® apparatus before wound widths were analyzed. Results are expressed as wound confluency, which is the ratio of the occupied area of the initially scratched area to the total area of the scratch.Wound Healing assay: cells were seeded at 3 × 106 cells in 100 μL of Matrigel (R & D)) was performed into the flank of 8-week-old male severe combined immunodeficient mice . Groups consisted of six mice. Tumor volume was monitored by measuring length (L) and width (W) using the formula (W2 × L). All procedures were evaluated by the animal care committee .Subcutaneous injection of CAPAN-1 LV-miR-neg or LV-miR-210 cells  and human Ki-67 (NLC Ki-67p Novocastra) were used for immunostaining as described previously ,23. The p < 0.05 was considered as statistically significant.At least three independent experiments were performed for every assay. ANOVA and Student’s t-test statistical analyses were performed using Graphpad Prism 4.0 software . In the present work, we showed for the first time the existence of a feedback regulation between MUC4 and miR-210-3p. We observed that both miR-210-3p and MUC4 expression levels are increased in pancreatic cancer. We deciphered the complex MUC4–miR-210-3p negative regulation loop in PDAC. Indeed, we observed that MUC4 activates miR-210-3p transcriptional expression via NF-κB pathway modulation and leads to miR-210-3p overexpression in pancreatic cancer cells and tissues. On the contrary, miR-210-3p inhibits MUC4 expression via its 3′ UTR in vitro. Finally, we demonstrated in vitro and in vivo anti-proliferative and anti-migratory effects of miR-210-3p in PDAC, suggesting a complex balance between oncogenic roles of MUC4 and anti-tumoral roles of miR-210-3p.


Covalent organic frameworks (COFs) have been at the forefront of porous-material research in recent years. With predictable structural compositions and controllable functionalities, the structures and properties of COFs could be controlled to achieve targeted materials. On the other hand, the predesigned structure of COFs allows fruitful postsynthetic modifications to introduce new properties and functions. In this review, the postsynthetic functionalizations of COFs are discussed and their impacts towards structural qualities and performances are comparatively elaborated on. The functionalization involves the formation of specific interactions  and chemical reactions (oxidation/reduction reaction) with pendant groups, skeleton and reactive linkages of COFs. The chemical stability and performance of COFs including catalytic activity, storage, sorption and opto-electronic properties might be enhanced by specific postsynthetic functionalization. The generality of these strategies in terms of chemical reactions and the range of suitable COFs places them as a pivotal role for the development of COF-based smart materials. N. N3) andx-TPB-DMTP-COFs by reacting alkynyl (-C≡CH) pendant groups with azide-containing pyrrolidine (N3-Py) [S)-Py]x-TPB-DMTP-COFs contained chiral and catalytic active pyrrolidine moieties. Thus, they have potential as a chiral organocatalyst. Remarkably, [(S)-Py]x-TPB-DMTP-COFs were crystalline with a significant porosity intake. As a heterogeneous chiral organocatalyst, [(S)-Py]x-TPB-DMTP-COFs could catalyse the organic Michael-addition reaction—a basic C–C bond-formation reaction for synthesizing various important synthetics and natural products [S)-Py]x-TBP-DMTP-COFs were able to promote asymmetric C–C bond formation in water under ambient temperature with prominent activity, enantioselectivity, recyclability and environmental benignity compared to the other metal-based catalysts. For instance, [(S)-Py]0.17-TBP-DMTP-COF proceeded smoothly and cleanly to C–C bond formation with 100% conversion in 12 h (e.e. = 92% and d.r. = 90/10), which largely exceeded the molecular (S)-Py catalyst. Furthermore, several other COFs have been functionalized via triazole linkage, including the transformation from [CH≡C]x-H2P-COF into [Pyr]x-H2P-COF and [CH≡C]0.5-TPB-DMTP-COF into ([uracil]0.5-TPB-DMTP-COF) with preservation of crystallinity and enhanced performance for catalyst and adsorbent applications, respectively [60) has also been anchored on the pore surface of predesigned X%[N3]-ZnPc-COF to afford donor–acceptor heterojunction-based [C60]y-ZnPc-COF via covalent triazole linkage [60]y-ZnPc-COF exhibited a photoelectric phenomenon with segregated donor–acceptor alignment in which electron-accepting bucky-balls were spatially confined within the nanochannel via triazole units. In addition, it performed photo-induced electron transfer facilitated by an electron-donating COF scaffold and electron-accepting bucky-ball molecules. COFs with high energy-storage performance were achieved by the covalent introduction of redox-active species  (N3-Py) . The resproducts . In addiectively . Meanwhi linkage . As pred species . In anotproaches .x%-H2P-COFs) were synthesized and employed as scaffolds to demonstrate the structural functionalization of COF via an ester bond [2C]x%-H2P-COFs). This functionalization allows structural rigidity preservation with a well-organized functional constituent within the pore channel and substantial porosity intake. At the end, [HO2C]x%-H2P-COFs were employed as solid sorbents for CO2 storage and separation. Remarkably, the [HO2C]x%-H2P-COF sample demonstrated dramatic CO2-uptake enhancement relative to [HO]x%-H2P-COF. In addition, the ideal absorbed solution theory (IAST) calculation was performed based on their CO2 and N2 sorption isotherms, revealing high CO2-absorption selectivity (adsS) with 323 for [HOOC]100%-H2P-COF (at 0.1 kPa)—much greater than [HO]100%-H2P-COF (only 18). Indeed, this performance was much higher than for other reported adsorbents. On another occasion, a predesigned hydroxyl-appended 3D COF (3D-OH-COF) was prepared and further functionalized via an ester bond  and Fe3+ ions (IAST selectivity = 18) for a solution containing 5% Nd3+ ion and 95% Sr2+ or Fe3+ ions.Ester bonds have judiciously been applied to functionalize COFs, which were efficiently obtained under a mild and metal-free ring-opening reaction between anhydride and hydroxyl (-OH) groups Fig. b 22]. H. H22]. Hter bond . The rea2) and carbonyl groups 2 was designed by reducing its parent nitro-functionalized COF (TpBD(NO2)2) x%-Py-COFs afforded [Et4NBr]x%-Py-COFs (x = 25–100)  exhibited elevated CO2-uptake capacities and higher isosteric heat absorption (stQ) than those [HO]x%-Py-COFs samples and was able to catalyse the transformation of CO2 into value-added chemicals (N-formylation reaction). For instance, [Et4NBr]50%-Py-COF accounted for high CO2-uptake capacity , which was the highest value compared to several reported COFs. Meanwhile, with 5 mol% [Et4NBr]50%-Py-COF, a solely formylated product of N-methylaniline was obtained with an isolated yield of 94% under mild conditions, while [HO]50%-Py-COF could only exhibit 32% conversion under similar conditions. The generality of this postsynthetic functionalization was also demonstrated on other 2D COFs. For example, the functionalization of [OH]x%-TD-COFs into [BE]x%-TD-COFs transformed the neutral skeleton of the COF scaffold into zwitterionic-functionalized COF and the design of ionic-based [SO3−]-DhaTab COF was obtained by reacting DhaTab COF with 1,3-propanesulton in toluene -COFs) was also reduced (treated with PPh3 in methanol) to give the corresponding amine-containing COF (X%[NH2]-COFs) -COFs) ,51.Unlike the covalent bond that was formed through sharing electrons, coordination-bond formation involves the donor–acceptor electron transfer between the active sites of the components .Meanwhile, ionic interaction occurs on the dipolar region between the two active sites, establishing engagement forces between the components ,54. Thess- or p-type orbitals may facilitate the formation of coordination bonds with metals . For example, imine linkages found in imine-linked COFs are able to govern coordination bonds with metals or metal ions . I. I56]. Ireaction . Meanwhimethanol . Howevere Re-COF . The res2 at room temperature to afford Ni-DBA-3D-COF  in which the Pd ions were assumed to be coordinatively bonded on solely nitrogen of a particular imine linkage within the skeleton  ethylenediamine  has been considered as an attractive moiety for coordination chemistry due to its ability to hold metal ions . T. T2 at rskeleton . The X-rtal ions . In linetal ions . This trtalation . The JUC′-BPyPh was performed via sequential impregnation treatment, where MnCl2 was previously impregnated to give Mn@Py-2,2′-BPyPh and followed by treatment with Pd(OAc)2 to afford bimetal Mn/Pd@Py-2,2′-BPyPh  with the ability to catalyse a one-pot addition–oxidation reaction 17-COF by simply mixing the COF with the chloride salt solution of the respective metals and afforded [MOOC]17-COFs . As predicted, the resultant hybrids exhibited enhanced ammonia uptakes relative to the bare COF.The incorporation of a multi-metal component into the pore of COFs has also been demonstrated. The successful docking of bimetallic salts of Mn/Pd into the pore of a dual-coordinative site containing Py-2,2yPh Fig. c 67]. B. B′-BPyPreaction . Furtheromponent . VariousxOy@COF hybrids. The Au nanoparticles (Au(0)) were deposited within the channel of imine-linked TpPa-1 by reducing (using NaBH4 solution) the deposited Au(III) ions in Au(III)@TpPa-1 to give Au(0)@TpPa-1  and no catalytic activity was observed for the COF-TpPa-1 sample. Meanwhile, the Pd(0)@TpPa-1 has also been obtained under similar procedures and further employed as a catalyst for both C–C Sonogashira/Heck coupling and C–H activation reactions . T. TxOy@COeactions . Furthereactions ,76.3CN solution of ruthenium chloride (RuCl3) and further reduced by NaBH4. Notably, the hybrid showed enhanced catalytic performance for a one-pot solvent-free tandem reaction of imine synthesis from alcohols ,9089,90]-300-AR) . The red3.Et2O, chloranil and toluene for 72 h . T. T92]. Tfor 72 h . DrasticOF) Fig. c 94,95],953.Et2O2SO4, 14.8 M H3PO4 and 9 M H2SO4 in dimethyl sulfoxide (DMSO)) for 1 day. Although the linkage transformation discussed in this section mainly utilizes imine linkage, other linkages may also possibly be functionalized via a certain reaction or treatment.give thiazole-linked COF (COF-921) or with 2,5-diaminohydroquinone dihydrochloride in DMF under oxygen atmosphere to obtain oxazole-linked COF (LZU-192), respectively . These lAs highly porous materials, COFs are perfect porous confinement materials acting as hosts to accommodate guest molecules with controlled quantities via the host–guest chemistry approach. This confinement property involves weak forces that have been well studied in general supramolecular chemistry including van der Walls, π–π interaction and hydrophobic/hydrophilic interactions .60 molecules were incorporated into the channel of the highly stable, electronically conjugated 2D phenazine-linked COF (CS-COF) via host–guest chemistry to obtain donor–acceptor-based CS-COF⊃C60 , [Co(η5-C5H5)2] and [Ru(cod)(cot)] were loaded into the pore of COF-102 to obtain (FeCp2)4@COF-102, (CoCp2)4@COF-102 and [Ru(cod)(cot)]@COF-102 [Another piece of sophisticated work was loading ors Fig. b 109]. . N-heter in COFs . Meanwhi@COF-102 . InteresCOFs materials with tunable structural topologies and high thermal/chemical stabilities provide a versatile postsynthetic functionalization platform to enhance their psychochemical properties and performances toward specific applications. The manageable porosities and functionalities via the bottom-up design of COFs allow judicious incorporation of constituents that can be bonded by strong covalent bonds, ionic/coordination forces and host–guest chemistry interactions. Nevertheless, their linkages can also be postsynthetically functionalized to improve their structural stability. Furthermore, the resultant functionalized COFs with particular hybrid or composite fashions show outstanding performances in the field of catalyst, gaseous uptake–release, optics, energy storage and so on. In the future development of COFs, we assume that there are still many gaps left for further explorations, especially in functionalization of the available linkages that are less studied. In addition, incorporations of functional constituents via host–guest chemistry so far have been mainly developed for electronic and energy-storage applications, thus investigations in wider sophisticated applications are still highly demanded. Last but not least, the postsynthetic functionalization of 3D COFs is still less studied; thus, continuous efforts in the development of 3D COFs may need special concern.


Oncorhynchus mykiss, and investigated its role in TLR response regulation. The deduced amino acid sequence of OmA20 contained a conserved N-terminal ovarian tumor (OTU) domain and seven C-terminal zinc-finger (ZnF) domains. Lipopolysaccharide (LPS) stimulation increased OmA20 expression in RTH-149 cells. In LPS-stimulated RTH-149 cells, gain- and loss-of-function experiments revealed that OmA20 inhibited MAPK and NF-κB activation, as well as the expression of pro-inflammatory cytokines. OmA20 interacted with TRAF6, a key molecule involved in the activation of TLR-mediated NF-κB signaling pathways. LPS treatment increased the K63-linked polyubiquitination of TRAF6 in RTH-149 cells, which was suppressed when OmA20 was forced expression. Furthermore, mutations in the OTU domain significantly decreased deubiquitination of the K63-linked ubiquitin chain on TRAF6, indicating that deubiquitinase activity is dependent on the OTU domain. These findings suggest that OmA20, like those of mammals, reduces LPS-induced inflammation in rainbow trout, most likely by regulating K63-linked ubiquitination of TRAF6.The ubiquitin-editing enzyme A20 is known to inhibit the NF-κB transcription factor in the Toll-like receptor (TLR) pathways, thereby negatively regulating inflammation. However, its role in the TLR signaling pathway in fish is still largely unknown. Here, we identified a gene encoding A20 (OmA20) in rainbow trout,  The innate immune system, an evolutionarily conserved component of the host defense, provides initial protection against infectious pathogens ,2. It reAmong the various PRRs, the TLR family is one of the most well-studied PRR families responsible for detecting invading pathogens both outside and inside the cell . Upon reSeveral mechanisms are responsible for the regulation of the TLR signaling pathways. These include physical interactions, conformational changes, phosphorylation, ubiquitination, and proteasome-mediated degradation of numerous regulatory proteins ,11,12. ADanio rerio) [Pimephales promelas) [Takifugu rubripes) [Oncorhynchus mykiss, one of the most significant cold-water fish species because it is essential for food production and sport fishing, as well as a research model. In particular, we investigated the role of OmA20 in the regulation of TLR responses in a rainbow trout hepatoma cell line RTH-149.Orthologs of human A20 have been identified in several fish species, including zebrafish (o rerio) , fatheadromelas) , and Japubripes) . Fish A2ubripes) . The funubripes) . In a prubripes) . Other subripes) . On the Salmo salar), channel catfish , zebrafish (D. rerio), human (Homo sapiens), and mouse (Mus musculus) revealed that OmA20 contains a highly conserved OTU domain, which is required for DUB activity .We identified an A20 candidate (OmA20) from the total RNA of rainbow trout using 5′- and 3′-RACE. The cDNA of OmA20 (GenBank Accession number MF671983) is 3367 bp long and contains an open reading frame (ORF) of 2415 bp, a 5′-untranslated region (UTR) of 162 bp, and a 3′-UTR of 790 bp. The ORF encodes a deduced protein of 804 amino acids with an estimated molecular mass of 90.2 kDa. Secondary structure prediction by SMART revealed that OmA20 contained an N-terminal ovarian tumor (OTU) domain (103–257 aa) and seven C-terminal zinc-finger (ZnF) domains  . The whoactivity . A phyloactivity . The resThe tissue-specific expression of OmA20 was examined to select a suitable cell line for in vitro studies. As shown in TLR coding sequences and functions are substantially conserved in vertebrates. Similarly, TLR-mediated signaling pathways are highly conserved ,35. In mNext, we investigated the effect of OmA20 overexpression or silencing on the expression of downstream target genes of MAPKs and NF-κB, such as IL-1β, TNF-α, IL-6, and IL-8 in RTH-149 cells stimulated with LPS. At 12 h after LPS stimulation, the expression of IL-1β, TNF-α, IL-6, and IL-8 in RTH-149 cells transfected with EGFP-His (or non-specific siRNA) was elevated by 9.13- (7.84-), 11.57- (10.6-), 7.45- (7.34-), and 7.64-fold (9.35-fold), respectively, compared to control cells that had not been activated . OmA20 oTRAF6 is a key transduction molecule downstream of the TLR pathways whose polyubiquitination can induce NF-κB activity and the subsequent secretion of inflammatory factors ,40. In mThe DUB activity of mammalian A20 towards key NF-κB signaling proteins, such as TRAF6, is conferred by the N-terminal OTU domain. Therefore, to determine the physiological significance of the OmA20 OTU domain in regulating the activity of TRAF6 by removing K63-linked chains, we examined the influence of overexpression of OmA20 mutants on the ubiquitination of TRAF6 in LPS-stimulated RTH-149 cells. Wild-type OmA20 and C-terminal ZnF domain deletion mutant OmA20-N disassembled K63-linked ubiquitin chains on TRAF6. In contrast, DUB-deficient mutants, the N-terminal OTU domain deletion mutant OmA20-C and a catalytically inactive DUB mutant OmA20 (C107A), markedly attenuated deubiquitination of K63-linked ubiquitin chains on TRAF6, implying that the OTU domain is required for DUB activity B. TRAF6 Because TLR signals commit organisms to inflammatory responses, they must be strictly regulated to protect hosts from excessive inflammation ,43,44. TIn mammals, it has been shown that A20 can target TRAF6 and downregulate NF-κB activity. TRAF6 is an E3 ubiquitin ligase that regulates the TLR signaling pathway in response to microbial products and cytokines ,48. TRAFEscherichia coli 0111:B4 LPS and MG132 were purchased from InvivoGen  and Sigma-Aldrich , respectively. The antibodies against JNK, phosphorylated JNK (Thr183/Tyr185), TAK1, phosphorylated TAK1 (Thr184/187), phosphorylated p38 MAPK (Thr180/Tyr182), K48-linkage specific polyubiquitin, K63-linkage specific polyubiquitin, HA-tag, and β-actin were purchased from Cell Signaling Technology . The antibodies against p38 MAPK (ab170099) and TRAF6 (ab33915) were from Abcam . The antibodies against 6×His tag and Flag-tag were from Invitrogen .The rainbow trout hepatoma cell line RTH-149 was obtained from the American Type Culture Collection  and cultured as previously described . EscheriA first-strand cDNA was synthesized from 1 μg of total RNA isolated from rainbow trout fingerlings  as previously described . Based ohttp://www.expasy.org/tools/blast/; last accessed date: 7 September 2021). An OTU domain and seven ZnF domains of OmA20 were predicted by the Simple Modular Architecture Research Tool (SMART) and Pfam servers . A multiple sequence alignment analysis was performed using the MegAlign program of the DNASTAR Lasergene software package . A phylogenetic tree analysis was carried out using the Neighbor-Joining (NJ) method with 10,000 bootstrap replicates in the MEGA-X software . Accession numbers of proteins used in the sequence analysis were listed in The deduced amino acid sequence of OmA20 was analyzed for similarity using the BLAST program at the ExPASy Molecular Biology Server (6 cells/well in 6-well plates) were stimulated with different concentrations of LPS for the indicated time periods. Reverse transcription of 1 μg total RNA from each sample was performed using an iScript cDNA synthesis kit  following the manufacturer’s instructions. qRT-PCR was performed using iQTM SYBR® Green Supermix  as previously described [Total RNA was extracted using the RNeasy plus mini kit  from tissue samples and RTH-149 cells: eight different tissues  were obtained from four fish separately; RTH-149 cells (1 × 10escribed .6 cells/well in 6-well plates) were stimulated with LPS (10 μg/mL) for the indicated time periods. Cells were harvested and lysed in RIPA buffer  containing protease and phosphatase inhibitors on ice for 30 min. Equal amounts of denatured protein samples (40 μg) were loaded onto 12% SDS-PAGE. Then, the gels were transferred to polyvinyl difluoride membranes , the transferred membranes were blocked with 5% (w/v) BSA in TBS containing 0.1% Tween-20 for 1 h, and immunoblotting was carried out with appropriate antibodies. Chemiluminescence was detected using an ECL kit  following the manufacturer’s protocol.RTH-149 cells  before being subcloned into pcDNA4.1 (Invitrogen) digested with the same restriction enzymes. The resultant plasmids were named His-OmA20, EGFP-His, His-OmA20-N, His-OmA20-C, His-OmA20 (C107A), Flag-TRAF6, HA-Ubiquitin, HA-Ubiquitin (K48R), HA-Ubiquitin (K63R), HA-Ubiquitin (K48O), and HA-Ubiquitin (K63O).ORFs of OmA20 and EGFP were amplified from OmA20 full-length cDNA clone and pEGFP-N2 plasmid , respectively, by PCR with the gene-specific primer sets . OmA20 deletion mutants (OmA20-N and OmA20-C) were generated by standard PCR methods with each primer set . To introduce a mutation at the Cys residue in the OTU domain of OmA20, which is essential for DUB function , OmA20 (6 cells/well in 6-well plates) were transfected with His-OmA20, His-OmA20-N, His-OmA20-C, His-OmA20 (C107A), or EGFP-His (control) using the Lipofectamine 2000 (Invitrogen). Overexpression of OmA20, OmA20 mutants, and EGFP was confirmed at the protein level by western blot analysis using a 6×His tag antibody (Invitrogen). At 48 h after transfection, cells (except control cells) were stimulated with LPS (10 μg/mL) for 12 h and collected to assess targets at the protein and gene expression level by western blot and qRT-PCR, respectively, as described above.RTH-149 cells (1 × 105 cells/well in 6-well plates) were transfected with 24 nM siRNA against OmA20 or control siRNA for 48 h using the Lipofectamine 2000 (Invitrogen). Then, cells (except control cells) were stimulated with LPS (10 μg/mL) for 12 h and collected to assess targets at the protein and gene expression level by western blot and qRT-PCR, respectively, as described above. siRNA sequences used in this study were as follows: OmA20, 5′-CGU UAC AGC ACC AUG AAU UGG UU-3′ (sense) and 5′-CCA AUU CAU GGU GCU GUA ACG UU-3′ (antisense) and control, 5′-AGA UCC GCU ACU GUC CGA AUU-3′ (sense) and 5′-UUC GGA CAG UAG CGG AUC UUU-3′ (antisense).OmA20 siRNA gene silencing was performed, as previously described . In brieRenilla luciferase activities were measured using the Dual-Luciferase Assay System (Promega). Firefly luciferase activity was normalized to that of Renilla. In some experiments, cells were co-transfected with His-OmA20, His-OmA20-N, His-OmA20-C, His-OmA20 (C107A), or EGFP-His along with pNF-κB-Luc and pRL-TK, in order to examine the effect of OmA20 and OmA20 mutants on the activation state of NF-κB.After co-transfecting RTH-149 cells with luciferase reporter plasmids pNF-κB-Luc  and pRL-TK  for 48 h, cells (except control cells) were stimulated with LPS (10 μg/mL) for the indicated time periods. Then, cells were lysed with lysis buffer (Promega), and firefly and N-hydroxysuccinimide-activated magnetic beads in an amine-free buffer. Then, 500 μL of cell lysates (2 mg/mL) were subjected to immunoprecipitation by incubation (overnight at 4 °C) with the prepared antibody-coupled beads. After extensive washing, the beads were spun down, resuspended, and boiled. Pull-down samples were detected using western blotting as described above.Immunoprecipitation assays were performed using a Pierce Direct Magnetic IP/Co-IP kit  following the manufacturer’s protocol. Briefly, RTH-149 cells were co-transfected with His-OmA20 and Flag-TRAF6 for 48 h. Then, cells were stimulated with LPS (10 μg/mL) for 12 h, lysed with IP lysis buffer, and protein levels were quantified. Five micrograms of 6×His tag or Flag tag antibodies were coupled to the RTH-149 cells were transfected for 48 h with Flag-TRAF6 and various constructs as indicated. Then, cells (except control cells) were stimulated with 10 μg/mL LPS for 12 h and next treated with or without MG132 (25 μM) for an additional 4 h to block proteasomal degradation. Cell lysates were collected and immunoprecipitated with Flag-tag antibody (5 μg). Pull-down samples were subjected to immunoblotting with K48-linkage specific polyubiquitin, K63-linkage specific polyubiquitin, and HA-tag antibodies to visualize polyubiquitinated TRAF6 protein bands.p < 0.05.GraphPad Prism software  was used for graphing and statistical analysis. One-way ANOVA followed by Tukey’s post hoc test was applied to multiple-group comparisons. All experiments were performed in triplicate, repeated at least three times, and the results were expressed as mean ± SEM. The statistical significance was accepted as O. mykiss. Gain- and loss-of-function experiments showed that OmA20 inhibits MAPK and NF-κB activation, as well as the expression of pro-inflammatory cytokines in RTH-149 cells stimulated with LPS. Mechanistically, OmA20 interacted directly with TRAF6 and deubiquitinated LPS-induced K63-linked polyubiquitination of TRAF6 in a DUB activity-dependent manner. Although additional in vivo studies are needed to clarify the current in vitro data, our findings indicate that OmA20 reduces LPS-induced inflammation in rainbow trout, most likely by regulating K63-linked ubiquitination of TRAF6. The knowledge of OmA20 as a negative regulator of the TLR signaling pathway will help to provide insights into the development of new control strategies for the inflammatory diseases of fish.In this study, we have identified and characterized A20 in rainbow trout,


Butyrate acts as a regulator in multiple inflammatory organ injuries. However, the role of butyrate in acute liver injury has not yet been fully explored. In the present study, we aimed to investigate the association between butyrate and lipopolysaccharide (LPS)-induced acute liver injury and the signaling pathways involved.LPS-induced acute liver injury was induced by intraperitoneal injection of LPS (5 mg/kg) in G-protein-coupled receptor 43 (GPR43)-knockout (KO) and wild-type female C57BL/6 mice. Sodium butyrate (500mg/kg) was administered intraperitoneally 30 min prior to LPS exposure. Liver injury was detected by serum markers, tissue morphology, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Pro-inflammatory-factor levels were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (RT-PCR). Cell models were first treated with sodium butyrate (4 μmol/mL), followed by LPS (1 μg/mL) half an hour later in GPR43 small interfering RNA (siRNA)-transfected or control RAW264.7 cells. Cell-inflammation status was evaluated through detecting pro-inflammatory-factor expression by RT-PCR and also through checking toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB)-element levels including TLR4, TRAF6, IKKβ, IкBα, phospho-IкBα, p65, and phospho-p65 by Western blot. The interaction between GPR43 and β-arrestin-2 was tested by co-immunoprecipitation.Sodium butyrate reversed the LPS-induced tissue-morphology changes and high levels of serum alanine aminotransferase, aspartate transaminase, myeloperoxidase, TUNEL, and pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The ameliorating effect of sodium butyrate was weakened in GPR43-KO mice and GPR43 siRNA RAW264.7 cells, compared with those of GPR43-positive controls. Sodium butyrate downregulated some elements of the TLR4/NF-κB pathway, including phospho-IκBα and phospho-p65, in RAW264.7 cells. Increased interactions between GPR43 and β-arrestin-2, and between β-arrestin-2 and IкBα were observed.Sodium butyrate significantly attenuated LPS-induced liver injury by reducing the inflammatory response partially via the GPR43/β-arrestin-2/NF-κB signaling pathway. Diet goes beyond nutritional value and profoundly influences human health. Lifestyle changes in recent years have paralleled the increasing incidence of various gastrointestinal and metabolic diseases, such as inflammatory bowel diseases (IBDs), intestinal cancers, and metabolic syndrome. Short-chain fatty acids (SCFAs) are produced from dietary fibers through fermentation by the microbiota in the lower intestine and are indigestible by intestinal enzymes . LocallySepsis is a critical disease with a mortality rate of ≤50% . EndotoxNuclear factor-κB (NF-κB) is a well-known major inflammatory factor that pushes the inflammatory process forward to eventually cause potential tissue damage and is activated in LPS-induced liver injury . NumerouTM (Cat. FSK-100) was obtained from Toyobo . Alanine transaminase  and aspartate transaminase  enzyme-linked immunosorbent assay (ELISA) kits were all obtained from Cusabio . The Pierce Classic IP Kit (Cat. 26146) was obtained from Thermo Scientific .LPS (Cat. L2630), sodium butyrate (Cat.B5887), and TRIzol reagent (Cat. T9424) were obtained from Sigma . Dulbecco’s modified Eagle’s medium , fetal bovine serum (Cat. 10270–106), penicillin and streptomycin (Cat. 15140122), and trypsin (Cat. 25200–056) were obtained from Gibco . The real-time PCR Master Mix kit-SYBR Green (Cat. AQ141-04) was obtained from TransGen . Small interfering RNA (siRNA)-GPR43 was designed by GenePharma . Anti-glyceraldehyde-3-phosphate dehydrogenase antibody , horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) secondary antibody (Cat. sc-2004), anti-p65 (Cat. sc-372), anti-phospho-IkBα (Cat. sc-8404), anti-IkBα (Cat. sc-371), and anti-TRAF6 (Cat. sc-7221) antibodies were all obtained from Santa Cruz . Anti-GPR43 (Cat. ABC299) antibodies were obtained from Merck . Anti-myeloperoxidase (MPO) primary antibody (Cat. ab9535), anti-β-arrestin-2 (Cat. ab54790), and anti-phospho-p65 (Cat. ab86299) were obtained from Abcam . Lipofectamine 3000 (Cat. L3000-015) was purchased from Invitrogen . The First Strand cDNA Synthesis Kit ReverTra Ace-α-+/− heterozygous C57BL/6 mice were purchased from Bioray Laboratories Inc.  and were bred, propagated, and identified at the Institutional Animal Care and Use Committee of Sun Yat-sen University . All experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (certification no.: IACUC-F3-17–1004). Animal studies were performed in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines  into the cytoplasm  and the National Natural Science Foundation of China [81470848]. None declared. goaa085_Supplementary_DataClick here for additional data file.


Naja atra (Taiwan cobra) cardiotoxin 1 (CTX1)–induced death of leukemia cell lines U937 and HL-60. CTX1 increased cytoplasmic Ca2+ and reactive oxygen species (ROS) production, leading to the death of U937 cells. It was found that Ca2+-induced NOX4 upregulation promoted ROS-mediated p38 MAPK phosphorylation, which consequently induced c-Jun and ATF-2 phosphorylation. Using siRNA knockdown, activated c-Jun and ATF-2 were demonstrated to regulate the expression of Fas and FasL, respectively. Suppression of Ca2+-mediated NOX4 expression or ROS-mediated p38 MAPK activation increased the survival of U937 cells exposed to CTX1. FADD depletion abolished CTX1-induced cell death, caspase-8 activation, and t-Bid production, supporting the correlation between the Fas death pathway and CTX1-mediated cytotoxicity. Among the tested N. atra CTX isotoxins, only CTX1 induced Fas and FasL expression. Chemical modification studies revealed that intact Met residues were essential for the activity of CTX1 to upregulate Fas and FasL expression. Taken together, the data in this study indicate that CTX1 induces c-Jun-mediated Fas and ATF-2-mediated FasL transcription by the Ca2+/NOX4/ROS/p38 MAPK axis, thereby activating the Fas death pathway in U937 cells. Furthermore, CTX1 activates Fas/FasL death signaling in the leukemia cell line HL-60.This study aimed to investigate the mechanistic pathway of  Elapidae contains a large amount of cardiotoxins (CTXs) i to a maximum level in U937 cells (2+ with BAPTA-AM eliminated ROS production induced by CTX1 (2+]i (2+ induced ROS production in CTX1-treated U937 cells. Compelling evidence has shown that mitochondria and NADPH oxidase (NOX) are the main sources of intracellular ROS [2+]i (2+ from intracellular depots), EGTA markedly inhibited CTX1-induced elevation of [Ca2+]i (2+. Pretreatment with BAPTA-AM, NAC, or GLX351322 inhibited CTX1-induced cell death (2+-induced ROS production is associated with the cytotoxicity of CTX1 in U937 cells. Since the above data showed NOX4-mediated ROS production in CTX1-treated cells, we further analyzed NOX4 expression in these cells. CTX1 treatment increased NOX4 protein and mRNA expression (2+-induced NOX4 expression increased ROS production in CTX1-treated cells.Since previous studies have indicated that Ca of CTXs ,17, we nreatment A, while 37 cells B. Chelat by CTX1 C, while X1 (2+]i D. These ular ROS ,32. NextOS [2+]i D. Compar [Ca2+]i E, suggesll death F. These pression G,H. Conspression I. Pretrepression J,K. AltoEmerging evidence indicates that ROS can elicit changes in MAPK phosphorylation . Yang etTo examine the mechanism of CTX1-induced upregulation of Fas and FasL, we analyzed their transcription in CTX1-treated cells. CTX1 treatment increased the transcription of Fas and FasL, as demonstrated by qRT-PCR A. Consis2+]i was observed after 3 h of CTX1 treatment (2+ and ROS in CTX1-induced HL-60 cell death. CTX1 induced p38 MAPK activation (2+/NOX4/ROS axis-mediated p38 MAPK activation in HL-60 cells.To examine whether CTX1 also increased Fas/FasL expression in other cell lines, we investigated its effect on HL-60 cells. CTX1 reduced the viability of HL-60 cells by 50% at a concentration of approximately 800 nM after 4 h of treatment A. A singreatment C. Pretrereatment D,E, suggtivation F, while tivation G,H. Moretivation I,J. ThesCTX1-treated HL-60 cells showed increased Fas and FasL expression A,B. Comp2+/NOX4/ROS/p38 MAPK axis, leading to ATF-2-mediated FasL expression and c-Jun-mediated Fas expression. Because c-Jun, c-Fos, and ATF-2 can form heterodimers or homodimers of AP-1 transcriptional factors [The data in this study demonstrated that CTX1 induced Fas and FasL expression in U937 and HL-60 cells, which thus activates FasL/Fas-mediated death signaling. Mechanistically, CTX1 activated the Ca factors , we infe factors . Other s factors . TherefoN. atra CTXs, only CTX1 upregulated Fas/FasL expression. This indicates that the cytotoxicity of CTX isotoxins is not necessarily mediated by the same mechanism. A previous study showed that N. atra CTX3 induces autophagy-dependent apoptosis in U937 cells by activating AMPK [N. mossambica mossambica CTXs and N. atra CTX3 induce mitochondrial fragmentation, leading to the death of neuroblastoma SH-SY5Y cells and H9C2 myoblast cells [N. atra CTX1 induces lysosomal membrane permeability, thereby leading to the death of leukemia K562 and breast cancer MCF-7 cells [N. haje, N. kaouthia, and N. oxiana CTXs in HL-60 cells and lung cancer A549 cells depends on their ability to destroy cell lysosomes [Among the tested st cells ,13. A pr-7 cells . Furtherysosomes . Taken tN. atra CTX3-induced disruption of the membrane is not exclusively related to its cytotoxicity [N.atra CTX2, CTX3, CTX4, and CTX5 actively induce the leakage of lipid bilayers [2+ influx in NIH3T3 cells, while CTX2 and CTX4 do not show such an effect [2+ influx in U937 cells. Collectively, these results emphasize that the cytotoxicity of different CTXs can be mediated through different mechanisms.Experimental evidence has shown that toxicity ,39. Comptoxicity . Althougbilayers , they wen effect . Our datN. atra CTXs induce hemolysis of RBCs [N. atra CTX1 causes necrosis of skeletal muscle cells after intramuscular injection into mice [Importantly, the results of this study do not imply that CTX1 can be used directly to treat leukemia. Previous studies have shown that CTX1 and other  of RBCs . Other snto mice . Therefonto mice , the optN. atra CTX1 activates Fas/FasL-mediated U937 and HL-60 cell death. Although N. atra CTXs share high degree of sequence conservation, the CTX1-induced death pathway was not observed with other N. atra CTXs. These results indicate that slight changes in the fine structure of CTXs greatly affect the mode of their cytotoxic mechanism, and suggest that the diverse activities of CTXs may be due to the activation of different cytotoxic mechanisms in different cells.In summary, our findings demonstrate that


N-methyl-D-aspartate receptors (NMDARs) from cerebellar granule cells (GC-GluN1 ko), but not from Purkinje cells (PC-GluN1 ko), impairs long-term depression (LTD) at parallel fiber (PF) to PC synapses and vestibuloocular reflex (VOR) phase reversal. NMDARs are postsynaptically expressed at climbing fiber (CF) synapses (Schonewille et al.  show thasynapses . Challensynapses , the auts GC-GluN ko, but 2+]o = 1.2 mM; [Mg2+]o = 1 mM; ref. 5For LTD, an older-generation protocol was used, in which PF bursts are paired with 400-Hz CF bursts . We receNonrealistic protocols remain informative about mechanistic plasticity aspects. For example, our finding that postsynaptic NMDAR blockade prevents LTD shows that NMDAR activation principally can initiate LTD . The phyFinally, it is unclear why the authors chose VOR adaptation as a motor learning test , which,


Solid organ transplant recipients (SOTR) have lower humoral responses following SARS-CoV-2 vaccination. Whether this equates to reduced vaccine effectiveness in SOTR or impacts disease severity is not yet known. We used the IDSA Emerging Infections Network (EIN) to identify SARS-CoV-2 cases in vaccinated SOTR. We describe their clinical characteristics and outcomes.th. Online data collection included patient demographics, dates of SARS-CoV-2 vaccine administration and clinical data related to COVID-19 infection. We performed a descriptive analysis of these patient factors and compared differences between early onset (< / = 21 days after completing vaccine series) and late onset infection ( > 21 days after completing vaccine series).On 4/7/21, we requested case reports via the EIN listserv of COVID-19 infection following SARS-CoV-2 vaccination in immunocompromised individuals. Case reports were collected until June 7As of 6/7/21, 34 cases of COVID-19 infection after vaccination in SOTR were submitted. Most cases (79%) occurred in individuals who were fully vaccinated. Only 3 cases (8.5%) occurred in SOTR within their first year after transplantation. Clinical characteristics are listed in Table 1. The vaccine administration date was known for 26 SOTR among whom symptoms occurred a median of 26.5 days  after completing the COVID-19 vaccine series. Twenty-three SOTR (68%) required hospitalization of which 12 had critical illness. Outcome data was available for 29 individuals of whom 20 (69%) demonstrated improvement. When comparing SOTR with early versus late onset COVID-19 infection in relation to vaccination timing, there were no differences in disease severity  or outcome .Table 1: Characteristics of Solid Organ Transplant Recipients with COVID-19 Infection Following SARS-CoV-2 VaccinationSARS-CoV-2 infections after vaccination are occurring in SOTR, including cases of critical illness, suggesting reduced vaccine effectiveness within this vulnerable population. We did not appreciate any correlation between time from vaccination and COVID-19 disease severity or outcome. Further studies evaluating the true incidence of and risk factors for breakthrough infections among vaccinated SOTR are needed.Matthew Kuehnert, M.D., American Association of Tissue Banks (Board Member)ICCBBA (Board Member)Musculoskeletal Transplant Foundation (Employee) John W. Baddley, M.D., Eli Lilly (Consultant)Pfizer (Consultant)R-Pharm (Consultant)Viela Bio (Consultant)


HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172–176.Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the Clathrin was co-immunoprecipitated by HPS3 antibodies from normal but not HPS3 null melanocytes. Normal melanocytes expressing a GFP-HPS3 fusion protein demonstrated partial co-localization of GFP-HPS3 with clathrin following a 20°C temperature block. GFP-HPS3 in which the predicted clathrin-binding domain of HPS3 was mutated (GFP-HPS3-delCBD) did not co-localize with clathrin under the same conditions. Immunoelectron microscopy of normal melanocytes expressing GFP-HPS3 showed co-localization of GFP-HPS3 with clathrin, predominantly on small vesicles in the perinuclear region. In contrast, GFP-HPS3-delCBD did not co-localize with clathrin and exhibited a largely cytoplasmic distribution.HPS3 associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region. This association most likely occurs directly via a functional clathrin-binding domain in HPS3. These results suggest a role for HPS3 and its protein complex, BLOC-2, in vesicle formation and trafficking. Hermansky-Pudlak syndrome (HPS MIM: 203300]) is an autosomal recessive disorder of vesicle biogenesis resulting in the dysfunction of lysosome-related organelles such as melanosomes and platelet dense bodies -4. Affec03300) isAP3B1) with a known function. AP3B1 codes for the β3A subunit of adaptor complex-3 (AP-3), a coat protein that is involved in sorting transmembrane proteins to lysosomes and lysosome-related organelles ), HPS4  606682; ,21]), HP 607521; ,23]), HP 607145; ]) is uni 607145; -26. The HPS3 gene, have absent platelet dense bodies, mild to moderate hypopigmentation of skin and hair, iris transillumination, and patchy hypopigmentation of the fundus  with sequence specific primers and subcloned into pEGFP-C1 . Site-directed mutagenesis to replace the clathrin-binding motif (LLDFE) at residues 172–176 with the non-conserved amino acids AAAPG was performed with the QuikChange Site-Directed Mutagenesis Kit  according to the manufacturer's recommendations, using the forward primer 5'-AATGAGGAATTCTCAGCAGCGGCCCCTGGACGTTCTTTAATTATAC-3' and its reverse complement. All constructs were verified by sequencing before use.The All transfections were performed by electroporation in an Amaxa nucleofector electroporator . Electroporation of melanocytes was performed as described . FibroblApproximately 16 h after transfection with either GFP-HPS3 or GFP-HPS3-delCBD normal melanocytes were incubated at 20°C for 2 h to block trafficking out of the trans-Golgi ,36. CellLive cell imaging was performed on HPS3 null fibroblasts expressing either GFP-HPS3 or GFP-HPS3-delCBD approximately 16 h after transfection. Cells were incubated at 20°C for 2 h followed by a 10 min incubation with the acidic vesicle dye Lysotracker Red . The cells were then placed in fresh, pre-warmed culture media and imaged on a 37°C warming stage. All cells were imaged with a Zeiss 510 META confocal laser-scanning microscope  using a 488 Argon and a 543 HeNe laser. Images were acquired using either a Plan Apochromat 63X/1.4 oil DIC or a 100× Plan Apochromat 100X/1.4 oil DIC objective.Normal melanocytes were fixed approximately 9 h or 24 h after transfection in 2% paraformaldehyde with 0.2% glutaraldehyde in PHEM buffer for 2 h. After embedding, cutting, cryoprotection and snap-freezing of the pellet, ultrathin cryosections were labeled with mouse monoclonal antibodies to clathrin (1:100) (BD Biosciences Pharmingen). The mouse monoclonal antibodies were indirectly labeled with 20-nm protein A-gold particles via a rabbit anti-mouse IgG bridging antibody (1:200) . The second labeling was performed with a rabbit polyclonal anti-GFP antibody (1:1000) , followe2HPS, Hermansky-Pudlak syndrome; BLOC, biogenesis of lysosome-related organelles complexes; GFP, green fluorescent protein; CBD, clathrin-binding domain; TGN, trans-Golgi network; DOPA, dihydroxyphenylalanine; PBS, phosphate buffered saline; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PHEM, PIPES, HEPES, EGTA, MgClAHW performed the immunoprecipitations. AHW and MH prepared the plasmid constructs. AHW and HD carried out the cell culture, transfection experiments, immunofluorescence and live cell confocal imaging. WAG recruited the HPS-3 patient and established the fibroblast cultures. RB established the HPS-3 patient melanocyte cultures. Immunoelectron microscopy and quantitation was performed by WW and MM. WAG, MH, WW and AHW prepared the manuscript.Time series of GFP-HPS3 and acidic vesicle live cell imaging. GFP-HPS3 expressing HPS3 null fibroblast was imaged at 37°C following a 2 h incubation at 20°C. Time series (from inset in Figure Click here for file


Drosophila. Recent studies suggest that Notch signaling may play a role in the physiological, rather than developmental, regulation of neurons. Here, we investigate a new non-developmental role for Caenorhabditis elegans lin-12 Notch signaling in neurons regulating the spontaneous reversal rate during locomotion.Notch signaling pathways are conserved across species and traditionally have been implicated in cell fate determination during embryonic development. Notch signaling components are also expressed postdevelopmentally in the brains of adult mice and C. elegans during normal locomotion is constant. Both lin-12 gain and loss of function mutant animals had significantly increased reversal rates compared to wild type controls. These defects were caused by lin-12 activity, because the loss of function defect could be rescued by a wild type lin-12 transgene. Furthermore, overexpression of lin-12 recapitulated the gain-of-function defect. Increasing or decreasing lin-12 activity in the postdevelopmental adult animal was sufficient to rapidly and reversibly increase reversals, thereby excluding a developmental role for lin-12. Although lin-12 is expressed in the vulval and somatic gonad lineages, we find that these tissues play no role in regulating reversal rates. In contrast, altering lin-12 activity specifically in the nervous system was sufficient to increase reversals. These behavioral changes require components of the canonical lin-12 signaling cascade, including the ligand lag-2 and the transcriptional effector lag-1. Finally, the C. elegans AMPA/kainate glutamate receptor homolog glr-1 shows strong genetic interactions with lin-12, suggesting that glr-1 and/or other glutamate gated channels may be targets of lin-12 regulation.The spontaneous reversal rate of lin-12 Notch in C. elegans and suggest that lin-12 acutely regulates neuronal physiology to modulate animal behavior, without altering neuronal cell fate specification or neurite outgrowth. This is consistent with a role for Notch signaling in neurological disease with late onset symptoms.Our results demonstrate a neuronal role for  Delta/Serrate/LAG-2) family ligands , mgIs18 [lin-15(+) ttx-3p::gfp], nuIs25 [lin-15(+) glr-1p::glr-1::gfp], nuIs1 [lin-15(+) glr-1p:::gfp], rtIs11 [osm-10p::gfp], and rtIs18 [elt-2p::gfp]. Transgenes were co-injected using pha-1(+) (pBX1), myo-2p::gfp (pPD48.33), and/or elt-2p::gfp (pJM67) as markers; details upon request. Heat shock induction occurred at 33°C for 2 hours. hsp::lin-12(RNAi) introduced at 8 ng/μl yielded inducible transgenic lines (hsp::lin-12(RNAi)); introduction at 50 ng/μl resulted in lines with increased reversal rates even in the absence of heat shock  in the text to distinguish them from the inducible hsp::lin-12(RNAi) lines. Transgenic lines overexpressing lin-12p::lin-12 at very high levels (100 ng/μl) often had extra vulvae and were difficult to generate and maintain; these animals were used only for expression analysis. Moderate overexpression (50 ng/μl) of lin-12p::lin-12 was not overtly deleterious and vulval perturbations were infrequent; these animals were used for behavioral analysis. The integrated transgene nuIs25 that overexpresses a GFP tagged glr-1 rescue construct [58] increased reversals .Strains used in this study: N2 Bristol wild type isolate, lin-12n17n460gfcsM.Y.C, J.L.-F., T.T., and A.C.H. all contributed to the genetic, molecular, and behavioral experiments. M.Y.C. and A.C.H. drafted the manuscript. All authors read and approved the final manuscript.


We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility.To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (P We also found that basal Ptet/Ptetbi activity yields permissive conditions for faster activation/re-activation, possibly by providing better accessibility of Ptet/Ptetbi to tTA/rtTA.Here we investigated the regulation of several Tet-responder genes integrated at different sites in the genome CaMK mouse lines. For measuring gene activation, we employed LC-1 reporter mice tetbi) with genes for luciferase (Luc) and Cre recombinase (Cre)  was apparent in the olfactory bulb and low transactivation in the forebrain by three routes of Dox delivery; water, food and intraperitoneal injection (i.p.) (CaMK lines could we detect the expected robust forebrain specific gene activation observed with the tTA system (tTACaMK/LC-1 mice) CaMK lines tested  . We treas tested . One posCaMK lines tested, but only a few Cre positive nuclei were seen in other forebrain regions (tetbi gene activation in different brain regions is clearly apparent in the striatum of rtTA2-M2CaMK-4 mice (CaMK-2 mice) in a larger fraction of DG granule cells when tested with the responder mouse line, LC-1 , which when compared to Dox has approximately 10-times higher binding affinity to the tet repressor  and thus it is also approximately 10-times more effective than Dox in rtTA2-M2 dependent gene activation in HeLa cells (data not shown). We found that granule cells of DG, the subregion with the highest rtTA2 mRNA level , showed  regions . The infK-4 mice . Increasne, LC-1 , but pooCaMK-2 mice, we used the transgenic line G3 which harbors under Ptetbi control the LacZ and GFP genes tetbi activation in G3 responder mice failed even when Dox availability was not a limiting factor.To investigate whether the BBB was the reason for inefficient Tet-controlled gene activation in hippocampus and cortex of our rtTA2-M2CaMK -2/LC-1 mice. We again used the Ptetbi–directed Cre expression of the responder LC-1 to exclude line specific effects. Dox added to the culture medium activated Ptetbi expression only in DG granule cells but again not in CA1/CA3 neurons  with different Dox concentrations (2 to 2000 µg/ml in drinking water) and measured down-regulation of Ptetbi-controlled luciferase expression by bioluminescence imaging of brain explants and biochemical measurements of whole brain extracts  carrying the Ptetbi responder genes Cre and GFP into the hippocampus and cortex of rtTA2-M2CaMK-2 mice. As AAV remains in an episomal state tetbi, possibly by increasing promoter accessibility CaMK-2 mouse brains after viral injection with rAAV-Ptetbi-GFP/Cre robust GFP expression by 9TB-Dox . A large number of cortical neurons were visible by live GFP fluorescence and in fixed brain slices after 10 days of 9TB-Dox treatment (tetbi activation (data not shown). Because non-integrated rAAV-Ptetbi-GFP/Cre, without tTA, have basal promoter activity, we used low virus titers to reduce background signal (data not shown). We estimate that 9TB-Dox induced, rtTA2-M2 dependent gene activation of rAAV-Ptetbi-GFP/Cre can be as high as 20-fold, as determined by two-photon imaging of GFP fluorescence in fixed brain slices (data not shown). Further, we find that gene activation occurred rapidly within hours after a single pulse of 9TB-Dox by i.p. injection  by injecting rAAV-hSyn-tTA2 into cortex and hippocampus of Tet-responder line MTH-Cg2-7 that showed robust Ptetbi promoter activity in the genetic background of tTACaMK mice  tetbi and antagonizes silencing. Moreover, even when tTACaMK/LC-1 and tTACN12/LC-1 mice are kept on Dox (2 mg/ml) for weeks, luciferase activity is not completely suppressed to levels observed in single-positive LC-1 mice  high tTA2 levels in neurons can overcome Ptetbi gene silencing . Individual DNA sequencing of clones of Ptetbi from brain tissue revealed that only 4% of the clones show strong methylation over the entire sequence of Ptetbi  CN12tetbi are all indicated here first by the line name and the two genes in parentheses; LC-1 (luciferase and Cre recombinase) Novel synthetic rtTA variants, rtTA2-M2, and, in addition, rtTA2-nM2, containing a simian virus 40 (SV40) 13 amino acid nuclear localization signal (nls) with optimal codons, lacking putative splice acceptor/donor sites In situ hybridization studies using 35S-labeled oligonucleotide probes were performed according to the method described previously tTA/rtTA-Oligo#1: 5′-TTTAGCTGTTTCTCCAGGCCACATATGATTAGTTCC-3′tTA/rtTA-Oligo#2: 5′-AGCTGATTTTCCAGGGTTTCGTACTGTTTCTCTGTT-3′tTA/rtTA-Oligo#3: 5′-ATAGAATCGGTGGTAGGTGTCTCTCTTTCCTCTTTT-3′To assess for specificity and rule out non-specific labeling of brain sections, each radiolabeled oligonucleotide was hybridized with a 100-fold excess of unlabelled oligonucleotide. Images were processed after 4 week exposure using Biomax MR (Kodak) X-ray films.Animals were singly housed in a cage in order to avoid competition for water and food intake. Doxycycline  was administered to animals by three different delivery routes; drinking water , food  and intraperitoneal injection (i.p) . Protocol 1: 9TB-Dox injected every 12 hours. Protocol 2: 9TB-Dox injected every other day. Experiments were performed according to animal guidelines .Mouse brains were saggitally cut into two halves. One half was fixed in 4% paraformaldehyde, the other half was used for the measurement of luciferase activity in different brain subregions. Fixed brain slices were cut to a thickness of 75–100 µm using vibratome . Luciferase activity was measured from brain extracts as described previously CaMK-2×LC-1, positively identified by genotyping) and were cultured at 7% CO2. Doxycycline treatment (1 µg/ml) was started on the day of slicing and was continued for 4 days. Cultures were then fixed for 15 minutes in 4% paraformaldehyde and stained with an anti-Cre rabbit polyclonal antibody (1∶1000) and a Cy3-labeled secondary antibody . In Approximately 300 µm thick hippocampus organotypic slice cultures from P3-P5 pups were prepared as described tetbi tetbi-GFP/Cre. Plasmids, rAAV-Ptetbi-GFP/Cre, rAAV-hSyn-tTA and rAAV-hSyn-rtTA2-nM2 were individually co-transfected with pDp1, pDp2 (ratio: 3∶1) helper plasmids Recombinant adeno-associated virus (rAAV) /Taq/ DNA polymerase (Qiagen), 200 µM dNTPs, 12.5 pmol each of forward and reverse primers, and 7 ng of bisulphite-treated genomic DNA. The amplification conditions were 95°C for 15 min and 40 cycles of 95°C for 1 min, 55°C for 45 sec and 72°C for 1 min and a final extension step of 10 min at 72°C. PCR products were purified using ExoSAP-IT  and sequenced employing the PCR primers and the ABI Big Dye Terminator v1.1 cycle sequencing chemistry  followed by capillary electrophoresis on an ABI 3100 genetic analyzer. AB1-files were interpreted using ESME, which normalizes sequence traces, corrects for incomplete bisulphite conversion and allows for quantification of methylation signals. The following primers were used both for PCR amplification of bisulfite converted genomic DNA and sequence reactions:Genomic DNA was prepared according to standard procedures. Sodium bisulphite treatment of genomic DNA was performed as described previously Amp806 fw: 5′-TATAGTTTTATGTAGTTGTTTTTTAG-3′ andAmp806 rev: 5′-AATAAATTAAACACCTTCCTC-3′.PCR products were cloned using the TOPO TA Cloning Kit (Invitrogen). Sequencing was carried out using the M13 Forward and M13 Reverse primers by 3100 Genetic Analyzer (Applied Biosystem). All bisulphite sequencing experiments were performed in duplicate and sequencing data was analyzed using SeqMan . The conversion rate was approxinately 95%. We have fully sequenced and characterized 71 clones.Figure S1CaMK mouse lines. Specific signal detected with radiolabeled oligonucleotides for rtTA2-M2 (left) and non-specific signal (right). (B) 9TB-Dox induced, rtTA-dependent Cre expression in different brain regions (rtTA2-M2CaMK lines crossed to LC-1 responders). Abbreviations: Cx (cortex), Hi (hippocampus), Ce (cerebellum), St (striatum) and Ob (olfactory bulb). Scale bars, 2 mm (A) and 1 mm (B).Expression of rtTA mRNA and Cre protein in the brain of different mouse lines. (A) Detection of rtTA-M2 mRNA in the brain slices of different rtTA2-M2(6.30 MB TIF)Click here for additional data file.Figure S2CaMK) (left panel) and double-positive (with tTACaMK) (right panel).Forebrain-specific, tTA-dependent gene activation in responder mice MTH-Cg2-17 and MTH-Cg2-19.  single-positive (without tTA(8.22 MB TIF)Click here for additional data file.Figure S3CaMK-2 and (B) with tTACaMK. Scale bar, 500 µm.9TB-Dox induced, rtTA-dependent gene activation in MTH-Cg2-19. (A) with rtTA-M2(9.49 MB TIF)Click here for additional data file.Figure S4tetbi. (A) The bidirectional tetracycline-responsive promoter (Ptetbi) is depicted and CpG sites are indicated as open circles. (B) Sequence analysis of 71 individual clones is plotted with the number of methylated CpG detected (y-axis) against the number of independent clones (x-axis). The actual data set collected is based on methylation detected from sequencing (red) and the calculated data is based on 5% C-to-T conversion loss (blue). Three of seventy-one clones (4%) are strongly methylated.Methylation of the P(0.47 MB TIF)Click here for additional data file.Figure S5tetbi methylation analyses.Notes on P(0.02 MB DOC)Click here for additional data file.


Drosophila olfactory system, projection neurons (PNs) send dendrites to single glomeruli in the antenna lobe (AL) based upon lineage and birth order and send axons with stereotyped terminations to higher olfactory centers. These decisions are likely specified by a PN-intrinsic transcriptional code that regulates the expression of cell-surface molecules to instruct wiring specificity.Precise connections of neural circuits can be specified by genetic programming. In the longitudinals lacking (lola), which encodes a BTB-Zn-finger transcription factor with 20 predicted splice isoforms, results in wiring defects in both axons and dendrites of all lineages of PNs. RNA in situ hybridization and quantitative RT-PCR suggest that most if not all lola isoforms are expressed in all PNs, but different isoforms are expressed at widely varying levels. Overexpression of individual lola isoforms fails to rescue the lola null phenotypes and causes additional phenotypes. Loss of lola also results in ectopic expression of Gal4 drivers in multiple cell types and in the loss of transcription factor gene lim1 expression in ventral PNs.We find that the loss of lola is required for wiring of axons and dendrites of most PN classes, and suggest a need for its molecular diversity. Expression pattern changes of Gal4 drivers in lola-/- clones imply that lola normally represses the expression of these regulatory elements in a subset of the cells surrounding the AL. We propose that Lola functions as a general transcription factor that regulates the expression of multiple genes ultimately controlling PN identity and wiring specificity.Our results indicate that  Drosophila olfactory system is achieved during pupal development before the onset of olfactory receptor expression . Int Figure . Lim1 exDrosophila olfactory projection neurons is regulated by intrinsic TFs    and injeUAS-mCD8a::GFP  hsFlpCD8; FRT42D, Gal80/FRT42D, lolax, GH146, UAS-mCD8a::GFP [where x denotes any lola allele] (control GH146 MARCM) hsFlpCD8; FRT42D, Gal80/FRT42D, GH146, UAS-mCD8a::GFP  hsFlpCD8; FRT42D, Gal80/FRT42D, lolaorc4, GH146, UAS-mCD8a::GFP (control Mz19 MARCM) hsFlpCD8; Gal4-Mz19, UAS-mCD8a::GFP, FRT42D, Gal80/FRT42D  hsFlpCD8; Gal4-Mz19, UAS-mCD8a::GFP, FRT42D, Gal80/FRT42D, lolax (UAS-lola A misexpression) UAS-lola A/hsFlp122, UAS-mCD8a::GFP; FRT42D, Gal80/FRT42D, Gal4-GH146, UAS-mCD8a::GFP (UAS-lola L (X) misexpression) UAS-lola T/hsFlp122, UAS-mCD8a::GFP; FRT42D, Gal80/FRT42D, Gal4-GH146, UAS-mCD8a::GFP (UAS-lola T (III) misexpression) hsFlp122, UAS-mCD8a::GFP; FRT42D, Gal80/FRT42D, Gal4-GH146, UAS-mCD8a::GFP; UAS-lola T (UAS-lola L misexpression) hsFlp122, UAS-mCD8a::GFP; FRT42D, Gal80/FRT42D, Gal4-GH146, UAS-mCD8a::GFP; UAS-lola L (UAS-lola A rescue) UAS-lola A/hsFlp122, UAS-mCD8a::GFP; FRT42D, Gal80/FRT42D, lolaORE76, Gal4-GH146, UAS-mCD8a::GFP (UAS-lola T rescue) UAS-lola T/hsFlp122, UAS-mCD8a::GFP; FRT42D, Gal80/FRT42D, lolaORE76, Gal4-GH146, UAS-mCD8a::GFP (UAS-lola L rescue) hsFlp122, UAS-mCD8a::GFP; FRT42D, Gal80/FRT42D, lolaORE76, Gal4-GH146, UAS-mCD8a::GFP; UAS-lola L  hsFlp122, UAS-mCD8a::GFP;FRT42D, Gal80/FRT42D, lolax;Gal4-NP3529.Genotypes of flies generated in experiments are as follows: (Lola protein expression time course and Lim1 WT staining) yw; Gal4-GH146, MARCM was performed as previously described ,35. ClonAntibody staining was performed as previously described ,39. The White y, w; Gal4-GH146, UAS-CD8::GFP pre-pupa (considered 0 h APF) were collected, covered with optimal cutting temperature compound , frozen on dry ice and stored at -80°C not longer than 1 month. Blocks were sectioned on a cryostat and sections containing ALs were mounted on PEN membrane slides (Leica #11505158) and stored on dry ice. Slides were put through an EtOH dehydration series , treated for 2 minutes with xylenes, dried and mounted for LCM capture. PN enriched samples as identified by GFP fluorescence were immediately captured into PCR tube caps on a Leica LCM microscope (model ASLMD) using Laser Microdissection software version 4.4. Tubes were frozen on dry ice and stored at -80°C up to one day. RNA was extracted using the Arcturus PicoPure™ RNA Isolation kit  as per the manufacturer's instructions.lola isoforms. The Taqman probes were synthesized by Integrated DNA Technologies, Inc. . Microfluidic matrix chips  were used to perform real time PCR assays in a high throughput fashion. Each chip allows complete combinatorial tests  between N independent cDNA samples and N distinct primer pairs [The cRNA samples were used as the template for another round of reverse transcription , producing a high yield of cDNA. The cDNA samples were aliquoted and stored at -20°C before use. Unique primer pairs and Taqman probes were designed for individual er pairs . A separlola were designed to the common region and isoform specific exons (for primers see Table S6 in additional file in situ hybridization is available in additional file Probes for S Quake is a co-founder, consultant, and equity holder of Fluidigm Corporation. All other authors declare that they have no competing interests.in situ protocol and participated in in situ hybridization experiments. Jian Liu helped conceive of and design and participated in LCM RT-PCR experiments. Stephen Quake helped conceive of and participated in the design and analysis of LCM RT-PCR experiments. Edward Giniger helped revise the manuscript and provided initial reagents and intellectual input for the study. Takaki Komiyama originally conceived of the study, performed initial PN phenotype characterization of the lolaORE5D2 allele and helped revise the manuscript. Liqun Luo participated in the overall design and coordination of the study and helped write the manuscript. All authors have read and approved the final manuscript.Maria Spletter designed the study, wrote the manuscript, and performed or participated in all experiments. Justin Liu participated in Lola antibody staining experiments, Gal4-GH146 driver characterization at larval time-points, and MARCM experiments. Helen Su helped develop the lola mutant allele MARCM analysis demonstrating dendritic targeting defects. Supplemental Figure S1 showing targeting defects in lolaore5D2, lolaorc4, and lolaore119 mutant PNs.Additional Click here for filelola-/- and lolaORE5D20-/-, a complete summary of DL1 dendrite and axon phenotypes by allele as percentage affected, a complete summary of vPN dendritic phenotypes by allele as percentage targeted correctly, clonal frequencies for MARCM experiments and primer sequences for in situ hybridization probes.Supplemental Tables S1-S6. Supplemental tables and legends referenced in the main text. This includes a semi-quantitative analysis of axon targeting defects in Click here for filelola isoform in situ in the Drosophila brain. Supplemental Figure S2 showing controls and verification for our in situ technique and additional lola isoform hybridization results.Additional Click here for filelola isoform RT-PCR analysis in the Drosophila brain. Supplemental Figure S3 showing addition LCM RT-PCR experiments comparing lola isoform expression at different developmental time points and in different brain tissues.Additional Click here for fileUAS-lola overexpression on adPN and lPN dendrites and axons. Supplemental Figure S4 showing adPN and lPN phenotypes in MARCM clones in a wild-type background expressing UAS-lola A, UAS-lola L and UAS-lola T.Effects of Click here for filelola-/-, UAS-lola MARCM clones. Supplemental Figure S5 showing adPN and lPN phenotypes in lola-/- clones expressing UAS-lola A, UAS-lola L and UAS-lola T.adNB and lNB phenotypes in Click here for filein situ hybridization in fly larval/pupal/adult tissues. Our detailed protocol for performing RNA in situ hybridization on Drosophila at later developmental time points.Protocol for RNA Click here for fileLegends for supplemental Figures S1-S5. All supplemental figure titles and legends.Click here for file


The pyrazolopyrimidine derivatives 7 and 10 are synthesized via reaction of the aminopyrazole 4b with phenylisothiocyanate and DMFDMA/NH4OAc respectively.3-Oxo-2-arylhydrazononitriles  Thus, compounds 1a-c, were prepared according to literature procedures via coupling of ethyl cyanoacetate with aromatic diazonium salts pyrimidine 10 on treatment with AcOH/NH4OAc mixture. (cf. Compound ure. cf. 1 reacted with hydroxylamine hydrochloride in ethanol/sodium acetate solution to yield amidooxime 11 as in the literature [12 utilizing the reaction conditions described earlier in literature [11 cyclizes smoothly via loss of ethanol in DMF and in presence of anhydrous sodium acetate into isoxazolone 13. (cf. Compound terature . Trials  13. cf. 1a-c are valuable precursors to 4-amino-5-substituted-1-aryl-1H-pyrazole-3-carboxylic acid ethyl ester which can be used for preparation of sildenafil analogues.We could show that arylhydrazononitriles File 1The experimental section. The experimental data and the results of analysis


Direct evidence of a role for the brain-RAS in Parkinson's disease (PD) comes from studies demonstrating the neuroprotective effect of RAS inhibitors in several neurotoxin based PD models. In this study, we show that an antagonist of the angiotensin II (Ang II) type 1 (AT+ (5 μM) induced DA neuronal loss by 72% in vitro. Mice challenged with MPTP showed a 62% reduction in the number of DA neurons in the SNpc and a 71% decrease in tyrosine hydroxylase (TH) immunostaining of the striatum, whereas daily treatment with losartan lessened MPTP-induced loss of DA neurons to 25% and reduced the decrease in striatal TH+ immunostaining to 34% of control.In the presence of exogenous Ang II, losartan reduced MPP1 receptor as a potential novel target for neuroprotection.Our study demonstrates that the brain-RAS plays an important neuroprotective role in the MPTP model of PD and points to AT Parkinson's disease (PD) was originally described in 1817 by James Parkinson and since then there has been much progress in determining the etiology of the disease . Insight1 and AT2. The AT1 receptor was originally identified by the selective binding of a non-peptide receptor antagonist, 2-n-butyl-4-chloro-5-hydroxy-methyl-1-[(2'-(1H)-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazol potassium salt , whereas the AT2, receptor was identified by the selective binding of a non-peptide receptor antagonist, PD123319 imidazol potassium salt  (1 μM) (AT1 receptor antagonist)  or PD123319 (1 μM) (AT2 receptor antagonist) (Sigma) or both, 15 min prior to the addition of Ang II [VM cultures were exposed to the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium  were used. Mice (n = 4–5 per group) received subcutaneous (200 μl) injections of 2-n-Butyl-4-chloro-5-hydroxy-methyl-1-[(2'-(1H)-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazol potassium salt  (90 mg/kg)  daily for 16 days, beginning two days prior to the first MPTP injection  using a Vectastain kit  or by immunofluorescence using an appropriate secondary antibody. Different cell types in the cultures were identified with a polyclonal (mouse) anti-glial fibrillary acidic protein (GFAP) (1:200) for astrocytes, a polyclonal (mouse) anti-nestin (1:400) for neural progenitors, and a polyclonal (mouse) anti-microtubule-associated protein 2 (MAP2) (1:200) or a polyclonal (mouse) anti-neuronal nuclear protein (NeuN) (1:200) for neuronal cell types . To determine AT receptor subtypes, a commercially available polyclonal (rabbit) anti-AT1 (N-10) (1:50) , polyclonal (goat) anti-AT2 (H-143) (1:50) , polyclonal anti-AT1(rabbit) (18801) (1:200) (Abcam), polyclonal (rabbit) anti-AT2 (C-18) (1:100)  or a polyclonal anti-AT2 (rabbit) (19134) (1:200) (Abcam) antibodies were used  DA. Nonspecific [3H] DA accumulation was determined in the presence of 1 mM (-) cocaine hydrochloride. After 10 min of incubation at 37°C, uptake was terminated by quickly washing the neurons three times with 1 ml of ice-cold KRH. Neurons were then solubilized in 0.5 ml of 3% trichloroacetic acid for 60 min with gentle shaking. Accumulated [3H] DA was determined by liquid scintillation counting (n = 3) [Dissociated mesencephalic neurons were grown as described above. The neurons were rinsed and then assayed at 37°C in Krebs-Ringer HEPES buffer  .Losartan (0.18 mg/mL) was administered through the drinking water and tail artery blood pressures were recorded non-invasively from awake animals. Animals were placed in a restraining tube heated to 37°C, and a pressure cuff and piezoelectric transducer were secured to the rostral base of the tail . Animals were conditioned for ten to fifteen minutes by inflating the pressure cuff to 200 mmHg once every minute. Post-conditioning pulse and cuff pressure recordings were simultaneously acquired for up to 45 min at one reading per minute. Motion artifact detected by the pulse transducer during cuff deflation served as grounds for elimination of data points. Systolic and diastolic pressures were determined offline . Systolic pressure was defined as cuff pressure upon first detection of pulse during deflation. Diastolic pressure was defined as cuff pressure when the pulse reading resumed normal amplitude .+ profiles that came into focus within the counting frame thickness (13 μm) between the guard zones were counted. Care was taken to ensure that the top and bottom forbidden planes were not included in the analysis. Adjacent sections were stained with cresyl violet acetate and cells in the SNpc were counted in a similar manner as with the TH+ immunostained sections, with the exception that large nuclear Nissl stained cells were counted instead of TH+ cells. Because Nissl staining was done separately from TH immunostaining, it is possible that non-dopaminergic neurons and glia were also included in the total cell counts. Because of the careful demarcation of the SNpc  densitometry readings were subtracted from the measured densitometry of striatal areas. Measurements are represented as percent of control densities from saline-treated animals and are represented as mean ± SEM. Densitometric quantification of striatal TH+ staining was done by a blinded observer.Striatal sections were stained for TH as described above. Digital images were taken of each striatal section  using a Nikon Eclipse 800 microscope and a CCD camera with Spot software. TH2 asphyxiation. The brains were removed and immediately frozen in isopentane on dry ice. Brains were then stored at -80°C until analysis. Brains were then cut on a cryostat and 10 μm coronal sections were mounted on uncoated slides and stored at -80°C. Slides were allowed to briefly reach room temperature and were stained for TH using a rapid staining protocol to minimize mRNA degradation. Briefly, slides were fixed in acetone (40 seconds), washed briefly 3 times in phosphate buffer , followed by a 5 min incubation with a mouse-anti-TH antibody  diluted in PB. Slides were washed 3 times with PB, followed by 5 minute incubation with a Cy3-conjugated goat anti-mouse IgG  diluted in PB. Slides were washed twice in PB and then dehydrated in ethanol , (10 seconds each) and delipidated in xylene (40 seconds). Slides were then allowed to air dry. All procedures were carried out in an RNAse-free environment.Twelve-week-old male C57BL/6 mice were euthanized using COTyrosine hydroxylase positive cells of the SNpc and VTA were visualized and dissected using a PixCell laser capture microscope under a 20× objective with an infrared diode laser . Approximately 500 TH positive cells per animal from the SNpc or VTA we isolated on a single LCM HS cap . RNAs were isolated using a PicoPure RNA Isolation kit  according to manufacturer's guidelines, and subjected to DNAse treatment .+ neurons from the SNpc or VTA. Primers used are listed in table Atgr2, TH and GAPDH mRNA expression profiles were done by quantitative real-time PCR on TH1 and AT2 receptors was examined in an immortalized mesencephalic dopaminergic cell line, N27 [1 receptors , AT2 receptors . Secondary, alkaline phosphatase-conjugated anti-rabbit  and anti-mouse  antibodies were followed by Lumi-Phos (Pierce) for chemiluminescence detection. Blots were stripped and reprobed for β-actin  to confirm equal protein loading on the gel.The expression of ATine, N27 . N27 celData were analyzed using Statistica software. Statistical significance was determined by one-way ANOVA followed by post-hoc Newman-Keuls multiple comparison tests. Differences in mean values were considered significant at p ≤ 0.05. All data were obtained from at least three independent experiments and are represented as a mean ± SEM.1 receptor, Ang II type 1 receptor; Agtr2, gene encoding AT2 receptor; BP, blood pressure; CE, coefficient of error; CV, coefficient of variance; DA, dopamine or dopaminergic; DAT, dopamine transporter; E15, embryonic day 15; GDNF, glial cell line-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; GPCR, G-protein coupled receptor; HBSS, Hank's buffered salt solution; LCM, laser capture microdissection; MANF, mesencephalic astrocytes-derived neurotrophic factor; MAP2, microtubule-associated protein 2; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPP+, 1-methyl-4-phenylpyridinium; NeuN, neuronal nuclear protein; NIH, National Institutes of Health; PD, Parkinson's disease; PKC, protein kinase C; PLA2, phospholipase 2; RAS, renin-angiotensin-system; RT-PCR, reverse transcriptase polymerase chain reaction; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase; VM, ventral mesencephalon; VTA, ventral tagmental area.ACE, angiotensin converting enzyme; Ang II, angiotensin II; ANOVA, analysis of variance; ATThe author(s) declare that they have no competing interests.All authors read and approved the final manuscript. TNG, WMZ and JAW designed the research. TNG and WMZ wrote the paper. TNG, SMJ, FAA, BRH, LDS, AMP, JS and VVJ performed the research.


Ki-67 is a monoclonal antibody which recognises a human nuclear antigen expressed in proliferating cells. The antibody was used to assess proliferation in primary human bladder tumours from 64 patients. Ki-67 index  was derived from 59 tumours. A wide range of Ki-67 indices were recorded, range 3.0-65.8%, mean 20.2%. The Ki-67 index correlated with known prognostic factors: T stage (P = 0.002) and histological grade (P less than 0.001), early stage disease and more differentiated tumours having lower Ki-67 indices. Patients with invasive disease (21 patients) had significantly higher Ki-67 indices than those with non-invasive disease (P = 0.01). Patients with metastatic disease at presentation (four cases) all had a Ki-67 index of greater than or equal to 29%. Ki-67 antibody staining is a simple technique for assessing the proliferation fraction than can be performed on a small amount of tissue taken at routine biopsy without prior injection of thymidine analogues.


Recent studies suggest a high specificity of 99mTc-galactosyl neoglycoalbumin (99mTc-NGA) receptor scanning in vivo by providing both morphological and functional diagnosis of liver disease. In 22 patients with advanced breast cancer 99mTc-NGA  was exclusively trapped by the liver, the images showing 'cold spots' in areas of liver metastases formation. A two-tailed analysis was performed: the time activity curves recorded for the liver and precordial area were subjected to a kinetic receptor-calculating model allowing an estimation of the NGA-receptor concentration of the liver  as well as calculation of the residual functional liver volume (RFLV) via the S.P.E.C.T.-study. In breast cancer patients with liver metastases a significantly (P < 0.01) lower HBP-concentration was estimated (0.65 +/- 0.16 vs 0.82 +/- 0.17 mumol l-1) as evidenced by a lower 99mTc-NGA-accumulation in the liver resulting also in a significantly (P < 0.001) lower RFLV (739 +/- 348 vs 1336 +/- 184 ml). In four amonafide-treated patients (800 mg m-2 intravenous infusion over 3 h) approximately one week after one chemotherapy cycle a significant (P < 0.05) increase in HBP-concentration (0.56 +/- 0.10 vs 0.72 +/- 0.06 mumol l-1) of the liver was found corresponding with an increase in RVLF (546 +/- 297 vs 670 +/- 265 ml). These regulatory mechanisms at the HBP level measured in vivo provide further evidence that 99mTc-NGA should have promise as a clinically useful receptor radiopharmaceutical for both quantification of liver function and assessment of liver morphology.


Hypoxia-induced mitogenic factor (HIMF), a lung-specific growth factor, promotes vascular tubule formation in a matrigel plug model. We initially found that HIMF enhances vascular endothelial growth factor (VEGF) expression in lung epithelial cells. In present work, we tested whether HIMF modulates expression of fetal liver kinase-1 (Flk-1) in endothelial cells, and dissected the possible signaling pathways that link HIMF to Flk-1 upregulation.Recombinant HIMF protein was intratracheally instilled into adult mouse lungs, Flk-1 expression was examined by immunohistochemistry and Western blot. The promoter-luciferase reporter assay and real-time RT-PCR were performed to examine the effects of HIMF on Flk-1 expression in mouse endothelial cell line SVEC 4–10. The activation of NF-kappa B (NF-κB) and phosphorylation of Akt, IKK, and IκBα were examined by luciferase assay and Western blot, respectively.Intratracheal instillation of HIMF protein resulted in a significant increase of Flk-1 production in lung tissues. Stimulation of SVEC 4–10 cells by HIMF resulted in increased phosphorylation of IKK and IκBα, leading to activation of NF-κB. Blocking NF-κB signaling pathway by dominant-negative mutants of IKK and IκBα suppressed HIMF-induced Flk-1 upregulation. Mutation or deletion of NF-κB binding site within Flk-1 promoter also abolished HIMF-induced Flk-1 expression in SVEC 4–10 cells. Furthermore, HIMF strongly induced phosphorylation of Akt. A dominant-negative mutant of PI-3K, Δp85, as well as PI-3K inhibitor LY294002, blocked HIMF-induced NF-κB activation and attenuated Flk-1 production.These results suggest that HIMF upregulates Flk-1 expression in endothelial cells in a PI-3K/Akt-NF-κB signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis. Flt-1-deficient embryos showed an overgrowth of endothelial cells, disorganization of blood vessels , IKKβ [IKKβ (K44A)], IκBα super-repressor [IκBα (S32A/S36A)] and phosphatidylinositol 3-kinase (PI-3K) dominant negative mutant (Δp85) were previously described ,19,20. HAF153057) were amplified by PCR from genomic DNA obtained from SVEC 4–10 and subcloned into the KpnI-HindIII site of pGL3-Basic , a firefly luciferase reporter vector. Mutagenesis and deletion of NF-κB binding site within Flk-1 promoter were performed using the GeneTailor Site-Directed Mutagenesis System (Invitrogen). Mutation and deletion oligonucleotides for NF-κB binding site were designed as follows: forward mutation 5'-TATCGATAGGTACCGGACGCACCGAGTCCCCACCCCT, forward deletion 5'-TATCGATAGGTACCGGACGCACCCCACCCCT, reverse 5'-TGCGTC CGGTACCTATCGATAGAG AAATGTT. The DNA constructs were verified by sequence analysis. The NF-κB firefly luciferase reporter vector, pNFκB-Luc , is designed to measure the binding of transcription factors to the κ enhancer. It contains five tandem repeats of NF-κB binding sites (TGGGGACTTTCCGC) as promoters upstream of the luciferase transcription start site in the vector. The expression of luciferase gene in the reporter plasmid is controlled by these NF-κB binding sequences. Only when there is activated NF-κB in the nucleus (translocated NF-κB), the luciferase transcription and translation start. By measuring the luciferase activity in the transfected cell lysats, it provides an indirect evidence of NF-κB activation in the nucleus. Cells were co-transfected with each reporter construct and the renilla luciferase vector pRL-TK , with or without HIMF protein stimulation, and then treated with passive lysis buffer according to the dual-luciferase assay manual (Promega). The luciferase activity was measured with a luminometer . The firefly luciferase signal was normalized to the renilla luciferase signal for each individual analysis to eliminate the variations of transfection efficiencies.Mouse Flk-1 5'-flanking regions  from each sample was subjected to 4–20% pre-cast polyacrylamide gel (Bio-Rad) electrophoresis and transferred to nitrocellulose membranes (Bio-Rad), and then probed with rabbit anti-mouse antibodies against phospho-specific and non-phosphorylated IKK, IκBα, Akt, ERK1/2, p38 kinase, and JNK mitogen-activated protein kinase (MAPK) , followed by 1:3000 dilution of goat anti-rabbit HRP-labeled antibody (Bio-Rad). ECL substrate kit (Amersham) was used for the chemiluminscent detection of the signals with autoradiography film (Amersham).P < 0.05) was determined by t test or analysis of variance (ANOVA) followed by assessment of differences using SigmaStat 2.03 software .Unless otherwise stated, all data were shown as mean ± standard error of the mean (SEM). Statistical significance , which contains a luciferase gene driven by the Flk-1 5'-upstream proximal promoter. The reporter plasmid was transiently transfected into SVEC-HIMF, which resulted in higher Flk-1 promoter activities than those of its counterparts Fig. . HIMF trSince HIMF enhances Flk-1 expression at transcriptional level, we further explored the possible transcription factor(s) involved in Flk-1 gene expression regulation. We generated a series of luciferase reporter constructs containing different deletion segments of mouse Flk-1 promoter sequence , includiIt has been reported that HIMF activates PI-3K/Akt signaling pathway in lung epithelial cells . It is uEndothelial cell tyrosine kinase receptors are of fundamental importance in transmission of both differentiation and angiogenic signals from the extracellular environment to the endothelium. Five endothelial cell-specific tyrosine kinase receptors, each of which has a specific role in blood vessel formation, have been identified. These include Tie-1, Tie-2 , Flt-1, Flt-4, and Flk-1/KDR . While tcis-regulatory elements, including Sp1-, AP-2-, NF-κB-, and GATA-binding sites [Both mouse (Flk-1) and human (KDR) genes reveal a class II promoter structure, characterized by the absence of a TATA box and by the presence of several conserved ng sites ,29. The ng sites . In adding sites . It has ng sites , while Tng sites . These fThe stimulating effects of HIMF on Flk-1 upregulation in SVEC 4–10 cells can only maintain for 24 hours. The dramatic decrease of NF-κB activity at 48 hour time point might be a result of HIMF degradation because we only administered the HIMF protein at the beginning of the experiment. These effects parallel with the activation of IKK and increased PI-3K activities as we showed that blocking IKK or PI-3K abolished HIMF-induced NF-κB activity and decreased Flk-1 mRNA production. The quick degradation or lost activity of HIMF further indicates that HIMF is a cytokine-like molecule and an early response gene to hypoxia, inflammation or other stress related stimuli ,14.NF-κB is composed of heterodimers of DNA-binding subunits (p50 and p52) and subunits with transcriptional activity  . In unstPhosphatidylinositol 3-kinase (PI-3K) is a heterodimer of an 85-kDa (p85) adaptor subunit and a 100-kDa (p110) catalytic subunit . PI-3K aIn summary, the current studies indicated that HIMF enhances Flk-1 expression in mouse lung tissues and endothelial cells in a PI-3K/Akt-NF-κB signaling pathway-dependent manner, which at least in part, elucidated the molecular mechanisms of transcriptional regulation of the Flk-1/KDR gene and contributed to our better understanding of the functions of HIMF in pulmonary angiogenesis and maintenance of pulmonary vascular homeostasis.


Immunoscintigraphy with radiolabelled anti-carcinoembryonic antigen monoclonal antibody was performed on 21 patients with active small-cell lung cancer. Patients received either In-111-labelled Mab F6 F(ab')2 fragments or Tc-99m-labelled BW 431/26 intact antibody. Tumour was imaged in 13 patients (62%). Of 38 known sites of disease, 18 sites were positively detected. Serum CEA levels were known in 19 patients and were abnormally elevated in three . Eight of 15 patients with normal serum CEA had positive imaging. Using the In-111-labelled antibody seven out of ten patients (nine out of 18 sites) gave positive scans; with the Tc-99m-labelled antibody these were obtained in nine out of 18 patients (nine out of 20 sites).