The role of monoammonium glycyrrhizinate as a methane inhibitor to limit the rumen methane emissions of Karakul sheep.

Methane (CH4) from ruminant production systems produces greenhouse gases that contribute to global warming. Our goal was to determine whether monoammonium glycyrrhizinate could inhibit CH4 emissions over the long term without affecting animal performance and immune indices in Karakul sheep. This study aimed to assess the effects of medium-term (60 days) addition of monoammonium glycyrrhizinate on growth performance, apparent digestibility, CH4 emissions, methanogens, fibre-degrading bacteria and blood characteristics in Karakul sheep. Twelve male Karakul sheep (40.1 ± 3.59 kg) with fistula were randomly divided into two groups (n = 6): the Control group received a basal diet + the same volume of distilled water (30 ml) and the Treatment group received a basal diet + 8.75 g/kg monoammonium glycyrrhizinate injected via fistula. The adaptation stage was 15 days, and the measurement stage was 60 days. The sampling during the measurement stage was divided into two stages, stage I (1 ∼ 30 d) and stage II (31 ∼ 60 d). The results showed that monoammonium glycyrrhizinate significantly reduced the relative abundance of Bacteroides caccae, daily CH4 emission and protozoa population, significantly increased the relative abundance of Lachnospiraceae bacterium AD3010, Lachnospiraceae bacterium FE2018, Lachnospiraceae bacterium NK3A20, Lachnospiraceae bacterium NK4A179 and Lachnospiraceae bacterium V9D3004 in stage I (P < 0.05); significantly increased the relative abundance of Lachnospiraceae bacterium AD3010, but significantly decreased the relative abundance of Lachnospiraceae bacterium NK4A179 and Lachnospiraceae bacterium C6A11 in stage II (P < 0.05). Therefore, monoammonium glycyrrhizinate could be used as a CH4 inhibitor to limit the rumen CH4 emissions of Karakul sheep in short-term period (30 days) without affecting the growth performance, fibre digestibility and blood parameters.

A comparison of the pregnancy outcomes between ultrasound-guided high-intensity focused ultrasound ablation and laparoscopic myomectomy for uterine fibroids: a comparative study.

Objective: To compare the pregnancy outcomes between ultrasound-guided high-intensity focused ultrasound (USgHIFU) ablation and laparoscopic myomectomy (LM).Materials and methods: This study included 676 women with symptomatic uterine fibroids who wished to become pregnant underwent USgHIFU or LM at three hospitals in China from 1 May 2009 to 31 May 2018. The related information of pregnancy and delivery were followed up and analyzed using the chi-square test and two-sided Student t-test.Results: The median follow-up duration was 5 (1-8) years; 20 patients (2.9%) were lost to follow-up. 320 patients were treated with UsgHIFU, and 336 were treated with LM. Two hundred nineteen (68.4%) women became pregnant after USgHIFU ablation, and 224 (66.7%) became pregnant after LM. Four hundred forty-three patients had 501 pregnancies (natural pregnancies, 405; in vitro fertilisation-embryo transfer pregnancies, 38). Average times to pregnancy were 13.6 ± 9.5 months after USgHIFU and 18.9 ± 7.3 months after LM (p < 0.05). The rate of cesarean delivery was lower in the USgHIFU group (41.6%) than in the LM group (54.9%) (p < 0.05). Incidences of placenta increta, placenta previa, and postpartum hemorrhage were low after USgHIFU compared with after LM. Incidences of preterm birth, fetal distress, fetal growth restriction, and puerperal infection were higher after USgHIFU than after LM. There was a risk of uterine rupture after both procedures.Conclusions: Compared with LM, USgHIFU ablation can significantly shorten the time to pregnancy, although pregnancy rates of the two procedures are similar. Some risks in pregnancy and delivery after HIFU should be evaluated and monitored.

Identification and preclinical development of an anti-proteolytic uPA antibody for rheumatoid arthritis.

Blocking the proteolytic capacity of urokinase-type plasminogen activator (uPA) with a monoclonal antibody (mAb) reduces arthritis progression in the collagen-induced mouse arthritis model to an extent that is on par with the effect of blocking tumor necrosis factor-alpha by etanercept. Seeking to develop a novel therapy for rheumatoid arthritis, a humanized mAb, NNC0266-0043, was selected for its dual inhibition of both the zymogen activation and the proteolytic capacity of human uPA. The antibody revealed nonlinear elimination kinetics in cynomolgus monkeys consistent with binding to and turnover of endogenous uPA. At a dose level of 20.6 mg kg-1, the antibody had a plasma half-life of 210 h. Plasma uPA activity, a pharmacodynamic marker of anti-uPA therapy, was reduced to below the detection limit during treatment, indicating that an efficacious plasma concentration was reached. Pharmacokinetic modeling predicted that sufficient antibody levels can be sustained in arthritis patients dosed subcutaneously once weekly. The anti-uPA mAb was also well tolerated in cynomolgus monkeys at weekly doses up to 200 mg kg-1 over 4 weeks. The data from cynomolgus monkeys and from human material presented here indicates that anti-uPA mAb NNC0266-0043 is suitable for clinical testing as a novel therapeutic for rheumatic diseases. KEY MESSAGES: Background: Anti-uPA therapy is on par with etanercept in a mouse arthritis model. A new humanized antibody blocks activation and proteolytic activity of human uPA. The antibody represents a radically novel mode-of-action in anti-rheumatic therapy. The antibody has PK/PD properties in primates consistent with QW clinical dosing.

Construction of a versatile expression library for all human single-pass transmembrane proteins for receptor pairings by high throughput screening.

Interactions between protein ligands and receptors play crucial roles in cell-cell signalling. Most of the human cell surface receptors have been identified in the post-Human Genome Project era but many of their corresponding ligands remain unknown. To facilitate the pairing of orphan receptors, 2762 sequences encoding all human single-pass transmembrane proteins were selected for inclusion into a mammalian-cell expression library. This expression library, consisting of all the individual extracellular domains (ECDs), was constructed as a Fab fusion for each protein. In this format, individual ECD can be produced as a soluble protein or displayed on cell surface, depending on the applied heavy-chain Fab configuration. The unique design of the Fab fusion concept used in the library led to not only superior success rate of protein production, but also versatile applications in various high-throughput screening paradigms including protein-protein binding assays as well as cell binding assays, which were not possible for any other existing expression libraries. The protein library was screened against human coagulation factor VIIa (FVIIa), an approved therapeutic for the treatment of hemophilia, for binding partners by AlphaScreen and ForteBio assays. Two previously known physiological ligands of FVIIa, tissue factor (TF) and endothelial protein C receptor (EPCR) were identified by both assays. The cell surface displayed library was screened against V-domain Ig suppressor of T-cell activation (VISTA), an important immune-checkpoint regulator. Immunoglobulin superfamily member 11 (IgSF11), a potential target for cancer immunotherapy, was identified as a new and previously undescribed binding partner for VISTA. The specificity of the binding was confirmed and validated by both fluorescence-activated cell sorting (FACS) and surface plasmon resonance (SPR) assays in different experimental setups.

GFRAL is the receptor for GDF15 and is required for the anti-obesity effects of the ligand.

Growth differentiation factor 15 (GDF15; also known as MIC-1) is a divergent member of the TGF-ß superfamily and is associated with body-weight regulation in humans and rodents. However, the cognate receptor of GDF15 is unknown. Here we show that GDF15 binds specifically to GDNF family receptor α-like (GFRAL) with high affinity, and that GFRAL requires association with the coreceptor RET to elicit intracellular signaling in response to GDF15 stimulation. We also found that GDF15-mediated reductions in food intake and body weight of mice with obesity were abolished in GFRAL-knockout mice. We further found that GFRAL expression was limited to hindbrain neurons and not present in peripheral tissues, which suggests that GDF15-GFRAL-mediated regulation of food intake is by a central mechanism. Lastly, given that GDF15 did not increase energy expenditure in treated mice with obesity, the anti-obesity actions of the cytokine are likely driven primarily by a reduction in food intake.

Sensitive and specific in situ hybridization for early drug discovery.

High-throughput analyses of gene expression such as microarrays and RNA-sequencing are widely used in early drug discovery to identify disease-associated genes. To further characterize the expression of selected genes, in situ hybridization (ISH) using RNA probes (riboprobes) is a powerful tool to localize mRNA expression at the cellular level in normal and diseased tissues, especially for novel drug targets, where research tools like specific antibodies are often lacking.We describe a sensitive ISH protocol using radiolabelled riboprobes suitable for both paraffin-embedded and cryo-preserved tissue. The riboprobes are generated by in vitro transcription using PCR products as templates, which is less time consuming compared to traditional transcription from linearized plasmids, and offers a relatively simple way to generate several probes per gene, e.g., for splice variant analyses. To ensure reliable ISH results, we have incorporated a number of specificity controls in our standard experimental setup. We design antisense probes to cover two non-overlapping parts of the gene of interest, and use the corresponding sense probes as controls for unspecific binding. Probes are furthermore tested on sections of paraffin-embedded or cryo-preserved positive and negative control cells with known gene expression. Our protocol thus provides a method for sensitive and specific ISH, which is suitable for target validation and characterization in early drug discovery.

The C-terminal region of activation-induced cytidine deaminase is responsible for a recombination function other than DNA cleavage in class switch recombination.

Activation-induced cytidine deaminase (AID) is an essential factor for the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. CSR and SHM are initiated by AID-induced DNA breaks in the S and V regions, respectively. Because truncation or frame-shift mutations at the carboxyl (C)-terminus of AID abolishes CSR but not SHM, the C-terminal region of AID likely is required for the targeting of DNA breaks in the S region. To test this hypothesis, we determined the precise location and relative amounts of AID-induced DNA cleavage using an in situ DNA end-labeling method. We established CH12F3-2 cell transfectants expressing the estrogen receptor (ER) fused with wild-type (WT) AID or a deletion mutant lacking the C-terminal 16 aa, JP8Bdel. We found that AID-ER, but not JP8Bdel-ER, caused a CSR to IgA from the addition of 4-hydroxy tamoxifen. In contrast, both WT AID and JP8Bdel induced DNA breaks in both the V and S regions. In addition, JP8Bdel enhanced c-myc/IgH translocations. Our findings indicate that the C-terminal domain of AID is not required for S-region DNA breaks but is required for S-region recombination after DNA cleavage. Therefore, AID does not distinguish between the V and S regions for cleavage, but carries another function specific to CSR.

The transcriptional repression activity of KyoT2 on the Notch/RBP-J pathway is regulated by PIAS1-catalyzed SUMOylation.

The LIM domain protein KyoT2 negatively regulates the Notch signaling pathway through interaction with RBP-J, the core element of the Notch signaling pathway in the nucleus. Here we show that PIAS1 (the protein inhibitor of activated STAT1) interacts with KyoT2 directly and attenuates KyoT2-mediated transcriptional repression. We demonstrate that KyoT2 is modified by SUMOylation at two lysine residues, K144 and K171. SUMOylation of the transfected KyoT2 is enhanced by PIAS1 but not hPc2, another KyoT2-interacting protein with SUMO E3 ligase activity, and is repressed by a PIAS1 mutant that is deficient of E3 ligase activity. Using mutants disrupting either or both of the SUMO sites, we show that SUMOylation of KyoT2 does not influence its expression, intracellular localization, or interaction with known partners. However, disruption of the K171 SUMOylation site does reinforce the transcriptional repression activity of KyoT2, suggesting that SUMOylation of this site counters the repression activity of KyoT2. Finally, we show that PIAS1 fails to attenuate the repression activity of the K171R mutant of KyoT2, suggesting that PIAS1 may potentially antagonize the transcriptional repression activity of KyoT2 through catalyzing its SUMOylation at K171. These results suggest that KyoT2 is a substrate of SUMO modification catalyzed by PIAS1, and that SUMOylation may modulate the transcriptional repression effect of KyoT2 on the Notch/RBP-J signaling pathway.

Identification of CD36 as a new surface marker of marginal zone B cells by transcriptomic analysis.

Follicular (FO) B cells and marginal zone (MZ) B cells belong to the mature B cell population in spleen of mice. To identify new surface markers of these mature B cell subsets, we compared gene expression profiles of FO and MZ B cells by DNA microarray using FACS-sorted mouse FO and MZ B cells. From 14,000 mouse genes, 27 membrane proteins were expressed mainly in MZ B cells while another 22 membrane proteins expressed largely in FO B cells. Using FACS analysis, we identified that CD36, CD68, and CD49e were expressed on MZ B cells but not on FO B cells. In addition, using semi-quantitative PCR, we found that the mRNA of CD131 were much more abundant in MZ B cells. These results revealed new phenotypic properties of MZ and FO B cells, and would facilitate further studies in the differentiation and functions of these mature B cells.

Identification of a specific domain required for dimerization of activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is essential to all three genetic alterations required for generation of antigen-specific immunoglobulin: class switch recombination, somatic hypermutation, and gene conversion. Here we demonstrate that AID molecules form a homodimer autonomously in the absence of RNA, DNA, other cofactors, or post-translational modifications. Studies on serial deletion mutants revealed the minimum region between Thr27 and His56 responsible for dimerization. Analyses of point mutations within this region revealed that the residues between Gly47 and Gly54 are most important for the dimer formation. Functional analyses of these mutations indicate that all mutations impairing the dimer formation are inefficient for class switching, suggesting that dimer formation is required for class switching activity. Dimer formation and its requirement for the function of AID are features that AID shares with APOBEC-1, an RNA editing enzyme of apolipoprotein B100 mRNA.

Mouse follicular and marginal zone B cells show differential expression of Dnmt3a and sensitivity to 5'-azacytidine.

Mature B cells in the spleen of mouse and human comprise of two main subsets, the follicular (Fo) B cells and the marginal zone (MZ) B cells. In this study, we report that Fo and MZ B cells express different levels of DNA methyltransferase Dnmt3a. By using RT-PCR and immunohistochemistry, we found that Fo B cells expressed high level of Dnmt3a while MZ B cells expressed little. Treatment of mice by in vivo administration of 5'-azacytidine, an inhibitor of DNA methyltransferases, induced B cell loss in both the bone marrow and the spleen. We noticed that this treatment resulted in a much faster and more severe disappearance of Fo B cells than MZ B cells in the spleen. Further analysis showed that MZ progenitors increased significantly in mice treated with 5'-azacytidine. These results suggest that epigenetic mechanisms involving Dnmt3a might participate in the development of B cells including the differentiation of Fo B cells and MZ B cells in the periphery.

The Spen homolog Msx2-interacting nuclear target protein interacts with the E2 ubiquitin-conjugating enzyme UbcH8.

The nuclear matrix protein Msx2-interacting nuclear target protein (MINT) is a transcription factor that regulates the expression of key transcriptional effectors in diverse signaling pathways. To further understand the function and mechanism of the MINT-mediated transcription regulation, the yeast two-hybrid system was employed to screen proteins that interact with the C-terminal fragment of MINT. From a cDNA library of human lymph nodes, a cDNA encoding the ubiquitin-conjugating enzyme UbcH8 was identified. Using different truncated versions of MINT, we show that the C-terminal Spen paralog and ortholog C-terminal domain (SPOC) domain, which has been demonstrated to mediate interactions between MINT and a panel of other molecules, might be responsible for interaction between MINT and UbcH8 in yeast, as confirmed by the beta-galactosidase assay. The interaction between MINT and UbcH8 in mammalian cells was further proved by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, and co-immunoprecipitation assay. Using a reporter system, we found that MINT-mediated transcription suppression was sensitive to MG132, an inhibitor of the proteosome system. These results suggest a novel mechanism of MINT-mediated transcription regulation, and might be helpful for understanding functions of MINT.

Structure of the polyketide cyclase SnoaL reveals a novel mechanism for enzymatic aldol condensation.

SnoaL belongs to a family of small polyketide cyclases, which catalyse ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. Several of these antibiotics are among the most used anti-cancer drugs currently in use. The crystal structure of SnoaL, involved in nogalamycin biosynthesis, with a bound product, has been determined to 1.35 A resolution. The fold of the subunit can be described as a distorted alpha+beta barrel, and the ligand is bound in the hydrophobic interior of the barrel. The 3D structure and site-directed mutagenesis experiments reveal that the mechanism of the intramolecular aldol condensation catalysed by SnoaL is different from that of the classical aldolases, which employ covalent Schiff base formation or a metal ion cofactor. The invariant residue Asp121 acts as an acid/base catalyst during the reaction. Stabilisation of the enol(ate) intermediate is mainly achieved by the delocalisation of the electron pair over the extended pi system of the substrate. These polyketide cyclases thus form of family of enzymes with a unique catalytic strategy for aldol condensation.

RING1 inhibits transactivation of RBP-J by Notch through interaction with LIM protein KyoT2.

The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1.

[Characterization of Notch gene involved in genetic regulatory networks in dental pulp stem cells].

OBJECTIVE: To investigate the characterization of Notch gene involved in genetic regulatory networks in dental pulp stem cells. METHODS: The pulp tissue was separated from mouse teeth and digested by collagenase type I. Single-cell suspensions of dental pulp were seeded into 6-well plates with alpha modification of Eagle's medium supplemented with ES cell qualified Fetal Bovine Serum. Colony-forming efficiency was assessed in 14ds culture. Transcripts for Notch were detected by reverse transcription-PCR by using total RNA isolated from cells. RESULTS: There were clonogenic cells in dental pulp cell and the incidence of colony-forming cells derived from mouse dental pulp cells was 1.6-2.5 colonies/10(4) plate. Mouse-specific Notch mRNA expressed in colony-forming cells. CONCLUSION: Notch mRNA expressing in colony-forming cells provided a more detailed understanding of mouse dental pulp stem cell biology.

[Interaction between HTLV-1 transcription activator tax and Taxreb107].

Tax is a transcription activator encoded by human T-cell leukemia virus (HTLV)-1. Ribosomal protein L6 was also defined as Taxreb107 (Tax responsible element binding protein 107) for its activity of binding to the long terminal repeats of HTLV-1. To investigate the relationship between Tax and Taxreb107/RpL6, yeast two hybrid and GST pull-down assays were used. Results suggest that Tax can interact with Taxreb107/RpL6 directly and Taxreb107/RpL6 may regulate the function of Tax in HTLV-1 proliferation.

Cloning of mouse genomic ribosomal protein L6 gene and analysis of its promoter.

Ribosomal protein (Rp) L6 is also defined as Taxreb107 (Tax responsive element binding protein 107) for its binding activity to the long terminal repeats of human T cell leukemia virus (HTLV)-I. We cloned the genomic gene of mouse RpL6/Taxreb107 and analyzed its exon/intron structures. The promoter of RpL6/Taxreb107 contains recognition sites for multiple transcription factors including nuclear factor (NF)-kappa B. Luciferase reporter assay showed that the RpL6/Taxreb107 promoter has a constitutive activity in transfected cells, and the constitutive activity depends on the intact promoter. Expression of HTLV-I viral protein Tax mildly but reproducibly induced RpL6/Taxreb107 mRNA and promoter activity. We provide evidence suggesting that induction of RpL6/Taxreb107 by Tax is at least partially mediated by the NF-kappa B site in the promoter of RpL6/Taxreb107. Taken together, Tax up-regulates RpL6/Taxreb107 and this may provide a feedback mechanism to facilitate proliferation of HTLV-I-infected cells and production of viral particles.

[Screening and detecting of proteins interaction with KyoT].

To study the function of KyoT2 in vivo, two-hybrid yeast, purification of KyoT2 protein, preparation of antibody and GST-pulldown methods were used in the experiments. 42 clones were obtained after 5 x 10(6) clones were screened by four kinds of nutrition limitation and beta-galactosidase assay, 22 clones were obtained after restriction of positive clones. Finally, 13 genes were obtained by sequence assay. Two of these were RBP-Jk and PIAS1. After they and KyoT2 changed vectors, negative two-hybrid yeast was finished. The result was positive; Using KyoT2 protein and antibody GST-pulldown of KyoT2 and RBP-Jk, KyoT2 and PIAS1 were done, the result was also positive. Therefore, KyoT2 interacted with RBP-Jk and PIAS1.

Expression of LIM Protein KyoT in Adult Mouse Testis.

The KyoT expression in the adult mouse was reported here for further investigation on the functions of KyoT in adult mouse. To study the expression of mRNA and protein of KyoT, Northern blot, RT-PCR, Immunohistochemical SABC methods and in situ hybridization methods were used in the experiments. Two kinds of KyoT were expressed at high levels in testis of adult mouse, and KyoT immunore activity was mainly located in Leydig's cells. The reactive substance was distributed in cytoplasm rather than in nuclei. KyoT mRNA hybridization signals were also detected in cytoplasm of Leydig's cells rather than in nuclei. The spermatogenic cells and negative controls showed negative results. These results suggest that KyoT was expressed in testis of adult mouse and mainly located in Leydig's cells.