Activation of human STING by a molecular glue-like compound.

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

[Performance Recoverability of Denitrifying Granular Sludge Under the Stressing Effect of Nanoscale Zero-valent Iron].

To explore the potential stressing effect of nanoscale zero-valent iron (nZVI) on denitrifying granular sludge (DGS), the evolution of DGS denitrifying performance under different C/N ratios was investigated in this study, by carrying out batch tests of eight successive periods with the nZVI shock-loading. The results showed that the specific denitrification rate of µ value decreased when the nZVI dosage was higher than 5 mg · L⁻¹. Meanwhile, a positive correlation between the inhibition ratio (IR) of µ value and substrate C/N ratios or nZVI dosage was observed. When the nZVI dosage reached 100 mg · L⁻¹, both extracellular protein and polysaccharides concentrations decreased obviously. It would be beneficial to promote the recovery of DGS denitrifying activity and reduce the COD demanding to remove unit mass of nitrate, by increasing external carbon source with C/N ratios of higher than 4. On the basis of Freundlich and Langmuir adsorption isotherms, when higher C/N ratio was provided, stronger bioadsorption of nZVI would be achieved. During the recovery period, a significant improvement of DCS denitrifying performance under the high C/N ratio was expected, due to the continuous washout of total iron in sludge phase (Qe), while the µ value would reach or approach the one of the control group when Qe was lower than 0.4 mg · g⁻¹.

(R)-2-Phenylpyrrolidine Substituted Imidazopyridazines: A New Class of Potent and Selective Pan-TRK Inhibitors.

Deregulated kinase activities of tropomyosin receptor kinase (TRK) family members have been shown to be associated with tumorigenesis and poor prognosis in a variety of cancer types. In particular, several chromosomal rearrangements involving TRKA have been reported in colorectal, papillary thyroid, glioblastoma, melanoma, and lung tissue that are believed to be the key oncogenic driver in these tumors. By screening the Novartis compound collection, a novel imidazopyridazine TRK inhibitor was identified that served as a launching point for drug optimization. Structure guided drug design led to the identification of (R)-2-phenylpyrrolidine substituted imidazopyridazines as a series of potent, selective, orally bioavailable pan-TRK inhibitors achieving tumor regression in rats bearing KM12 xenografts. From this work the (R)-2-phenylpyrrolidine has emerged as an ideal moiety to incorporate in bicyclic TRK inhibitors by virtue of its shape complementarity to the hydrophobic pocket of TRKs.

Discovery of GNF-5837, a Selective TRK Inhibitor with Efficacy in Rodent Cancer Tumor Models.

Neurotrophins and their receptors (TRKs) play key roles in the development of the nervous system and the maintenance of the neural network. Accumulating evidence points to their role in malignant transformations, chemotaxis, metastasis, and survival signaling and may contribute to the pathogenesis of a variety of tumors of both neural and non-neural origin. By screening the GNF kinase collection, a series of novel oxindole inhibitors of TRKs were identified. Optimization led to the identification of GNF-5837 (22), a potent, selective, and orally bioavailable pan-TRK inhibitor that inhibited tumor growth in a mouse xenograft model derived from RIE cells expressing both TRKA and NGF. The properties of 22 make it a good tool for the elucidation of TRK biology in cancer and other nononcology indications.

Epithelial progenitor 1, a novel factor associated with epithelial cell growth and differentiation.

The growth and renewal of epithelial tissue is a highly orchestrated and tightly regulated process occurring in different tissue types under a variety of circumstances. We have been studying the process of pancreatic regeneration in mice. We have identified a cell surface protein, named EP1, which is expressed on the duct epithelium during pancreatic regeneration. Whereas it is not detected in the pancreas of normal mice, it is found in the intestinal epithelium of normal adult mice, as well as during pancreatic repair following cerulein-induced destruction of the acinar tissue. The distinctive situations in which EP1 is expressed, all of which share in common epithelial cell growth in the gastrointestinal tract, suggest that EP1 is involved in the growth and renewal of epithelial tissues in both the intestine and the pancreas.

The ITK-SYK fusion oncogene induces a T-cell lymphoproliferative disease in mice mimicking human disease.

Peripheral T-cell lymphomas (PTCL) constitute a major treatment problem with high mortality rates due to the minimal effectiveness of conventional chemotherapy. Recent findings identified ITK-SYK as the first recurrent translocation in 17% of unspecified PTCLs and showed the overexpression of SYK in more than 90% of PTCLs. Here, we show that the expression of ITK-SYK in the bone marrow of BALB/c mice causes a T-cell lymphoproliferative disease in all transplanted mice within 8 weeks after transplantation. The disease was characterized by the infiltration of spleen, lymph nodes, bone marrow, and skin with CD3+CD4+CD8- and CD3+CD4-CD8- ITK-SYK-positive T-cells accompanied by a systemic inflammatory reaction with upregulation of interleukin 5 and INF-gamma. ITK-SYK-positive T-cells showed enhanced apoptosis resistance and INF-gamma production in vitro. The disease was serially transplantable, inducing clonal T-cell expansion in secondary recipients. The action of ITK-SYK in vivo was dependent on SYK kinase activity and disease development could be inhibited by the treatment of mice with SYK inhibitors. Interestingly, the translocation of ITK-SYK from the membrane to the cytoplasm, using a point mutation in the pleckstrin homology domain (ITK-SYK R29C), did not abolish, but rather, enhanced disease development in transplanted mice. CBL binding was strongly enhanced in membrane-associated ITK-SYK E42K and was causative for delayed disease development. Our results show that ITK-SYK causes a T-cell lymphoproliferative disease in mice, supporting its role in T-cell lymphoma development in humans. Therefore, pharmacologic inhibition of SYK in patients with U-PTCLs carrying the ITK-SYK fusion protein might be an effective treatment strategy.

Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors.

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

FGFR3 is a negative regulator of the expansion of pancreatic epithelial cells.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are key signaling molecules for pancreas development. Although FGFR3 is a crucial developmental gene, acting as a negative regulator of bone formation, its participation remains unexplored in pancreatic organogenesis. We found that FGFR3 was expressed in the epithelia in both mouse embryonic and adult regenerating pancreata but was absent in normal adult islets. In FGFR3 knockout mice, we observed an increase in the proliferation of epithelial cells in neonates, leading to a marked increase in islet areas in adults. In vitro studies showed that FGF9 is a very potent ligand for FGFR3 and activates extracellular signal-related kinases (ERKs) in pancreatic cell lines. Moreover, FGFR3 blockade or FGFR3 deficiency led to increased proliferation of pancreatic epithelial cells in vivo. This was accompanied by an increase in the proportion of potential islet progenitor cells. Thus, our results show that FGFR3 signaling inhibits the expansion of the immature pancreatic epithelium. Consequently, this study suggests that FGFR3 participates in regulating pancreatic growth during the emergence of mature islet cells.

PYY in the expanding pancreatic epithelium.

Gut peptide YY (PYY) plays an important role in regulating metabolism and is expressed during the ontogeny of the pancreas. However, its biological role during endocrine cell formation is not fully understood, and its role, if any, during pancreatic regeneration in the adult has not yet been explored. The knowledge of factors involved in beta cell renewal in adult animals is clearly relevant for the design of treatment strategies for type 1 diabetes. We therefore sought to determine if observations during fetal pancreas formation also apply to pancreatic growth in adult animals. Indeed, we have found marked expansion of the PYY-expressing population during pancreatic regeneration. In addition, we demonstrate the presence of cells co-expressing PYY and the critical pancreatic transcription factor pancreatic duodenal homeobox1 (PDX-1). Interestingly, these cells also co-expressed specific islet hormones during pancreatic development and re-growth, suggesting a developmental relationship. Furthermore, we have found that PYY can act in concert with IGF-1 to stimulate cellular responsiveness in pancreatic epithelial cells in vitro. Our data suggest that PYY may be a mediator of islet cell development, as well as a cofactor for growth factor responses, not only during fetal pancreas formation but also during regeneration in adult animals.

[Association of fibrinogen B beta -1420G/A, -993C/T and -854G/A gene polymorphism with coronary heart disease].

OBJECTIVE: To analyze the frequency of FGB gene -1420G/A, -993C/T and -854G/A polymorphisms, and their association with plasma fibrinogen levels in patients with coronary heart disease and in health adults. METHODS: The FGB gene -1420G/A, -993C/T and -854G/A polymorphisms were analyzed with restriction fragment length polymorphisms, polymerase chain reaction with allele-specific primer and nucleotide sequencing methods. Plasma fibrinogen levels were determined by turbidimetry. RESULTS: The frequencies of -1420A were 0.33 in patients with coronary heart disease and 0.26 in health adults. The frequencies of -1420A in coronary heart disease were apparently higher than that in health adults. There were no difference in frequencies of other two alleles. The logistic study suggested -1420G/A polymorphism was associated with coronary heart disease. There are significantly difference in plasma fibrinogen levels in two groups. Plasma fibrinogen levels were significantly increased in patients with coronary heart disease. CONCLUSION: This study suggests -1420G/A polymorphism may be associated with occurrence of coronary heart disease.

[Changes and clinical significance of serum soluble Apo-1/Fas in pancreatic cancer].

OBJECTIVE: To detect changes of serum soluble Apo-1/Fas (sApo-1/Fas) in pancreatic cancer patients and to investigate its clinical value in assessing the effect of chemotherapy. METHODS: The serum level of sApo-1/Fas in 30 normal control subjects and 58 pancreatic cancer patients were detected using enzyme-linked immunosorbent assay (ELISA), and the sApo-1/Fas level of 48 pancreatic cancer patients, before and after chemotherapy was compared. RESULTS: Compared with the level of the control group, the level of serum soluble Apo-1/Fas was significantly correlated with clinical stage but not with age, sex or pathologic type of pancreatic cancer. It was elevated gradually from stage II to IV (P < 0.01). However, it would obviously decrease in pancreatic cancer patients after chemotherapy (P < 0.01). CONCLUSION: The serum soluble Apo-1/Fas may be involved in the development of pancreatic cancer, and it may be used as one parameter to assess the disease status and prognosis of pancreatic cancer patient.

[A related analysis for alpha, beta fibrinogen gene haplotypes and nucleotide polymorphisms associated with the ischemic stroke in Hainan Han population].

OBJECTIVE: To analyze the association of that the polymorphisms and haplotypes of Taq I site in beta fibrinogen gene and the single nucleotide sites -455 G/A, -249 C/T, -148 C/T, +1689T/G, Bsm A I G/C, 448 G/A, Bcl I G/A, Hinf I A/C in beta-fibrinogen gene are linked up with the ischemic stroke(IS). METHODS: Turbidmetric assay was used to measure the plasma fibrinogen level of one hundred and sixty cases with ischemic stroke and one hundred and thirty healthy individuals from Hainanese Han population. The polymorphisms and genotypes were characterized by PCR-RFLP. Hardy-Weinberg equilibrium and statistical differences of allelic, genotype and haplotype frequencies were obtained by Chi-square test. Pairwise linkage disequilibrium was calculated and haplotypes of nine or four polymorphisms were estimated by the EH + program. RESULTS: There were highly significant differences in genotype frequencies and allelic frequencies of the polymorphisms -455 G/A, -148 C/T, 448 G/A, which happened between the IS group and control subjects (P< 0.01). However, the significant differences of the allelic frequencies in the other six polymorphisms were not found between the IS group and the control (P> 0.05). The odds ratio(OR) with the rare alleles of A -455, T -148 and A 448 is 2.46, 2.30 and 2.08 (95% confidence interval 1.153%-3.924%, 1.429%-3.694% and 1.298%-3.329%) respectively. No definite haplotype block was found by linkage disequilibrium analysis in the control group and the IS group. Association of haplotypes constructed from the nine polymorphisms with IS was not found. Among the haplotypes constructed from four polymorphisms including -455 G/A, -148 C/T, 448 G/A alleles, haplotype differences were found between the control group and the IS group. Haplotypes with G -455, C -148, G448 alleles appeared more frequently in control group(P< or = 0.01), whereas haplotypes with A -455, T -148, A 448 occurred more frequently in the IS group(P< 0.01). CONCLUSION: The results of multi-allele and haplotype analysis indicated that the polymorphisms -455 G/A, -148 C/T, 448 G/A in beta fibrinogen gene were the possible risk factors associated with the occurrence of ischemic stroke in Hainan Han population.

[Nine polymorphisms of fibrinogen gene and their association with plasma fibrinogen levels in Hainan Han population].

OBJECTIVE: To investigate the allelic frequencies of polymorphisms of alpha Taq I and beta Bcl I, Hinf I A/C, 448 G/A, beta BsmA I G/C, +1689T/G, -148C/T, -249C/T, -455G/A in Hainan Han population and their association with plasma fibrinogen level. METHODS: Turbidmetric assay was used to measure plasma fibrinogen level of two hundred and thirty-eight healthy individuals. The genotypes were characterized by PCR-RFLP and sequence analysis. The relationships between the genotypes and plasma fibrinogen levels were analyzed by t test and ANOVA. RESULTS: The frequencies of the rare alleles of alpha Taq I and beta Bcl I, Hinf I A/C, 448 G/A, beta BsmA I G/C, +1689T/G, -148C/T, -249C/T, -455G/A polymorphisms were 0.445, 0.239, 0.134, 0.235, 0.273, 0.241, 0.265, 0.441, 0.254 respectively. In the general population, the plasma fibrinogen level is significantly higher in the groups of genotypes -455GA and AA, -148CT and TT, alpha Taq I T1T1 than in the group of wild types(P=0.004, 0.015 and 0.043 respectively). In the men, plasma fibrinogen level is significantly higher in the groups of genotypes -455GA and AA, -148CT and TT, alpha Taq I T1T1, alpha Taq I T1T2 than in the group of wild types(P=0.001, 0.023, 0.003 and 0.032 respectively). In the women, no significant genotype association with plasma fibrinogen level was detected. CONCLUSION: There was linkage disequilibrium between the fibrinogen gene loci. The beta -455G/A beta 448G/A, alpha Fg Taq I polymorphisms were associated with the difference in plasma fibrinogen in men. A(-455), T(-148) and alpha Taq I T1 alleles were associated with higher fibrinogen levels.

Inhibition of activin signaling induces pancreatic epithelial cell expansion and diminishes terminal differentiation of pancreatic beta-cells.

Activins regulate the growth and differentiation of a variety of cells. During pancreatic islet development, activins are required for the specialization of pancreatic precursors from the gut endoderm during midgestation. In this study, we probed the role of activin signaling during pancreatic islet cell development and regeneration. Indeed, we found that both activins and activin receptors are upregulated in duct epithelial cells during islet differentiation. Interestingly, the expression of endogenous cellular inhibitors of activin signaling, follistatin and Cripto, were also found to be augmented. Inhibition of activins significantly enhanced survival and expansion of pancreatic epithelial cells but decreased the numbers of differentiated beta-cells. Our results suggest that the homeostasis of growth and terminal differentiation requires a precise context-dependent regulation of activin signaling. Follistatin participates in this process by promoting expansion of precursor cells during pancreas growth.

The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas.

The SDF-1alpha/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1alpha and CXCR4 expression in fetal pancreas. We have found that SDF-1alpha and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) gamma-nonobese diabetic mouse. We show that SDF-1alpha stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1alpha on ductal cell migration. Importantly, blocking the SDF-1alpha/CXCR4 axis in IFNgamma-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1alpha-CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration.

[Clinical value of serum soluble Apo-1/Fas for predicting biological behaviors and prognosis of gastric carcinoma].

BACKGROUND & OBJECTIVE: Literatures reported that the soluble Apo-1/Fas(sApo-1/Fas) levels in serum of patients with malignant carcinoma were higher than that in normal control subject, but there were fewer studies was seldom to detect the level of sApo-1/Fas in patients with malignancy carcinoma and effect of chemotherapy; the subject is to detect the level of sApo-1/Fas in patients with gastric carcinoma and effect of chemotherapy on it, and to investigate its clinical value. METHODS: Enzyme linked immunosorbent assays(ELISA) was available to detect the level of sApo-1/Fas in 42 case of patients with gastric carcinoma before and after chemotherapy, as compared with 30 case of normal control subject. RESULTS: Levels of sApo-1/Fas were elevated in all subgroups of patients with gastric carcinoma as compared to the controls (P < 0.01), sApo-1/Fas was correlated with clinical stage and histological grade, and not with sex, age; the sApo-1/Fas level in stage IV was higher in comparison with stage III and II (P < 0.05-0.01), and in stage III it was higher than in stage II (P < 0.05); being lower in the well differentiated and moderately differentiated than the poorly differentiated(P < 0.05-0.01), that the sApo-1/Fas levels were remarkably reduced in complete remission or partial remission patients(P < 0.01) after chemotherapy. CONCLUSION: sApo-1/Fas may play closely reflect growth and regulation in gastric carcinoma, it may be a predictor for biological behaviors and prognosis of gastric carcinoma; sApo-1/Fas may be a new target in treating gastric carcinoma.

[Effect of beta-Fibrinogen-455 Gene Polymorphism on Plasma Fibrinogen Levels in Patients with Ischemic Stroke]

In large prospective studies, plasma fibrinogen levels have been shown to be an independent risk factor of vascular disease, including ischemic stroke. Elevated plasma fibrinogen in an individual could be due to the presence of predisposing genetic and/or environmental factors, such as smoking. Of the polymorphisms studies to date, the beta-fibrinogen-455 (beta-Fg-455) G-->A substitution in the 5' flanking region is associated with the most consistent difference in plasma fibrinogen levels in both case-control studies and in selected groups of healthy individuals. In order to further elucidate the role of the beta-Fg-455 G-->A substitution in determining fibrinogen levels and susceptibility to ischemic stroke in case-control population, including 104 individuals with verified ischemic stroke and 156 healthy individuals. Turbidimetriy assays were used to measure plasma fibrinogen levels of all samples. The beta-Fg-455 G-->A mutation was identified by the polymerase chain reaction followed by restriction enzyme digestion of the amplified DNA with HaeIII. The plasma fibrinogen level in patients with ischemic stroke [(3.51 +/- 1.09) g/L] was significantly higher than that in the control [(3.08 +/- 0.71) g/L] (P < 0.01). The A-allele is associated with elevated fibrinogen levels in both patients and controls. The plasma fibrinogen levels in controls with A-allele in elder people were higher than in younger people (P < 0.05). Those with A allele in males of ischemic stroke had significantly higher plasma fibrinogen levels in smokers than in non-smokers and ex-smokers (P < 0.05), but it was not significantly difference in subjects of GG genotype (P > 0.05). Our data demonstrates an association of the beta-Fg promoter A-455 allele with higher fibrinogen levels in the general population, and suggests that the A-allele may be a susceptible predictor of ischemic stroke, particularly in aging and smoking.