Competitive Microarray Screening Reveals Functional Ligands for the DHX15 RNA G-Quadruplex.

RNAs are increasingly considered valuable therapeutic targets, and the development of methods to identify and validate both RNA targets and ligands is more important than ever. Here, we utilized a bioinformatic approach to identify a hairpin-containing RNA G-quadruplex (rG4) in the 5' untranslated region (5' UTR) of DHX15 mRNA. By using a novel competitive small molecule microarray (SMM) approach, we identified a compound that specifically binds to the DHX15 rG4 (K D = 12.6 ± 1.0 µM). This rG4 directly impacts translation of a DHX15 reporter mRNA in vitro, and binding of our compound (F1) to the structure inhibits translation up to 57% (IC50 = 22.9 ± 3.8 µM). This methodology allowed us to identify and target the mRNA of a cancer-relevant helicase with no known inhibitors. Our target identification method and the novelty of our screening approach make our work informative for future development of novel small molecule cancer therapeutics for RNA targets.

Mechanistic Analysis of Riboswitch Ligand Interactions Provides Insights into Pharmacological Control over Gene Expression.

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

Group 2 innate lymphoid cells constrain type 3/17 lymphocytes in shared stromal niches to restrict liver fibrosis.

Group 2 innate lymphoid cells (ILC2s) cooperate with adaptive Th2 cells as key organizers of tissue type 2 immune responses, while a spectrum of innate and adaptive lymphocytes coordinate early type 3/17 immunity. Both type 2 and type 3/17 lymphocyte associated cytokines are linked to tissue fibrosis, but how their dynamic and spatial topographies may direct beneficial or pathologic organ remodelling is unclear. Here we used volumetric imaging in models of liver fibrosis, finding accumulation of periportal and fibrotic tract IL-5 + lymphocytes, predominantly ILC2s, in close proximity to expanded type 3/17 lymphocytes and IL-33 high niche fibroblasts. Ablation of IL-5 + lymphocytes worsened carbon tetrachloride-and bile duct ligation-induced liver fibrosis with increased niche IL-17A + type 3/17 lymphocytes, predominantly γδ T cells. In contrast, concurrent ablation of IL-5 + and IL-17A + lymphocytes reduced this progressive liver fibrosis, suggesting a cross-regulation of type 2 and type 3 lymphocytes at specialized fibroblast niches that tunes hepatic fibrosis.

Interferon gamma constrains type 2 lymphocyte niche boundaries during mixed inflammation.

Allergic immunity is orchestrated by group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells prominently arrayed at epithelial- and microbial-rich barriers. However, ILC2s and Th2 cells are also present in fibroblast-rich niches within the adventitial layer of larger vessels and similar boundary structures in sterile deep tissues, and it remains unclear whether they undergo dynamic repositioning during immune perturbations. Here, we used thick-section quantitative imaging to show that allergic inflammation drives invasion of lung and liver non-adventitial parenchyma by ILC2s and Th2 cells. However, during concurrent type 1 and type 2 mixed inflammation, IFNγ from broadly distributed type 1 lymphocytes directly blocked both ILC2 parenchymal trafficking and subsequent cell survival. ILC2 and Th2 cell confinement to adventitia limited mortality by the type 1 pathogen Listeria monocytogenes. Our results suggest that the topography of tissue lymphocyte subsets is tightly regulated to promote appropriately timed and balanced immunity.

B cell-activating factor enhances interleukin-6 and interleukin-10 production by ODN-activated human B cells.

We aim to investigate the additive value of B cell-activating factor (BAFF) when added to oligodeoxynucleotides (ODN)-activated B cells with respect to TLR-9, CD69, MHC-II expression, IL-6 and IL-10 secretion and B cell cycling. Therefore, B cells from healthy individuals were incubated under the following conditions: (1) B cells with medium, (2) B cells with ODN 0.5 µm, (3) B cells with BAFF 20 µm and (4) B cells with both ODN 0.5 µm and BAFF 20 µm. We found that addition of BAFF did not enhance the expression of TLR-9, CD69 and MHC-II in ODN-activated B cells. Incubation of B cells with BAFF and ODN together leads to a marked elevation of IL-6 and IL-10 levels compared to ODN alone. Synthesis and mitosis were higher in B cells stimulated by BAFF than in B cells stimulated by ODN. These findings suggest that both BAFF and TLR-9 contribute independently to B cell function.

The autoreactivity of B cells in hereditary angioedema due to C1 inhibitor deficiency.

Patients with hereditary angioedema (HAE) tend to produce autoantibodies and have a propensity to develop immunoregulatory disorders. We characterize the profile of autoantibodies in a group of HAE patients and investigate their memory B cells' phenotype and activation status. We studied the activity status phenotype, Toll-like receptor (TLR)-9 expression and total phosphotyrosine in B cells isolated from HAE patients. Additionally, the following autoantibodies were assessed in the serum of 61 HAE patients: anti-nuclear, rheumatoid factor, anti-cardiolipin, anti-tissue transglutaminase, anti-endomysial, anti-Saccharomyces cerevisiae, anti-thyroid and anti-neutrophil cytoplasmic antibodies. In 47·5% of HAE patients we detected at least one of the tested autoantibodies. Expression of CD69, CD5 and CD21 was found to be significantly higher on memory B cells from HAE patients compared to healthy controls (4·59 ± 4·41 versus 2·06 ± 1·81, P = 0·04, 8·22 ± 7·17 versus 3·65 ± 3·78, P = 0·05, 2·43 ± 0·54 versus 1·92 ± 0·41, P = 0·01, respectively). Total phosphotyrosine in B cells from HAE patients was significantly higher compared to healthy controls (4·8 ± 1·1 versus 2·7 ± 1·3, P = 0·0003). Memory B cells isolated from the HAE group contained higher amounts of TLR-9 compared to healthy controls (8·17 ± 4·1 versus 4·56 ± 1·6, P = 0·0027). Furthermore, the expression of TLR-9 in memory B cells from HAE patients with autoantibodies was significantly higher than the control group (10 ± 4·7 versus 4·56 ± 1·6, P = 0·0002) and from that in HAE patients without autoantibodies (10 ± 4·7 versus 5·8 ± 0·9, P = 0·036). HAE patients have enhanced production of autoantibodies due most probably to the increased activation of B cells, which was found to be in association with a high expression of TLR-9.

The role of regulatory T cells in familial Mediterranean fever (FMF).

The role of regulatory T cells (T-regs) in familial Mediterranean fever (FMF) was never evaluated. Preliminary studies that we have conducted suggested a rise in the number of regulatory T cells after FMF attacks reaching a maximal level at 7 days. The aim of this study was to evaluate the percentage and activity of regulatory T cells in FMF. Six patients with refractory FMF and six healthy controls were evaluated. The percentage of T-reg cells and forkhead box protein 3 (Foxp3) expression was evaluated and compared between four states: FMF in remission, FMF at the first day of an attack, FMF 7 days after the start of the attack, and healthy controls. Four females and two males were included. All patients had FMF with high severity score, 2.8 ± 0.4 (0-3). The mean age was 31.6 ± 6.2. The mean age at onset was 9.3 ± 9.3. The mean colchicine dose was 2.6 mg ± 0.4. The expression of Foxp3 7 days after the attacks was significantly higher than in FMF at the first day of the attack, FMF in remission, and healthy controls 10.08 ± 2.36 vs. 7.005 ± 0.3 vs. 5.3 ± 1.06 vs. 4.44 ± 1.8; p < 0.05 (Fig.1). The percentage of T-regs in peripheral blood was not statistically different between the four groups. Theexpression of Foxp3 by T-regs increases 7 days after attacks of FMF. Anti-inflammatory cytokines interleukin-10 and TGF-ß are known to activate T-regs and have been reported to increase in FMF attacks in line with the present findings. It is suggested that T-regs may have a role in terminating FMF attacks.

Increased susceptibility of cord blood B lymphocytes to undergo spontaneous apoptosis.

In this study, we compared the rate of spontaneous apoptosis of B cells from umbilical cord blood with adult B cells and assessed the role of Bcl-2, CD5, interleukin (IL)-4 and B cell-activating factor in B cell spontaneous apoptosis. We found that spontaneous apoptosis of cultured B cells, as assessed by utilizing annexin-V binding, was significantly higher in cord blood than in healthy adult individuals (77.5; 95 CI, 73.5-81.5 versus 59.2; 95 CI, 54-64, respectively, P < 0.0001) and further confirmed by 4' 6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining. Whereas the expression of B cell-activating factor from the tumour necrosis factor family (BAFF) receptor mRNA was similar in B cells from adults and cord blood, we detected lower levels of circulating BAFF in the serum of cord blood (0.68 +/- 0.13 ng versus 1.83 +/- 0.54 ng, P = 0.01). The latter may explain, in part, our observation of lower levels of mean fluorescence intensity of Bcl-2 in cord B cells compared with adults (1.6 +/- 0.9 versus 2.85 +/- 1.3, P = 0.033). CD19(+) CD5(+) B cells from cord blood underwent a lower rate of apoptosis in comparison to CD19(+) CD5(-) B cells (25.1 +/- 9.3%versus 58.5 +/- 12.5%, P < 0.0001). This pattern of sensitivity was comparable in adult blood (15 +/- 5.5%versus 22.7 +/- 9.3%, P = 0.01). Nevertheless, the rate of apoptosis was higher in CD19(+) CD5(+) from cord blood compared to CD19(+) CD5(+) from adults (25.1 +/- 9.3%versus 15 +/- 5.5%, P = 0.0013). The addition of rIL-4 (10 u/ml) to cultured cells decreased B cell apoptosis in a similar fashion in both cord and adults blood. This rescue was strengthened when BAFF (100 microg/ml) was further added. Thus, alterations in Bcl-2 or serum BAFF level may explain the increased rate of cord blood B cell apoptosis.

Effect of injected sample amount on the shape of chromatographic peaks under condition of linear partition isotherm.

In a previous paper a model function was tested in order to approximate the peak shape obtained on non-polar column by injecting different compounds. The simulation of the symmetrical or non-symmetrical shape of gas chromatographic peaks was satisfactory. In this paper, the influence of the amount of injected substance was investigated at different values of inlet pressure and carrier gas velocity, in order to evaluate the relative contribution to the total peak area and shape of the symmetrical distribution due to partition phenomena and of the non-symmetrical and tailing distribution due to adsorption-desorption kinetics. The effect of the molecular mass and of the chain length of compounds belonging to the homologous series of 1-alcohols and n-alkanes on the adsorption phenomena was evaluated.

Regulation of L-type calcium channels in pituitary GH(4)C(1) cells by depolarization.

The neurosecretory anterior pituitary GH(4)C(1) cells exhibit the high voltage-activated dihydropyridine-sensitive L-type and the low voltage-activated T-type calcium currents. The activity of L-type calcium channels is tightly coupled to secretion of prolactin and other hormones in these cells. Depolarization induced by elevated extracellular K(+) reduces the dihydropyridine (+)-[(3)H]PN200-110 binding site density and (45)Ca(2+) uptake in these cells (). This study presents a functional analysis by electrophysiological techniques of short term regulation of L-type Ca(2+) channels in GH(4)C(1) cells by membrane depolarization. Depolarization of GH(4)C(1) cells by 50 mm K(+) rapidly reduced the barium currents through L-type calcium channels by approximately 70% and shifted the voltage dependence of activation by 10 mV to more depolarized potentials. Down-regulation depended on the strength of the depolarizing stimuli and was reversible. The currents recovered to near control levels on repolarization. Down-regulation of the calcium channel currents was calcium-dependent but may not have been due to excessive accumulation of intracellular calcium. Membrane depolarization by voltage clamping and by veratridine also produced a down-regulation of calcium channel currents. The down-regulation of the currents had an autocrine component. This study reveals a calcium-dependent down-regulation of the L-type calcium channel currents by depolarization.

Mutations in the Kv beta 2 binding site for NADPH and their effects on Kv1.4.

Kv beta 2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kv beta 2 binds NADP(+), and it has been suggested that Kv beta 2 is an oxidoreductase enzyme (). To investigate how this function might relate to channel modulation, we made point mutations in Kv beta 2 in either the NADPH docking or putative catalytic sites. Using the yeast two-hybrid system, we found that these mutations did not disrupt the interaction of Kv beta 2 with Kv alpha 1 channels. To characterize the Kv beta 2 mutants functionally, we coinjected wild-type or mutant Kv beta 2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kv beta 2 increased both the amplitude and rate of inactivation of Kv1.4 currents. The cellular content of Kv1.4 protein was unchanged on Western blot, but the amount in the plasmalemma was increased. Mutations in either the orientation or putative catalytic sites for NADPH abolished the expression-enhancing effect on Kv1.4 current. Western blots showed that both types of mutation reduced Kv1.4 protein. Like the wild-type Kv beta 2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kv beta 2 subunits with Kv1.4. Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kv beta 2 on Kv1.4 currents from its effect on the rate of inactivation. These results suggest that the binding of NADPH and the putative oxidoreductase activity of Kv beta 2 may play a role in the processing of Kv1.4.

Permanently charged chiral 1,4-dihydropyridines: molecular probes of L-type calcium channels. Synthesis and pharmacological characterization of methyl(omega-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate iodide, calcium channel antagonists.

We report the synthesis of the single enantiomers of permanently charged dihydropyridine derivatives (DHPs with alkyl linker lengths of two and eight carbon atoms) and their activities on cardiac and neuronal L-type calcium channels. Permanently charged chiral 1,4-dihydropyridines and methyl (omega)-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate iodides were synthesized in high optical purities from (R)-(-) and (S)-(+)-1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-+ ++pyridinecarboxylic acid, obtained by resolution of racemic 1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-pyridi necarboxylic acid. Competition binding experiments with radioligand [3H]-(+)-PN200-110 and the block of whole cell barium currents through L-type calcium channels in GH4C1 cells show that the compounds with the eight-carbon alkyl linker optimally block the L-type Ca2+ channels, and that the S-enantiomer is more potent than the R-enantiomer.

Adherence properties of enterotoxigenic Escherichia coli (ETEC) isolated in the Tel-Aviv area.

We tested the expression of adherence properties of Enterotoxigenic Escherichia coli (ETEC) strains isolated in the Tel-Aviv area by examining their hemagglutination (HA) patterns, and their adherence in vitro to Human Kidney (Huk) cells in tissue culture. Hemagglutination patterns of 75 ETEC and 63 non-ETEC strains isolated from stool samples were examined. The strains were tested for mannose-sensitive hemagglutination (MSHA) capacity of guinea pig erythrocytes, and for mannose-resistant hemagglutination (MRHA) capacity of human and bovine erythrocytes. The distribution of HA patterns among groups of non-ETEC and ETEC predominating strains was compared. (The latter contained strains which were isolated from 21 stool samples as at least two identical isolates.) The ratio of strains containing at least one of MS and MR hemagglutinins was found to be significantly higher in the group of ETEC-predominating strains than in non-ETEC. Eleven of 21 ETEC- predominating strains showed MRHA, and in ten of them Colonization Factor Antigens, CFA/I or CFA/II were identified by HA inhibition and agglutination using specific antisera. The influence of the bacterial culture conditions on expression of MSHA and MRHA and on adherence capacity of the bacteria to HuK cell culture was tested. Results show that ETEC strains exhibited a wide range of phenotypic manifestations of type and strength of adhesions. The variations depend not only on strain but also on culture condition and the type of target cells used in the experiments. In some strains a correlation was observed in the expression of adhesions using various experimental models; in others this correlation was not found. These findings suggest that, although CFAs were relatively common among ETEC-predominating strains isolated in the Tel-Aviv area, other adhesions could be involved in the pathogenic process.

An outbreak (?) caused by Escherichia coli O126:K71:H5 in premature infants.

The strain E. coli O126:K71:H5 was recovered during a 6 week period, from stool samples of 13 premature infants hospitalized in the Premature Infants Nursery Unit. Of a total of 147 tested stool samples, 69 were positive. This strain was also recovered for a long period from the respiratory tract of one of the infants. The strain had a low virulence for the infants. No clear relationship could be demonstrated between clinical status and recovery of the strain. The strain did not possess any of the well defined pathogenic mechanisms, namely enterotoxins LT and ST, and invasiveness. The production of a thermolabile substance toxic for Vero cells was demonstrated. The strain possessed both guinea pig mannose-sensitive and human mannose resistant hemagglutinins. In spite of the production of thermolabile toxic substance for Vero cells and in spite of its contagiousness and intestinal tract colonization capacity, the strain displayed the characteristics of an opportunistic pathogen rather than classic EPEC.

Enterotoxigenic Escherichia coli (ETEC) isolated in the Tel-Aviv (Israel) area.

The prevalence of enterotoxigenic E coli (ETEC) as a pathogenic agent of diarrhoea in the Tel-Aviv (Israel) area was determined, and the isolated E. coli strains characterized. During three periods (summer 1977, summer 1978, and summer 1979), a total of 335 specimens were tested for the presence of E. coli producing LT and ST toxin. Most of the specimens were from sporadic ambulatory diarrhoea cases (children and adults) attending a number of health care clinics in Tel-Aviv. Two to five colonies were tested from each sample. ETEC was detected in 69 cases (20%): LT/ST strains were isolated from 9 cases (2.7%); LT from 7 cases (2.1%); and ST from 53 cases (15.2%). ETEC was isolated in all age groups. In 19 specimens, 2 or more of 4 colonies tested were enterotoxigenic and were identical according to biotype, antibiotic sensitivity, and serogroup. These findings suggest that enterotoxigenic strains predominated in the bacterial population of the stool specimen. Part of the isolated ETEC strains belonged to serotypes already known as enterotoxigenic in different geographic areas of the world. The most frequently encountered were serogroups O8 (9 cases) represented by at least three serotypes, among them O8:K40:H9, and serotype O6:K15:H16 (5 cases); a number of serotypes were represented only by two cases or by single cases. Among 16 LT-producing stains (LT/ST and LT-only), 13 belonged to 3 serogroups, while ST-only strains represented a large spectrum of serotypes, some of which are now known as enterotoxigenic. Several serotypes common in other geographical locations were not detected.