Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (sORNA-soaked M incognita eggs Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH2)-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s. which recovered within 24 h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48 h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-en-I transcript which encodes an RNAi-inhibiting siRNA exonuclease We observe a marked up-regulation of MI-en-I transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RN/NI-based reverse genetic screens in plant parasitic nematodes. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

N-terminally modified glucagon-like peptide-1(7-36) amide exhibits resistance to enzymatic degradation while maintaining its antihyperglycaemic activity in vivo

Glucagon-like peptide-1 (7-36)amide (tGLP-1) is inactivated by dipeptidyl peptidase (DPP) IV by removal of the NH2-terminal dipeptide His(7)-Ala(8). We examined the degradation of NH2-terminally modified His(7)-glucitol tGLP-1 and its insulin-releasing and antihyperglycaemic activity in vivo, tGLP-1 was degraded by purified DPP IV after 4 h (43% intact) and after 12 hi 89% was converted to GLP-1(9-36)amide. In contrast > 99% of His(7)-glucitol tGLP-1 remained intact at 12 h. His(7)-glucitol tGLP-1 was similarly resistant to plasma degradation in vitro. His7-glucitol tGLP-1 showed greater resistance to degradation in vivo (92% intact) compared to tGLP-1 (27% intact) 10 min after i.p. administration to Wistar rats. Glucose homeostasis was examined following i.p. injection of both peptides (12 nmol/kg) together with glucose (18 mmol/kg). Plasma glucose concentrations were significantly reduced and insulin concentrations elevated following peptides administration compared with glucose alone. The area under the curve (AUC) for glucose for controls (AUC 691 +/- 35 mM/min) was significantly lower after administration of tGLP-1 and His7-glucitol tGLP-1 (36 and 49% less; AUC; 440 +/- 40 and 353 +/- 31 mM/min, respectively; P

Dissociative electron attachment to the highly reactive difluoromethylene molecule–importance of CF2 for negative ion formation in fluorocarbon plasmas

Dissociative electron attachment to the highly reactive difluoromethylene molecule, CF2, produced in a C3F6/He microwave plasma and stepwise via the fast atom reaction CF3I+H?CF3+HI and CF3+H?CF2+HF, has been investigated. The upper limit for the cross section of formation of F- via dissociative electron attachment to CF2 is estimated to be 5×10-4?Å2. This value is four orders of magnitude smaller than the cross section previously predicted from scattering calculations. It is concluded that difluoromethylene plays a negligible role in negative ion formation in fluorocarbon plasmas.

Promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster:structural and functional characterization of an upstream untranslated mRNA sequence

We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7). Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes. We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes. wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.

Molecular cloning of the Haemophilus influenzae gmhA (lpcA) gene encoding a phosphoheptose isomerase required for lipooligosaccharide biosynthesis

We have determined that gene HI#1181 of Haemophilus influenzae is a homolog of Escherichia coli gmhA (previously designated lpcA) (J. S. Brooke and M. A. Valvano, J. Biol. Chem. 271:3608-3614, 1996), which encodes a phosphoheptose isomerase catalyzing the first step of the biosynthesis of ADP-L-glycero-D-manno heptose. Mutations in this gene are associated with a heptoseless core lipopolysaccharide which determines an increased outer membrane permeability to hydrophobic compounds. The cloned H. influenzae gmhA restored the synthesis of a complete core in the gmhA-deleted E. coli strain chi711. Amino acid sequence comparisons of the GmhA proteins of E. coli and H. influenzae with other proteins in the databases revealed the existence of a novel family of phosphosugar a1do-keto isomerases.

Two-dimensional Monte Carlo simulations of H i line formation in massive young stellar object disc winds

Massive young stellar objects (YSOs) are powerful infrared Hi line emitters. It has been suggested that these lines form in an outflow from a disc surrounding the YSO. Here, new two-dimensional Monte Carlo radiative transfer calculations are described which test this hypothesis. Infrared spectra are synthesized for a YSO disc wind model based on earlier hydrodynamical calculations. The model spectra are in qualitative agreement with the observed spectra from massive YSOs, and therefore provide support for a disc wind explanation for the Hi lines. However, there are some significant differences: the models tend to overpredict the Bra/Br? ratio of equivalent widths and produce line profiles which are slightly too broad and, in contrast to typical observations, are double-peaked. The interpretation of these differences within the context of the disc wind picture and suggestions for their resolution via modifications to the assumed disc and outflow structure are discussed. © 2005 RAS.

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of <1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions

Nepsilon-(Carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined nonenzymatic glycation and oxidation (glycoxidation) reactions. We now report that CML is also formed during metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of protein. During copper-catalyzed oxidation in vitro, the CML content of low density lipoprotein increased in concert with conjugated dienes but was independent of the presence of the Amadori compound, fructoselysine, on the protein. CML was also formed in a time-dependent manner in RNase incubated under aerobic conditions in phosphate buffer containing arachidonate or linoleate; only trace amounts of CML were formed from oleate. After 6 days of incubation the yield of CML in RNase from arachidonate was approximately 0.7 mmol/mol lysine compared with only 0.03 mmol/mol lysine for protein incubated under the same conditions with glucose. Glyoxal, a known precursor of CML, was also formed during incubation of RNase with arachidonate. These results suggest that lipid peroxidation, as well as glycoxidation, may be an important source of CML in tissue proteins in vivo and that CML may be a general marker of oxidative stress and long term damage to protein in aging, atherosclerosis, and diabetes.

Multi-epoch high-resolution spectroscopy of SN2011fe - Linking the progenitor to its environment

This study attempts to establish a link between the reasonably well known nature of the progenitor of SN2011fe and its surrounding environment. This is done with the aim of enabling the identification of similar systems in the vast majority of the cases, when distance and epoch of discovery do not allow a direct approach. To study the circumstellar environment of SN2011fe we have obtained high-resolution spectroscopy of SN2011fe on 12 epochs, from 8 to 86 days after the estimated date of explosion, targeting in particular at the time evolution of CaII and NaI. Three main absorption systems are identified from CaII and NaI, one associated to the Milky Way, one probably arising within a high-velocity cloud, and one most likely associated to the halo of M101. The Galactic and host galaxy reddening, deduced from the integrated equivalent widths (EW) of the NaI lines are E(B-V)=0.011+/-0.002 and E(B-V)=0.014+/-0.002 mag, respectively. The host galaxy absorption is dominated by a component detected at the same velocity measured from the 21-cm HI line at the projected SN position (~180 km/s). During the ~3 months covered by our observations, its EW changed by 15.6+/-6.5 mA. This small variation is shown to be compatible with the geometric effects produced by therapid SN photosphere expansion coupled to the patchy fractal structure of the ISM. The observed behavior is fully consistent with ISM properties similar to those derived for our own Galaxy, with evidences for structures on scales

Isotretinoin therapy changes the expression of antimicrobial peptides in acne vulgaris

In acne vulgaris, antimicrobial peptides (AMPs) could play a dual role; i.e., protective by acting against Propionibacterium acnes, pro-inflammatory by acting as signalling molecules. The cutaneous expression of 15 different AMPs was investigated in acne patients; furthermore, the impact of isotretinoin therapy on AMP expression was analysed in skin biopsies from 13 patients with acne vulgaris taken before, during and after a 6-month treatment cycle with isotretinoin using quantitative real-time polymerase chain reaction. Cutaneous expression of the AMPs cathelicidin, human β-defensin-2 (HBD-2), lactoferrin, lysozyme, psoriasin (S100A7), koebnerisin (S100A15), and RNase 7 was upregulated in untreated acne vulgaris, whereas α-defensin-1 (HNP-1) was downregulated compared to controls. While relative expression levels of cathelicidin, HBD-2, lactoferrin, psoriasin (S100A7), and koebnerisin (S100A15) decreased during isotretinoin treatment, only those of cathelicidin and koebnerisin returned to normal after 6 months of isotretinoin therapy. The increased expression of lysozyme and RNase 7 remained unaffected by isotretinoin treatment. The levels of granulysin, RANTES (CCL5), perforin, CXCL9, substance P, chromogranin B, and dermcidin were not regulated in untreated acne patients and isotretinoin had no effect on these AMPs. In conclusion, the expression of various AMPs is altered in acne vulgaris. Isotretinoin therapy normalizes the cutaneous production of distinct AMPs while the expression of others is still increased in healing acne. Considering the antimicrobial and pro-inflammatory role of AMPs, these molecules could serve as specific targets for acne therapy and maintenance of clinical remission.

Line-driven radiative outflows in luminous quasars

An analysis of ≃19 500 narrow (≲200 km s^-1) CIV λλ1548.2,1550.8 absorbers in ≃34 000 Sloan Digital Sky Survey quasar spectra is presented. The statistics of the number of absorbers as a function of outflow velocity shows that in approximately two-thirds of outflows, with multiple C IV absorbers present, absorbers are line-locked at the 500 km s^-1 velocity separation of the C IV absorber doublet; appearing as 'triplets' in the quasar spectra. Line-locking is an observational signature of radiative line-driving in outflowing material, where the successive shielding of 'clouds' of material in the outflow locks the clouds together in outflow velocity. Line-locked absorbers are seen in both broad absorption line (BAL) quasars and non-BAL quasars with comparable frequencies and with velocities out to at least 20 000 km s^-1. There are no detectable differences in the absorber properties and the dust content of single C IV doublets and line-locked C IV doublets. The gas associated with both single and line-locked CIV absorption systems includes material with a wide range of ionization potential (14-138 eV). Both single and line-locked CIV absorber systems show strong systematic trends in their ionization as a function of outflow velocity, with ionization decreasing rapidly with increasing outflow velocity. Initial simulations, employing CLOUDY, demonstrate that a rich spectrum of line-locked signals at various velocities may be expected due to significant opacities from resonance lines of Li-, He- and H-like ions of O, C and N, along with contributions from He II and HI resonance lines. The simulations confirm that line-driving can be the dominant acceleration mechanism for clouds with N(H I) ≃ 10¹⁹ cm^-2.