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At least 469 records · Page 26Linked to original sources
PubMed1996

A quantitative method for comparing and evaluating manual prosthetic socket modifications.

Manually designed prosthetic sockets are difficult to evaluate since the hand-sculpting modification process does not retain the initial shape for comparison. A quantitative method for defining and comparing manual socket modifications was developed and integrated into the CADVIEW software package. The numerical comparison procedure consisted of a) digitizing premodification and post-modification models of a prosthetic socket, b) aligning these two shapes to a common axis (calculate cross section centroids, determine line of best fit through the centroids, rotate and move the line of best fit to a common axis), and c) displaying the differences in shape both numerically and using a color-coded three dimensional image. Alignment technique testing showed a between-radial-point average error of 2.5 mm using automatic alignment and 1.4 mm after further manual alignment adjustment. The between socket difference values were used to outline individual modifications and save these outlines to disk. Modification outlines from a series of patients were averaged to determine a prosthetist's general modification style. Averaged results from two prosthetists qualitatively supported the effectiveness of this procedure. This alignment and comparison system should help transfer hand-sculpting skills to prosthetic CAD/CAM systems, clinical research, and education for new clinicians.

Amputation Stumps↗
PubMed1985

Chemical modification of the bifunctional human serum pseudocholinesterase. Effect on the pseudocholinesterase and aryl acylamidase activities.

The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenylglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation. Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imipramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N-acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non-active-site tyrosine residues.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1997

Inappropriate sinus tachycardia (IST): management by radiofrequency modification of sinus node.

BACKGROUND: Inappropriate sinus tachycardia (IST) is a rare form of supraventricular arrhythmia. It can cause disabling symptoms and may be refractory to medical treatment. In symptomatic drug refractory patients, sinus node excision or total ablation of the sinus node with permanent pacemaker implantation was the only therapeutic option. Recently, radiofrequency (RF) modification of the sinus node has been reported to be an effective treatment for this condition. AIM: To present our experience with sinus node modification using RF energy in the management of IST. METHODS: Between 1989 to 1996 three patients (two females and one male), aged 28-36 years were diagnosed with symptomatic IST. All had failed multiple drugs and hence underwent sinus node modification using RF. In the first two patients, the site of RF application was guided by anatomical landmarks using fluoroscopy to localise the presumed most superior portion of the crista terminalis and also the earliest site of atrial activation. In the third patient, a 20 pole electrode catheter was used to map the crista terminalis and guide the ablation. Success was defined by 20-30% reduction in the heart rate with normal atrial activation sequence after ablation. RESULTS: The three patients described here had IST by clinical, electrocardiographic and electrophysiological criteria and were refractory to multiple antiarrhythmic drugs. The number of RF applications were 11, 15, and three applied at the site of earliest atrial activation for the control of heart rates. Patient 3 had a early recurrence at one month and underwent repeat sinus node modification (five RF applications). All three patients who underwent RF modification of the sinus node had a successful outcome. The procedure was uncomplicated and the patients remain asymptomatic during follow up (20, 12 and three months) with satisfactory control of heart rate, although one patient requires atenolol which was previously ineffective. CONCLUSIONS: RF modification of the sinus node is feasible and effective for IST, and should be the treatment of choice in patients refractory to medical therapy.

Adult↗
PubMed1999

Low temperature and low energy radiofrequency modification of atrioventricular nodal slow pathways in pediatric patients.

OBJECTIVES: To report our experience using low temperature and energy in the modification of the slow pathway in pediatric patients with atrioventricular nodal reentrant tachycardia. BACKGROUND: A concern in performing a slow pathway modification is the possible damage of the normal AV conduction system. Lesion size has been shown to have a linear relationship with temperature. Previous reports have used energy of 25-50 W that generate temperatures of 60 degrees C -70 degrees C for successful procedures. METHODS: Report of results of attempted AV nodal slow pathway modification in 17 consecutive pediatric patients < 15 years of age at The Children's Hospital of Philadelphia from April 1995 to November 1997 using low temperature and energy. RESULTS: There were 18 successful slow pathway modifications with 1 recurrence in 17 patients. The maximum energy used during successful lesions was 32.7 +/- 13.8 W (range 15-50 W) with a mean energy of 26.4 +/- 13.3 W (range 12-48 W). The peak temperature during these lesions was 55.1 degrees C +/- 4.1 degrees C (range 48 degrees C-64 degrees C) with a mean temperature of 47.9 degrees C +/- 2.7 degrees C (range 44 C-540C). The mean number of radiofrequency lesions required for a successful modification was 5.8 +/- 6.7 (median 4.0, range 1-26). Patients have been followed for 2.08 +/- 0.79 years. CONCLUSIONS: Slow pathway modification can be performed successfully with a low incidence of recurrence in the pediatric patient using low energy and temperature. It is possible that this may lead to smaller lesions.

Adolescent↗
PubMed1981

Evidence for Plasmid Linkage of Restriction and Modification in Streptococcus cremoris KH.

Restriction and modification have been demonstrated in Streptococcus cremoris KH cells when infected by Streptococcus lactis C2 phage (designated c2) at an efficiency of plating of 2 x 10. The growth of c2 phage through KH cells produces modified progeny phage capable of unrestricted growth on KH cells. The ability of single-colony isolates of S. cremoris KH cultures to restrict and modify c2 phage was found to be variable. From 2 to 6.5% of colonies isolated were partially deficient in restrictive capacity, permitting a greater plaquing ability by c2 phage of 1.8 to 2.9 log cycles. No completely restrictionless mutants were isolated from 1,000 colonies examined. Mutants were shown to be deficient in both restriction and modification capabilities of the same specificity. The frequent occurrence of a genotypic change that resulted in the loss of both restriction and modification capacities indicated the involvement of plasmid deoxyribonucleic acid in genetically determining this specific restriction and modification system. S. cremoris KH was found to harbor 11 plasmid molecules, with molecular weights (x10) estimated to be 50, 41, 24, 18, 10, 7.4, 3.3, 3.0, 2.8, 2.5, and 1.5. Of the 27 mutants examined, 25 were missing the 10-megadalton plasmid. This consistent plasmid difference among the majority of mutants isolated supports the involvement of this plasmid in restriction and modification. Plasmid linkage of restriction and modification systems provides a genetic mechanism for the rapid development of phage-sensitive starter cultures due to the inherent instability of extrachromosomal elements.

Journal Article↗
PubMed1997

Posttranscriptional modification of tRNA in psychrophilic bacteria.

Posttranscriptional modification in tRNA is known to play a multiplicity of functional roles, including maintenance of tertiary structure and cellular adaptation to environmental factors such as temperature. Nucleoside modification has been studied in unfractionated tRNA from three psychrophilic bacteria (ANT-300 and Vibrio sp. strains 5710 and 29-6) and one psychrotrophic bacterium (Lactobacillus bavaricus). Based on analysis of total enzymatic hydrolysates by liquid chromatography-mass spectrometry, unprecedented low amounts of modification were found in the psychrophiles, particularly from the standpoint of structural diversity of modifications observed. Thirteen to 15 different forms of posttranscriptional modification were found in the psychrophiles, and 10 were found in L. bavaricus, compared with approximately 29 known to occur in bacterial mesophiles and 24 to 31 known to occur in the archaeal hyperthermophiles. The four most abundant modified nucleosides in tRNA from each organism were dihydrouridine, pseudouridine, 7-methylguanosine, and 5-methyluridine. The molar abundances of the latter three nucleosides were comparable to those found in tRNA from Escherichia coli. By contrast, the high levels of dihydrouridine observed in all three psychrophiles are unprecedented for any organism in any of the three phylogenetic domains. tRNA from these organisms contains 40 to 70% more dihydrouridine, on average, than that of the mesophile E. coli or the psychrotroph L. bavaricus. This finding supports the concept that a functional role for dihydrouridine is in maintenance of conformational flexibility of RNA, especially important to organisms growing under conditions where the dynamics of thermal motion are severely compromised. This is in contrast to the role of modifications contained in RNA from thermophiles, which is to reduce regional RNA flexibility and provide structural stability to RNA for adaptation to high temperature.

Base Composition↗
PubMed1999

The acidic domain and first immunoglobulin-like loop of fibroblast growth factor receptor 2 modulate downstream signaling through glycosaminoglycan modification.

Fibroblast growth factor receptors (FGFRs) are membrane-spanning tyrosine kinases that have been implicated in a variety of biological processes including mitogenesis, cell migration, development, and differentiation. We identified a unique isoform of FGFR2 expressed as a diffuse band with an unusually large molecular mass. This receptor is modified by glycosaminoglycan at a Ser residue located immediately N terminal to the acidic box, a stretch of acidic amino acids. The acidic box and the glycosaminoglycan modification site are encoded by an alternative exon of the FGFR2 gene. The acidic box appears to play an important role in glycosaminoglycan modification, and the presence of this domain is required for modification by heparan sulfate glycosaminoglycan. Moreover, the presence of the first immunoglobulin-like domain encoded by another alternative exon abrogated the modification. The high-affinity receptor with heparan sulfate modification enhanced receptor autophosphorylation, substrate phosphorylation, and ternary complex factor-independent gene expression. It also sustained mitogen-activated protein kinase activity and increased eventual DNA synthesis, a long-term response to fibroblast growth factor stimulation, at physiological ligand concentrations. We propose a novel regulation mechanism of FGFR2 signal transduction through glycosaminoglycan modification.

Alternative Splicing↗
PubMed2005

Contribution of individual spikes in burst-induced long-term synaptic modification.

Long-term synaptic modification depends on the relative timing of individual pre- and postsynaptic spikes, but the rules governing the effects of multispike bursts remain to be fully understood. In particular, some studies suggest that the spike timing dependence of synaptic modification breaks down with high-frequency bursts. In this study, we characterized the effects of pre- and postsynaptic bursts on long-term modification of layer 2/3 synapses in visual cortical slices from young rats. We found that, while pairing-induced synaptic modification depends on the burst frequency, this dependence can be explained in terms of the timing of individual pre- and postsynaptic spikes. Later spikes in each burst are less effective in synaptic modification, but spike efficacy is regulated differently in pre- and postsynaptic bursts. Presynaptically, spike efficacy is progressively weakened, in parallel with short-term synaptic depression. Postsynaptically, spike efficacy is suppressed to a lesser extent, and it depends on postsynaptic potassium channel activation. Such timing-dependent interaction among multiple spikes can account for synaptic modifications induced by a variety of spike trains, including the frequency-dependent transition from depression to potentiation induced by a postsynaptic burst preceding a presynaptic burst.

Action Potentials↗
PubMedDate not supplied

Varying susceptibility of different low density lipoproteins to oxidative modification.

The oxidative modification of low density lipoprotein (LDL) may provide a crucial link between plasma LDL and the atherosclerotic lesion. The studies presented herein define time-dependent modifications of LDL constituents caused by CuSO4-catalyzed oxidation. Measurement of the cholesterol content of oxidized LDL by the cholesterol esterase-oxidase assay was found to be inaccurate. The enzymatic assay detected oxysterols as well as cholesterol and thus substantially overestimated the actual cholesterol content. Alteration of electrophoretic mobility and conversion of sterols into oxysterols increased in a parallel, time-dependent manner. Lipid peroxidation, judged by the thiobarbituric acid-reacting substances assay, increased early to maximal values but was not linearly related to either electrophoretic mobility or to oxysterol formation. Neither electrophoretic mobility nor oxysterol formation varied much between repeated oxidative modifications of any given LDL preparation but varied markedly among LDLs from different normolipidemic individuals, suggesting that LDL particles contain some factor conferring susceptibility or resistance to oxidation. Indeed, LDL preparations from different individuals have varying susceptibilities to oxidative modification as evidenced by the three indexes used. The major oxysterol generated was 7-ketocholesterol. Macrophage modification of LDL also resulted in the generation of oxysterols. Thus, measurement of oxysterols may afford an additional index of the oxidative modification of LDL. Since incubation of macrophages with oxidized LDL but not native LDL resulted in the accumulation of oxysterols, this could account for some of the toxic and metabolic effects of oxidized LDL on cells.

Adult↗
PubMed1989

Percutaneous catheter modification of the atrioventricular node. A potential cure for atrioventricular nodal reentrant tachycardia.

Our purpose was to describe a technique of atrioventricular (AV) node modification for patients with drug refractory AV nodal reentrant tachycardia (AVNRT). Nine patients (mean age, 45 +/- 20; range, 14-82) with recurrent drug refractory AVNRT (n = 8) or sudden cardiac death thought to be precipitated by AVNRT (n = 1) underwent a percutaneous catheter procedure to modify AV nodal function. The area between the electrode recording the maximal His-bundle electrogram and the ostium of the coronary sinus was divided into three zones. Perinodal direct current shocks of 100-300 J were delivered to one (n = 2), two (n = 3), or three (n = 4) zones without complications. The procedure endpoints were modification of AV conduction (either first degree AV block or complete retrograde ventriculo-atrial [VA] block) and failure to induce AVNRT before or after isoproterenol and/or atropine administration. Six of nine patients (67%) have had no inducible or spontaneous AVNRT over a mean follow-up of 12.3 +/- 4.1 months (range, 4.5-17). One of the six underwent repeat, successful modification, because AVNRT was inducible at restudy 2 days after the initial procedure. AVNRT recurred in three patients (33%), one early (3 days) and two late (3-4 months). Two of these patients underwent complete ablation of the AV junction and permanent pacemaker placement, whereas one is controlled with drug therapy. Therefore, AV nodal modification resulted in tachycardia control without antiarrhythmic drugs in six of nine (67%) and obviated the need for complete AV junctional ablation in seven of nine patients (78%). Elimination of AVNRT appears to result from either block in the retrograde fast pathway or modification of the antegrade slow pathway, such that AVNRT cannot be sustained. Additional findings suggest that an atrio-Hisian accessory connection may not be involved in AVNRT in some of these patients. Percutaneous catheter AV nodal modification appears to be a promising technique for treatment of refractory AVNRT and may obviate need for complete AV junctional ablation in a substantial number of patients with drug/pacemaker refractory AVNRT.

Adult↗
PubMed1996

Electrophysiological mechanisms in successful radiofrequency catheter modification of atrioventricular junction for patients with medically refractory paroxysmal atrial fibrillation.

BACKGROUND: Mechanisms and changes of electrophysiological (EP) characteristics in successful radiofrequency (RF) modification of right midseptal and posteroseptal areas for controlling rapid ventricular response to atrial fibrillation (Af) are not clear. METHODS AND RESULTS: We studied 50 patients with medically refractory paroxysmal Af. Group 1 consisted of 40 patients without dual atrioventricular (AV) node physiology with modification sites located in the mid/posteroseptal area. Of the 40 patients, 36 had successful modification (follow-up of 14 +/- 8 months), and 3 had AV block. Late follow-up electrophysiological study (98 +/- 10 days) showed pattern 1 (67%) with prolongation of AV node effective refractory period (ERP, > or =40 milliseconds) and Wenckebach block cycle length (WBCL, > or =40 milliseconds); pattern 2 (22%) with prolongation of AH interval (> or =20 milliseconds), ERP, and WBCL; and pattern 3 (11%) without any change in AV node conduction parameter. Change in ventricular rate negatively correlated with change of WBCL in patterns 1 (r=-.691, P=.019) and 2 (r=-.90, P=.01). Group 2 consisted of 10 patients with dual AV node pathway; elimination of slow pathway property was performed. Late follow-up electrophysiological study (92+/-7 days) showed that change in ventricular rate negatively correlated with change in AV node ERP (r=-.926, P=.0001) and WBCL (r=-.969, P=.0001). Four patients without significant modification effect had success after RF energy was delivered to higher levels (follow-up, 15+/-7 months). CONCLUSIONS: RF modification of right mid/posteroseptal area is feasible in 92% of patients with paroxysmal Af. Mechanisms of successful modification might be elimination of posterior input and/or partial injury of the compact node. Furthermore, simple elimination of slow pathway might be inadequate for control of ventricular rate in patients with little difference in conduction properties between fast and slow pathways.

Adult↗
PubMed1996

Surface modification of chitosan membranes by complexation-interpenetration of anionic polysaccharides for improved blood compatibility in hemodialysis.

Chitosan membrane surface was modified by complexation and interpenetration of anionic polysaccharides--heparin and dextran sulfate--for improved blood compatibility in hemodialysis. Electron spectroscopy for chemical analysis results showed a characteristic sulfur (S) and sodium (Na) peaks after modification with dextran sulfate. The sulfur/carbon (S/C) atomic composition ratio increased from 0.03 to 0.08 when the bulk dextran sulfate concentration used for modification was increased from 2.5 to 10 mg ml-1. The permeability of urea and creatinine did not change significantly upon modification with heparin or dextran sulfate. Surface modification, however, did decrease the permeability coefficients of glucose, vitamin B-2, and vitamin B-12. Unlike Cuprophan, chitosan and surface-modified chitosan membranes did not significantly activate the complement system as measured by the serum iC3b concentration. Compared to forty and sixty fully-activated platelets present on control surfaces, surface modification with heparin and dextran sulfate significantly reduced the number of adherent platelets per 25,000 microns 2 area and the extent of platelet activation. Surface modification with anionic polysaccharides, however, did significantly shorten the plasma recalcification time leading to fibrin clot formation. The results of this study show that chitosan membrane surface can be modified by complexation-interpenetration of anionic modifying agents. The modified membranes do resist complement activation and platelet adhesion and activation.

Adult↗
PubMed2005

Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis.

The production of ROS is an inevitable consequence of metabolism. However, high levels of ROS within a cell can be lethal and so the cell has a number of defences against oxidative cell stress. Occasionally the cell's antioxidant mechanisms fail and oxidative stress occurs. High levels of ROS within a cell have a number of direct and indirect consequences on cell signalling pathways and may result in apoptosis or necrosis. Although some of the indirect effects of ROS are well known, limitations in technology mean that the direct effects of the cell's redox environment upon proteins are less understood. Recent work by a number of groups has demonstrated that ROS can directly modify signalling proteins through different modifications, for example by nitrosylation, carbonylation, di-sulphide bond formation and glutathionylation. These modifications modulate a protein's activity and several recent papers have demonstrated their importance in cell signalling events, especially those involved in cell death/survival. Redox modification of proteins allows for further regulation of cell signalling pathways in response to the cellular environment. Understanding them may be critical for us to modulate cell pathways for our own means, such as in cytotoxic drug treatments of cancer cells. Protein modifications mediated by oxidative stress can modulate apoptosis, either through specific protein modifications resulting in regulation of signalling pathways, or through a general increase in oxidised proteins resulting in reduced cellular function. This review discusses direct oxidative protein modifications and their effects on apoptosis.

Antioxidants↗
PubMed2016

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.

We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.

5-Methylcytosine↗
PubMed1986

Therapeutic efficacy of multiagent chemotherapy with drug delivery enhancement by blood-brain barrier modification in glioblastoma.

Reversible osmotic blood-brain barrier (BBB) modification was used in 38 patients with glioblastoma to enhance the delivery of chemotherapeutic agents. The patients ranged in age from 14 to 70 years (mean, 43), and all had prior surgery and radiation; 5 had also received systemic chemotherapy. Karnofsky Performance Status (KPS) scores ranged from 60 to 100% (mean, 79) on admission to the treatment program. Barrier modification was achieved by intracarotid or intravertebral artery infusion of mannitol, and a chemotherapy regimen of methotrexate, cytoxan, and procarbazine was given in conjunction with barrier modification. The 38 glioblastoma patients were compared to two control groups of patients with glioblastoma; these encompassed 14 patients treated with surgery and radiation and 8 treated with surgery, radiation, and systemic chemotherapy. Survival analysis using the Cox Proportional Hazards Regression Model (corrected for age, sex, presence or absence of necrosis, and functional status) showed that patients receiving chemotherapy with BBB modification had a statistically significant (P = 0.0006) longer expected survival (17.5 months) than the control groups (12.8 and 11.4 months, respectively). Presently 16 patients of the barrier-enhanced treatment group are alive at 5 to 42 months from diagnosis (median, 20) with KPS scores ranging from 40 to 90% (median, 65). The neurological complications seen included a stroke-like syndrome in 3 patients (1 with decreased motor movement in the hand, 1 with marked hemiparesis, and 1 with hemiplegia), transient exacerbation of preexisting neurological deficits lasting 2 to 3 days, and a 15% incidence of seizures during or within 24 hours of the BBB modification. In 2 of the 38 patients, radiographic documentation of central nervous system tumor regression concurrent with the development of new tumor nodule(s) in portions of the brain distant from the region of osmotic BBB opening was seen. These studies indicate that chemotherapeutic drug delivery to tumors (as well as surrounding brain) can be augmented by osmotic BBB modification and that such therapy can result in a prolongation of survival.

Adult↗
PubMed2003

siRNA function in RNAi: a chemical modification analysis.

Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.

HeLa Cells↗
PubMed1983

Induction of the base displacement or Z conformation in DNA by N-2-acetylaminofluorene modification.

Modification of deoxyguanosine at the C(8) position by the carcinogen N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) has been shown to result in two different conformational changes dependent on the nucleotide sequence of the modified polymer. AAF modification of random sequence DNA results in a large distortion of the helix which is termed base displacement. In this conformation, the carcinogen is inserted into the DNA perpendicular to the helix axis with the guanosine displaced to the outside. Large single-stranded regions are generated which are susceptible to S(1) nuclease digestion and react with anti-cytidine antibodies.A different conformation has been observed when the alternating purine pyrimidine copolymer, poly(dG-dC).poly(dG-dC) is modified. At a modification level of 28% this polymer shows a CD spectrum characteristic of the left-handed Z-DNA seen in the unmodified polymer at high ethanol or salt concentrations. Base pairing of the modified polymer remains intact as demonstrated by its resistance to digestion with S(1) nuclease and lack of reactivity with anti-cytidine antibodies. Modification of poly(dG-m(5)dC).poly(dG-m(5)dC) with AAF was also shown to induce the Z conformation. However, for this polymer, inversion of the CD spectrum takes place at a much lower modification level (10%) than for the nonmethylated polymer (>20%). This polymer is also resistant to S(1) nuclease digestion consistent with its adoption of the Z conformation with AAF modification. A possible role in gene expression for the Z conformation of AAF modified regions is discussed.

2-Acetylaminofluorene↗
PubMed2025

N6-methyladenosine modification of the subgroup J avian leukosis viral RNAs attenuates host innate immunity via MDA5 signaling.

Subgroup J avian leukosis virus (ALV-J), a retrovirus, elicits immunosuppression and persistent infections in chickens. Although it is widely acknowledged that ALV-J can evade the host's innate immune defenses, the mechanisms behind this immune evasion remain elusive. N6-methyladenosine (m6A), the most prevalent internal RNA modification, plays a role in innate immune evasion. Our research identified ALV-J as an inefficient stimulator of innate immunity in vitro and in vivo, with its genomic RNA featuring m6A modifications predominantly in the envelope protein (Env) region and 3' untranslated region (3'UTR). To elucidate the functional consequences of m6A modification, we subsequently generated m6A-deficient ALV-J through its culturing in the DF-1 overexpressing fat mass and obesity-associated protein (FTO) cells. The m6A-deficient ALV-J virus, or its RNAs significantly enhanced IFN-&#x3b2; production compared to the wild-type (wt) ALV-J, suggesting a pivotal regulatory function of m6A modifications in modulating innate immune response. Mechanistically, the m6A modification of the ALV-J genomic RNA directly impacted its recognition by MDA5, weakening its binding and ubiquitination and attenuating IFN-&#x3b2; activation. Moreover, m6A-deficient ALV-J, created by inducing mutations in m6A sites within Env and 3'UTR, exhibited reduced replication capacity and elevated IFN-&#x3b2; expression in host cells. Importantly, this phenomenon was abolished in MDA5-knockout DF-1 cells, further demonstrating the core role of MDA5. These data demonstrate that m6A modification of ALV-J genomic RNA dampens the host's innate immune response through MDA5 signaling pathway.

Animals↗