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PubMed1997

Differences in the prevalence of IS6110 insertion sites in Mycobacterium tuberculosis strains: low and high copy number of IS6110.

SETTING: Mycobacterium tuberculosis (M. tuberculosis) isolates from various parts of the USA which have few copies of the insertion sequence IS6110. OBJECTIVES: To characterize the sites of insertion of IS6110 among M. tuberculosis isolates that have one to six copies of the insertion sequence. DESIGN: The mixed-linker polymerase chain reaction (ML-PCR) procedure was used to amplify the terminal repeats on the ends of IS6110 and adjacent flanking sequences. From the ML-PCR products, sequences flanking 14 copies of IS6110 in strains containing less than seven copies of the insertion were determined. Sequence information from the flanking deoxyribonucleic acid was used to construct flanking primers that can be used to indicate the presence of IS6110 at a particular site when paired with outbound IS6110 primers in a PCR. Over 200 strains of diverse origin were screened for the insertion of IS6110 at several distinct sites using this procedure. RESULTS: The direct repeat (DR) locus has been described as a highly preferred site for insertion of IS6110 in strains of M. tuberculosis. Another highly preferred site of insertion of IS6100, DK1, is herein described. Insertions at DK1 are highly prevalent in M. tuberculosis strains harboring two to six copies of IS6110. The prevalence of insertions at this site decreases in strains with more than six copies of IS6110, even though the sequence itself is present in strains lacking a copy of IS6110 at this site. CONCLUSION: In addition to the DR locus there are other conserved sites of insertion among M. tuberculosis strains. The data further suggest a separate lineage for the high copy and the low copy strains, and a possible sequential insertion of IS6110 in strains of M. tuberculosis with less than seven copies.

Base Sequence↗
PubMedDate not supplied

Interventional radiologic placement of peripherally inserted central catheters.

PURPOSE: This study examines the difference in success between bedside insertions of peripherally inserted central catheters (PICCs) performed by clinicians and fluoroscopically assisted insertions by cardiovascular/interventional (CV/I) radiologists. PATIENTS AND MATERIALS: Four hundred four PICCs were inserted in 305 patients, with a mean age of 48 years (range, 8-78 years), who required intermediate and long-term central venous access. One hundred fifty patients underwent bedside insertion, and 155 patients underwent fluoroscopically assisted insertion. Dual- and single-lumen PICC devices were used depending on venous access needs. RESULTS: Central venous access with the PICC device was achieved in 111 (74%) of 150 bedside insertions and in 153 (98.7%) of 155 fluoroscopic insertions. Average PICC dwell time was 72.7 days (range, 2-307 days). Of the 305 patients, 244 (73.4%) completed planned therapy with a single catheter, 54 patients (17.7%) required two PICCs at the same site, 15 patients (4.9%) required three or more PICCs at the same site, and 12 patients (4.0%) required PICC replacement to a different site to complete therapy. Complications with bedside insertions included upper extremity thrombophlebitis (n = 4), entry site infection (n = 6), and PICC shear with migration (n = 2), while complications with fluoroscopic insertions included entry site infection (n = 2). CONCLUSION: Because of the far greater placement success achieved by CV/I radiologists, all PICCs are now inserted fluoroscopically. The devices complete favorably with surgically placed central catheters with regard to service interval, morbidity, and cost. Use of fluoroscopic techniques enables placement of these devices in virtually all patients, even those with seemingly no peripheral veins.

Adolescent↗
PubMed2001

Substance loss caused by scaling with different sonic scaler inserts--an in vitro study.

BACKGROUND, AIMS: Depending on the phase of periodontal treatment, power-driven inserts with different power levels are necessary: during initial treatment and flap operations, it must be possible to remove calculus which firmly adheres to the root surface with great efficiency; in contrast, gentle root-surface instrumentation has a high priority during maintenance treatment. Recently, modified sonic scaler inserts have been introduced: diamond-coated inserts for open flap operations and slim probe-shaped sonic scaler inserts for deplaqueing during maintenance. METHOD: We compared the substance removal by these inserts to that of a conventional insert. In a bench system which allows application force, stroke height, and width and frequency of instrumentation to be monitored and also permits assessment of the topography before and after instrumentation (yielding depth removal and removed volume), we instrumented aluminum rods in triplicate and bovine roots in a 5-fold repetition. RESULTS: With high application forces (1.0 N), the diamond-coated inserts removed 30 to 50% more substance than the conventional one. Except for the slim inserts, there was a significant linear relationship between increasing force and removed volume. Probe-shaped inserts do not remove considerable substance even with increasing pressures. CONCLUSIONS: Diamond-coated inserts remove tooth substance very effectively and must thus be handled with care: more pressure effects more removal. Probe-shaped inserts can be used without risk during maintenance visits.

Animals↗
PubMed2001

Insertion of PsaK into the thylakoid membrane in a "Horseshoe" conformation occurs in the absence of signal recognition particle, nucleoside triphosphates, or functional albino3.

The photosystem I subunit PsaK spans the thylakoid membrane twice, with the N and C termini both located in the lumen. The insertion mechanism of a thylakoid membrane protein adopting this type of topology has not been studied before, and we have used in vitro assays to determine the requirements for PsaK insertion into thylakoids. PsaK inserts with high efficiency and we show that one transmembrane span (the C-terminal region) can insert independently of the other, indicating that a "hairpin"-type mechanism is not essential. Insertion of PsaK does not require stromal extract, indicating that signal recognition particle (SRP) is not involved. Removal of nucleoside triphosphates inhibits insertion only slightly, both in the presence and absence of stroma, suggesting a mild stimulatory effect of a factor in the translation system and again ruling out an involvement of SRP or its partner protein, FtsY. We, furthermore, find no evidence for the involvement of known membrane-bound translocation apparatus; proteolysis of thylakoids destroys the Sec and Tat translocons but does not block PsaK insertion, and antibodies against the Oxa1/YidC homolog, Alb3, block the SRP-dependent insertion of Lhcb1 but again have no effect on PsaK insertion. Because YidC is required for the efficient insertion of every membrane protein tested in Escherichia coli (whether SRP-dependent or -independent), PsaK is the first protein identified as being independent of YidC/Alb3-type factors in either thylakoids or bacteria. The data raise the possibility of a wholly spontaneous insertion pathway.

Amino Acid Sequence↗
PubMed2002

Prevention of bloodstream infections with central venous catheters treated with anti-infective agents depends on catheter type and insertion time: evidence from a meta-analysis.

OBJECTIVE: To test the evidence that the risk of infection related to central venous catheters (CVCs) is decreased by anti-infective coating or cuffing. DESIGN: Systematic review of randomized, controlled trials comparing anti-infective with inactive (control) CVCs. INTERVENTIONS: Average insertion times were taken as a measurement of the length of insertion. Dichotomous data were combined using a fixed effect model and expressed as odds ratio (OR) with 95% confidence interval (CI95). RESULTS: Two trials on antibiotic coating (343 CVCs) had an average insertion time of 6 days; the risk of BSI decreased from 5.1% with control to 0% with anti-infective catheters. There were no trials with longer average insertion times. In three trials on silver collagen cuffs (422 CVCs), the average insertion time ranged from 5 to 8.2 days (median, 7 days); the risk of BSI was 5.6% with control and 3.2% with anti-infective catheters. In another trial on silver collagen cuffs (101 CVCs), the average insertion time was 38 days; the risk of BSI was 3.7% with control and 4.3% with anti-infective catheters. In five trials on chlorhexidine-silver sulfadiazine coating (1,269 CVCs), the average insertion time ranged from 5.2 to 7.5 days (median, 6 days); the risk of BSI decreased from 4.1% with control to 1.9% with anti-infective catheters. In five additional trials on chlorhexidine-silver sulfadiazine coating (1,544 CVCs), the average insertion time ranged from 7.8 to 20 days (median, 12 days); the risk of BSI was 4.5% with control and 4.2% with anti-infective catheters. CONCLUSIONS: Antibiotic and chlorhexidine-silver sulfadiazine coatings are anti-infective for short (approximately 1 week) insertion times. For longer insertion times, there are no data on antibiotic coating, and there is evidence of lack of effect for chlorhexidine-silver sulfadiazine coating. For silver-impregnated collagen cuffs, there is evidence of lack of effect for both short- and long-term insertion.

Administration, Topical↗
PubMed2001

Reassessment of the location of the thyrotropin receptor 50 amino acid "insertion" provides evidence in favor of a second downstream cleavage site.

Cleavage of thyrotropin receptors (TSHR) on the cell surface into disulfide-linked A and B subunits involves deletion of an intervening region that corresponds approximately to a 50 amino acid "insertion" in the TSHR relative to the noncleaving luteinizing hormone/choriogonadotropin receptor (LH/CGR). The location of this insertion is imprecise because of the relatively low homology between the two receptors in this region. We tested the hypothesis that the TSHR 50 amino acid insertion was further downstream than we previously concluded, a possibility that would relocate the crucial LH/CGR glycan at N291 relative to the position of the TSHR insertion, and that would mitigate against the 50 amino acid insertion playing a role in TSHR intramolecular cleavage. Thus, we transferred the LH/CGR glycan at amino acid 291 from downstream (N367) to upstream of the 50 amino acid insertion (N317) in the TSHR, leaving this insertion intact. TSHR cleavage persisted. Moreover, deletion of amino acid residues 320-366 in addition to the upstream N291 substitution (ALN317-319NET) also did not prevent cleavage. On the other hand, deletion of three contiguous downstream residues (GQE367-369) in the TSHR 50 amino acid insertion abolished receptor cleavage into subunits. In summary, the present data are consistent with our previous location of the TSHR 50 amino acid insertion and, therefore, do not undermine evidence for the involvement of this insertion in TSHR cleavage. In addition, the data regarding TSHR residues GQE367-369 (far downstream of cleavage site 1) support the controversial possibility of a secondary cleavage site downstream of the insertion.

Amino Acid Sequence↗
PubMed1994

Influence of drug release rate on systemic timolol absorption from polymeric ocular inserts in the pigmented rabbit.

There is an expectation that ocular inserts, regardless of the nature of the polymer, will faithfully reduce systemic drug absorption. This may not necessarily be so, however, since not all polymers would release drug at the same rate and to the same extent. The objective of the present study was to determine how drug release rate from various polymeric ocular inserts may influence systemic timolol absorption in the pigmented rabbit. The inserts tested were made of polyvinyl alcohol (PVA), hydroxypropylcellulose (HPC), or partial ethyl ester of poly(vinyl methyl ether/maleic anhydride) (PVMMA), approximately 89.4% w/w in all cases. Some polyvinyl alcohol inserts contained timolol in salt form with Carbopol 940 (PVA-C940), 8.6% w/w. The time course of timolol in plasma over 6 hr was monitored using reversed phase HPLC. While all inserts reduced the peak timolol concentration in plasma (Cmax), only the PVA and HPC inserts, which released timolol rapidly in vitro, reduced the extent of systemic timolol absorption (AUC). On the other hand, both PVA-C940 and PVMMA inserts, which released timolol relatively slowly in vitro, increased the extent of systemic timolol absorption. Moreover, the time at which peak timolol concentration was achieved in the plasma was much delayed, raising the possibility of delayed timolol absorption until discharge of the presumably viscous and/or mucoadhesive solutions of PVA-C940 and PVMMA inserts into the nasal cavity. It may be concluded that not all polymeric ocular inserts reduce systemic timolol absorption. Whether an insert would do so depends on the interplay of residence time in the conjunctival sac and rate of drug release from the insert.

Absorption↗
PubMed2003

Patterns of selection against transposons inferred from the distribution of Tc1, Tc3 and Tc5 insertions in the mut-7 line of the nematode Caenorhabditis elegans.

To identify the factors (selective or mutational) that affect the distribution of transposable elements (TEs) within a genome, it is necessary to compare the pattern of newly arising element insertions to the pattern of element insertions that have been fixed in a population. To do this, we analyzed the distribution of recent mutant insertions of the Tc1, Tc3, and Tc5 elements in a mut-7 background of the nematode Caenorhabditis elegans and compared it to the distribution of element insertions (presumably fixed) within the sequenced genome. Tc1 elements preferentially insert in regions with high recombination rates, whereas Tc3 and Tc5 do not. Although Tc1 and Tc3 both insert in TA dinucleotides, there is no clear relationship between the frequency of insertions and the TA dinucleotide density. There is a strong selection against TE insertions within coding regions: the probability that a TE will be fixed is at least 31 times lower in coding regions than in noncoding regions. Contrary to the prediction of theoretical models, we found that the selective pressure against TE insertions does not increase with the recombination rate. These findings indicate that the distribution of these three transposon families in the genome of C. elegans is determined essentially by just two factors: the pattern of insertions, which is a characteristic of each family, and the selection against insertions within coding regions.

Animals↗
PubMed2004

Locked internal fixator: sensitivity of screw/plate stability to the correct insertion angle of the screw.

OBJECTIVE: Internal fixators with angular stability have been developed to provide high stability without compression of the plate on to the bone. Angular and axial stability of a plate-screw construct can be achieved using a conically threaded screw head undersurface and a corresponding conically threaded plate hole. Furthermore, the insertion angle of the screw must correspond precisely to the axis of the screw hole. This is not always achieved in clinical practice and may result in screw loosening. The objective of this study was to examine the relationship between the stability of the locked screw-plate on the insertion angle of the screw. METHODS: Locking screws were inserted in an isolated (Point Contact Fixator, PC-Fix) or combined (Locking Compression Plate, LCP 4.5) locking hole with the use of an aiming device. The optimal insertion angle for these plates is perpendicular to the plate surface. The screws were inserted with an axis deviation of 0 degrees (optimal condition), 5 degrees , and 10 degrees respective to the optimal angle (variance +/- 1 degrees ). The samples were tested under shear or axial (push out) loading conditions until failure occurred. An Instron materials testing machine was used. RESULTS: Locking screws inserted in the isolated locking hole (PC-Fix) showed a significant decrease of failure load if inserted at 5 degrees and 10 degrees angle. Using an optimal insertion angle (0 degrees ), failure load was 1480 +/- 390 N, with 5 degrees axis deviation 780 +/- 160 N, P = 0.0001, and with 10 degrees axis deviation 550 +/- 110 N, P = 0.0001. Screws inserted in the combined locking hole (LCP) also showed a significant decrease of push-out force of 77% (4960 +/- 1000 N versus 1120 +/- 400 N) with 10 degrees axis deviation. Compared to optimal insertion angle (0 degrees ), bending load to failure did decrease up to 69% (1240 +/- 210 N vs. 390 +/- 100 N) with 10 degrees axis deviation. CONCLUSION: A locking head screw exhibits high stability with a moderate axis deviation in the angle of insertion of up to 5 degrees . However, there is a significant decrease in stability with increasing axis deviation (>5 degrees ). An aiming device is recommended to provide optimal fixation with angular stability.

Bone Screws↗
PubMed1999

Shoe inserts and orthotics for sport and physical activities.

The purposes of this paper were to discuss the perceived benefits of inserts and orthotics for sport activities and to propose a new concept for inserts and orthotics. There is evidence that inserts or orthotics reduce or prevent movement-related injuries. However, there is limited knowledge about the specific functioning an orthotic or insert provides. The same orthotic or insert is often proposed for different problems. Changes in skeletal movement due to inserts or orthotics seem to be small and not systematic. Based on the results of a study using bone pins, one may question the idea that a major function of orthotics or inserts consists in aligning the skeleton. Impact cushioning with shoe inserts or orthotics is typically below 10%. Such small reductions might not be important for injury reduction. It has been suggested that changes in material properties might produce adjustments in the muscular response of the locomotor system. The foot has various sensors to detect input signals with subject specific thresholds. Subjects with similar sensitivity threshold levels seem to respond in their movement pattern in a similar way. Comfort is an important variable. From a biomechanical point of view, comfort may be related to fit, additional stabilizing muscle work, fatigue, and damping of soft tissue vibrations. Based on the presented evidence, the concept of minimizing muscle work is proposed when using orthotics or inserts. A force signal acts as an input variable on the shoe. The shoe sole acts as a first filter, the insert or orthotic as a second filter, the plantar surface of the foot as a third filter for the force input signal. The filtered information is transferred to the central nervous system that provides a subject specific dynamic response. The subject performs the movement for the task at hand. For a given movement task, the skeleton has a preferred path. If an intervention supports/counteracts the preferred movement path, muscle activity can/must be reduced/increased. Based on this concept, an optimal insert or orthotic would reduce muscle activity, feel comfortable, and should increase performance.

Athletic Injuries↗
PubMed2000

Can insertional torque predict screw loosening and related failures? An in vivo study of pedicle screw fixation augmenting posterior lumbar interbody fusion.

STUDY DESIGN: An investigation of the relation between intraoperative insertional torque of pedicle screws, bone mineral density of the vertebra, and development of screw loosening in vivo. OBJECTIVES: To determine the usefulness of intraoperative measurement of the insertional torque of pedicle screws. SUMMARY OF BACKGROUND DATA: Some biomechanical studies have demonstrated that the insertional torque is highly correlated with bone mineral density and the stability of pedicle screws in vitro. METHODS: Pedicle screw fixation was performed with posterior lumbar interbody fusion in 62 consecutive patients. The mean age of the patients at the time of surgery was 58 years. The insertional torque of pedicle screws was measured intraoperatively in all patients. The mean follow-up period was 2.7 years. RESULTS: The mean insertional torque was 1.28 +/- 0.37 Nm in patients with screw loosening and 1.50 +/- 0. 40 Nm in patients without the problem. The mean insertional torque in patients with compression fractures in the upper vertebra adjacent to the fixed segment was 0.83 +/- 0.23 Nm. There was no significant difference between the mean insertional torque in patients with screw loosening and those without the condition. The mean insertional torque in patients without screw loosening was significantly greater than in patients with compression fractures (P < 0.01). A high correlation was found between insertional torque and bone mineral density (P < 0.01). CONCLUSIONS: Although a high correlation was found between the insertional torque of pedicle screws and bone mineral density in vivo, the insertional torque could not objectively predict screw loosening.

Bone Density↗
PubMed2005

Standard multiplanar fluoroscopy versus a fluoroscopically based navigation system for the percutaneous insertion of iliosacral screws: a cadaver model.

OBJECTIVES: To compare the safety and efficiency of standard multiplanar fluoroscopy (StdFluoro) and virtual fluoroscopy (VirtualFluoro) for use in the percutaneous insertion of iliosacral screws. DESIGN: : Human cadaver study comparing 2 imaging modalities during iliosacral screw insertion; imaging randomized from side to side. SETTING: Bioskills laboratory in a medical school. PARTICIPANTS: Twenty-nine embalmed whole human cadavers without prior hip or pelvic surgery. INTERVENTION: Iliosacral screws were inserted into the S1 bodies using a percutaneous insertion technique. Screws were inserted on one side using StdFluoro, and on the other side, screws were placed using VirtualFluoro. MAIN OUTCOME MEASUREMENTS: Time necessary for imaging preparation, screw insertion, and actual fluoroscopy were recorded. Accuracy and safety of screw placement was assessed using computed tomography and an anatomic dissection of the pelvis. RESULTS: : Fifty-six of 58 iliosacral screws were placed within the desired bony corridor of the posterior pelvis. One screw placed using each method was inserted erroneously, but both were relatively minor deviations. There were no obvious injuries to major vessels or nerve roots. The total surgical time required for preparation of imaging and screw insertion averaged 7.3 minutes using StdFluoro and 6.7 minutes using VirtualFluoro (P = 0.4). Although the time necessary for screw insertion using VirtualFluoro averaged only 3.5 minutes, compared to 7.0 minutes for StdFluoro (P < 0.05), this time savings was offset by that required for application and calibration of tracking devices when using VirtualFluoro. The average fluoroscopy time using StdFluoro method was 26 seconds, whereas that for the VirtualFluoro was only 6 seconds (P < 0.01). CONCLUSIONS: Most of the percutaneous iliosacral screws were safely inserted using StdFluoro and VirtualFluoro, and total surgical times were similar using both methods. As VirtualFluoro continues to evolve, improved efficiency in operative times may be expected. Currently, the most beneficial aspect of using VirtualFluoro during the insertion of percutaneous iliosacral screws appears to be significantly decreased use of fluoroscopy when compared to StdFluoro.

Aged↗
PubMed2003

Use of ultrasound guidance in the insertion of radial artery catheters.

OBJECTIVE: To assess the role of a portable ultrasound device in the insertion of radial artery catheters. DESIGN: Prospective, randomized, comparative study. SETTING: Tertiary university hospital. PATIENTS: Elective surgery patients requiring arterial catheter insertion for intraoperative monitoring. INTERVENTIONS: A portable ultrasound device was used to visualize the radial artery at the wrist and to direct arterial catheter insertion. This new technique of arterial catheter insertion was compared with the classic palpation technique. MEASUREMENTS AND MAIN RESULTS: A total of 69 patients requiring an arterial catheter were randomized to either the ultrasound (34 patients) or palpation technique (35 patients). The time taken from skin puncture to successful arterial catheter insertion, the time taken per insertion attempt, the number of attempts required, and the number of cannulae used were recorded for each group. The arterial cannula was inserted on the first attempt in 21 (62%) cases using ultrasound vs. 12 (34%) cases by palpation (p =.03). Significantly fewer attempts were required for catheter insertion using ultrasound as compared with palpation (mean +/- sd, 1.6 +/- 1.0 vs. 3.1 +/- 2.4; p=.003); however, the time taken for each successful attempt was longer (26.1 +/- 2.0 vs. 17.3 +/- 1.6 secs, p=.001). A trend toward shorter overall time required for catheter insertion was found for the ultrasound group (55.5 +/- 63.8 vs. 111.5 +/- 121.5 secs, p=.17). There were four failures in the ultrasound group and one in the palpation group (not significant). CONCLUSIONS: Ultrasound is a useful adjunct to arterial catheter insertion and increases the rate of success at first attempt. The technique is easy to learn and may reduce the time taken to insert the catheter.

Aged↗
PubMed2001

In vivo controlled release of PGE2 from a vaginal insert (0.8 mm, 10 mg) during induction of labour.

OBJECTIVE: To measure the release rate of prostaglandin E2 (PGE2) in vivo from a controlled-release vaginal insert used for cervical ripening and induction of labour at term in women with intact membranes or pre-labour rupture of membranes (PROM). DESIGN: Open-label, single centre study. POPULATION: Women at term (> or = 37 gestational weeks) with unripe cervices (Bishop score < or = 6) scheduled for labour induction for mainly medical reasons. METHODS: sixty-eight women (47 with intact membranes and 21 with PROM) had the PGE2 vaginal insert placed in the posterior fornix of the vagina. Each insert was removed from the women at a predetermined time interval between 0.5 h and 24 h, or earlier if labour was induced, fetal distress was detected or maternal complications occurred. After removal, the vaginal insert was frozen and stored for subsequent assay of residual PGE2. Blood samples were collected immediately before insertion and at 4-hour intervals until removal of the vaginal insert to determine plasma concentrations of PGE2 and the major PGE metabolite, 15-Keto-13, 14-dihydro-PGF2alpha (PGEm). Vaginal pH was measured immediately before insertion and directly after removal of the vaginal insert. Bishop score was assessed before induction, after 8 h, 12 h and immediately after removal of the vaginal insert. RESULTS: There was a positive linear relationship between the amount of PGE2 released from the insert and the duration of treatment in women with intact membranes (rp = 0.95, P = 0.0001), with a calculated PGE2 release rate of 0.52 +/- 0.33 mg/h over 24 h. The PGE2 release rate in women with PROM was not linear. The PGE2 release rate was dependent on vaginal pH, with a faster release rate at higher vaginal pH. Forty-seven women (69.1%) had the insert removed due to the successful induction of labour and consequently discontinued study treatment before their allocated time period. At vaginal delivery, the released amount of PGE2 at onset of labour was 4.0 +/- 3.0 mg and 2.4 +/- 2.1 mg for nulliparous women with PROM and intact membranes, respectively (P = 0.1). In multiparous women, the equivalent mean released amount was 3.2 +/- 2.6 mg and 1.9 +/- 1.4 mg, respectively (P = 0.14). In women with intact membranes, the mean plasma concentrations of PGE2 and PGEm after treatment were not statistically different to those women with PROM (P = 0.27 and 0.64, respectively). In women who were delivered vaginally, the median induction to delivery time interval was 17.0 h (range 4-42) in nulliparous women and 8.7 h (range 5-19) in multiparous women (P = 0.003). Ten (14.7%) women, who were all nulliparous, were delivered by caesarean section. CONCLUSIONS: In women with intact membranes, the PGE2 release rate was linear over 24 hours. There was a positive linear relationship between vaginal pH and PGE2 release rate. The metabolite analysis revealed no evidence of dose dumping neither in women with intact membranes or in women with PROM.

Adult↗
PubMed1986

Large, unstable inserts in the chromosome affect virulence properties of uropathogenic Escherichia coli O6 strain 536.

The hemolytic, uropathogenic Escherichia coli 536 (O6:K15:H31) contains two inserts in its chromosome (insert I and insert II), both of which carried hly genes, were rather unstable, and were deleted spontaneously with a frequency of 10(-3) to 10(-4). These inserts were not found in the chromosome of two nonhemolytic E. coli strains, whereas the chromosomal sequences adjacent to these inserts appeared to be again homologous in the uropathogenic and two other E. coli strains. Insert I was 75 kilobases in size and was flanked at both ends by 16 base pairs (bp) (TTCGACTCCTGTGATC) which were arranged in direct orientation. For insert I it was demonstrated that deletion occurred by recombination between the two 16-bp flanking sequences, since mutants lacking this insert still carried a single copy of the 16-bp sequence in the chromosome. Both inserts contained a functional hemolysin determinant. However, the loss of the inserts not only affected the hemolytic phenotype but led to a considerable reduction in serum resistance and the loss of mannose-resistant hemagglutination, caused by the presence of S-type fimbriae (sfa). It is shown that the Sfa-negative phenotype is due to a block in transcription of the sfa genes. Mutants of strain 536 which lacked both inserts were entirely avirulent when tested in several animal model systems.

Adhesins, Escherichia coli↗
PubMed1996

Random removal of inserts from an RNA genome: selection against single-stranded RNA.

We have monitored the evolution of insertions in two MS2 RNA regions of known secondary structure where coding pressure is negligible or absent. Base changes and shortening of the inserts proceed until the excessive nucleotides can be accommodated in the original structure. The stems of hairpins can be dramatically extended but the loops cannot, revealing natural selection against single-stranded RNA. The 3' end of the MS2 A-protein gene forms a small hairpin with an XbaI sequence in the loop. This site was used to insert XbaI fragments of various sizes. Phages produced by these MS2 cDNA clones were not wild type, nor had they retained the full insert. Instead, every revertant phage had trimmed the insert in a different way to leave a four- to seven-membered loop to the now extended stem. Similar results were obtained with inserts in the 5' untranslated region. The great number of different revertants obtained from a single starting mutant as well as sequence inspection of the crossover points suggest that the removal of redundant RNA occurs randomly. The only common feature among all revertants appears the potential to form a hairpin with a short loop, suggesting that single-stranded RNA negatively affects the viability of the phage. To test this hypothesis, we introduced XbaI fragments of 34 nucleotides that could form either a long stem with a small loop or a short stem with a large loop (26 nucleotides). The base-paired inserts were perfectly maintained for many generations, whereas the unpaired versions were quickly trimmed back to reduce the size of the loop. These data confirm that single-stranded RNA adversely affects phage fitness and is strongly selected against. The repair of the RNA genome that we describe here appears as the result of random recombination. Of the plethora of recombinants, only those able to adopt a base-paired structure survive. The frequency with which our inserts are removed seems higher than measured by others for small inserts in a reading frame in Q beta RNA. To account for this higher frequency, we suggest models in which the single-stranded nature of our inserts induces random recombination at the site of the insertion.

Amino Acid Sequence↗
PubMed2005

Increased multinucleoside drug resistance and decreased replicative capacity of a human immunodeficiency virus type 1 variant with an 8-amino-Acid insert in the reverse transcriptase.

Resistance to antiretroviral drugs is generally conferred by specific amino acid substitutions, rather than insertions or deletions, in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The exception to these findings is the amino acid insertions found in the beta3-beta4 loop of the RT enzyme in response to treatment with nucleoside reverse transcriptase inhibitors. This insert consists most commonly of two amino acids, but we describe in detail the evolution of a variant with an 8-amino-acid (aa) insert in a patient treated with zidovudine (ZDV) and 2'-3'-dideoxycytidine (ddC). The 24-nucleotide insert is a partial duplication of local sequences but also contains a sequence segment of unknown origin. Extensive sequence analysis of longitudinal patient samples indicated that the HIV-1 population prior to the start of therapy contained not the wild-type amino acid 215T in RT but a mixture with 215D and 215C. Treatment with ZDV and subsequent ZDV-ddC combination therapy resulted in the evolution of an HIV-1 variant with a typical ZDV resistance genotype (41L, 44D, 67N, 69D, 210W, 215Y), which was slowly replaced by the insert-containing variant (41L, 44D, insert at position 69, 70R, 210W, 215Y). The latter variant demonstrated increased resistance to a wide range of drugs, indicating that the 8-aa insert augments nucleoside analogue resistance. The gain in drug resistance of the insert variant came at the expense of a reduction in replication capacity when assayed in the absence of drugs. We compared these data with the resistance and replication properties of 133 insert-containing sequences of different individuals present in the ViroLogic database and found that the size and actual sequence of the insert at position 69 influence the level of resistance to nucleoside analogues.

Adult↗
PubMed2002

Gum-elastic bougie-guided insertion of the ProSeal laryngeal mask airway: a new technique.

We determined the success rates, cardiovascular responses and airway morbidity for gum-elastic bougie-guided insertion of the ProSeal laryngeal mask airway. One hundred anaesthetized, non-paralyzed adults (ASA 1-2 aged 18 to 80 years) were studied. The ProSeal LMA drainage tube was primed with a well-lubricated 16 French gauge gum-elastic bougie with the curved end proximal and the straight end protruding 30 cm beyond the drainage tube tip. The straight end of the gum-elastic bougie was inserted into the oesophagus under laryngoscopic guidance, the laryngoscope removed and the ProSeal LMA inserted using the standard insertion technique and the gum-elastic bougie as a guide. The following variables were recorded: ease of insertion, oropharyngeal leak pressure, ventilatory capability, ease of gastric tube insertion, blood staining on the bougie or LMA at removal, and postoperative airway morbidity. Haemodynamic data were recorded immediately pre-insertion and every minute for five minutes after insertion. Gum-elastic bougie and ProSeal LMA insertion was successful at the first attempt in all patients within 50 seconds. There were no significant increases in heart rate or blood pressure. Oropharyngeal leak pressure was 33 (17-40) cmH2O and ventilation was possible without leak in all patients at 9.5 ml x kg(-1) tidal volume. There were no drainage tube or gastric air leaks. Gastric tube insertion was successful at the first attempt in all patients. Blood staining at removal was not detected on the gum-elastic bougie, but was detected in 3% of ProSeal LMAs. The incidence of sore throat, dysphagia and dysarthria was 21%, 9% and 1% respectively. We conclude that gum-elastic bougie-guided insertion of the ProSeal LMA has a high success rate and is associated with minimal haemodynamic change and a low incidence of trauma.

Adolescent↗