QuickMap: a public tool for large-scale gene therapy vector insertion site mapping and analysis.

PubMed

Appelt, J-U; Giordano, F A; Ecker, M; Roeder, I; Grund, N; Hotz-Wagenblatt, A; Opelz, G; Zeller, W J; Allgayer, H; Fruehauf, S; Laufs, S

2009-07-01

Several events of insertional mutagenesis in pre-clinical and clinical gene therapy studies have created intense interest in assessing the genomic insertion profiles of gene therapy vectors. For the construction of such profiles, vector-flanking sequences detected by inverse PCR, linear amplification-mediated-PCR or ligation-mediated-PCR need to be mapped to the host cell's genome and compared to a reference set. Although remarkable progress has been achieved in mapping gene therapy vector insertion sites, public reference sets are lacking, as are the possibilities to quickly detect non-random patterns in experimental data. We developed a tool termed QuickMap, which uniformly maps and analyzes human and murine vector-flanking sequences within seconds (available at www.gtsg.org). Besides information about hits in chromosomes and fragile sites, QuickMap automatically determines insertion frequencies in +/- 250 kb adjacency to genes, cancer genes, pseudogenes, transcription factor and (post-transcriptional) miRNA binding sites, CpG islands and repetitive elements (short interspersed nuclear elements (SINE), long interspersed nuclear elements (LINE), Type II elements and LTR elements). Additionally, all experimental frequencies are compared with the data obtained from a reference set, containing 1 000 000 random integrations ('random set'). Thus, for the first time a tool allowing high-throughput profiling of gene therapy vector insertion sites is available. It provides a basis for large-scale insertion site analyses, which is now urgently needed to discover novel gene therapy vectors with 'safe' insertion profiles.

Comparing schizophrenia symptoms in the Iban of Sarawak with other populations to elucidate clinical heterogeneity.

PubMed

McLean, Duncan; Barrett, Robert; Loa, Peter; Thara, Rangaswamy; John, Sujit; McGrath, John; Gratten, Jake; Mowry, Bryan

2015-03-01

The symptom profile of schizophrenia can vary between ethnic groups. We explored selected symptom variables previously reported to be characteristic of schizophrenia in the Iban of Sarawak in transethnic populations from Australia, India, and Sarawak, Malaysia. We tested site differences to confirm previous research, and to explore implications of differences across populations for future investigations. We recruited schizophrenia samples in Australia (n = 609), India (n = 310) and Sarawak (n = 205) primarily for the purposes of genetic studies. We analyzed seven identified variables and their relationship to site using logistic regression, including: global delusions, bizarre delusions, thought broadcast/insertion/withdrawal delusions, global hallucinations, auditory hallucinations, disorganized behavior, and prodromal duration. We identified a distinct symptom profile in our Sarawak sample. Specifically, the Iban exhibit: low frequency of thought broadcast/insertion/withdrawal delusions, high frequency of auditory hallucinations and disorganized behavior, with a comparatively short prodrome when compared with Australian and Indian populations. Understanding between-site variation in symptom profile may complement future transethnic genetic studies, and provide important clues as to the nature of differing schizophrenia expression across ethnically distinct groups. A comprehensive approach to subtyping schizophrenia is warranted, utilizing comprehensively ascertained transethnic samples to inform both schizophrenia genetics and nosology. Copyright © 2013 Wiley Publishing Asia Pty Ltd.

NASA's Planned Return to the Moon: Global Access and Anytime Return Requirement Implications on the Lunar Orbit Insertion Burns

NASA Technical Reports Server (NTRS)

Garn, Michelle; Qu, Min; Chrone, Jonathan; Su, Philip; Karlgaard, Chris

2008-01-01

Lunar orbit insertion LOI is a critical maneuver for any mission going to the Moon. Optimizing the geometry of this maneuver is crucial to the success of the architecture designed to return humans to the Moon. LOI burns necessary to meet current NASA Exploration Constellation architecture requirements for the lunar sortie missions are driven mainly by the requirement for global access and "anytime" return from the lunar surface. This paper begins by describing the Earth-Moon geometry which creates the worst case (delta)V for both the LOI and the translunar injection (TLI) maneuvers over the full metonic cycle. The trajectory which optimizes the overall (delta)V performance of the mission is identified, trade studies results covering the entire lunar globe are mapped onto the contour plots, and the effects of loitering in low lunar orbit as a means of reducing the insertion (delta)V are described. Finally, the lighting conditions on the lunar surface are combined with the LOI and TLI analyses to identify geometries with ideal lighting conditions at sites of interest which minimize the mission (delta)V.

Effect of skin disinfection with octenidine dihydrochloride on insertion site colonization of intravascular catheters.

PubMed

Dettenkofer, M; Jonas, D; Wiechmann, C; Rossner, R; Frank, U; Zentner, J; Daschner, F D

2002-10-01

We investigated the efficacy of two commercially available, alcohol-based antiseptic solutions in decontaminating the insertion site of central lines. One solution contained the bispyridine octenidine dihydrochloride. Inpatients receiving either a central venous catheter (CVC) or a peripherally inserted central catheter (PICC) were alternately assigned to different skin disinfection regimens at the insertion site: (A) 0.1% octendine dihydrochloride with 30% 1-propanol and 45% 2-propanol, (B) 74% ethanol with 10% 2-propanol. Quantitative skin cultures were obtained from the insertion site at predetermined intervals. A total of 60 patients received 12 CVCs and 47 PICCs (no significant difference with respect to gender, age and catheter type). In total, 90 cultures were assessed in each group. The median colony-forming unit (cfu) counts per 24 cm(2) (group A vs B) were 2,270 vs 2,950 before, 20 vs 40 following and 860 vs 1,210 24 h after catheter insertion, respectively. A statistically significant difference in the efficacy of skin decontamination was seen between groups in culture set (3) and in the difference between culture sets (2) and (3) (Wilcoxon rank sum test). Octenidine/propanol appears to be more effective than alcohol (ethanol/propanol) alone in reducing microflora of the skin at the PICC/CVC insertion site over a 24-h period.

Soft tissue graft interference fit fixation: observations on graft insertion site healing and tunnel remodeling 2 years after ACL reconstruction in sheep.

PubMed

Hunt, Patrick; Rehm, Oliver; Weiler, Andreas

2006-12-01

Using soft tissue grafts for anterior cruciate ligament (ACL) reconstruction, insertion site healing plays a crucial role in the long-term fate of the graft. It has been shown in an experimental animal study that using a soft tissue graft and anatomic graft fixation, a direct ligamentous insertion alike the native ACL developed 24 weeks postoperatively. Yet there are no reports on the long-term insertion site healing of anatomically fixed soft tissue grafts. The objective of this study was to evaluate graft insertion site healing, the intra-tunnel fate of the graft and its osseous replacement 2 years after ACL reconstruction in sheep. The left ACLs of six sheep were replaced by an autologous flexor tendon split graft and anatomically fixed with biodegradable poly-(D, L-lactide) interference screws. Animals received polychromic sequential labeling at different points in time to determine bone apposition per period. For evaluation of the insertion site healing and intra-tunnel changes, MRI scans were taken in vivo. Following sacrifice, radiographic imaging, conventional histology and fluorescence microscopy was undertaken. Most of the specimens showed a wide direct ligamentous insertion. It showed patterns alike the direct ligament insertion seen in intact ACLs. The intra-tunnel part of the graft had completely lost its tendon-like structure and in two cases, it was separated from the graft insertion by a thick bony layer. The biodegradable interference screw was fully degraded in all specimens. Ossification of the former drill tunnels was intense, showing only partial-length tunnel remnants in one femoral and three tibial specimens. As the graft heals to the joint surface and the aperture site is closed with soft tissue, mechanical stress of the intra-tunnel part of the graft is eliminated and the bone tunnel is protected from synovial fluid, resulting in osseous bridging of the tunnel aperture site, accelerated intra-tunnel graft resorption and its osseous replacement.

The P gene of bovine parainfluenza virus 3 expresses all three reading frames from a single mRNA editing site.

PubMed Central

Pelet, T; Curran, J; Kolakofsky, D

1991-01-01

The P gene of bovine parainfluenza virus 3 (bPIV3) contains two downstream overlapping ORFs, called V and D. By comparison with the mRNA editing sites of other paramyxoviruses, two editing sites were predicted for bPIV3; site a to express the D protein, and site b to express the V protein. Examination of the bPIV3 mRNAs, however, indicates that site b is non-functional whereas site a operates frequently. Insertions at site a give rise to both V and D protein mRNAs, because a very broad distribution of Gs is added when insertions occur. This broad distribution is very different from the editing sites of Sendai virus or SV5, where predominantly one form of edited mRNA containing either a one or two G insertion respectively is created, to access the single overlapping ORF of these viruses. A model is proposed to explain how paramyxoviruses control the range of G insertions on that fraction of the mRNAs where insertions occur. The bPIV3 P gene is unique as far as we know, in that a sizeable portion of the gene expresses all 3 reading frames as protein. bPIV3 apparently does this from a single editing site by removing the constraints which control the number of slippage rounds which take place. Images PMID:1846805

Clinical implications of acute pelvicaliceal hematoma formation during percutaneous catheter nephrostomy insertion.

PubMed

Stewart, Jessica K; Smith, Tony P; Kim, Charles Y

To determine the clinical implications of acute pelvicaliceal hematoma formation during percutaneous catheter nephrostomy (PCN) insertion. Collecting system hematoma burden was retrospectively assessed for 694 PCN insertions in 502 patients. Pelvicaliceal hematoma formation occurred in 146 kidneys (21%) in 136 patients. Clinically significant blood loss occurred in 3 patients with hematomas within one week compared to 4 patients without hematomas (p=0.39). Twenty-four patients with hematomas underwent catheter exchange within one week, compared to 55 patients without hematomas (p=0.49). Pelvicaliceal hematoma formation after PCN insertion is not uncommon and is associated with very rare clinical sequelae. Copyright © 2017 Elsevier Inc. All rights reserved.

Does a conservative tibial cut in conventional total knee arthroplasty violate the deep medial collateral ligament?

PubMed

Maes, Michael; Luyckx, Thomas; Bellemans, Johan

2014-11-01

Based on the anatomy of the deep medial collateral ligament (MCL), it was hypothesized that at least part of its cross-sectional insertion area is jeopardized while performing a standard tibial cut in conventional total knee arthroplasty (TKA). The aim of this study was to determine whether it is anatomically possible to preserve the tibial deep MCL insertion during conventional TKA. Thirty-three unpaired cadaveric knee specimens were used for this study. Knees with severe varus/valgus deformity or damage to the medial structures of the knee were excluded. In the first part of the study, the dimensions of the tibial insertion of the deep MCL and its relationship to the joint line were recorded. Next, the cross-sectional area of the deep MCL insertion was determined using calibrated digital photographic analysis. In the second part, the effect of a standard 9-mm 3° sloped tibial cut on the structural integrity of the deep MCL cross-sectional insertion area was determined using conventional instrumentation. The proximal border of the deep MCL insertion site on the tibia was located on average 4.7 ± 1.2 mm distally to the joint line. After performing a standard 9-mm 3° sloped tibial cut, on average 54% of the deep MCL insertion area was resected. In 29% of the cases, the deep MCL insertion area was completely excised. The deep MCL cannot routinely be preserved in conventional TKA. The deep MCL insertion is at risk and may be jeopardized in case of a tibial cut 9 mm below the native joint line. As the deep MCL is a distinct medial stabilizer and plays an important role in rotational stability, this may have implications in future designs of both unicondylar and total knee arthroplasty, but further research is necessary.

Identifying transposon insertions and their effects from RNA-sequencing data.

PubMed

de Ruiter, Julian R; Kas, Sjors M; Schut, Eva; Adams, David J; Koudijs, Marco J; Wessels, Lodewyk F A; Jonkers, Jos

2017-07-07

Insertional mutagenesis using engineered transposons is a potent forward genetic screening technique used to identify cancer genes in mouse model systems. In the analysis of these screens, transposon insertion sites are typically identified by targeted DNA-sequencing and subsequently assigned to predicted target genes using heuristics. As such, these approaches provide no direct evidence that insertions actually affect their predicted targets or how transcripts of these genes are affected. To address this, we developed IM-Fusion, an approach that identifies insertion sites from gene-transposon fusions in standard single- and paired-end RNA-sequencing data. We demonstrate IM-Fusion on two separate transposon screens of 123 mammary tumors and 20 B-cell acute lymphoblastic leukemias, respectively. We show that IM-Fusion accurately identifies transposon insertions and their true target genes. Furthermore, by combining the identified insertion sites with expression quantification, we show that we can determine the effect of a transposon insertion on its target gene(s) and prioritize insertions that have a significant effect on expression. We expect that IM-Fusion will significantly enhance the accuracy of cancer gene discovery in forward genetic screens and provide initial insight into the biological effects of insertions on candidate cancer genes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Variation in the shape of the tibial insertion site of the anterior cruciate ligament: classification is required.

PubMed

Guenther, Daniel; Irarrázaval, Sebastian; Nishizawa, Yuichiro; Vernacchia, Cara; Thorhauer, Eric; Musahl, Volker; Irrgang, James J; Fu, Freddie H

2017-08-01

To propose a classification system for the shape of the tibial insertion site (TIS) of the anterior cruciate ligament (ACL) and to demonstrate the intra- and inter-rater agreement of this system. Due to variation in shape and size, different surgical approaches may be feasible to improve reconstruction of the TIS. One hundred patients with a mean age of 26 ± 11 years were included. The ACL was cut arthroscopically at the base of the tibial insertion site. Arthroscopic images were taken from the lateral and medial portal. Images were de-identified and duplicated. Two blinded observers classified the tibial insertion site according to a classification system. The tibial insertion site was classified as type I (elliptical) in 51 knees (51 %), type II (triangular) in 33 knees (33 %) and type III (C-shaped) in 16 knees (16 %). There was good agreement between raters when viewing the insertion site from the lateral portal (κ = 0.65) as well as from the medial portal (κ = 0.66). Intra-rater reliability was good to excellent. Agreement in the description of the insertion site between the medial and lateral portals was good for rater 1 and good for rater 2 (κ = 0.74 and 0.77, respectively). There is variation in the shape of the ACL TIS. The classification system is a repeatable and reliable tool to summarize the shape of the TIS using three common patterns. For clinical relevance, different shapes may require different types of reconstruction to ensure proper footprint restoration. Consideration of the individual TIS shape is required to prevent iatrogenic damage of adjacent structures like the menisci. III.

A first-principles comparative study of lithium, sodium, and magnesium storage in pure and gallium-doped germanium: Competition between interstitial and substitutional sites

NASA Astrophysics Data System (ADS)

Legrain, Fleur; Manzhos, Sergei

2017-01-01

Thermodynamics and kinetics of Li, Na, and Mg storage in Ge are studied ab initio. The most stable configurations can consist of tetrahedral, substitutional, or a combination of the two types of sites. In the dilute limit, Li and Na prefer interstitial, while Mg prefers substitutional sites. At higher concentrations of Li, Na, and Mg, there is a combination of interstitial and substitutional sites. This is an important finding, as most previous ab initio studies of alloying type electrode materials ignored substitutional sites. Insertion energies computed at dilute concentration (x = 1/64) show that Na and Mg insertion are not thermodynamically favored in Ge vs. the formation of bulk Na and Mg, as opposed to Li insertion which is favored. We investigate the effect of p-doping of Ge (with Ga) on the thermodynamics and find that it considerably lowers the defect formation energies associated with the insertion of Li/Na/Mg at tetrahedral sites. On the other hand, the energetics associated with Li/Na/Mg insertion at substitutional sites are not significantly affected. In addition, we compute the migration energy barriers for Li/Na/Mg diffusion between two tetrahedral sites (0.38/0.79/0.66 eV), between two substitutional sites (0.77/0.93/1.83 eV), and between two sites of different types (2.15/1.75/0.85 eV).

A first-principles comparative study of lithium, sodium, and magnesium storage in pure and gallium-doped germanium: Competition between interstitial and substitutional sites.

PubMed

Legrain, Fleur; Manzhos, Sergei

2017-01-21

Thermodynamics and kinetics of Li, Na, and Mg storage in Ge are studied ab initio. The most stable configurations can consist of tetrahedral, substitutional, or a combination of the two types of sites. In the dilute limit, Li and Na prefer interstitial, while Mg prefers substitutional sites. At higher concentrations of Li, Na, and Mg, there is a combination of interstitial and substitutional sites. This is an important finding, as most previous ab initio studies of alloying type electrode materials ignored substitutional sites. Insertion energies computed at dilute concentration (x = 1/64) show that Na and Mg insertion are not thermodynamically favored in Ge vs. the formation of bulk Na and Mg, as opposed to Li insertion which is favored. We investigate the effect of p-doping of Ge (with Ga) on the thermodynamics and find that it considerably lowers the defect formation energies associated with the insertion of Li/Na/Mg at tetrahedral sites. On the other hand, the energetics associated with Li/Na/Mg insertion at substitutional sites are not significantly affected. In addition, we compute the migration energy barriers for Li/Na/Mg diffusion between two tetrahedral sites (0.38/0.79/0.66 eV), between two substitutional sites (0.77/0.93/1.83 eV), and between two sites of different types (2.15/1.75/0.85 eV).

Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.

PubMed

Li, Jun; Meng, Xiangbing; Zong, Yuan; Chen, Kunling; Zhang, Huawei; Liu, Jinxing; Li, Jiayang; Gao, Caixia

2016-09-12

Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants(1), but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.

P and M gene junction is the optimal insertion site in Newcastle disease virus vaccine vector for foreign gene expression

USDA-ARS?s Scientific Manuscript database

Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy purposes. However, the optimal insertion site for foreign gene expression remained to be determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five different intergenic ...

Recurrence, submicroscopic complexity, and potential clinical relevance of copy gains detected by array CGH that are shown to be unbalanced insertions by FISH.

PubMed

Neill, Nicholas J; Ballif, Blake C; Lamb, Allen N; Parikh, Sumit; Ravnan, J Britt; Schultz, Roger A; Torchia, Beth S; Rosenfeld, Jill A; Shaffer, Lisa G

2011-04-01

Insertions occur when a segment of one chromosome is translocated and inserted into a new region of the same chromosome or a non-homologous chromosome. We report 71 cases with unbalanced insertions identified using array CGH and FISH in 4909 cases referred to our laboratory for array CGH and found to have copy-number abnormalities. Although the majority of insertions were non-recurrent, several recurrent unbalanced insertions were detected, including three der(Y)ins(Y;18)(q?11.2;p11.32p11.32)pat inherited from parents carrying an unbalanced insertion. The clinical significance of these recurrent rearrangements is unclear, although the small size, limited gene content, and inheritance pattern of each suggests that the phenotypic consequences may be benign. Cryptic, submicroscopic duplications were observed at or near the insertion sites in two patients, further confounding the clinical interpretation of these insertions. Using FISH, linear amplification, and array CGH, we identified a 126-kb duplicated region from 19p13.3 inserted into MECP2 at Xq28 in a patient with symptoms of Rett syndrome. Our results demonstrate that although the interpretation of most non-recurrent insertions is unclear without high-resolution insertion site characterization, the potential for an otherwise benign duplication to result in a clinically relevant outcome through the disruption of a gene necessitates the use of FISH to determine whether copy-number gains detected by array CGH represent tandem duplications or unbalanced insertions. Further follow-up testing using techniques such as linear amplification or sequencing should be used to determine gene involvement at the insertion site after FISH has identified the presence of an insertion.

HIV Integration at Certain Sites in Host DNA is Linked to the Expansion and Persistence of Infected Cells | Center for Cancer Research

Cancer.gov

When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with

Quantifying particulate matter deposition in Niwot Ridge, Colorado: Collection of dry deposition using marble inserts and particle imaging using the FlowCAM

NASA Astrophysics Data System (ADS)

Goss, Natasha R.; Mladenov, Natalie; Seibold, Christine M.; Chowanski, Kurt; Seitz, Leslie; Wellemeyer, T. Barret; Williams, Mark W.

2013-12-01

Atmospheric wet and dry deposition are important sources of carbon for remote alpine lakes and soils. The carbon inputs from dry deposition in alpine National Atmospheric Deposition Program (NADP) collectors, including aeolian dust and biological material, are not well constrained due to difficulties in retaining particulate matter in the collectors. Here, we developed and tested a marble insert for dry deposition collection at the Niwot Ridge Long Term Ecological Research Station (NWT LTER) Soddie site (3345 m) between 24 May and 8 November 2011. We conducted laboratory tests of the insert's effect on particulate matter (PM) mass and non-purgeable organic carbon (DOC) and found that the insert did not significantly change either measurement. Thus, the insert may enable dry deposition collection of PM and DOC at NADP sites. We then developed a method for enumerating the collected wet and dry deposition with the Flow Cytometer and Microscope (FlowCAM), a dynamic-image particle analysis tool. The FlowCAM has the potential to establish morphology, which affects particle settling and retention, through particle diameter and aspect ratio. Particle images were used to track the abundance of pollen grains over time. Qualitative image examination revealed that most particles were biological in nature, such as intact algal cells and pollen. Dry deposition loading to the Soddie site as determined by FlowCAM measurements was highly variable, ranging from 100 to >230 g ha-1 d-1 in June-August 2011 and peaking in late June. No significant difference in diameter or aspect ratio was found between wet and dry deposition, suggesting fundamental similarities between those deposition types. Although FlowCAM statistics and identification of particle types proved insightful, our total-particle enumeration method had a high variance and underestimated the total number of particles when compared to imaging of relatively large volumes (60-125 mL) from a single sample. We recommend use of the FlowCAM, especially for subclasses of particles, but in light of uncertainty in particle counts, believe that it should be paired with traditional methods such as microscopy in this stage of the technique's development. Analysis of well-mixed samples produced lower variability than settling methods used for algae samples. Use of the marble inserts in the dry deposition collector in the NADP context is recommended, and the implications of various particle counting and identification methods are explored.

Intravenous insertion site protection: moisture accumulation in intravenous site protectors.

PubMed

Lee, W E; Vallino, L M

1996-01-01

Stabilizing the intravenous catheter after insertion is a significant part of intravenous therapy. Dislodgments of the cannula from its optimal position in the vein can lead to complications such as phlebitis, thrombophlebitis, infiltration, and infection. Intravenous site protector shields are designed to protect the catheter from impact and tissue trauma at the insertion site. Nurses have requested ventilation in these shields to avoid moisture build up that may increase the risk of infections. To address this issue, experimental laboratory testing was performed to determine if moisture accumulation as evidenced by increased weight of the shield and visible evidence of condensation occurred. No moisture condensation problems with the ventilated intravenous site protectors were found.

Surgical implications of perimodiolar cochlear implant electrode design: avoiding intracochlear damage and scala vestibuli insertion.

PubMed

Briggs, R J; Tykocinski, M; Saunders, E; Hellier, W; Dahm, M; Pyman, B; Clark, G M

2001-09-01

To review the mechanisms and nature of intracochlear damage associated with cochlear implant electrode array insertion, in particular, the various perimodiolar electrode designs. Make recommendations regarding surgical techniques for the Nucleus Contour electrode to ensure correct position and minimal insertion trauma. The potential advantages of increased modiolar proximity of intracochlear multichannel electrode arrays are a reduction in stimulation thresholds, an increase in dynamic range and more localized neural excitation. This may improve speech perception and reduce power consumption. These advantages may be negated if increased intracochlear damage results from the method used to position the electrodes close to the modiolus. A review of the University of Melbourne Department of Otolaryngology experience with temporal bone safety studies using the Nucleus standard straight electrode array and a variety of perimodiolar electrode array designs; comparison with temporal bone insertion studies from other centres and postmortem histopathology studies reported in the literature. Review of our initial clinical experience using the Nucleus Contour electrode array. The nature of intracochlear damage resulting from electrode insertion trauma ranges from minor, localized, spiral ligament tear to diffuse organ of Corti disruption and osseous spiral lamina fracture. The type of damage depends on the mechanical characteristics of the electrode array, the stiffness, curvature and size of the electrode in relation to the scala, and the surgical technique. The narrow, flexible, straight arrays are the least traumatic. Pre-curved or stiffer arrays are associated with an incidence of basilar membrane perforation. The cochleostomy must be correctly sited in relation to the round window to ensure scala tympani insertion. A cochleostomy anterior to the round window rather than inferior may lead to scala media or scala vestibuli insertion. Proximity of electrodes to the modiolus can be achieved without intracochlear damage provided the electrode array is a free fit within the scala, of appropriate size and shape, and accurate scala tympani insertion is performed.

National Biocontainment Training Center

DTIC Science & Technology

2014-06-01

of novel antimicrobial compounds with antibacterial activity against TB (Vijayakumar, et al., Dec. 2013, Tuberculosis). She has also developed...Nanoluc. (a) insert the Nanoluc after the signal peptide , (b) insert the Nanoluc before the RSKR site, (c) insert the Nanoluc before the RRLL site...biological safe techniques. This training took place in Dallas, Texas. Trainers: Vickie Jones and Jason Hardcastle. Food and Drug Administration (FDA

Characterization of insertion sites in Rainbow papaya, the first commercialized transgenic tree-fruit crop

USDA-ARS?s Scientific Manuscript database

Inserts and insert sites in transgenic, commercial papaya line 55-1 derivatives Rainbow and SunUp were characterized as part of a petition to Japan to allow import of fresh fruit of these cultivars from the U.S. and to provide data for a larger study aimed at understanding the global impact of DNA t...

Identification of multiple sites suitable for insertion of foreign genes in herpes simplex virus genomes.

PubMed

Morimoto, Tomomi; Arii, Jun; Akashi, Hiroomi; Kawaguchi, Yasushi

2009-03-01

Information on sites in HSV genomes at which foreign gene(s) can be inserted without disrupting viral genes or affecting properties of the parental virus are important for basic research on HSV and development of HSV-based vectors for human therapy. The intergenic region between HSV-1 UL3 and UL4 genes has been reported to satisfy the requirements for such an insertion site. The UL3 and UL4 genes are oriented toward the intergenic region and, therefore, insertion of a foreign gene(s) into the region between the UL3 and UL4 polyadenylation signals should not disrupt any viral genes or transcriptional units. HSV-1 and HSV-2 each have more than 10 additional regions structurally similar to the intergenic region between UL3 and UL4. In the studies reported here, it has been demonstrated that insertion of a reporter gene expression cassette into several of the HSV-1 and HSV-2 intergenic regions has no effect on viral growth in cell culture or virulence in mice, suggesting that these multiple intergenic regions may be suitable HSV sites for insertion of foreign genes.

Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing

PubMed Central

Lepage, Étienne; Zampini, Éric; Boyle, Brian; Brisson, Normand

2013-01-01

Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity. PMID:23951038

Insights into the surface topology of polyhydroxyalkanoate synthase: self-assembly of functionalized inclusions.

PubMed

Hooks, David O; Rehm, Bernd H A

2015-10-01

The polyhydroxyalkanoate (PHA) synthase catalyzes the synthesis of PHA and remains attached to the hydrophobic PHA inclusions it creates. Although this feature is actively exploited to generate functionalized biobeads via protein engineering, little is known about the structure of the PHA synthase. Here, the surface topology of Ralstonia eutropha PHA synthase was probed to inform rational protein engineering toward the production of functionalized PHA beads. Surface-exposed residues were detected by conjugating biotin to inclusion-bound PHA synthase and identifying the biotin-conjugated lysine and cysteine residues using peptide fingerprinting analysis. The identified sites (K77, K90, K139, C382, C459, and K518) were investigated as insertion sites for the generation of new protein fusions. Insertions of FLAG epitopes into exposed sites K77, K90, K139, and K518 were tolerated, retaining >65 % of in vivo activity. Sites K90, K139, and K518 were also tested by insertion of the immunoglobulin G (IgG)-binding domain (ZZ), successfully producing PHA inclusions able to bind human IgG in vitro. Although simultaneous insertions of the ZZ domain into two sites was permissive, insertion at all three lysine sites inactivated the synthase. The K90/K139 double ZZ insertion had the optimum IgG-binding capacity of 16 mg IgG/g wet PHA beads and could selectively purify the IgG fraction from human serum. Overall, this study identified surface-exposed flexible regions of the PHA synthase which either tolerate protein/peptide insertions or are critical for protein function. This further elucidates the structure and function of PHA synthase and provides new opportunities for generating functionalized PHA biobeads.

Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patra, Amritaj; Zhang, Qianqian; Lei, Li

2015-02-09

The most prevalent lesion in DNA is an abasic site resulting from glycolytic cleavage of a base. In a number of cellular studies, abasic sites preferentially code for dATP insertion (the “A rule”). In some cases frameshifts are also common. X-ray structures with abasic sites in oligonucleotides have been reported for several microbial and human DNA polymerases (pols), e.g. Dpo4, RB69, KlenTaq, yeast pol ι, human (h) pol ι, and human pol β. We reported previously that hpol η is a major pol involved in abasic site bypass (Choi, J.-Y., Lim, S., Kim, E. J., Jo, A., and Guengerich, F.more » P. (2010 J. Mol. Biol. 404, 34–44). hpol η inserted all four dNTPs in steady-state and pre-steady-state assays, preferentially inserting A and G. In LC-MS analysis of primer-template pairs, A and G were inserted but little C or T was inserted. Frameshifts were observed when an appropriate pyrimidine was positioned 5' to the abasic site in the template. In x-ray structures of hpol η with a non-hydrolyzable analog of dATP or dGTP opposite an abasic site, H-bonding was observed between the phosphate 5' to the abasic site and water H-bonded to N1 and N6 of A and N1 and O6 of G nucleoside triphosphate analogs, offering an explanation for what appears to be a “purine rule.” A structure was also obtained for an A inserted and bonded in the primer opposite the abasic site, but it did not pair with a 5' T in the template. Finally, we conclude that hpol η, a major copying enzyme with abasic sites, follows a purine rule, which can also lead to frameshifts. The phenomenon can be explained with H-bonds.« less

Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

PubMed Central

Lim, Kwang-il; Klimczak, Ryan; Yu, Julie H.; Schaffer, David V.

2010-01-01

Retroviral vectors offer benefits of efficient delivery and stable gene expression; however, their clinical use raises the concerns of insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSS). We have shifted the integration preferences of retroviral vectors by generating a library of viral variants with a DNA-binding domain inserted at random positions throughout murine leukemia virus Gag-Pol, then selecting for variants that are viable and exhibit altered integration properties. We found seven permissive zinc finger domain (ZFD) insertion sites throughout Gag-Pol, including within p12, reverse transcriptase, and integrase. Comprehensive genome integration analysis showed that several ZFD insertions yielded retroviral vector variants with shifted integration patterns that did not favor TSS. Furthermore, integration site analysis revealed selective integration for numerous mutants. For example, two retroviral variants with a given ZFD at appropriate positions in Gag-Pol strikingly integrated primarily into four common sites out of 3.1 × 109 possible human genome locations (P = 4.6 × 10-29). Our findings demonstrate that insertion of DNA-binding motifs into multiple locations in Gag-Pol can make considerable progress toward engineering safer retroviral vectors that integrate into a significantly narrowed pool of sites on human genome and overcome the preference for TSS. PMID:20616052

Nicotinamide dependence of uropathogenic Escherichia coli UTI89 and application of nadB as a neutral insertion site.

PubMed

Li, Zhaoli; Bouckaert, Julie; Deboeck, Francine; De Greve, Henri; Hernalsteens, Jean-Pierre

2012-03-01

NAD and NADP are ubiquitous in the metabolism of Escherichia coli K-12. NAD auxotrophy can be rendered by mutation in any of the three genes nadB, nadA and nadC. The nadB and nadA genes were defined as antivirulence loci in Shigella spp., as a mutation (mainly in nadB) disrupting the synthesis of quinolinate is required for virulence. Uropathogenic E. coli (UPEC) isolates from acute cystitis patients, exhibiting nicotinamide auxotrophy, were of serotype O18 : K1 : H7. E. coli UTI89, the model uropathogenic and O18 : K1 : H7 strain, requires nicotinamide or quinolinate for growth. A mutation in the nadB gene, encoding L-aspartate oxidase, was shown to be responsible for the nicotinamide requirement of UTI89. This was further confirmed by complementation of UTI89 with a recombinant plasmid harbouring the nadB gene of E. coli K-12. An Ala28Val point mutant of the recombinant plasmid failed to support the growth of UTI89 in minimal medium. This proves that the Ala28Val mutation in the NadB gene of UTI89 completely impedes de novo synthesis of nicotinamide. In spontaneous prototrophic revertants of UTI89, the nadB gene has a Val28Ala mutation. Both analyses implicate that the nicotinamide auxotrophy of UTI89 is caused by a single Ala28Val mutation in NadB. We showed that the same mutation is also present in other NAD auxotrophic E. coli O18 strains. No significant differences were observed between the virulence of isogenic NAD auxotrophic and prototrophic strains in the murine ascending urinary tract infection model. Considering these data, we applied the nadB locus as a neutral site for DNA insertions in the bacterial chromosome. We successfully restored the parental phenotype of a fimH mutant by inserting fimH, with a synthetic em7 promoter, into the nadB gene. This neutral insertion site is of significance for further research on the pathogenicity of UPEC.

Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

PubMed

Hoque, Md Anarul; Zhang, Yong; Chen, Liuqing; Yang, Guangyu; Khatun, Mst Afroza; Chen, Haifeng; Hao, Liu; Feng, Yan

2017-05-19

The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 7 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.

Hemlock woolly adelgid (Homoptera: Adelgidae): stylet bundle insertion and feeding sites

Treesearch

Rebecca F. Young; Kathleen S. Shields; Graeme P. Berlyn

1995-01-01

Stylet bundle insertion site, path traveled, and feeding site were examined for the hemlock woolly adelgid, Adelges tsugae Annand, on needles from current and previous years of eastern hemlock, Tsuga canadensis Carriere. The stylet bundle is composed of 4 individual stylets--2 outer mandibular stylets and 2 inner maxillary stylets...

Morphological size evaluation of the mid-substance insertion areas and the fan-like extension fibers in the femoral ACL footprint.

PubMed

Suruga, Makoto; Horaguchi, Takashi; Iriuchishima, Takanori; Yahagi, Yoshiyuki; Iwama, Genki; Tokuhashi, Yasuaki; Aizawa, Shin

2017-08-01

The purpose of this study was to evaluate the detailed anatomy of the femoral anterior cruciate ligament (ACL) insertion site, with special attention given to the morphology of the mid-substance insertion areas and the fan-like extension fibers. Twenty-three non-paired human cadaver knees were used (7 Males, 16 Females, median age 83, range 69-96). All soft tissues around the knee were resected except the ligaments. The ACL was divided into antero-medial (AM) and postero-lateral (PL) bundles according to the difference in macroscopic tension patterns. The ACL was carefully dissected and two outlines were made of the periphery of each bundle insertion site: those which included and those which excluded the fan-like extension fibers. An accurate lateral view of the femoral condyle was photographed with a digital camera, and the images were downloaded to a personal computer. The area of each bundle, including and excluding the fan-like extension fibers, was measured with Image J software (National Institution of Health). The width and length of the mid-substance insertion sites were also evaluated using same image. The femoral ACL footprint was divided into four regions (mid-substance insertion sites of the AM and PL bundles, and fan-like extensions of the AM and PL bundles). The measured areas of the mid-substance insertion sites of the AM and PL bundles were 35.5 ± 12.5, and 32.4 ± 13.8 mm 2 , respectively. Whole width and length of the mid-substance insertion sites were 5.3 ± 1.4, and 15.5 ± 2.9 mm, respectively. The measured areas of the fan-like extensions of the AM and PL bundles were 27 ± 11.5, and 29.5 ± 12.4 mm 2 , respectively. The femoral ACL footprint was divided into quarters of approximately equal size (mid-substance insertion sites of the AM and PL bundles, and fan-like extensions of the AM and PL bundles). For clinical relevance, to perform highly reproducible anatomical ACL reconstruction, the presence of the fan-like extension fibers should be taken into consideration.

Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins

PubMed Central

Moore, Desmond A.; Whatley, Zakiya N.; Joshi, Chandra P.; Osawa, Masaki

2016-01-01

ABSTRACT FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo. One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro. Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging. PMID:27795325

Structural insights into translational recoding by frameshift suppressor tRNASufJ

PubMed Central

Fagan, Crystal E.; Maehigashi, Tatsuya; Dunkle, Jack A.; Miles, Stacey J.

2014-01-01

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5′ or 3′ direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNASufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNASufJ contains an insertion 5′ to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASLSufJ or tRNASufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASLSufJ and ASLThr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASLSufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNASufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting. PMID:25352689

The highly pathogenic H7N3 avian influenza strain from July 2012 in Mexico acquired an extended cleavage site through recombination with host 28S rRNA.

PubMed

Maurer-Stroh, Sebastian; Lee, Raphael T C; Gunalan, Vithiagaran; Eisenhaber, Frank

2013-05-01

A characteristic difference between highly and non-highly pathogenic avian influenza strains is the presence of an extended, often multibasic, cleavage motif insertion in the hemagglutinin protein. Such motif is found in H7N3 strains from chicken farm outbreaks in 2012 in Mexico. Through phylogenetic, sequence and structural analysis, we try to shed light on the role, prevalence, likelihood of appearance and origin of the inserted cleavage motifs in these H7N3 avian influenza strains. The H7N3 avian influenza strain which caused outbreaks in chicken farms in June/July 2012 in Mexico has a new extended cleavage site which is the likely reason for its high pathogenicity in these birds. This cleavage site appears to have been naturally acquired and was not present in the closest low pathogenic precursors. Structural modeling shows that insertion of a productive cleavage site is quite flexible to accept insertions of different length and with sequences from different possible origins. Different from recent cleavage site insertions, the origin of the insert here is not from the viral genome but from host 28S ribosomal RNA (rRNA) instead. This is a novelty for a natural acquisition as a similar insertion has so far only been observed in a laboratory strain before. Given the abundance of viral and host RNA in infected cells, the acquisition of a pathogenicity-enhancing extended cleavage site through a similar route by other low-pathogenic avian strains in future does not seem unlikely. Important for surveillance of these H7N3 strains, the structural sites known to enhance mammalian airborne transmission are dominated by the characteristic avian residues and the risk of human to human transmission should currently be low but should be monitored for future changes accordingly. This highly pathogenic H7N3 avian influenza strain acquired a novel extended cleavage site which likely originated from recombination with 28S rRNA from the avian host. Notably, this new virus can infect humans but currently lacks critical host receptor adaptations that would facilitate human to human transmission.

Response Changes During Insertion of a Cochlear Implant Using Extracochlear Electrocochleography.

PubMed

Giardina, Christopher K; Khan, Tatyana E; Pulver, Stephen H; Adunka, Oliver F; Buchman, Craig A; Brown, Kevin D; Pillsbury, Harold C; Fitzpatrick, Douglas C

2018-03-16

Electrocochleography is increasingly being utilized as an intraoperative monitor of cochlear function during cochlear implantation (CI). Intracochlear recordings from the advancing electrode can be obtained through the device by on-board capabilities. However, such recordings may not be ideal as a monitor because the recording electrode moves in relation to the neural and hair cell generators producing the responses. The purposes of this study were to compare two extracochlear recording locations in terms of signal strength and feasibility as intraoperative monitoring sites and to characterize changes in cochlear physiology during CI insertion. In 83 human subjects, responses to 90 dB nHL tone bursts were recorded both at the round window (RW) and then at an extracochlear position-either adjacent to the stapes or on the promontory just superior to the RW. Recording from the fixed, extracochlear position continued during insertion of the CI in 63 cases. Before CI insertion, responses to low-frequency tones at the RW were roughly 6 dB larger than when recording at either extracochlear site, but the two extracochlear sites did not differ from one another. During CI insertion, response losses from the promontory or adjacent to the stapes stayed within 5 dB in ≈61% (38/63) of cases, presumably indicating atraumatic insertions. Among responses which dropped more than 5 dB at any time during CI insertion, 12 subjects showed no response recovery, while in 13, the drop was followed by partial or complete response recovery by the end of CI insertion. In cases with recovery, the drop in response occurred relatively early (<15 mm insertion) compared to those where there was no recovery. Changes in response phase during the insertion occurred in some cases; these may indicate a change in the distributions of generators contributing to the response. Monitoring the electrocochleography during CI insertion from an extracochlear site reveals insertions that are potentially atraumatic show interaction with cochlear structures followed by response recovery or show interactions such that response losses persist to the end of recording.

Smoke-derived karrikin perception by the α/β-hydrolase KAI2 from Arabidopsis

PubMed Central

Guo, Yongxia; Zheng, Zuyu; La Clair, James J.; Chory, Joanne; Noel, Joseph P.

2013-01-01

Genetic studies in Arabidopsis implicate an α/β-hydrolase, KARRIKIN-INSENSITIVE 2 (KAI2) as a receptor for karrikins, germination-promoting butenolide small molecules found in the smoke of burned plants. However, direct biochemical evidence for the interaction between KAI2 and karrikin and for the mechanism of downstream signaling by a KAI2–karrikin complex remain elusive. We report crystallographic analyses and ligand-binding experiments for KAI2 recognition of karrikins. The karrikin-1 (KAR1) ligand sits in the opening to the active site abutting a helical domain insert but distal from the canonical catalytic triad (Ser95-His246-Asp217) of α/β-hydrolases, consistent with the lack of detectable hydrolytic activity by purified KAI2. The closest approach of KAR1 to Ser95-His246-Asp217 is 3.8 Å from His246. Six aromatic side chains, including His246, encapsulate KAR1 through geometrically defined aromatic–aromatic interactions. KAR1 binding induces a conformational change in KAI2 at the active site entrance. A crevice of hydrophobic residues linking the polar edge of KAR1 and the helical domain insert suggests that KAI2–KAR1 creates a contiguous interface for binding signaling partners in a ligand-dependent manner. PMID:23613584

Smoke-derived karrikin perception by the α/β-hydrolase KAI2 from Arabidopsis.

PubMed

Guo, Yongxia; Zheng, Zuyu; La Clair, James J; Chory, Joanne; Noel, Joseph P

2013-05-14

Genetic studies in Arabidopsis implicate an α/β-hydrolase, KARRIKIN-INSENSITIVE 2 (KAI2) as a receptor for karrikins, germination-promoting butenolide small molecules found in the smoke of burned plants. However, direct biochemical evidence for the interaction between KAI2 and karrikin and for the mechanism of downstream signaling by a KAI2-karrikin complex remain elusive. We report crystallographic analyses and ligand-binding experiments for KAI2 recognition of karrikins. The karrikin-1 (KAR1) ligand sits in the opening to the active site abutting a helical domain insert but distal from the canonical catalytic triad (Ser95-His246-Asp217) of α/β-hydrolases, consistent with the lack of detectable hydrolytic activity by purified KAI2. The closest approach of KAR1 to Ser95-His246-Asp217 is 3.8 Å from His246. Six aromatic side chains, including His246, encapsulate KAR1 through geometrically defined aromatic-aromatic interactions. KAR1 binding induces a conformational change in KAI2 at the active site entrance. A crevice of hydrophobic residues linking the polar edge of KAR1 and the helical domain insert suggests that KAI2-KAR1 creates a contiguous interface for binding signaling partners in a ligand-dependent manner.

A novel eight amino acid insertion contributes to the hemagglutinin cleavability and the virulence of a highly pathogenic avian influenza A (H7N3) virus in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Xiangjie; Belser, Jessica A.; Tumpey, Terrence M., E-mail: tft9@cdc.gov

In 2012, an avian influenza A H7N3 (A/Mexico/InDRE7218/2012; Mx/7218) virus was responsible for two confirmed cases of human infection and led to the death or culling of more than 22 million chickens in Jalisco, Mexico. Interestingly, this virus acquired an 8-amino acid (aa)-insertion (..PENPK-DRKSRHRR-TR/GLF) near the hemagglutinin (HA) cleavage site by nonhomologous recombination with host rRNA. It remains unclear which specific residues at the cleavage site contribute to the virulence of H7N3 viruses in mammals. Using loss-of-function approaches, we generated a series of cleavage site mutant viruses by reverse genetics and characterized the viruses in vitro and in vivo. Wemore » found that the 8-aa insertion and the arginine at position P4 of the Mx/7218 HA cleavage site are essential for intracellular HA cleavage in 293T cells, but have no effect on the pH of membrane fusion. However, we identified a role for the histidine residue at P5 position in viral fusion pH. In mice, the 8-aa insertion is required for Mx/7218 virus virulence; however, the basic residues upstream of the P4 position are dispensable for virulence. Overall, our study provides the first line of evidence that the insertion in the Mx/7218 virus HA cleavage site confers its intracellular cleavability, and consequently contributes to enhanced virulence in mice. - Highlights: • An avian influenza H7N3 virus acquired a unique 8-amino acid (aa) insertion. • The role of specific basic residues in the HA insertion in viral pathogenesis was determined. • In mice, the 8-aa insertion is required for H7N3 virus virulence. • The R residue at position P4 is essential for HA intracellular cleavage and virus virulence.« less

Correlation between Initial BIC and the Insertion Torque/Depth Integral Recorded with an Instantaneous Torque-Measuring Implant Motor: An in vivo Study.

PubMed

Capparé, Paolo; Vinci, Raffaele; Di Stefano, Danilo Alessio; Traini, Tonino; Pantaleo, Giuseppe; Gherlone, Enrico Felice; Gastaldi, Giorgio

2015-10-01

Quantitative intraoperative evaluation of bone quality at implant placement site and postinsertion implant primary stability assessment are two key parameters to perform implant-supported rehabilitation properly. A novel micromotor has been recently introduced allowing to measure bone density at implant placement site and to record implant insertion-related parameters, such as the instantaneous, average and peak insertion torque values, and the insertion torque/depth integral. The aim of this study was to investigate in vivo if any correlation existed between initial bone-to-implant contact (BIC) and bone density and integral values recorded with the instrument. Twenty-five patients seeking for implant-supported rehabilitation of edentulous areas were consecutively treated. Before implant placement, bone density at the insertion site was measured. For each patient, an undersized 3.3 × 8-mm implant was placed, recording the insertion torque/depth integral values. After 15 minutes, the undersized implant was retrieved with a 0.5 mm-thick layer of bone surrounding it. Standard implants were consequently placed. Retrieved implants were analyzed for initial BIC quantification after fixation, dehydration, acrylic resin embedment, sections cutting and grinding, and toluidine-blue and acid fuchsine staining. Correlation between initial BIC values, bone density at the insertion site, and the torque/depth integral values was investigated by linear regression analysis. A significant linear correlation was found to exist between initial BIC and (a) bone density at the insertion site (R = 0.96, explained variance R(2)  = 0.92) and (b) torque/depth integral at placement (R = 0.81, explained variance R(2)  = 0.66). The system provided quantitative, reliable data correlating significantly with immediate postinsertion initial BIC, and could therefore represent a valuable tool both for clinical research and for the oral implantologist in his/her daily clinical practice. © 2015 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Young-Min; Kim, Chan-Young; Yang, Doo-Hyun

Purpose. To evaluate the feasibility and effectiveness of feeding tube insertion and enteral feeding for the treatment of postoperative gastrointestinal anastomotic obstruction and leakage. Materials and Methods. From June 1999 to June 2002, thirty-four cases of postoperative gastrointestinal anastomotic obstruction and leakage after surgery for gastric carcinoma were treated by insertion of a feeding tube under fluoroscopic guidance. Twenty-one patients were male and 13 were female. The patients' ages ranged from 39 to 74 years (mean age: 61 years). All the patients experienced vomiting, and 15 patients had anastomotic site or duodenal stump leakage. We evaluated the feasibility of feedingmore » tube insertion for enteral feeding to improve the obstruction and facilitate leakage site closure, and the patients' nutritional benefit was also evaluated by checking the serum albumin level between pre- and post-enteral feeding via the feeding tube.Results. Thirty-two patients (94%) were successfully managed by feeding tube insertion, but the remaining two were not managed, and this was due to severe angulations at the anastomotic site. The procedure times for feeding tube insertion ranged from 15 to 60 minutes (mean time: 45 minutes). Twenty-eight patients experienced symptomatic relief of gastrointestinal obstruction, and they were able to resume a normal regular diet after feeding tube removal. Three patients underwent stent insertion due to recurrent symptoms, and one patient underwent jejunostomy feeding due to the presence of a persistent leakage site. Eleven patients achieved leakage site closure after enteral feeding via a feeding tube. The serum albumin level was significant, increased from pre-enteral feeding (2.65 {+-} 0.37 g/dL) to the post-enteral feeding (3.64 {+-} 0.58 g/dL) via the feeding tube (p < 0.001). The duration of follow-up ranged from one to 53 months (mean: 23 months). Conclusion. The insertion of a feeding tube for enteral feeding under fluoroscopic guidance is safe, and it provides effective relief from gastrointestinal anastomotic site obstruction and leakage after gastric surgery. Moreover, our findings indicate that feeding tube insertion for enteral feeding may be used as the primary procedure to treat postoperative anastomotic obstruction and leakage.« less

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

PubMed Central

Venken, Koen J. T.; Schulze, Karen L.; Haelterman, Nele A.; Pan, Hongling; He, Yuchun; Evans-Holm, Martha; Carlson, Joseph W.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

2011-01-01

We demonstrate the versatility of a collection of insertions of the transposon Minos mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp system. Insertions within coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the Drosophila melanogaster toolkit. PMID:21985007

Prospective comparison of two management strategies of central venous catheters in burn patients.

PubMed

Kealey, G P; Chang, P; Heinle, J; Rosenquist, M D; Lewis, R W

1995-03-01

Central venous catheters (CVCs) are associated with sepsis in burn patients. This study was undertaken to compare two strategies of CVC management in patients with major burn injuries. Forty-two burn patients with major burn injuries were randomly assigned to undergo site change every 48 hours of the CVC or to undergo wire guide exchange of the CVC every 48 hours at the same site. Catheter insertion site, distance from the burn wound, cultures of catheter tips, and blood cultures were obtained from all patients in a prospective manner. There was no difference in the incidence of CVC sepsis between the two groups studied. CVCs inserted less than 5 cm from the burn wound developed bacterial contamination at an earlier time than CVCs inserted more than 5 cm from the burn wound. There was no advantage to changing the CVC insertion site every 48 hours. Changing the CVC using the wire guide technique did not prevent, nor predict, CVC bacterial contamination.

HIV Integration at Certain Sites in Host DNA Is Linked to the Expansion and Persistence of Infected Cells | Poster

Cancer.gov

Editor’s note: This article was originally published on the Center for Cancer Research website. When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites

Mitochondrial DNA transfer to the nucleus generates extensive insertion site variation in maize.

PubMed

Lough, Ashley N; Roark, Leah M; Kato, Akio; Ream, Thomas S; Lamb, Jonathan C; Birchler, James A; Newton, Kathleen J

2008-01-01

Mitochondrial DNA (mtDNA) insertions into nuclear chromosomes have been documented in a number of eukaryotes. We used fluorescence in situ hybridization (FISH) to examine the variation of mtDNA insertions in maize. Twenty overlapping cosmids, representing the 570-kb maize mitochondrial genome, were individually labeled and hybridized to root tip metaphase chromosomes from the B73 inbred line. A minimum of 15 mtDNA insertion sites on nine chromosomes were detectable using this method. One site near the centromere on chromosome arm 9L was identified by a majority of the cosmids. To examine variation in nuclear mitochondrial DNA sequences (NUMTs), a mixture of labeled cosmids was applied to chromosome spreads of ten diverse inbred lines: A188, A632, B37, B73, BMS, KYS, Mo17, Oh43, W22, and W23. The number of detectable NUMTs varied dramatically among the lines. None of the tested inbred lines other than B73 showed the strong hybridization signal on 9L, suggesting that there is a recent mtDNA insertion at this site in B73. Different sources of B73 and W23 were examined for NUMT variation within inbred lines. Differences were detectable, suggesting either that mtDNA is being incorporated or lost from the maize nuclear genome continuously. The results indicate that mtDNA insertions represent a major source of nuclear chromosomal variation.

Establishment of an Arbitrary PCR for Rapid Identification of Tn917 Insertion Sites in Staphylococcus epidermidis: Characterization of Biofilm-Negative and Nonmucoid Mutants

PubMed Central

Knobloch, Johannes K.-M.; Nedelmann, Max; Kiel, Kathrin; Bartscht, Katrin; Horstkotte, Matthias A.; Dobinsky, Sabine; Rohde, Holger; Mack, Dietrich

2003-01-01

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis. PMID:14532029

Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations.

PubMed

Erdemir, Aysegul; Mutlu, Ozal

2017-06-01

Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Octenidine hydrochloride for the care of central venous catheter insertion sites in severely immunocompromised patients.

PubMed

Tietz, Andreas; Frei, Reno; Dangel, Marc; Bolliger, Dora; Passweg, Jakob R; Gratwohl, Alois; Widmer, Andreas E

2005-08-01

To determine the efficacy and tolerability of octenidine hydrochloride, a non-alcoholic skin antiseptic, for the care of central venous catheter (CVC) insertion sites. Prospective, observational study. Bone marrow transplantation unit of a university hospital. All consecutive patients with a nontunneled CVC were enrolled prospectively after informed consent. Octenidine hydrochloride (0.1%) was applied for disinfection at the CVC insertion site during dressing changes. The following cultures were performed weekly as well as at the occurrence of any systemic inflammatory response syndrome criteria: cultures of the skin surrounding the CVC entry site, cultures of the three-way hub connected to the CVC, blood cultures, and cultures of the CVC tip on removal. Enhanced microbiological methods (skin swabs of a 24-cm2 standardized area, roll plate, and sonication of catheter tips) were applied. One hundred thirty-five CVCs were inserted in 62 patients during the study period and remained for a mean period of 19.1 days, corresponding to 2,462 catheter-days. Bacterial density at the insertion site declined substantially over time, and most cultures became negative 2 weeks after insertion. Only 6 patients had a documented catheter-related bloodstream infection. The incidence density was 2.39 catheter infections per 1,000 catheter-days. No side effects were noted with application of the antiseptic. Disinfection with a skin antiseptic that contains octenidine hydrochloride is highly active and well tolerated. It leads to a decrease in skin colonization over time and may be a new option for CVC care.

Efforts to deregulate Rainbow papaya in Japan: Molecular Characterization of Transgene and Vector Inserts

USDA-ARS?s Scientific Manuscript database

Transformation plasmid-derived insert number and insert site sequence in 55-1 line papaya derivatives Rainbow and SunUp was determined as part of a larger petition to allow its import into Japan (Suzuki, et al., 2007, 2008). Three insertions were detected by Southern analysis and their correspondin...

Histopathology of tympanic membranes from patients with ventilation tubes.

PubMed

Oktay, Mehmet Faruk; Tansuker, Hasan Deniz; Fukushima, Hisaki; Paparella, Michael M; Schachern, Patricia A; Cureoglu, Sebahattin

2018-06-01

To evaluate the histopathologic changes in tympanic membranes (TMs) with ventilation tubes (VTs). In this retrospective human temporal bone study our overall study group included 4 subgroups of TMs from deceased donors as follows: 24 with a history of VT insertion for chronic otitis media with effusion (COME-VT); 5 with a history of VT insertion for Meniere's disease (MD-VT); 33 without a history of VT insertion for chronic otitis media with effusion (COME); and 14 without a history of VT insertion for Meniere's disease (MD). We classified the extent of migration of the outer keratinized squamous epithelium onto the inner surface of TM perforations and noted the presence and location of tympanosclerosis, of atrophy, of perforation, and/or of cholesteatoma formation. Tympanosclerosis occurred in 14/24 TMs in the COME-VT subgroup; 2/5, MD-VT; 7/33, COME; and 0/14, MD. The VT insertion site was mostly in the anteroinferior (63%) quadrant of the TM; tympanosclerosis occurred more frequently in the posteroinferior (42%) and posterosuperior (33%) quadrants. We found no significant correlation between the location of tympanosclerosis and the VT insertion site (P>0.05). Atrophy occurred in 7/24 TMs in the COME-VT subgroup; 3/5, MD-VT; 8/33, COME; and 2/14, MD. We found no significant correlation between the location of atrophy and the VT insertion site; however, atrophy was located mostly in the anteroinferior quadrant (one of the most common VT insertion sites) of the TM. Regarding the ingrowth of keratinized epithelium, the mucocutanous junction was detected at any point at the inner surface of the TM in 50% of the specimens. We observed intratympanic cholesteatoma formation in 2/24 TMs in the COME-VT subgroup. TM changes due to VT insertion are more common than previously realized. Meticulous otomicroscopic evaluation of the TM is necessary during tympanomastoidectomies in order to prevent the intratympanic inclusion pearls and squamous epithelial ingrowth to prevent any further cholesteatoma formation. Copyright © 2017. Published by Elsevier B.V.

Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae

PubMed Central

Mesarich, Carl H.; Rees-George, Jonathan; Gardner, Paul P.; Ghomi, Fatemeh Ashari; Gerth, Monica L.; Andersen, Mark T.; Rikkerink, Erik H. A.; Fineran, Peter C.

2017-01-01

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a ‘phenotype of interest’ (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with ‘Fuzzy-Spreader’-like morphologies were also identified through a visual screen. The second, a ‘mutant of interest’ (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid. PMID:28249011

Structural insights into translational recoding by frameshift suppressor tRNA SufJ

DOE PAGES

Fagan, Crystal E.; Maehigashi, Tatsuya; Dunkle, Jack A.; ...

2014-10-28

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5' or 3' direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA SufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA SufJ contains an insertion 5' to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL SufJ ormore » tRNA SufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL SufJ and ASL Thr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL SufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA SufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.« less

Repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome.

PubMed

Onozawa, Masahiro; Zhang, Zhenhua; Kim, Yoo Jung; Goldberg, Liat; Varga, Tamas; Bergsagel, P Leif; Kuehl, W Michael; Aplan, Peter D

2014-05-27

We used the I-SceI endonuclease to produce DNA double-strand breaks (DSBs) and observed that a fraction of these DSBs were repaired by insertion of sequences, which we termed "templated sequence insertions" (TSIs), derived from distant regions of the genome. These TSIs were derived from genic, retrotransposon, or telomere sequences and were not deleted from the donor site in the genome, leading to the hypothesis that they were derived from reverse-transcribed RNA. Cotransfection of RNA and an I-SceI expression vector demonstrated insertion of RNA-derived sequences at the DNA-DSB site, and TSIs were suppressed by reverse-transcriptase inhibitors. Both observations support the hypothesis that TSIs were derived from RNA templates. In addition, similar insertions were detected at sites of DNA DSBs induced by transcription activator-like effector nuclease proteins. Whole-genome sequencing of myeloma cell lines revealed additional TSIs, demonstrating that repair of DNA DSBs via insertion was not restricted to experimentally produced DNA DSBs. Analysis of publicly available databases revealed that many of these TSIs are polymorphic in the human genome. Taken together, these results indicate that insertional events should be considered as alternatives to gross chromosomal rearrangements in the interpretation of whole-genome sequence data and that this mutagenic form of DNA repair may play a role in genetic disease, exon shuffling, and mammalian evolution.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik

A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appearmore » to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.« less

Diversity of Shiga Toxin-Producing Escherichia coli (STEC) O26:H11 Strains Examined via stx Subtypes and Insertion Sites of Stx and EspK Bacteriophages

PubMed Central

Bonanno, Ludivine; Loukiadis, Estelle; Mariani-Kurkdjian, Patricia; Oswald, Eric; Garnier, Lucille; Michel, Valérie

2015-01-01

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that may be responsible for severe human infections. Only a limited number of serotypes, including O26:H11, are involved in the majority of serious cases and outbreaks. The main virulence factors, Shiga toxins (Stx), are encoded by bacteriophages. Seventy-four STEC O26:H11 strains of various origins (including human, dairy, and cattle) were characterized for their stx subtypes and Stx phage chromosomal insertion sites. The majority of food and cattle strains possessed the stx1a subtype, while human strains carried mainly stx1a or stx2a. The wrbA and yehV genes were the main Stx phage insertion sites in STEC O26:H11, followed distantly by yecE and sbcB. Interestingly, the occurrence of Stx phages inserted in the yecE gene was low in dairy strains. In most of the 29 stx-negative E. coli O26:H11 strains also studied here, these bacterial insertion sites were vacant. Multilocus sequence typing of 20 stx-positive or stx-negative E. coli O26:H11 strains showed that they were distributed into two phylogenetic groups defined by sequence type 21 (ST21) and ST29. Finally, an EspK-carrying phage was found inserted in the ssrA gene in the majority of the STEC O26:H11 strains but in only a minority of the stx-negative E. coli O26:H11 strains. The differences in the stx subtypes and Stx phage insertion sites observed in STEC O26:H11 according to their origin might reflect that strains circulating in cattle and foods are clonally distinct from those isolated from human patients. PMID:25819955

Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma

PubMed Central

Weiser, Keith C.; Liu, Bin; Hansen, Gwenn M.; Skapura, Darlene; Hentges, Kathryn E.; Yarlagadda, Sujatha; Morse III, Herbert C.

2007-01-01

AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NFκB, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision. PMID:17926094

Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma.

PubMed

Weiser, Keith C; Liu, Bin; Hansen, Gwenn M; Skapura, Darlene; Hentges, Kathryn E; Yarlagadda, Sujatha; Morse Iii, Herbert C; Justice, Monica J

2007-10-01

AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NFkappaB, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision .

Effect-site concentration of propofol required for LMA-Supreme™ insertion with and without remifentanil: a randomized controlled trial.

PubMed

Zaballos, Matilde; Bastida, Emilia; Agustí, Salomé; Portas, Maite; Jiménez, Consuelo; López-Gil, Maite

2015-10-06

A new supraglottic device, the LMA-Supreme™, has recently become available for clinical use. Information on anaesthetic and co-adjuvant requirements for insertion of the LMA-Supreme™ is limited. The present study aimed to evaluate the optimal effect-site concentration of propofol in 50 % (EC50) of adults necessary for successful insertion of the LMA-Supreme™ and to examine remifentanil's effect on propofol requirements. Fifty-eight elective patients (aged 18-60 years; ASA (American Society Anaesthesiologists) physical status classification I and II) scheduled for day surgery were randomly assigned to one of two groups: propofol with saline or propofol with remifentanil. Anaesthesia was induced by target-controlled infusion according to predetermined effect-site concentrations of propofol and remifentanil (5 ng.mL(-1)). The EC50 was calculated using Dixon's up-and-down method. Ten minutes following drug administration, LMA-Supreme™ insertion was attempted without the use of muscle relaxant drugs. In the propofol + saline group, the EC50 of propofol required for LMA-Supreme™ insertion was 6.32 ± 0.67 μg.mL(-1) (95 % CI, 5.69-6.94 μg.mL(-1)). With the addition of remifentanil at an effect-site concentration of 5 ng.mL(-1), the EC50 of propofol required for LMA-Supreme™ insertion was 2.50 ± 0.80 μg.mL(-1) (95 % CI, 1.82-3.17 μg.mL(-1); p < 0.0001). The propofol requirement for smooth insertion of the LMA-Supreme™ was 60 % less when remifentanil (5 ng.mL(-1)) was co-administered. Identified as NCT01974648 at www.clinicaltrials.gov .

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method.

PubMed

Guo, Bingfu; Guo, Yong; Hong, Huilong; Qiu, Li-Juan

2016-01-01

Molecular characterization of sequence flanking exogenous fragment insertion is essential for safety assessment and labeling of genetically modified organism (GMO). In this study, the T-DNA insertion sites and flanking sequences were identified in two newly developed transgenic glyphosate-tolerant soybeans GE-J16 and ZH10-6 based on whole genome sequencing (WGS) method. More than 22.4 Gb sequence data (∼21 × coverage) for each line was generated on Illumina HiSeq 2500 platform. The junction reads mapped to boundaries of T-DNA and flanking sequences in these two events were identified by comparing all sequencing reads with soybean reference genome and sequence of transgenic vector. The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis. All these analyses supported that exogenous T-DNA fragments were integrated in positions of Chr19: 50543767-50543792 and Chr17: 7980527-7980541 in these two transgenic lines. Identification of genomic insertion sites of G2-EPSPS and GAT transgenes will facilitate the utilization of their glyphosate-tolerant traits in soybean breeding program. These results also demonstrated that WGS was a cost-effective and rapid method for identifying sites of T-DNA insertions and flanking sequences in soybean.

Male patients require higher optimal effect-site concentrations of propofol during i-gel insertion with dexmedetomidine 0.5 μg/kg.

PubMed

Choi, Jung Ju; Kim, Ji Young; Lee, Dongchul; Chang, Young Jin; Cho, Noo Ree; Kwak, Hyun Jeong

2016-03-22

The pharmacokinetics and pharmacodynamics of an anesthetic drug may be influenced by gender. The purpose of this study was to compare effect-site half maximal effective concentrations (EC50) of propofol in male and female patients during i-gel insertion with dexmedetomidine 0.5 μg/kg without muscle relaxants. Forty patients, aged 20-46 years of ASA physical status I or II, were allocated to one of two groups by gender (20 patients per group). After the infusion of dexmedetomidine 0.5 μg/kg over 2 min, anesthesia was induced with a pre-determined effect-site concentration of propofol by target controlled infusion. Effect-site EC50 values of propofol for successful i-gel insertion were determined using the modified Dixon's up-and-down method. Mean effect-site EC50 ± SD of propofol for successful i-gel insertion was significantly higher for men than women (5.46 ± 0.26 μg/ml vs. 3.82 ± 0.34 μg/ml, p < 0.01). The EC50 of propofol in men was approximately 40% higher than in women. Using isotonic regression with a bootstrapping approach, the estimated EC50 (95% confidence interval) of propofol was also higher in men [5.32 (4.45-6.20) μg/ml vs. 3.75 (3.05-4.43) μg/ml]. The estimated EC95 (95% confidence interval) of propofol in men and women were 5.93 (4.72-6.88) μg/ml and 4.52 (3.02-5.70) μg/ml, respectively. During i-gel insertion with dexmedetomidine 0.5 μg/kg without muscle relaxant, male patients had higher effect-site EC50 for propofol using Schnider's model. Based on the results of this study, patient gender should be considered when determining the optimal dose of propofol during supraglottic airway insertion. ClinicalTrials.gov identifier: NCT02268656. Registered August 26, 2014.

Transdiaphragmatic Approach to Attenuate Porto-Azygos Shunts Inserting in the Thorax.

PubMed

Or, Matan; Kitshoff, Adriaan; Devriendt, Nausikaa; De Ridder, Marianne; Quist-Rybachuk, Galena; de Rooster, Hilde

2016-11-01

To describe the surgical technique and document the application of a transdiaphragmatic approach to attenuate porto-azygos shunts inserting in the thoracic section of the azygos vein. Cadaveric study and prospective case series. Canine cadavers (n=6) and client-owned dogs with porto-azygos shunts inserting in the thoracic section of the azygos vein (n=9). In the cadavers, the azygos vein was filled with aqueous latex. Landmarks were established for creating a safe transdiaphragmatic approach to the caudal intrathoracic portion of the azygos vein. In the clinical cases, porto-azygos communication was diagnosed by trans-splenic portal scintigraphy. All shunts were attenuated close to their insertion site via ventral midline celiotomy and a transdiaphragmatic approach to the shunt. Perioperative complications were recorded. A 3-5 cm incision, 0.5-1 cm ventral and lateral to the level of the aortic hiatus, was made in the pars lumbalis part of the diaphragm. Stay sutures at both sides of the diaphragmatic incision were placed to open up the incision and a retractor was used to push the esophagus away from the aorta. Intrathoracic insertion of the shunt was confirmed intraoperative. Exposure of the shunt insertion site at the azygos vein was excellent in all clinical cases. No intraoperative or postoperative complications were encountered. If thoracic attenuation of a porto-azygos shunt is considered, a transdiaphragmatic approach exposes the insertion site for shunt attenuation. This approach is straightforward, without unnecessary abdominal organ manipulation, and since attenuates at the insertion, avoids missing additional contributing branches. © Copyright 2016 by The American College of Veterinary Surgeons.

Unusual Properties of Regulatory DNA from the Drosophila Engrailed Gene: Three ``pairing-Sensitive'' Sites within a 1.6-Kb Region

PubMed Central

Kassis, J. A.

1994-01-01

We have previously shown that a 2-kb fragment of engrailed DNA can suppress expression of a linked marker gene, white, in the P element vector CaSpeR. This suppression is dependent on the presence of two copies of engrailed DNA-containing P elements (P[en]) in proximity in the Drosophila genome (either in cis or in trans). In this study, the 2-kb fragment was dissected and found to contain three fragments of DNA which could mediate white suppression [called ``pairing-sensitive sites'' (PS)]. A PS site was also identified in regulatory DNA from the Drosophila escargot gene. The eye colors of six different P[en] insertions in the escargot gene suggest an interaction between P[en]-encoded and genome-encoded PS sites. I hypothesize that white gene expression from P[en] is repressed by the formation of a protein complex which is initiated at the engrailed PS sites and also requires interactions with flanking genomic DNA. Genes were sought which influence the function of PS sites. Mutations in some Polycomb and trithorax group genes were found to affect the eye color from some P[en] insertion sites. However, different mutations affected expression from different P[en] insertion sites and no one mutation was found to affect expression from all P[en] insertion sites examined. These results suggest that white expression from P[en] is not directly regulated by members of the Polycomb and trithorax group genes, but in some cases can be influenced by them. I propose that engrailed PS sites normally act to promote interactions between distantly located engrailed regulatory sites and the engrailed promoter. PMID:8005412

A High-Throughput Arabidopsis Reverse Genetics System

PubMed Central

Sessions, Allen; Burke, Ellen; Presting, Gernot; Aux, George; McElver, John; Patton, David; Dietrich, Bob; Ho, Patrick; Bacwaden, Johana; Ko, Cynthia; Clarke, Joseph D.; Cotton, David; Bullis, David; Snell, Jennifer; Miguel, Trini; Hutchison, Don; Kimmerly, Bill; Mitzel, Theresa; Katagiri, Fumiaki; Glazebrook, Jane; Law, Marc; Goff, Stephen A.

2002-01-01

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from ∼100,000 transformed lines. A total of 85,108 TAIL-PCR products from 52,964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org. PMID:12468722

Design of high-fidelity haptic display for one-dimensional force reflection applications

NASA Astrophysics Data System (ADS)

Gillespie, Brent; Rosenberg, Louis B.

1995-12-01

This paper discusses the development of a virtual reality platform for the simulation of medical procedures which involve needle insertion into human tissue. The paper's focus is the hardware and software requirements for haptic display of a particular medical procedure known as epidural analgesia. To perform this delicate manual procedure, an anesthesiologist must carefully guide a needle through various layers of tissue using only haptic cues for guidance. As a simplifying aspect for the simulator design, all motions and forces involved in the task occur along a fixed line once insertion begins. To create a haptic representation of this procedure, we have explored both physical modeling and perceptual modeling techniques. A preliminary physical model was built based on CT-scan data of the operative site. A preliminary perceptual model was built based on current training techniques for the procedure provided by a skilled instructor. We compare and contrast these two modeling methods and discuss the implications of each. We select and defend the perceptual model as a superior approach for the epidural analgesia simulator.

Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus.

PubMed

Zhu, Li-Ping; Yue, Xin-Jing; Han, Kui; Li, Zhi-Feng; Zheng, Lian-Shuai; Yi, Xiu-Nan; Wang, Hai-Long; Zhang, You-Ming; Li, Yue-Zhong

2015-07-22

Exotic genes, especially clustered multiple-genes for a complex pathway, are normally integrated into chromosome for heterologous expression. The influences of insertion sites on heterologous expression and allotropic expressions of exotic genes on host remain mostly unclear. We compared the integration and expression efficiencies of single and multiple exotic genes that were inserted into Myxococcus xanthus genome by transposition and attB-site-directed recombination. While the site-directed integration had a rather stable chloramphenicol acetyl transferase (CAT) activity, the transposition produced varied CAT enzyme activities. We attempted to integrate the 56-kb gene cluster for the biosynthesis of antitumor polyketides epothilones into M. xanthus genome by site-direction but failed, which was determined to be due to the insertion size limitation at the attB site. The transposition technique produced many recombinants with varied production capabilities of epothilones, which, however, were not paralleled to the transcriptional characteristics of the local sites where the genes were integrated. Comparative transcriptomics analysis demonstrated that the allopatric integrations caused selective changes of host transcriptomes, leading to varied expressions of epothilone genes in different mutants. With the increase of insertion fragment size, transposition is a more practicable integration method for the expression of exotic genes. Allopatric integrations selectively change host transcriptomes, which lead to varied expression efficiencies of exotic genes.

Lesion bypass activity of DNA polymerase θ (POLQ) is an intrinsic property of the pol domain and depends on unique sequence inserts.

PubMed

Hogg, Matthew; Seki, Mineaki; Wood, Richard D; Doublié, Sylvie; Wallace, Susan S

2011-01-21

DNA polymerase θ (POLQ, polθ) is a large, multidomain DNA polymerase encoded in higher eukaryotic genomes. It is important for maintaining genetic stability in cells and helping protect cells from DNA damage caused by ionizing radiation. POLQ contains an N-terminal helicase-like domain, a large central domain of indeterminate function, and a C-terminal polymerase domain with sequence similarity to the A-family of DNA polymerases. The enzyme has several unique properties, including low fidelity and the ability to insert and extend past abasic sites and thymine glycol lesions. It is not known whether the abasic site bypass activity is an intrinsic property of the polymerase domain or whether helicase activity is also required. Three "insertion" sequence elements present in POLQ are not found in any other A-family DNA polymerase, and it has been proposed that they may lend some unique properties to POLQ. Here, we analyzed the activity of the DNA polymerase in the absence of each sequence insertion. We found that the pol domain is capable of highly efficient bypass of abasic sites in the absence of the helicase-like or central domains. Insertion 1 increases the processivity of the polymerase but has little, if any, bearing on the translesion synthesis properties of the enzyme. However, removal of insertions 2 and 3 reduces activity on undamaged DNA and completely abrogates the ability of the enzyme to bypass abasic sites or thymine glycol lesions. Copyright © 2010 Elsevier Ltd. All rights reserved.

Comparison of Insertional RNA Editing in Myxomycetes

PubMed Central

Chen, Cai; Frankhouser, David; Bundschuh, Ralf

2012-01-01

RNA editing describes the process in which individual or short stretches of nucleotides in a messenger or structural RNA are inserted, deleted, or substituted. A high level of RNA editing has been observed in the mitochondrial genome of Physarum polycephalum. The most frequent editing type in Physarum is the insertion of individual Cs. RNA editing is extremely accurate in Physarum; however, little is known about its mechanism. Here, we demonstrate how analyzing two organisms from the Myxomycetes, namely Physarum polycephalum and Didymium iridis, allows us to test hypotheses about the editing mechanism that can not be tested from a single organism alone. First, we show that using the recently determined full transcriptome information of Physarum dramatically improves the accuracy of computational editing site prediction in Didymium. We use this approach to predict genes in the mitochondrial genome of Didymium and identify six new edited genes as well as one new gene that appears unedited. Next we investigate sequence conservation in the vicinity of editing sites between the two organisms in order to identify sites that harbor the information for the location of editing sites based on increased conservation. Our results imply that the information contained within only nine or ten nucleotides on either side of the editing site (a distance previously suggested through experiments) is not enough to locate the editing sites. Finally, we show that the codon position bias in C insertional RNA editing of these two organisms is correlated with the selection pressure on the respective genes thereby directly testing an evolutionary theory on the origin of this codon bias. Beyond revealing interesting properties of insertional RNA editing in Myxomycetes, our work suggests possible approaches to be used when finding sequence motifs for any biological process fails. PMID:22383871

Cloning and expression in Escherichia coli of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.

PubMed

Zalacain, M; Malpartida, F; Pulido, D; Jiménez, A

1987-01-15

The Streptomyces hygroscopicus hyg gene encoding a hygromycin B phosphotransferase has been introduced into different sites of both the Escherichia coli plasmid pBR322 and the Escherichia coli-Saccharomyces cerevisiae shuttle vector YRp7. When this gene was inserted into the BamHI site of pBR322 and then cloned in E. coli phosphorylating activity was not detected, indicating that the hyg gene promoter was not functional in this bacterium. However, when the hyg gene was inserted into either the unique PstI site of pBR322 or into each of the two PstI sites of YRp7, phosphotransferase activity was observed. Analysis of the translation products from these constructions by coupled in vitro transcription-translation systems suggested that in all cases transcrition was regulated by a promoter not provided by the inserted hyg gene and that the synthesized polypeptide was identical to that present in S. hygroscopicus.

Radiological Tenckhoff catheter insertion for peritoneal dialysis: A cost-effective approach.

PubMed

Lee, James; Mott, Nigel; Mahmood, Usman; Clouston, John; Summers, Kara; Nicholas, Pauline; Gois, Pedro Henrique França; Ranganathan, Dwarakanathan

2018-04-01

Radiological insertion of Tenckhoff catheters can be an alternative option for peritoneal dialysis access creation, as compared to surgical catheter insertion. This study will review the outcomes and complications of radiological Tenckhoff catheter insertion in a metropolitan renal service and compare costs between surgical and radiological insertion. Data were collected prospectively for all patients who had a Tenckhoff catheter insertion for peritoneal dialysis (PD) under radiological guidance at our hospital from May 2014 to November 2016. The type of catheter used and complications, including peri-catheter leak, exit site infection and peritonitis were reviewed. Follow-up data were also collected at points 3, 6 and 12 months from catheter insertion. Costing data were obtained from Queensland Health Electronic Reporting System (QHERS) data, average staff salaries and consumable contract price lists. In the 30-month evaluation period, 70 catheters were inserted. Two patients had an unsuccessful procedure due to the presence of abdominal adhesions. Seven patients had an episode of peri-catheter leak, and four patients had an exit site infection following catheter insertion. Peritonitis was observed in nine patients during the study period. The majority of patients (90%) remained on peritoneal dialysis at 3-month follow-up. The average costs of surgical and radiological insertion were noted to be AUD$7788.34 and AUD$1597.35, respectively. Radiological Tenckhoff catheter insertion for peritoneal dialysis appears to be an attractive and cost-effective option given less waiting periods for the procedure, the relatively low cost of insertion and comparable rates of complications. © 2017 The Royal Australian and New Zealand College of Radiologists.

Effect of single DNA lesions on in vitro replication with DNA polymerase III holoenzyme. Comparison with other polymerases.

PubMed

Belguise-Valladier, P; Maki, H; Sekiguchi, M; Fuchs, R P

1994-02-11

In the present work, we have studied in vitro replication of N-2-acetylaminofluorene (AAF) or cis-diamminedichloroplatinum II (cis-DDP) single modified DNA templates. We used the holoenzyme (pol III HE) or the alpha subunit of DNA polymerase III, which is involved in SOS mutagenesis, and other DNA polymerases in order to compare enzymes having different biological roles and properties. Single-stranded oligonucleotides (63-mer) bearing a single AAF adduct at one of the different guanine residues of the NarI sequence (-G1G2CG3CC-) have been used in primer extension assays. Site-specifically platinated 5'd(ApG) or 5'd(GpG) oligonucleotides were constructed and similarly used in primer extension assays. In all cases, irrespective of both the chemical nature of the lesion (i.e. AAF or cis-DDP) and its local sequence context (i.e. the 3 different sites for AAF adducts within the NarI site) replication by pol III HE and pol I Klenow fragment (pol I Kf) stops one base prior to the adduct site. Removal of the 3'-->5' proofreading activity alone was not sufficient to trigger bypass of DNA lesions. Indeed, when proofreading activity of pol I is inactivated by a point mutation (pol I Kf (exo-)), the major replication product corresponds to the position opposite the adduct site showing that incorporation across from the AAF adduct is possible. These results suggest that a polymerase with proofreading activity is actually found to stop one nucleotide before the adduct not because it is unable to insert a nucleotide opposite the adduct but most likely because elongation past the adduct is strongly impaired, giving thus an increased time frame for the proofreading exonuclease to remove the base inserted across from the adduct. These results are discussed in terms of their implications for error-free and error-prone bypass in vivo.

Thermodynamic impact of abasic sites on simulated translesion DNA synthesis.

PubMed

Malina, Jaroslav; Brabec, Viktor

2014-06-16

Loss of a base in DNA and the creation of an abasic (apurinic/apyrimidinic, AP) site is a frequent lesion that may occur spontaneously, or as a consequence of the action of DNA-damaging agents. The AP lesion is mutagenic or lethal if not repaired. We report a systematic thermodynamic investigation by differential scanning calorimetry on the evolution, during primer extension, of a model AP site in chemically simulated DNA translesion synthesis. Incorporation of dAMP (deoxyadenosine monophosphate), as well as dTMP (deoxythymidine monophosphate), opposite an AP site is enthalpically unfavorable, although incorporation of dTMP is more enthalpically unfavorable than that of dAMP. This finding is in a good agreement with experimental data showing that AP sites block various DNA polymerases of eukaryotic and prokaryotic origin and that, if bypassed, dAMP is preferentially inserted, whereas insertion of dTMP is less likely. The results emphasize the importance of thermodynamic contributions to the insertion of nucleotides opposite an AP site by DNA polymerases. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of insertion sites in a Newcastle disease virus vector on foreign gene expression through an internal ribosomal entry site

USDA-ARS?s Scientific Manuscript database

Newcastle disease virus (NDV), avian paramyxovirus type 1, has been developed as a vector to express foreign genes for vaccine and gene therapy purposes. The foreign genes are usually inserted into a non-coding region of the NDV genome as an independent transcription unit (ITU), which potentially a...

Current microbiology of percutaneous endoscopic gastrostomy tube (PEG tube) insertion site infections in patients with cancer.

PubMed

Rolston, Kenneth V I; Mihu, Coralia; Tarrand, Jeffrey J

2011-08-01

Percutaneous endoscopic gastrostomy (PEG) is frequently used to provide enteral access in cancer patients who are unable to swallow. Infection is an important complication in this setting. Current microbiological data are needed to guide infection prevention and treatment strategies. The microbiological records of our institution (a 550-bed comprehensive cancer center) were retrospectively reviewed over an 8-month study period in order to identify patients who developed PEG tube insertion site infections, and review their microbiological details and susceptibility/resistance data. Fifty-eight episodes of PEG tube insertion site infections were identified. Of these, 31 (53%) were monomicrobial, and the rest were polymicrobial. The most common organisms isolated were Candida species, Staphylococcus aureus, and Pseudomonas aeruginosa. All infections were local (cellulitis, complicated skin, and skin structure infections including abdominal wall abscess) with no cases of concomitant bacteremia being documented. Most of the organisms isolated were susceptible to commonly used antimicrobial agents, although some quinolone-resistant and some multidrug-resistant organisms were isolated. This retrospective study provides descriptive data regarding PEG tube insertion site infections. These data have helped us update institutional guidelines for infection prevention and treatment as part of our focus on antimicrobial stewardship.

Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula.

PubMed

d'Erfurth, Isabelle; Cosson, Viviane; Eschstruth, Alexis; Lucas, Helene; Kondorosi, Adam; Ratet, P

2003-04-01

The tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation-regeneration process. Two hundred and twenty-five primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and efficiently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences flanking the insertion sites revealed that Tnt1 had no insertion site specificity and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an efficient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.

Alu Insertions and Genetic Diversity: A Preliminary Investigation by an Undergraduate Bioinformatics Class

ERIC Educational Resources Information Center

Elwess, Nancy L.; Duprey, Stephen L.; Harney, Lindesay A.; Langman, Jessie E.; Marino, Tara C.; Martinez, Carolina; McKeon, Lauren L.; Moss, Chantel I. E.; Myrie, Sasha S.; Taylor, Luke Ryan

2008-01-01

"Alu"-insertion polymorphisms were used by an undergraduate Bioinformatics class to study how these insertion sites could be the basis for an investigation in human population genetics. Based on the students' investigation, both allele and genotype "Alu" frequencies were determined for African-American and Japanese populations as well as a…

Reconstitutional Mutagenesis of the Maize P Gene by Short-Range Ac Transpositions

PubMed Central

Moreno, M. A.; Chen, J.; Greenblatt, I.; Dellaporta, S. L.

1992-01-01

The tendency for Ac to transpose over short intervals has been utilized to develop insertional mutagenesis and fine structure genetic mapping strategies in maize. We recovered excisions of Ac from the P gene and insertions into nearby chromosomal sites. These closely linked Ac elements reinserted into the P gene, reconstituting over 250 unstable variegated alleles. Reconstituted alleles condition a variety of variegation patterns that reflect the position and orientation of Ac within the P gene. Molecular mapping and DNA sequence analyses have shown that reinsertion sites are dispersed throughout a 12.3-kb chromosomal region in the promoter, exons and introns of the P gene, but in some regions insertions sites were clustered in a nonrandom fashion. Transposition profiles and target site sequence data obtained from these studies have revealed several features of Ac transposition including its preference for certain target sites. These results clearly demonstrate the tendency of Ac to transpose to nearby sites in both proximal and distal directions from the donor site. With minor modifications, reconstitutional mutagenesis should be applicable to many Ac-induced mutations in maize and in other plant species and can possibly be extended to other eukaryotic transposon systems as well. PMID:1325389

Identification of Mycobacterial Genes Involved in Antibiotic Sensitivity: Implications for the Treatment of Tuberculosis with β-Lactam-Containing Regimens

PubMed Central

Viswanathan, Gopinath; Yadav, Sangya

2017-01-01

ABSTRACT In a Mycobacterium smegmatis mutant library screen, transposon mutants with insertions in fhaA, dprE2, rpsT, and parA displayed hypersusceptibility to antibiotics, including the β-lactams meropenem, ampicillin, amoxicillin, and cefotaxime. Sub-MIC levels of octoclothepin, a psychotic drug inhibiting ParA, phenocopied the parA insertion and enhanced the bactericidal activity of meropenem against Mycobacterium tuberculosis in combination with clavulanate. Our study identifies novel factors associated with antibiotic resistance, with implications in repurposing β-lactams for tuberculosis treatment. PMID:28438925

Guidewire exchange vs new site placement for temporary dialysis catheter insertion in ICU patients: is there a greater risk of colonization or dysfunction?

PubMed

Coupez, Elisabeth; Timsit, Jean-François; Ruckly, Stéphane; Schwebel, Carole; Gruson, Didier; Canet, Emmanuel; Klouche, Kada; Argaud, Laurent; Bohe, Julien; Garrouste-Orgeas, Maïté; Mariat, Christophe; Vincent, François; Cayot, Sophie; Cointault, Olivier; Lepape, Alain; Darmon, Michael; Boyer, Alexandre; Azoulay, Elie; Bouadma, Lila; Lautrette, Alexandre; Souweine, Bertrand

2016-07-30

Intensive care unit (ICU) patients require dialysis catheters (DCs) for renal replacement therapy (RRT). They carry a high risk of developing end-stage renal disease, and therefore their vascular access must be preserved. Guidewire exchange (GWE) is often used to avoid venipuncture insertion (VPI) at a new site. However, the impact of GWE on infection and dysfunction of DCs in the ICU is unknown. Our aim was to compare the effect of GWE and VPI on DC colonization and dysfunction in ICU patients. Using data from the ELVIS randomized controlled trial (RCT) (1496 ICU adults requiring DC for RRT or plasma exchange) we performed a matched-cohort analysis. Cases were DCs inserted by GWE (n = 178). They were matched with DCs inserted by VPI. Matching criteria were participating centre, simplified acute physiology score (SAPS) II +/-10, insertion site (jugular or femoral), side for jugular site, and length of ICU stay before DC placement. We used a marginal Cox model to estimate the effect of DC insertion (GWE vs. VPI) on DC colonization and dysfunction. DC colonization rate was not different between GWE-DCs and VPI-DCs (10 (5.6 %) for both groups) but DC dysfunction was more frequent with GWE-DCs (67 (37.6 %) vs. 28 (15.7 %); hazard ratio (HR), 3.67 (2.07-6.49); p < 0.01). Results were similar if analysis was restricted to DCs changed for dysfunction. GWE for DCs in ICU patients, compared with VPI did not contribute to DC colonization or infection but was associated with more than twofold increase in DC dysfunction. This study is registered with ClinicalTrials.gov, number NCT00563342 . Registered 2 April 2009.

Influence of Stepped Osteotomy on Primary Stability of Implants Inserted in Low-Density Bone Sites: An In Vitro Study.

PubMed

Degidi, Marco; Daprile, Giuseppe; Piattelli, Adriano

The aims of this study were to evaluate the ability of a stepped osteotomy to improve dental implant primary stability in low-density bone sites and to investigate possible correlations between primary stability parameters. The study was performed on fresh humid bovine bone classified as type III. The test group consisted of 30 Astra Tech EV implants inserted following the protocol provided by the manufacturer. The first control group consisted of 30 Astra Tech EV implants inserted in sites without the underpreparation of the apical portion. The second control group consisted of 30 Astra Tech TX implants inserted following the protocol provided by the manufacturer. Implant insertion was performed at the predetermined 30 rpm. The insertion torque data were recorded and exported as a curve; using a trapezoidal integration technique, the area underlying the curve was calculated: this area represents the variable torque work (VTW). Peak insertion torque (pIT) and resonance frequency analysis (RFA) were also recorded. A Mann-Whitney test showed that the mean VTW was significantly higher in the test group compared with the first control and second control groups; furthermore, statistical analysis showed that pIT also was significantly higher in the test group compared with the first and second control groups. Analyzing RFA values, only the difference between the test group and second control group showed statistical significance. Pearson correlation analysis showed a very strong positive correlation between pIT and VTW values in all groups; furthermore, it showed a positive correlation between pIT and RFA values and between VTW and RFA values only in the test group. Within the limitations of an in vitro study, the results show that stepped osteotomy can be a viable method to improve implant primary stability in low-density bone sites, and that, when a traditional osteotomy method is performed, RFA presents no correlation with pIT and VTW.

Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins.

PubMed

Harr, Jennifer C; Luperchio, Teresa Romeo; Wong, Xianrong; Cohen, Erez; Wheelan, Sarah J; Reddy, Karen L

2015-01-05

Nuclear organization has been implicated in regulating gene activity. Recently, large developmentally regulated regions of the genome dynamically associated with the nuclear lamina have been identified. However, little is known about how these lamina-associated domains (LADs) are directed to the nuclear lamina. We use our tagged chromosomal insertion site system to identify small sequences from borders of fibroblast-specific variable LADs that are sufficient to target these ectopic sites to the nuclear periphery. We identify YY1 (Ying-Yang1) binding sites as enriched in relocating sequences. Knockdown of YY1 or lamin A/C, but not lamin A, led to a loss of lamina association. In addition, targeted recruitment of YY1 proteins facilitated ectopic LAD formation dependent on histone H3 lysine 27 trimethylation and histone H3 lysine di- and trimethylation. Our results also reveal that endogenous loci appear to be dependent on lamin A/C, YY1, H3K27me3, and H3K9me2/3 for maintenance of lamina-proximal positioning. © 2015 Harr et al.

Photochemical Reactions of (n(5)-Pentamethylcyclpentadienyl)-Dicarbonyliron-Alkyl and -Silyl Complexes: Reversible Ethylene Insertion into an Iron-Silicon Bond and Implications for the Mechanism of Transition Metal-Catalyzed Hydrosilation of Alkenes.

DTIC Science & Technology

1985-12-11

RD-R162 462 PHOTOCHEMICAL REACTIONS OF(N(S)-P NTANETNYLCVCLPENTADIENYL)-DICARRONVLIR.. (U) MASSACHUSETTS INST OF TECH CAMBRIDGE DEPT OF CHEMISTRY...34 Photochemical Reactions of (n5-Pentamethylcyclpentadienyl)- Dicarbonyliron-Alkyl and -Silyl Complexes: Reversible Ethylene Insertion into an Iron-Silicon Bond...Chemical Society) PHOTOCHEMICAL REACTIONS OF (n5-PENTAMETHYLCYCLOPENTADIENYL)- DICARBONYLIRON-ALKYL AND -SILYL COMPLEXES: REVERSIBLE ETHYLENE INSERTION INTO

Structural Effect of the Asp345a Insertion in Penicillin-Binding Protein 2 from Penicillin-Resistant Strains of Neisseria gonorrhoeae

PubMed Central

2015-01-01

A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 Å resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the β2c−β2d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the β-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the β-lactam ring during acylation because of the negative charge of its side chain. PMID:25403720

Structural effect of the Asp345a insertion in penicillin-binding protein 2 from penicillin-resistant strains of Neisseria gonorrhoeae.

PubMed

Fedarovich, Alena; Cook, Edward; Tomberg, Joshua; Nicholas, Robert A; Davies, Christopher

2014-12-09

A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 Å resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the β2c-β2d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the β-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the β-lactam ring during acylation because of the negative charge of its side chain.

Osvaldo and Isis retrotransposons as markers of the Drosophila buzzatii colonisation in Australia.

PubMed

Guerreiro, María Pilar García; Fontdevila, Antonio

2011-04-24

Transposable elements (TEs) constitute an important source of genetic variability owing to their jumping and regulatory properties, and are considered to drive species evolution. Several factors that are able to induce TE transposition in genomes have been documented (for example environmental stress and inter- and intra-specific crosses) but in many instances the reasons for TE mobilisation have yet to be elucidated. Colonising populations constitute an ideal model for studying TE behaviour and distribution as they are exposed to different environmental and new demographic conditions. In this study, the distribution of two TEs, Osvaldo and Isis, was examined in two colonising populations of D. buzzatii from Australia. Comparing Osvaldo copy numbers between Australian and Old World (reported in previous studies) colonisations provides a valuable tool for elucidating the colonisation process and the effect of new conditions encountered by colonisers on TEs. The chromosomal distributions of Osvaldo and Isis retrotransposons in two colonising populations of D. buzzatii from Australia revealed sites of high insertion frequency (>10%) and low frequency sites. Comparisons between Osvaldo insertion profiles in colonising populations from the Old World and Australia demonstrate a tendency towards a higher number of highly occupied sites with higher insertion frequency in the Old World than in Australian populations. Tests concerning selection against deleterious TE insertions indicate that Isis is more controlled by purifying selection than Osvaldo. The distribution of both elements on chromosomal arms follows a Poisson distribution and there are non-significant positive correlations between highly occupied sites and chromosomal inversions. The occupancy profile of Osvaldo and Isis retrotransposons is characterised by the existence of high and low insertion frequency sites in the populations. These results demonstrate that Australian D. buzzatii populations were subjected to a founder effect during the colonisation process. Moreover, there are more sites with high insertion frequency in the Old World colonisation than in the Australian colonisation, indicating a probable stronger bottleneck effect in Australia. The results suggest that selection does not seem to play a major role, compared to demography, in the distribution of transposable elements in the Australian populations.

Osvaldo and Isis retrotransposons as markers of the Drosophila buzzatii colonisation in Australia

PubMed Central

2011-01-01

Background Transposable elements (TEs) constitute an important source of genetic variability owing to their jumping and regulatory properties, and are considered to drive species evolution. Several factors that are able to induce TE transposition in genomes have been documented (for example environmental stress and inter- and intra-specific crosses) but in many instances the reasons for TE mobilisation have yet to be elucidated. Colonising populations constitute an ideal model for studying TE behaviour and distribution as they are exposed to different environmental and new demographic conditions. In this study, the distribution of two TEs, Osvaldo and Isis, was examined in two colonising populations of D. buzzatii from Australia. Comparing Osvaldo copy numbers between Australian and Old World (reported in previous studies) colonisations provides a valuable tool for elucidating the colonisation process and the effect of new conditions encountered by colonisers on TEs. Results The chromosomal distributions of Osvaldo and Isis retrotransposons in two colonising populations of D. buzzatii from Australia revealed sites of high insertion frequency (>10%) and low frequency sites. Comparisons between Osvaldo insertion profiles in colonising populations from the Old World and Australia demonstrate a tendency towards a higher number of highly occupied sites with higher insertion frequency in the Old World than in Australian populations. Tests concerning selection against deleterious TE insertions indicate that Isis is more controlled by purifying selection than Osvaldo. The distribution of both elements on chromosomal arms follows a Poisson distribution and there are non-significant positive correlations between highly occupied sites and chromosomal inversions. Conclusions The occupancy profile of Osvaldo and Isis retrotransposons is characterised by the existence of high and low insertion frequency sites in the populations. These results demonstrate that Australian D. buzzatii populations were subjected to a founder effect during the colonisation process. Moreover, there are more sites with high insertion frequency in the Old World colonisation than in the Australian colonisation, indicating a probable stronger bottleneck effect in Australia. The results suggest that selection does not seem to play a major role, compared to demography, in the distribution of transposable elements in the Australian populations. PMID:21513573

Pes anserinus and anserine bursa: anatomical study

PubMed Central

Lee, Je-Hun; Kim, Kyung-Jin; Jeong, Young-Gil; Lee, Nam Seob; Han, Seung Yun; Lee, Chang Gug; Kim, Kyung-Yong

2014-01-01

This study investigated the boundary of anserine bursa with the recommended injection site and shape on the insertion area of pes anserinus (PA), with the aim of improving clinical practice. Eighty six legs from 45 Korean cadavers were investigated. The mixed gelatin solution was injected to identify the shape of anserine bursa, and then the insertion site of the PA tendons was exposed completely and carefully dissected to identify the shape of the PA. The sartorius was inserted into the superficial layer and gracilis, and the semitendinosus was inserted into the deep layer on the medial surface of the tibia. The number of the semitendinosus tendons at the insertion site varied: 1 in 66% of specimens, 2 in 31%, and 3 in 3%. The gracilis and semitendinosus tendons were connected to the deep fascia of leg. Overall, the shape of the anserine bursa was irregularly circular. Most of the anserine bursa specimens reached the proximal line of the tibia, and some of the specimens reached above the proximal line of the tibia. In the medial view of the tibia, the anserine bursa was located posteriorly and superiorly from the tibia's midline, and it followed the lines of the sartorius muscle. The injection site for anserine bursa should be carried out at 20° from the vertical line medially and inferiorly, 15 or 20 mm deeply, and at the point of about 20 mm medial and 12 mm superior from inferomedial point of tibial tuberosity. PMID:24987549

Relationship between peripheral insertion site and catheter-related phlebitis in adult hospitalized patients: a systematic review.

PubMed

Comparcini, Dania; Simonetti, Valentina; Blot, Stijn; Tomietto, Marco; Cicolini, Giancarlo

2017-01-01

To explore the relationship between the anatomical site of peripheral venous catheterization and risk of catheter-related phlebitis. Peripheral venous catheterization is frequently associated with phlebitis. Recent guidelines, recommend the use of an upper-extremity site for catheter insertion but no univocal consensus exists on the anatomical site with lower risk of phlebitis. Systematic review. We searched Medline (PubMed) and CINAHL (EBSCOhost) databases until the end of January 2017. We also reviewed the reference lists of retrieved articles and gray literature was excluded. Searches were limited to articles published in English with no restriction imposed to date of publication. The primary outcome was the incidence of phlebitis associated with anatomical site of peripheral catheterization. We included randomized controlled trials and observational studies on adult patients who required a peripheral catheter for the administration of medi- cation, intermittent or continuous fluid infusion. Antecubital fossa veins are associated with lower phlebitis rates, while hands veins are the most risky sites to develop phlebitis. There is no consensus regarding vein in forearm. Choosing the right anatomical site to insert a peripheral venous catheter is important to decrease phlebitis rate. Further studies should compare indwelling time in different anatomical sites with phlebitis rate. A more standardized approach in defining and assessing phlebitis among studies is recommended.

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.

PubMed

Oakes, Benjamin L; Nadler, Dana C; Flamholz, Avi; Fellmann, Christof; Staahl, Brett T; Doudna, Jennifer A; Savage, David F

2016-06-01

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

Peritoneal Dialysis Catheter Insertion.

PubMed

Crabtree, John H; Chow, Kai-Ming

2017-01-01

The success of peritoneal dialysis as renal-replacement therapy depends on a well-functioning peritoneal catheter. Knowledge of best practices in catheter insertion can minimize the risk of catheter complications that lead to peritoneal dialysis failure. The catheter placement procedure begins with preoperative assessment of the patient to determine the most appropriate catheter type, insertion site, and exit site location. Preoperative preparation of the patient is an instrumental step in facilitating the performance of the procedure, avoiding untoward events, and promoting the desired outcome. Catheter insertion methods include percutaneous needle-guidewire with or without image guidance, open surgical dissection, peritoneoscopic procedure, and surgical laparoscopy. The insertion technique used often depends on the geographic availability of material resources and local provider expertise in placing catheters. Independent of the catheter implantation approach, adherence to a number of universal details is required to ensure the best opportunity for creating a successful long-term peritoneal access. Finally, appropriate postoperative care and catheter break-in enables a smooth transition to dialysis therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel approach for Escherichia coli genome editing combining in vivo cloning and targeted long-length chromosomal insertion.

PubMed

Hook, Ch D; Samsonov, V V; Ublinskaya, A A; Kuvaeva, T M; Andreeva, E V; Gorbacheva, L Yu; Stoynova, N V

2016-11-01

Despite the abundance of genetic manipulation approaches, particularly for Escherichia coli, new techniques and increased flexibility in the application of existing techniques are required to address novel aims. The most widely used approaches for chromosome editing are based on bacteriophage site-specific and λRed/RecET-mediated homologous recombination. In the present study, these techniques were combined to develop a novel approach for in vivo cloning and targeted long-length chromosomal insertion. This approach permits direct λRed-mediated cloning of DNA fragment with lengths of 10kb or greater from the E. coli chromosome into the plasmid vector pGL2, which carries the ori of pSC101, the ϕ80-attP site of ϕ80 phage, and an excisable Cm R marker bracketed by λ-attL/attR sites. In pGL2-based recombinant plasmids, the origin of replication can be eliminated in vitro via hydrolysis by SceI endonuclease and recircularization by DNA ligase. The resulting ori-less circular recombinant DNA can be used for targeted insertion of the cloned sequence into the chromosome at a selected site via ϕ80 phage-specific integrase-mediated recombination using the Dual-In/Out approach (Minaeva et al., 2008). At the final stage of chromosomal editing, the Cm R -marker can be excised from the chromosome due to expression of the λint/xis genes. Notably, the desired fragment can be inserted as multiple copies in the chromosome by combining insertions at different sites in one strain using the P1 general transduction technique (Moore, 2011). The developed approach is useful for the construction of plasmidless, markerless recombinant strains for fundamental and industrial purposes. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Skin disinfection with octenidine dihydrochloride for central venous catheter site care: a double-blind, randomized, controlled trial.

PubMed

Dettenkofer, M; Wilson, C; Gratwohl, A; Schmoor, C; Bertz, H; Frei, R; Heim, D; Luft, D; Schulz, S; Widmer, A F

2010-06-01

To compare the efficacy of two commercially available, alcohol-based antiseptic solutions for preparation and care of central venous catheter (CVC) insertion sites, with and without octenidine dihydrochloride, a double-blind, randomized, controlled trial was undertaken in the haematology units and in one surgical unit of two university hospitals. Adult patients with a non-tunnelled CVC were randomly assigned to two different skin disinfection regimens at the insertion site: 0.1% octenidine with 30% 1-propanol and 45% 2-propanol, and as control 74% ethanol with 10% 2-propanol. Endpoints were (i) skin colonization at the insertion site; (ii) positive culture from the catheter tip (> or = 15 CFU); and (iii) occurrence of CVC-associated bloodstream infection (defined according to criteria set by the CDC). Four hundred patients with inserted CVC were enrolled from May 2002 through April 2005. Both groups were similar in respect of patient characteristics and co-morbidities. Skin colonization at the CVC insertion site during the first 10 days was significantly reduced by octenidine treatment (relative difference octenidine vs. control: 0.21; 95%CI: 0.11-0.39, p <0.0001). Positive culture of the catheter tip was significantly less frequent in the octenidine group (7.9%) than in the control group (17.8%): OR = 0.39 (95%CI: 0.20-0.80, p 0.009). Patients treated with octenidine had a non-significant reduction in catheter-associated bloodstream infections (4.1% vs. 8.3%; OR = 0.44; 95%CI: 0.18-1.08, p 0.081). Side effects were similar in both groups. This randomized controlled trial supports the results of two observational studies demonstrating octenidine in alcoholic solution to be a better option than alcohol alone for the prevention of CVC-associated infections.

Crystallographic and Molecular Dynamics Analysis of Loop Motions Unmasking the Peptidoglycan-Binding Site in Stator Protein MotB of Flagellar Motor

PubMed Central

Nahar, Musammat F.; Buckle, Ashley M.; Roujeinikova, Anna

2011-01-01

Background The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues. Methodology/Principal Findings We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the β-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan. Conclusion/Significance Our structural analysis provides a new insight into the mechanism by which MotB inserts into the peptidoglycan mesh, thus anchoring the power-generating complex to the cell wall. PMID:21533052

Comparison of success rates of orthodontic mini-screws by the insertion method.

PubMed

Kim, Jung Suk; Choi, Seong Hwan; Cha, Sang Kwon; Kim, Jang Han; Lee, Hwa Jin; Yeom, Sang Seon; Hwang, Chung Ju

2012-10-01

The aim of this study was to compare the success rates of the manual and motor-driven mini-screw insertion methods according to age, gender, length of mini-screws, and insertion sites. We retrospectively reviewed 429 orthodontic mini-screw placements in 286 patients (102 in men and 327 in women) between 2005 and 2010 at private practice. Age, gender, mini-screw length, and insertion site were cross-tabulated against the insertion methods. The Cochran-Mantel-Haenszel test was performed to compare the success rates of the 2 insertion methods. The motor-driven method was used for 228 mini-screws and the manual method for the remaining 201 mini-screws. The success rates were similar in both men and women irrespective of the insertion method used. With respect to mini-screw length, no difference in success rates was found between motor and hand drivers for the 6-mm-long mini-screws (68.1% and 69.5% with the engine driver and hand driver, respectively). However, the 8-mm-long mini-screws exhibited significantly higher success rates (90.4%, p < 0.01) than did the 6-mm-long mini-screws when placed with the engine driver. The overall success rate was also significantly higher in the maxilla (p < 0.05) when the engine driver was used. Success rates were similar among all age groups regardless of the insertion method used. Taken together, the motor-driven insertion method can be helpful to get a higher success rate of orthodontic mini-screw placement.

Many P-Element Insertions Affect Wing Shape in Drosophila melanogaster

PubMed Central

Weber, Kenneth; Johnson, Nancy; Champlin, David; Patty, April

2005-01-01

A screen of random, autosomal, homozygous-viable P-element insertions in D. melanogaster found small effects on wing shape in 11 of 50 lines. The effects were due to single insertions and remained stable and significant for over 5 years, in repeated, high-resolution measurements. All 11 insertions were within or near protein-coding transcription units, none of which were previously known to affect wing shape. Many sites in the genome can affect wing shape. PMID:15545659

Many P-element insertions affect wing shape in Drosophila melanogaster.

PubMed

Weber, Kenneth; Johnson, Nancy; Champlin, David; Patty, April

2005-03-01

A screen of random, autosomal, homozygous-viable P-element insertions in D. melanogaster found small effects on wing shape in 11 of 50 lines. The effects were due to single insertions and remained stable and significant for over 5 years, in repeated, high-resolution measurements. All 11 insertions were within or near protein-coding transcription units, none of which were previously known to affect wing shape. Many sites in the genome can affect wing shape.

Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence.

PubMed

Kim, Dong Seon; Hahn, Yoonsoo

2012-11-13

Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors) in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

The Effect of Insertion Torque on the Clinical Outcome of Single Implants: A Randomized Clinical Trial.

PubMed

Barone, Antonio; Alfonsi, Fortunato; Derchi, Giacomo; Tonelli, Paolo; Toti, Paolo; Marchionni, Saverio; Covani, Ugo

2016-06-01

The insertion torque value has been extensively used as an indicator for implant primary stability, which is considered a determining parameter for the implants success. The primary goal of the present randomized clinical trial was to evaluate and compare the clinical outcome for implants placed with high insertion torque (between 50 Ncm and 100 Ncm) and regular insertion torque (within 50 Ncm) in healed ridges. Partially edentulous patients, missing one or more mandibular or maxillary teeth, having an adequate amount of bone, requiring implant placement, were randomized to receive Blossom CT implants with regular insertion torque (<50 Ncm) or CT implants with high insertion torque (≥50 Ncm). Implants were left to heal submerged for 3 months. Implants were restored with individualized abutments and cemented metal-ceramic crowns. Acquired measurements were: insertion torque values (IT), thickness of buccal bone plate after implant osteotomy preparation (BBT), marginal bone level (MBL), and facial soft tissue level (FST). All patients were followed 12 months after implant placement. One hundred sixteen implants were placed in one hundred sixteen patients and enrolled for the study. Fifty-eight implants were randomly allocated in regular-IT and high-IT groups with a mean insertion torque ranging from 20 Ncm to 50 Ncm and from 50 Ncm to 100 Ncm, respectively. Three implants failed, and another five implants showed at the 12-month evaluation a marginal bone loss (ΔMBL) greater than 1.5 mm, being considered unsuccessful. The findings suggested that implants inserted with high-IT (≥50 Ncm) in healed bone ridges showed more peri-implant bone remodeling and buccal soft tissue recession than implants inserted with a regular-IT (<50 Ncm). Moreover, sites with a thick buccal bone wall (≥1 mm) - after implant osteotomy site preparation - seemed to be less prone to buccal soft tissue recession after 12 months than sites with a thin buccal bone wall (<1 mm). © 2015 Wiley Periodicals, Inc.

Initial and long-term use of inserts by red-cockaded woodpeckers

Treesearch

Daniel Saenz; Richard N. Conner; Christopher S. Collins; D. Craig Rudolph

2001-01-01

Artificial cavities have become a standard management technique for red-cockaded woodpeckers (Picoides borealis). Seventy cavity inserts were installed in our study sites on the Angelina National Forest in eastern Texas from 1990 to 1995. Eighty-two percent of the inserts were used for at least one year. It is still too early to make a direct...

The Effect of Intravenous Infiltration Management Program for Hospitalized Children.

PubMed

Park, Soon Mi; Jeong, Ihn Sook; Kim, Kyoung Lae; Park, Kyung Ju; Jung, Moon Ju; Jun, Seong Suk

2016-01-01

This study aimed to identify the effect of IV infiltration management program among hospitalized children. This was a quasi-experimental study with history comparison group design with 2,894 catheters inserted during 3 months comparison phase and 3,651 catheters inserted during 4 months experimental phase. The intervention was composed of seven activities including applying poster, documentation of catheter insertion, parents education, making infiltration report, assessment of vein condition before inserting catheter, appropriate site selection, and documentation of catheter insertion, and assessment of peripheral catheter insertion site every shift. Data were analyzed using of X2-test, Fisher's exact test. The infiltration incidence rate was 0.9% for experimental group and 4.4% for comparison group, which was significantly different (x2=80.42, p<.001). The catheter maintenance period (p=.035) and infiltration state (p=.039) were significantly different among participants with infiltration between comparison and experimental groups. IV Infiltration management program was founded to be effective in reducing the IV infiltration incidence rate and increasing early detection of IV infiltration. Considering the effect of IV Infiltration management program, we recommend that this infiltration management program would be widely used in the clinical settings. Copyright © 2016 Elsevier Inc. All rights reserved.

Fatal outcome arising from use of a sutureless "corkscrew" epicardial pacing electrode inserted into apex of left ventricle.

PubMed Central

Vecht, R J; Fontaine, C J; Bradfield, J W

1976-01-01

A 59-year-old man is described in whom the insertion of an epicardial sutureless "corkscrew" electrode resulted in fatal ventricular perforation. Fatal myocardial perforation can occur with this electrode and the apex of the left ventricle should never be used as the site of insertion. Necropsy also showed that the transvenous right ventricular electrode, inserted one year previously, had penetrated a tricuspid leaflet. This could have accounted for the ensuing pacing failure. Images PMID:1008980

CRISPR/Cas9 for genome editing: progress, implications and challenges.

PubMed

Zhang, Feng; Wen, Yan; Guo, Xiong

2014-09-15

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system provides a robust and multiplexable genome editing tool, enabling researchers to precisely manipulate specific genomic elements, and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas9 comprises of a nonspecific Cas9 nuclease and a set of programmable sequence-specific CRISPR RNA (crRNA), which can guide Cas9 to cleave DNA and generate double-strand breaks at target sites. Subsequent cellular DNA repair process leads to desired insertions, deletions or substitutions at target sites. The specificity of CRISPR/Cas9-mediated DNA cleavage requires target sequences matching crRNA and a protospacer adjacent motif locating at downstream of target sequences. Here, we review the molecular mechanism, applications and challenges of CRISPR/Cas9-mediated genome editing and clinical therapeutic potential of CRISPR/Cas9 in future. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Using synthetic bacterial enhancers to reveal a looping-based mechanism for quenching-like repression

PubMed Central

Brunwasser-Meirom, Michal; Pollak, Yaroslav; Goldberg, Sarah; Levy, Lior; Atar, Orna; Amit, Roee

2016-01-01

We explore a model for ‘quenching-like' repression by studying synthetic bacterial enhancers, each characterized by a different binding site architecture. To do so, we take a three-pronged approach: first, we compute the probability that a protein-bound dsDNA molecule will loop. Second, we use hundreds of synthetic enhancers to test the model's predictions in bacteria. Finally, we verify the mechanism bioinformatically in native genomes. Here we show that excluded volume effects generated by DNA-bound proteins can generate substantial quenching. Moreover, the type and extent of the regulatory effect depend strongly on the relative arrangement of the binding sites. The implications of these results are that enhancers should be insensitive to 10–11 bp insertions or deletions (INDELs) and sensitive to 5–6 bp INDELs. We test this prediction on 61 σ54-regulated qrr genes from the Vibrio genus and confirm the tolerance of these enhancers' sequences to the DNA's helical repeat. PMID:26832446

Using synthetic bacterial enhancers to reveal a looping-based mechanism for quenching-like repression.

PubMed

Brunwasser-Meirom, Michal; Pollak, Yaroslav; Goldberg, Sarah; Levy, Lior; Atar, Orna; Amit, Roee

2016-02-02

We explore a model for 'quenching-like' repression by studying synthetic bacterial enhancers, each characterized by a different binding site architecture. To do so, we take a three-pronged approach: first, we compute the probability that a protein-bound dsDNA molecule will loop. Second, we use hundreds of synthetic enhancers to test the model's predictions in bacteria. Finally, we verify the mechanism bioinformatically in native genomes. Here we show that excluded volume effects generated by DNA-bound proteins can generate substantial quenching. Moreover, the type and extent of the regulatory effect depend strongly on the relative arrangement of the binding sites. The implications of these results are that enhancers should be insensitive to 10-11 bp insertions or deletions (INDELs) and sensitive to 5-6 bp INDELs. We test this prediction on 61 σ(54)-regulated qrr genes from the Vibrio genus and confirm the tolerance of these enhancers' sequences to the DNA's helical repeat.

Cholorhexidine, octenidine or povidone iodine for catheter related infections: A randomized controlled trial.

PubMed

Bilir, Ayten; Yelken, Birgül; Erkan, Ayse

2013-06-01

Protection of the catheter site by antimicrobial agents is one of the most important factors in the prevention of infection. Povidone iodine and chlorhexidine gluconate are the most common used agents for dressing. The purpose of this study was to compare the effects of povidone iodine, chlorhexidine gluconate and octenidine hydrochloride in preventing catheter related infections. Patients were randomized to receive; 4% chlorhexidine gluconate, 10% povidone iodine or octenidine hydrochlorodine for cutaneous antisepsis. Cultures were taken at the site surrounding catheter insertion and at the catheter hub after removal to help identify the source of microorganisms. Catheter related sepsis was 10.5% in the povidone iodine and octenidine hydrochlorodine groups. Catheter related colonization was 26.3% in povidone iodine group and 21.5% in octenidine hydrochlorodine group. 4% chlorhexidine or octenidine hydrochlorodine for cutaneous disinfection before insertion of an intravascular device and for post-insertion site care can reduce the catheter related colonization.

HIV Integration at Certain Sites in Host DNA is Linked to the Expansion and Persistence of Infected Cells | Center for Cancer Research

Cancer.gov

When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

HIV Integration at Certain Sites in Host DNA Is Linked to the Expansion and Persistence of Infected Cells | Poster

Cancer.gov

Editor’s note: This article was originally published on the Center for Cancer Research website. When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

Site-controlled GaN nanocolumns with InGaN insertions grown by MBE

NASA Astrophysics Data System (ADS)

Nechaev, D. V.; Semenov, A. N.; Koshelev, O. A.; Jmerik, V. N.; Davydov, V. Yu; Smirnov, A. N.; Pozina, G.; Shubina, T. V.; Ivanov, S. V.

2017-11-01

The site-controlled plasma-assisted molecular beam epitaxy (PA MBE) has been developed to fabricate the regular array of GaN nanocolumns (NCs) with InGaN insertions on micro-cone patterned sapphire substrates (μ-CPSSs). Two-stage growth of GaN NCs, including a nucleation layer grown at metal-rich conditions and high temperature GaN growth in strong N-rich condition, has been developed to achieve the selective growth of the NCs. Microcathodoluminescence measurements have demonstrated pronounced emission from the InGaN insertions in 450-600 nm spectral range. The optically isolated NCs can be used as effective nano-emitters operating in the visible range.

Scala vestibuli insertion in cochlear implantation: a valuable alternative for cases with obstructed scala tympani.

PubMed

Kiefer, J; Weber, A; Pfennigdorff, T; von Ilberg, C

2000-01-01

Insertion of a sufficient number of electrodes is important for a successful use of cochlear implants. We investigated the results of scala vestibuli insertion for cochlear implantation in cases of obstructed scala tympani. In a series of 200 cochlear implantations, scala vestibuli insertion was successfully performed in 4 cases with obstruction of the scala tympani. Etiologies included a temporal bone fracture, severe otosclerosis and malformations of the cochlea. The maximum insertion depth obtained via the scala vestibuli was 30 mm. Postoperative results were comparable to patients in whom conventional scala tympani insertion was performed. No adverse effects related to the site of insertion were observed. Scala vestibuli insertion offers a valuable alternative in cases of obstructed scala tympani that can be employed for a variety of etiologies. Copyright 2000 S. Karger AG, Basel

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

PubMed

Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun; Ferrazzoli, Alexander E; Kuo, James; Anand, Ranjith P; Waterman, David P; Haber, James E

2018-02-27

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

Quantitative Mapping of Matrix Content and Distribution across the Ligament-to-Bone Insertion

PubMed Central

Spalazzi, Jeffrey P.; Boskey, Adele L.; Pleshko, Nancy; Lu, Helen H.

2013-01-01

The interface between bone and connective tissues such as the Anterior Cruciate Ligament (ACL) constitutes a complex transition traversing multiple tissue regions, including non-calcified and calcified fibrocartilage, which integrates and enables load transfer between otherwise structurally and functionally distinct tissue types. The objective of this study was to investigate region-dependent changes in collagen, proteoglycan and mineral distribution, as well as collagen orientation, across the ligament-to-bone insertion site using Fourier transform infrared spectroscopic imaging (FTIR-I). Insertion site-related differences in matrix content were also evaluated by comparing tibial and femoral entheses. Both region- and site-related changes were observed. Collagen content was higher in the ligament and bone regions, while decreasing across the fibrocartilage interface. Moreover, interfacial collagen fibrils were aligned parallel to the ligament-bone interface near the ligament region, assuming a more random orientation through the bulk of the interface. Proteoglycan content was uniform on average across the insertion, while its distribution was relatively less variable at the tibial compared to the femoral insertion. Mineral was only detected in the calcified interface region, and its content increased exponentially across the mineralized fibrocartilage region toward bone. In addition to new insights into matrix composition and organization across the complex multi-tissue junction, findings from this study provide critical benchmarks for the regeneration of soft tissue-to-bone interfaces and integrative soft tissue repair. PMID:24019964

Insertion torque in different bone models with different screw pitch: an in vitro study.

PubMed

Orlando, Bruno; Barone, Antonio; Giorno, Thierry M; Giacomelli, Luca; Tonelli, Paolo; Covani, Ugo

2010-01-01

Orthopedic surgeons use different types of screws for bone fixation. Whereas hard cortical bone requires a screw with a fine pitch, in softer cancellous bone a wider pitch might help prevent micromotion and eventually lead to greater implant stability. The aim of this study was to validate the assumption that fine-pitch implants are appropriate for cortical bone and wide-pitch implants are appropriate for cancellous bone. Wide-pitch and fine-pitch implants were inserted in both hard (D1 and D2) bone and soft (D3 and D4) bone, which was simulated by separate experimental blocks of cellular rigid polyurethane foam. A series of insertion sites in D1-D2 and D3-D4 experimental blocks were prepared using 1.5-mm and 2.5-mm drills. The final torque required to insert each implant was recorded. Wide-pitch implants displayed greater insertion torque (20% more than the fine-pitch implants) in cancellous bone and were therefore more suitable than fine-pitch implants. It is more appropriate to use a fine pitch design for implants, in conjunction with a 2.5-mm osteotomy site, in dense cortical bone (D1 or D2), whereas it is recommended to choose a wide-pitch design for implants, in conjunction with a 1.5-mm osteotomy site, in softer bone (D3 or D4).

A single gene mutation that increases maize seed weight

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giroux, M.J.; Shaw, J.; Hannah, L.C.

1996-06-11

The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3 feet to the insertion site andmore » involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts. 20 refs., 5 figs., 5 tabs.« less

Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles.

PubMed

Rueda, P; Hurtado, A; del Barrio, M; Martínez-Torrecuadrada, J L; Kamstrup, S; Leclerc, C; Casal, J I

1999-10-10

An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four loops and the C terminus of the CPV VP2 on the particle structure and immunogenicity. Epitope insertions in the four loops allowed the recovery of capsids in all of the mutants. However, only insertions of the C3:B epitope in VP2 residue 225 of the loop 2 were able to elicit a significant anti-peptide antibody response, but not poliovirus-neutralizing antibodies, probably because residue 225 is located in an small depression of the surface. To fine modulate the insertion site in loop 2, a cassette-mutagenesis was carried out to insert the epitope in adjacent positions 226, 227, and 228. The epitope C3:B inserted into these positions was well recognized by the specific monoclonal antibody C3 by immunoelectron microscopy. BALB/c mice immunized with these chimeric C3:B CPV:VLPs were able to elicit an strong neutralizing antibody response (>3 log(10) units) against poliovirus type 1 (Mahoney strain). Therefore, minor displacements in the insertion place cause dramatic changes in the accessibility of the epitope and the induction of antibody responses. Copyright 1999 Academic Press.

Randomized clinical trial of pigtail catheter versus chest tube in injured patients with uncomplicated traumatic pneumothorax.

PubMed

Kulvatunyou, N; Erickson, L; Vijayasekaran, A; Gries, L; Joseph, B; Friese, R F; O'Keeffe, T; Tang, A L; Wynne, J L; Rhee, P

2014-01-01

Small pigtail catheters appear to work as well as the traditional large-bore chest tubes in patients with traumatic pneumothorax, but it is not known whether the smaller pigtail catheters are associated with less tube-site pain. This study was conducted to compare tube-site pain following pigtail catheter or chest tube insertion in patients with uncomplicated traumatic pneumothorax. This prospective randomized trial compared 14-Fr pigtail catheters and 28-Fr chest tubes in patients with traumatic pneumothorax presenting to a level I trauma centre from July 2010 to February 2012. Patients who required emergency tube placement, those who refused and those who could not respond to pain assessment were excluded. Primary outcomes were tube-site pain, as assessed by a numerical rating scale, and total pain medication use. Secondary outcomes included the success rate of pneumothorax resolution and insertion-related complications. Forty patients were enrolled. Baseline characteristics of 20 patients in the pigtail catheter group were similar to those of 20 patients in the chest tube group. No patient had a flail chest or haemothorax. Pain scores related to chest wall trauma were similar in the two groups. Patients with a pigtail catheter had significantly lower mean(s.d.) tube-site pain scores than those with a chest tube, at baseline after tube insertion (3.2(0.6) versus 7.7(0.6); P < 0.001), on day 1 (1.9(0.5) versus 6.2(0.7); P < 0.001) and day 2 (2.1(1.1) versus 5.5(1.0); P = 0.040). The decreased use of pain medication associated with pigtail catheter was not significantly different. The duration of tube insertion, success rate and insertion-related complications were all similar in the two groups. For patients with a simple, uncomplicated traumatic pneumothorax, use of a 14-Fr pigtail catheter is associated with reduced pain at the site of insertion, with no other clinically important differences noted compared with chest tubes. NCT01537289 (http://clinicaltrials.gov). © 2013 BJS Society Ltd. Published by John Wiley & Sons Ltd.

Prevention of Device-Related Healthcare-Associated Infections

PubMed Central

Septimus, Edward J.; Moody, Julia

2016-01-01

Healthcare-associated infections (HAIs) are a leading cause of morbidity and mortality in hospitalized patients. Up to 15% of patients develop an infection while hospitalized in the United States, which accounts for approximately 1.7 million HAIs, 99,000 deaths annually and over 10 billion dollars in costs per year. A significant percentage of HAIs are preventable using evidenced-based strategies. In terms of device-related HAIs it is estimated that 65-70% of catheter-line associated bloodstream infections (CLABSIs) and catheter-associated urinary tract infections (CAUTIs) are preventable. To prevent CLABSIs a bundle which includes hand hygiene prior to insertion and catheter manipulation, use of chlorhexidene alcohol for site preparation and maintenance, use of maximum barrier for catheter insertion, site selection, removing nonessential lines, disinfect catheter hubs before assessing line, and dressing changes are essential elements of basic practices. To prevent CAUTIs a bundle that includes hand hygiene for insertion and catheter or bag manipulation, inserting catheters for appropriate indications, insert using aseptic technique, remove catheters when no longer needed, maintain a close system keeping bag and tubing below the bladder are the key components of basic practices. PMID:26918162

A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase.

PubMed

Shang, Pengcheng; Misra, Saurav; Hause, Ben; Fang, Ying

2017-07-15

Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3C pro cleavage sites at the 5' and 3' ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-β expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event. IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we identified a special case of cross-order recombination between enterovirus G (order Picornavirales ) and torovirus (order Nidovirales ). This naturally occurring recombination event may have broad implications for other picornaviral and/or nidoviral species. Importantly, we demonstrated that the exogenous ToV-PLP gene that was inserted into the EVG genome encodes a deubiquitinase/deISGylase and potentially suppresses host cellular innate immune responses. Our results provide insights into how a gain of function through genetic recombination, in particular cross-order recombination, may improve the ability of a virus to evade host immunity. Copyright © 2017 American Society for Microbiology.

A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase

PubMed Central

Shang, Pengcheng; Misra, Saurav

2017-01-01

ABSTRACT Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3Cpro cleavage sites at the 5′ and 3′ ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-β expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event. IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we identified a special case of cross-order recombination between enterovirus G (order Picornavirales) and torovirus (order Nidovirales). This naturally occurring recombination event may have broad implications for other picornaviral and/or nidoviral species. Importantly, we demonstrated that the exogenous ToV-PLP gene that was inserted into the EVG genome encodes a deubiquitinase/deISGylase and potentially suppresses host cellular innate immune responses. Our results provide insights into how a gain of function through genetic recombination, in particular cross-order recombination, may improve the ability of a virus to evade host immunity. PMID:28490584

Effects of FlAsH/Tetracysteine (TC) tag on PrP proteolysis and PrPres formation by TC-scanning

PubMed Central

Taguchi, Yuzuru; Hohsfield, Lindsay A.; Hollister, Jason R.

2014-01-01

The FlAsH/tetracysteine (FlAsH/TC) tag is a powerful tool for fluorescent labeling of proteins. However, even small tags such as FlAsH/TC could alter the behavior of the tagged proteins, especially if the insertion occurs at internal sites. Defining the influence of FlAsH/TC on nearby protein-protein interactions might aid in selecting appropriate positions for internal TC insertions and allow the exploitation of serial FlAsH/TC insertions (TC-scanning) as a probe to characterize sites of protein-protein interaction. To explore this application in the context of substrate-protease interactions, we analyzed the effect of FlAsH/TC insertions on proteolysis of cellular prion protein (PrPsen) in in vitro reactions and generation of the C1 metabolic fragment of PrPsen in live neuroblastoma cells. The influence of FlAsH/TC insertion was evaluated by TC-scanning across the cleavage sites of each protease. The results showed that FlAsH/TC inhibited protease cleavage only within limited ranges of the cleavage sites that varied from about 1 to 6 residues-wide depending on the protease, providing an estimate of the PrP residues interacting with each protease. TC-scanning was also used to probe a different type of protein-protein interaction, the conformational conversion of FlAsH-PrPsen to the prion disease-associated isoform, PrPres. PrP constructs with FlAsH/TC insertions at residues 90–96 but not 97–101 were converted to FlAsH-PrPres, identifying a boundary separating loosely versus compactly folded regions of PrPres. Our observations demonstrate that TC-scanning with the FlAsH/TC tag can be a versatile method for probing protein-protein interactions and folding processes. PMID:23943295

A Metagenome-Wide Association Study and Arrayed Mutant Library Confirm Acetobacter Lipopolysaccharide Genes Are Necessary for Association with Drosophila melanogaster.

PubMed

White, K Makay; Matthews, Melinda K; Hughes, Rachel C; Sommer, Andrew J; Griffitts, Joel S; Newell, Peter D; Chaston, John M

2018-03-28

A metagenome wide association (MGWA) study of bacterial host association determinants in Drosophila predicted that LPS biosynthesis genes are significantly associated with host colonization. We were unable to create site-directed mutants for each of the predicted genes in Acetobacter , so we created an arrayed transposon insertion library using Acetobacter fabarum DsW_054 isolated from Drosophila Creation of the A. fabarum DsW_054 gene knock-out library was performed by combinatorial mapping and Illumina sequencing of random transposon insertion mutants. Transposon insertion locations for 6,418 mutants were successfully mapped, including hits within 63% of annotated genes in the A. fabarum DsW_054 genome. For 45/45 members of the library, insertion sites were verified by arbitrary PCR and Sanger sequencing. Mutants with insertions in four different LPS biosynthesis genes were selected from the library to validate the MGWA predictions. Insertion mutations in two genes biosynthetically upstream of Lipid-A formation, lpxC and lpxB , show significant differences in host association, whereas mutations in two genes encoding LPS biosynthesis functions downstream of Lipid-A biosynthesis had no effect. These results suggest an impact of bacterial cell surface molecules on the bacterial capacity for host association. Also, the transposon insertion mutant library will be a useful resource for ongoing research on the genetic basis for Acetobacter traits. Copyright © 2018 White et al.

A practical review of the muscles of facial mimicry with special emphasis on the superficial musculoaponeurotic system.

PubMed

Hutto, Justin R; Vattoth, Surjith

2015-01-01

In this article, we elaborate a practical approach to superficial facial anatomy enabling easy identification of the facial mimic muscles by classifying them according to their shared common insertion sites. The facial mimic muscles are often difficult to identify on imaging. By tracing them from their common group insertion sites back to their individual origins as well as understanding key anatomic relationships, radiologists can more accurately identify these muscles.

Biomechanical evaluation of oversized drilling technique on primary implant stability measured by insertion torque and resonance frequency analysis

PubMed Central

Santamaría-Arrieta, Gorka; Brizuela-Velasco, Aritza; Fernández-González, Felipe J.; Chávarri-Prado, David; Chento-Valiente, Yelko; Solaberrieta, Eneko; Diéguez-Pereira, Markel; Yurrebaso-Asúa, Jaime

2016-01-01

Background This study evaluated the influence of implant site preparation depth on primary stability measured by insertion torque and resonance frequency analysis (RFA). Material and Methods Thirty-two implant sites were prepared in eight veal rib blocks. Sixteen sites were prepared using the conventional drilling sequence recommended by the manufacturer to a working depth of 10mm. The remaining 16 sites were prepared using an oversize drilling technique (overpreparation) to a working depth of 12mm. Bone density was determined using cone beam computerized tomography (CBCT). The implants were placed and primary stability was measured by two methods: insertion torque (Ncm), and RFA (implant stability quotient [ISQ]). Results The highest torque values were achieved by the conventional drilling technique (10mm). The ANOVA test confirmed that there was a significant correlation between torque and drilling depth (p<0.05). However, no statistically significant differences were obtained between ISQ values at 10 or 12 mm drilling depths (p>0.05) at either measurement direction (cortical and medullar). No statistical relation between torque and ISQ values was identified, or between bone density and primary stability (p >0.05). Conclusions Vertical overpreparation of the implant bed will obtain lower insertion torque values, but does not produce statistically significant differences in ISQ values. Key words:Implant stability quotient, overdrilling, primary stability, resonance frequency analysis, torque. PMID:27398182

Genome-wide analysis of Tol2 transposon reintegration in zebrafish.

PubMed

Kondrychyn, Igor; Garcia-Lecea, Marta; Emelyanov, Alexander; Parinov, Sergey; Korzh, Vladimir

2009-09-08

Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. We performed a large-scale enhancer trap (ET) screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

Worldwide distribution of transposable element copy number in natural populations of Drosophila simulans.

PubMed

Biémont, Christian; Nardon, Christiane; Deceliere, Grégory; Lepetit, David; Loevenbruck, Catherine; Vieira, Cristina

2003-01-01

Transposable elements (TEs), which promote various kinds of mutations, constitute a large fraction of the genome. How they invade natural populations and species is therefore of fundamental importance for understanding the dynamics of genetic diversity and genome composition. On the basis of 85 samples of natural populations of Drosophila simulans, we report the distributions of the genome insertion site numbers of nine TEs that were chosen because they have a low average number of sites. Most populations were found to have 0-3 insertion sites, but some of them had a significantly higher number of sites for a given TE. The populations located in regions outside Africa had the highest number of sites for all elements except HMS Beagle and Coral, suggesting a recent increase in the activity of some TEs associated with the colonization patterns of Drosophila simulans. The element Tirant had a very distinctive pattern of distribution: it was identified mainly in populations from East Africa and some islands in the Indian Ocean, and its insertion site number was low in all these populations. The data suggest that the genome of the entire species of Drosophila simulans may be being invaded by TEs from populations in which they are present in high copy number.

A multi-landing pad DNA integration platform for mammalian cell engineering

PubMed Central

Gaidukov, Leonid; Wroblewska, Liliana; Teague, Brian; Nelson, Tom; Zhang, Xin; Liu, Yan; Jagtap, Kalpana; Mamo, Selamawit; Tseng, Wen Allen; Lowe, Alexis; Das, Jishnu; Bandara, Kalpanie; Baijuraj, Swetha; Summers, Nevin M; Zhang, Lin; Weiss, Ron

2018-01-01

Abstract Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three ‘landing pad’ recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing. PMID:29617873

Management of post-gastrectomy anastomosis site obstruction with a self-expandable metallic stent.

PubMed

Cha, Ra Ri; Lee, Sang Soo; Kim, Hyunjin; Kim, Hong Jun; Kim, Tae-Hyo; Jung, Woon Tae; Lee, Ok Jae; Bae, Kyung Soo; Jeong, Sang-Ho; Ha, Chang Yoon

2015-04-28

Post-gastrectomy anastomosis site obstruction is a relatively rare complication after a subtotal gastrectomy. We present a case of a 75-year-old man who underwent a truncal vagotomy, omental patch, gastrojejunostomy, and Braun anastomosis for duodenal ulcer perforation and a gastric outlet obstruction. Following the 10(th) postoperative day, the patient complained of abdominal discomfort and vomiting. We diagnosed post-gastrectomy anastomosis site obstruction by an upper gastrointestinal series and an upper endoscopic examination. We inserted a self-expandable metallic stent (SEMS) at the anastomosis site. The stent was fully expanded after deployment. On the day following the stent insertion, the patient began to eat, and his abdominal discomfort was resolved. This paper describes the successful management of post-gastrectomy anastomosis site obstruction with temporary placement of a SEMS.

Etonogestrel implant migration to the vasculature, chest wall, and distant body sites: cases from a pharmacovigilance database.

PubMed

Kang, Sarah; Niak, Ali; Gada, Neha; Brinker, Allen; Jones, S Christopher

2017-12-01

To describe clinical outcomes of etonogestrel implant patients with migration to the vasculature, chest wall and other distant body sites spontaneously reported to the US Food and Drug Administration Adverse Event Reporting System (FAERS) database. We performed a standardized Medical Dictionary for Regulatory Activities (MedDRA) query in the FAERS database (through November 15, 2015), with reports coded with one or more MedDRA preferred terms that indicate complications with device placement or migration of the device from the original site of insertion to the vasculature, chest wall and other distant body sites. We excluded any cases previously described in the medical literature. We identified 38 cases of pronounced etonogestrel implant migration. Migration locations included the lung/pulmonary artery (n=9), chest wall (n=1), vasculature at locations other than the lung/pulmonary artery (n=14) and extravascular migrations (n=14) to other body sites (e.g., the axilla and clavicle/neck line/shoulder). The majority of cases were asymptomatic and detected when the patient desired implant removal; however, seven cases reported symptoms such as pain, discomfort and dyspnea in association with implant migration. Three cases also describe pulmonary fibrosis and skin reactions as a result of implant migration to the vasculature, chest wall and other distant body sites. Sixteen cases reported surgical removal in an operating room setting. Our FAERS case series demonstrates etonogestrel implant migration to the vasculature, chest wall and other body sites distant from the site of original insertion. As noted by the sponsor in current prescribing information, a key determinant in the risk for etonogestrel contraceptive implant migration appears to be improper insertion technique. Although migration of etonogestrel implants to the vasculature is rare, awareness of migration and education on proper insertion technique may reduce the risk. Published by Elsevier Inc.

In vivo modification of a maize engineered minichromosome.

PubMed

Gaeta, Robert T; Masonbrink, Rick E; Zhao, Changzeng; Sanyal, Abhijit; Krishnaswamy, Lakshminarasimhan; Birchler, James A

2013-06-01

Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.

GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database.

PubMed

Kleinboelting, Nils; Huep, Gunnar; Kloetgen, Andreas; Viehoever, Prisca; Weisshaar, Bernd

2012-01-01

T-DNA insertion mutants are very valuable for reverse genetics in Arabidopsis thaliana. Several projects have generated large sequence-indexed collections of T-DNA insertion lines, of which GABI-Kat is the second largest resource worldwide. User access to the collection and its Flanking Sequence Tags (FSTs) is provided by the front end SimpleSearch (http://www.GABI-Kat.de). Several significant improvements have been implemented recently. The database now relies on the TAIRv10 genome sequence and annotation dataset. All FSTs have been newly mapped using an optimized procedure that leads to improved accuracy of insertion site predictions. A fraction of the collection with weak FST yield was re-analysed by generating new FSTs. Along with newly found predictions for older sequences about 20,000 new FSTs were included in the database. Information about groups of FSTs pointing to the same insertion site that is found in several lines but is real only in a single line are included, and many problematic FST-to-line links have been corrected using new wet-lab data. SimpleSearch currently contains data from ~71,000 lines with predicted insertions covering 62.5% of the 27,206 nuclear protein coding genes, and offers insertion allele-specific data from 9545 confirmed lines that are available from the Nottingham Arabidopsis Stock Centre.

Effects of the adenovirus 2 late promoter on simian virus 40 transcription and replication.

PubMed Central

Grass, D S; Manley, J L

1986-01-01

A 100-base-pair fragment of adenovirus 2 (Ad2) DNA encompassing the major late transcriptional promoter was inserted into the simian virus 40 (SV40) late promoter region at SV40 nucleotide 294 to study the effects of a strong TATA box-containing promoter on SV40 late transcription. pSVAdE contains the insert in an orientation such that it would promote transcription towards the origin and early region of SV40, while the insert is in the opposite orientation in pSVAdL. Nuclease S1 analysis with 5'-end-labeled probes showed that in cells transfected with pSVAdE, the late mRNA initiation sites are essentially the same as in wild type, demonstrating that an insert of 100 base pairs can have no effect on utilization of the SV40 late start sites. In pSVAdL-transfected cells, however, the major late viral initiation site is now in the insert at +1 with respect to the Ad2 major late cap site. However, all of the SV40 initiation sites are still utilized and with the same efficiency relative to each other as in wild type. Thus, it appears that the Ad2 late promoter and the SV40 late promoter can function independently on the same DNA molecule, even when one promoter is embedded within the other. By using cytosine arabinoside to block DNA replication and thereby inhibit the onset of late expression, it has been shown that both the Ad2 late promoter and the SV40 late promoter have similar requirement for DNA replication in this context. In addition, pSVAdL showed dramatically diminished virus viability and VPI expression compared with both wildtype and pSVAdE. Possible explanations for this unexpected finding are discussed. Images PMID:3001338

From the Cover: Understanding nature's design for a nanosyringe

NASA Astrophysics Data System (ADS)

Lopez, Carlos F.; Nielsen, Steve O.; Moore, Preston B.; Klein, Michael L.

2004-03-01

Synthetic and natural peptide assemblies can possess transport or conductance activity across biomembranes through the formation of nanopores. The fundamental mechanisms of membrane insertion necessary for antimicrobial or synthetic pore formation are poorly understood. We observe a lipid-assisted mechanism for passive insertion into a model membrane from molecular dynamics simulations. The assembly used in the study, a generic nanotube functionalized with hydrophilic termini, is assisted in crossing the membrane core by transleaflet lipid flips. Lipid tails occlude a purely hydrophobic nanotube. The observed insertion mechanism requirements for hydrophobic-hydrophilic matching have implications for the design of synthetic channels and antibiotics.

Two open reading frames (ORF1 and ORF2) within the 2.0-kilobase latency-associated transcript of herpes simplex virus type 1 are not essential for reactivation from latency.

PubMed Central

Fareed, M U; Spivack, J G

1994-01-01

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are dispensable for establishment and maintenance of latent infection. However, the LATs have been implicated in reactivation of the virus from its latent state. Since the reported LAT deletion and/or insertion variants that are reactivation impaired contain deletions in the putative LAT promoter, it is not known which LAT sequences are involved in reactivation. To examine the role of the 2.0-kb LAT in the process of reactivation and the functional importance of the putative open reading frames (ORF1 and ORF2) contained within the 2.0-kb LAT, we have constructed an HSV-1 variant that contains a precise deletion and insertion within the LAT-specific DNA sequences using site-directed mutagenesis. The HSV-1 variant FS1001K contains an 1,186-bp deletion starting precisely from the 5' end of the 2.0-kb LAT and, for identification, a XbaI restriction endonuclease site insertion. The FS1001K genome contains no other deletions and/or insertions as analyzed by a variety of restriction endonucleases. The deletion in FS1001K removes the entire 556-bp intron within the 2.0-kb LAT, the first 229 nucleotides of ORF1, and the first 159 nucleotides of ORF2 without having an affect on the RL2 (ICP0) gene. Explant cocultivation reactivation assays indicated that this deletion had a minimal effect on reactivation of the variant FS1001K compared with the parental wild-type virus using a mouse eye model. As expected, Northern (RNA) blot analyses have shown that the variant virus (FS1001K) does not produce the 2.0-kb LAT or the 1.45- to 1.5-kb LAT either in vitro or in vivo; however, FS1001K produces an intact RL2 transcript in tissue culture. These data suggest that the 2.0-kb LAT putative ORF1 and ORF2 (or the first 1,186 bp of the 2.0-kb LAT) are dispensable for explant reactivation of latent HSV-1. Images PMID:7966597

Flat midsubstance of the anterior cruciate ligament with tibial "C"-shaped insertion site.

PubMed

Siebold, Rainer; Schuhmacher, Peter; Fernandez, Francis; Śmigielski, Robert; Fink, Christian; Brehmer, Axel; Kirsch, Joachim

2015-11-01

This anatomical cadaver study was performed to investigate the flat appearance of the midsubstance shape of the anterior cruciate ligament (ACL) and its tibial "C"-shaped insertion site. The ACL midsubstance and the tibial ACL insertion were dissected in 20 cadaveric knees (n = 6 fresh frozen and n = 14 paraffined). Magnifying spectacles were used for all dissections. Morphometric measurements were performed using callipers and on digital photographs. In all specimens, the midsubstance of the ACL was flat with a mean width of 9.9 mm, thickness of 3.9 mm and cross-sectional area of 38.7 mm(2). The "direct" "C"-shaped tibial insertion runs from along the medial tibial spine to the anterior aspect of the lateral meniscus. The mean width (length) of the "C" was 12.6 mm, its thickness 3.3 mm and area 31.4 mm(2). The centre of the "C" was the bony insertion of the anterior root of the lateral meniscus overlayed by fat and crossed by the ACL. No posterolateral (PL) inserting ACL fibres were found. Together with the larger "indirect" part (area 79.6 mm(2)), the "direct" one formed a "duck-foot"-shaped footprint. The tibial ACL midsubstance and tibial "C"-shaped insertion are flat and are resembling a "ribbon". The centre of the "C" is the bony insertion of the anterior root of the lateral meniscus. There are no central or PL inserting ACL fibres. Anatomical ACL reconstruction may therefore require a flat graft and a "C"-shaped tibial footprint reconstruction with an anteromedial bone tunnel for single bundle and an additional posteromedial bone tunnel for double bundle.

Mini-Tn7 Insertion in Bacteria With Multiple glmS-Linked attTn7 Sites: Example Burkholderia Mallei ATCC 23344

DTIC Science & Technology

2006-06-27

INTRODUCTION Burkholderia mallei is the etiological agent of glanders and a highly evolved obligate zoonotic mammalian pathogen that naturally affects horses...mini-Tn7 insertion in bacteria with multiple glmS-linked attTn7 sites: example Burkholderia mallei ATCC 23344 Kyoung-Hee Choi1, David DeShazer2... glanders is a rare disease, B. mallei has received renewed attention because of its listing as a category B agent by the Centers for Disease Control

Fission yeast retrotransposon Tf1 integration is targeted to 5' ends of open reading frames.

PubMed

Behrens, R; Hayles, J; Nurse, P

2000-12-01

Target site selection of transposable elements is usually not random but involves some specificity for a DNA sequence or a DNA binding host factor. We have investigated the target site selection of the long terminal repeat-containing retrotransposon Tf1 from the fission yeast Schizosaccharomyces pombe. By monitoring induced transposition events we found that Tf1 integration sites were distributed throughout the genome. Mapping these insertions revealed that Tf1 did not integrate into open reading frames, but occurred preferentially in longer intergenic regions with integration biased towards a region 100-420 bp upstream of the translation start site. Northern blot analysis showed that transcription of genes adjacent to Tf1 insertions was not significantly changed.

Fission yeast retrotransposon Tf1 integration is targeted to 5′ ends of open reading frames

PubMed Central

Behrens, Ralf; Hayles, Jacky; Nurse, Paul

2000-01-01

Target site selection of transposable elements is usually not random but involves some specificity for a DNA sequence or a DNA binding host factor. We have investigated the target site selection of the long terminal repeat-containing retrotransposon Tf1 from the fission yeast Schizosaccharomyces pombe. By monitoring induced transposition events we found that Tf1 integration sites were distributed throughout the genome. Mapping these insertions revealed that Tf1 did not integrate into open reading frames, but occurred preferentially in longer intergenic regions with integration biased towards a region 100–420 bp upstream of the translation start site. Northern blot analysis showed that transcription of genes adjacent to Tf1 insertions was not significantly changed. PMID:11095681

Inferior vena cava filter insertion through the popliteal vein: enabling the percutaneous endovenous intervention of deep vein thrombosis with a single venous access approach in a single session

PubMed Central

Kim, Hyoung Ook; Kim, Jae Kyu; Park, Jin Gyoon; Yim, Nam Yeol; Kang, Yang Jun; Jung, Hye Doo

2016-01-01

PURPOSE We aimed to evaluate the efficiency of placing an inferior vena cava (IVC) filter through the same popliteal vein access site used for percutaneous endovenous intervention in patients with extensive lower extremity deep vein thrombosis. METHODS This retrospective study included 21 patients who underwent IVC filter insertion through the popliteal vein over a three-year period. Patient medical records were reviewed for the location of the deep vein thrombosis, result of filter removal, and total number of endovascular procedures needed for filter insertion and recanalization of the lower extremity venous system. Follow-up lower extremity computed tomography (CT) venography was also reviewed in each patient to assess the degree of filter tilt in the IVC. RESULTS All patients had extensive lower extremity deep vein thrombosis involving the iliac vein and/or femoral vein. Seventeen patients showed deep vein thrombosis of the calf veins. In all patients, IVC filter insertion and the recanalization procedure were performed during a single procedure through the single popliteal vein access site. In the 17 patients undergoing follow-up CT, the mean tilt angle of the filter was 7.14°±4.48° in the coronal plane and 8.77°±5.49° in the sagittal plane. Filter retrieval was successful in 16 of 17 patients (94.1%) in whom filter retrieval was attempted. CONCLUSION Transpopliteal IVC filter insertion is an efficient technique that results in low rates of significant filter tilt and enables a single session procedure using a single venous access site for filter insertion and percutaneous endovenous intervention. PMID:27559713

Interkinase domain of kit contains the binding site for phosphatidylinositol 3' kinase.

PubMed Central

Lev, S; Givol, D; Yarden, Y

1992-01-01

Our previous analysis of the signal transduction pathway used by the c-kit-encoded receptor for the stem cell factor (SCF) indicated efficient coupling to the type I phosphatidylinositol 3' kinase (PI3K). In an attempt to localize the receptor's site of interaction with PI3K, we separately deleted either the noncatalytic 68-amino-acid-long interkinase domain or the carboxyl-terminal portion distal to the catalytic sequences. Loss of ligand-induced association of PI3K with the former deletion mutant and retention of the PI3K association by the carboxyl-terminally deleted receptor implied interactions of PI3K with the kinase insert. This was further supported by partial inhibition of the association by an anti-peptide antibody directed against the kinase insert and lack of effect of an antibody directed to the carboxyl tail of the SCF receptor. A bacterially expressed kinase insert domain was used as a fusion protein to directly test its presumed function as a PI3K association site. This protein bound PI3K from cell lysate as demonstrated by PI3K activity and by an associated phosphoprotein of 85 kDa. The association was dependent on phosphorylation of the tyrosine residues on the expressed kinase insert. On the basis of these observations, we conclude that the kinase insert domain of the SCF receptor selectively interacts with the p85 regulatory subunit of PI3K and that this association requires phosphorylation of tyrosine residues in the kinase insert region, with apparently no involvement of the bulk cytoplasmic structure or tyrosine kinase function of the receptor. Images PMID:1370584

Group B streptococci in women fitted with intrauterine devices.

PubMed Central

Mitchell, R G; Guillebaud, J; Day, D G

1977-01-01

A survey was made of group B streptococcal carriage at various sites in 100 women attending a clinic for the insertion of an intrauterine contraceptive device (IUD). Twenty-three women carried streptococci at one or more sites at the preinsertion visit, the vaginal carriage rate being 16%. Six months after insertion changes in carrier status were noted and there was evidence of a change of strain in four patients. Twenty-nine women were carriers at one or more sites at some stage of the study. There was no evidence that symptoms attributable to infection in patients fitted with an IUD were caused by group B streptococci. PMID:338639

Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci.

PubMed

Sabri, Suriana; Steen, Jennifer A; Bongers, Mareike; Nielsen, Lars K; Vickers, Claudia E

2013-06-24

Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic. A series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (β-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous 'arms' target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional. The KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal insertion provides stable expression without the need for continuous antibiotic selection. Three non-essential loci have been characterised as insertion loci; combinatorial insertion at all three loci can be performed in one strain. The largest insertion at a single site described here was 5.4 kb; we have used this method in other studies to insert a total of 7.3 kb at one locus and 11.3 kb across two loci. These vectors are particularly useful for integration of multigene cassettes for metabolic engineering applications.

Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

PubMed Central

2012-01-01

Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors) in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution. PMID:23148531

Risks of Preterm Premature Rupture of Membranes and Preterm Birth Post Fetoscopy Based on Location of Trocar Insertion Site.

PubMed

Chmait, Ramen H; Chon, Andrew H; Korst, Lisa M; Llanes, Arlyn; Kontopoulos, Eftichia V; Quintero, Ruben A

2018-07-01

 The objective of this study was to assess whether the location of the trocar insertion site for laser treatment of twin-twin transfusion syndrome was associated with preterm-premature rupture of membranes (PPROM) and preterm birth (PTB).  In this study trocar location was documented in the operating room. Lower uterine segment (LUS) location was defined as any insertion <10 cm vertically from the pubic symphysis. Lateral location was defined as ≥5 cm horizontally from the midline. Patient characteristics were tested against three outcomes: PPROM ≤ 21 days postoperative, PTB < 28 weeks, and PTB < 32 weeks. For each outcome, multiple logistic models were fitted to examine the effect of trocar location, controlling for potential risk factors.  A total of 743 patients were studied. Patients with LUS location were twice as likely as those with a more superior location to have PPROM ≤ 21 days (OR = 2.33, 1.12-4.83, p  = 0.0236). Patients with both a LUS and Lateral location were over six times more likely to have PPROM ≤ 21 days (OR = 6.66, 2.36-18.78, p  = 0.0003). Trocar insertion site was not associated with PTB.  We found that trocar insertion in the LUS, particularly the lateral LUS, was associated with an increased risk of PPROM. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

The Wnt-1 (int-1) oncogene promoter and its mechanism of activation by insertion of proviral DNA of the mouse mammary tumor virus.

PubMed Central

Nusse, R; Theunissen, H; Wagenaar, E; Rijsewijk, F; Gennissen, A; Otte, A; Schuuring, E; van Ooyen, A

1990-01-01

Wnt-1 (int-1) is a cellular oncogene often activated by insertion of proviral DNA of the mouse mammary tumor virus. We have mapped the 5' end and the promoter area of the Wnt-1 gene by nuclease protection and primer extension assays. In differentiating P19 embryonal carcinoma cells, in which Wnt-1 is naturally expressed, two start sites of transcription were found, one preceded by two TATA boxes and one preceded by several GC boxes. In P19 cells, a 1-kilobase upstream sequence of Wnt-1 was able to confer differentiation-specific expression on a heterologous gene. We have investigated how Wnt-1 transcription was affected by mouse mammary tumor virus proviral integrations in various configurations near the promoters of the gene. One provirus has been inserted in the 5' nontranslated part of Wnt-1, in the same transcriptional orientation, and has functionally replaced the Wnt-1 promoters. Wnt-1 transcription in this tumor starts in the right long terminal repeat of the provirus, with considerable readthrough transcription from the left long terminal repeat. Another provirus has been inserted in the orientation opposite that of Wnt-1 into a GC box, disrupting the first Wnt-1 transcription start site but not the downstream start site. Most insertions have not structurally altered the Wnt-1 transcripts and have enhanced the activity of the normal two promoters. Images PMID:1695322

Dental implants inserted in fresh extraction sockets versus healed sites: a systematic review and meta-analysis.

PubMed

Chrcanovic, Bruno Ramos; Albrektsson, Tomas; Wennerberg, Ann

2015-01-01

To test the null hypothesis of no difference in the implant failure rates, postoperative infection and marginal bone loss for the insertion of dental implants in fresh extraction sockets compared to the insertion in healed sites, against the alternative hypothesis of a difference. Main search terms used in combination: dental implant, oral implant, resh extraction socket, immediate placement, immediate insertion, immediate implant. An electronic search was undertaken in July/2014, in PubMed, Web of Science, Cochrane Oral Health Group Trials Register plus hand-searching. Eligibility criteria included clinical human studies, either randomized or not. The search strategy resulted in 73 publications, with 8,241 implants inserted in sockets (330 failures, 4.00%), and 19,410 in healed sites (599 failures, 3.09%). It is suggested that the insertion of implants in fresh extraction sockets affects the failure rates (RR 1.58, 95% CI 1.27-1.95, P<0.0001). The difference was not statistically significant when studies evaluating implants inserted in maxillae or in mandibles were pooled, or when the studies using implants to rehabilitate patients with full-arch prostheses were pooled; however, it was significant for the studies that rehabilitated patients with implant-supported single crowns and for the controlled studies. There was no apparent significant effect on the occurrence of postoperative infection or on the magnitude of marginal bone loss. The results should be interpreted with caution due to the potential for biases and to the presence of uncontrolled confounding factors in the included studies, most of them not randomized. The question whether immediate implants are more at risk for failure than implants placed in mature bone has received increasing attention in the last years. As the philosophies of treatment alter over time, a periodic review of the different concepts is necessary to refine techniques and eliminate unnecessary procedures. This would form a basis for optimum treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.

Maturation of the [Ni-4Fe-4S] active site of carbon monoxide dehydrogenases.

PubMed

Merrouch, Mériem; Benvenuti, Martino; Lorenzi, Marco; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

2018-02-14

Nickel-containing enzymes are diverse in terms of function and active site structure. In many cases, the biosynthesis of the active site depends on accessory proteins which transport and insert the Ni ion. We review and discuss the literature related to the maturation of carbon monoxide dehydrogenases (CODH) which bear a nickel-containing active site consisting of a [Ni-4Fe-4S] center called the C-cluster. The maturation of this center has been much less studied than that of other nickel-containing enzymes such as urease and NiFe hydrogenase. Several proteins present in certain CODH operons, including the nickel-binding proteins CooT and CooJ, still have unclear functions. We question the conception that the maturation of all CODH depends on the accessory protein CooC described as essential for nickel insertion into the active site. The available literature reveals biological variations in CODH active site biosynthesis.

Quantitative In Situ Analysis of the Anterior Cruciate Ligament: Length, Midsubstance Cross-sectional Area, and Insertion Site Areas.

PubMed

Fujimaki, Yoshimasa; Thorhauer, Eric; Sasaki, Yusuke; Smolinski, Patrick; Tashman, Scott; Fu, Freddie H

2016-01-01

Quantification of the cross-sectional area (CSA) of the anterior cruciate ligament (ACL) in different loading conditions is important for understanding the native anatomy and thus achieving anatomic reconstruction. The ACL insertion sites are larger than the ACL midsubstance, and the isthmus (region of the smallest CSA) location may vary with the load or flexion angle. To (1) quantify the CSA along the entire ACL, (2) describe the location of the ACL isthmus, (3) explore the relationship between ACL length and CSA, and (4) validate magnetic resonance imaging (MRI) for assessing the CSA of the midsubstance ACL. Descriptive laboratory study. Eight cadaveric knees were dissected to expose the ACL and its attachments. Knees were positioned using a robotic loading system through a range of flexion angles in 3 loading states: (1) unloaded, (2) anterior tibial translation, and (3) combined rotational load of valgus and internal torque. Laser scanning quantified the shape of the ACL and its insertion site boundaries. The CSA of the ACL was measured, and the location of the isthmus was determined; the CSA of the ACL was also estimated from MRI and compared with the laser-scanned data. The CSA of the ACL varied along the ligament, and the isthmus existed at an average (±SD) of 53.8% ± 5.5% of the distance from the tibial insertion center to the femoral insertion center. The average CSA at the isthmus was smallest in extension (39.9 ± 13.7 mm(2)) and increased with flexion (43.9 ± 12.1 mm(2) at 90°). The ACL length was shortest at 90° of flexion and increased by 18.8% ± 10.1% in unloaded extension. Application of an anterior load increased the ACL length by 5.0% ± 3.3% in extension, and application of a combined rotational load increased its length by 4.1% ± 3.0% in extension. The ACL isthmus is located almost half of the distance between the insertion sites. The CSA of the ACL at the isthmus is largest with the knee unloaded and at 90° of flexion, and the area decreases with extension and applied loads. The CSA at the isthmus represents less than half the area of the insertion sites. These results may aid surgical planning, specifically for choosing a graft size and fixation angle that most closely matches the native anatomy and function across the entire range of knee motion. © 2015 The Author(s).

Three Group-I introns in 18S rDNA of Endosymbiotic Algae of Paramecium bursaria from Japan

NASA Astrophysics Data System (ADS)

Hoshina, Ryo; Kamako, Shin-ichiro; Imamura, Nobutaka

2004-08-01

In the nuclear encoded small subunit ribosomal DNA (18S rDNA) of symbiotic alga of Paramecium bursaria (F36 collected in Japan) possesses three intron-like insertions (Hoshina et al., unpubl. data, 2003). The present study confirmed these exact lengths and insertion sites by reverse transcription-PCR. Two of them were inserted at Escherichia coli 16S rRNA genic position 943 and 1512 that are frequent intron insertion positions, but another insertion position (nearly 1370) was the first finding. Their secondary structures suggested they belong to Group-I intron; one belongs to subgroup IE, others belong to subgroup IC1. Similarity search indicated these introns are ancestral ones.

Non-mineralized fibrocartilage shows the lowest elastic modulus in the rabbit supraspinatus tendon insertion: measurement with scanning acoustic microscopy.

PubMed

Sano, Hirotaka; Saijo, Yoshifumi; Kokubun, Shoichi

2006-01-01

The acoustic properties of rabbit supraspinatus tendon insertions were measured by scanning acoustic microscopy. After cutting parallel to the supraspinatus tendon fibers, specimens were fixed with 10% neutralized formalin, embedded in paraffin, and sectioned. Both the sound speed and the attenuation constant were measured at the insertion site. The 2-dimensional distribution of the sound speed and that of the attenuation constant were displayed with color-coded scales. The acoustic properties reflected both the histologic architecture and the collagen type. In the tendon proper and the non-mineralized fibrocartilage, the sound speed and attenuation constant gradually decreased as the predominant collagen type changed from I to II. In the mineralized fibrocartilage, they increased markedly with the mineralization of the fibrocartilaginous tissue. These results indicate that the non-mineralized fibrocartilage shows the lowest elastic modulus among 4 zones at the insertion site, which could be interpreted as an adaptation to various types of biomechanical stress.

Latissimus dorsi transfer to restore external rotation with reverse shoulder arthroplasty: a biomechanical study.

PubMed

Favre, Philippe; Loeb, Michael D; Helmy, Naeder; Gerber, Christian

2008-01-01

In patients with pseudoparesis of the shoulder resulting from irreparable rotator cuff tears, reverse shoulder arthroplasty (RSA) can restore active elevation, but external rotation remains less predictable. Latissimus dorsi transfer (LDT) has been shown to be effective in restoring external rotation in patients with posterosuperior tears of the rotator cuff. The aim of this study is to determine the capacity of the LDT to restore external rotation in combination with RSA and to investigate the mechanical advantage produced by 3 different insertion sites. A biomechanical model was created using a reverse total shoulder prosthesis with 3 different transfer insertions. Moment arms were measured for 2 static positions and 1 motion of the humerus. The moment arm analysis showed that LDT can improve active external rotation in the setting of a reverse prosthesis. An insertion site on the posterior side of the greater tuberosity (adjacent to the teres minor insertion) produced a greater external rotation moment arm.

Cholorhexidine, octenidine or povidone iodine for catheter related infections: A randomized controlled trial

PubMed Central

Bilir, Ayten; Yelken, Birgül; Erkan, Ayse

2013-01-01

Background: Protection of the catheter site by antimicrobial agents is one of the most important factors in the prevention of infection. Povidone iodine and chlorhexidine gluconate are the most common used agents for dressing. The purpose of this study was to compare the effects of povidone iodine, chlorhexidine gluconate and octenidine hydrochloride in preventing catheter related infections. Materials and Methods: Patients were randomized to receive; 4% chlorhexidine gluconate, 10% povidone iodine or octenidine hydrochlorodine for cutaneous antisepsis. Cultures were taken at the site surrounding catheter insertion and at the catheter hub after removal to help identify the source of microorganisms. Results: Catheter related sepsis was 10.5% in the povidone iodine and octenidine hydrochlorodine groups. Catheter related colonization was 26.3% in povidone iodine group and 21.5% in octenidine hydrochlorodine group. Conclusion: 4% chlorhexidine or octenidine hydrochlorodine for cutaneous disinfection before insertion of an intravascular device and for post-insertion site care can reduce the catheter related colonization. PMID:24250702

The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition

PubMed Central

Servant, Geraldine; Streva, Vincent A.; Derbes, Rebecca S.; Wijetunge, Madushani I.; Neeland, Marc; White, Travis B.; Belancio, Victoria P.; Roy-Engel, Astrid M.; Deininger, Prescott L.

2017-01-01

Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a “copy-and-paste” mechanism termed retrotransposition, and de novo insertions related to these elements have been reported to cause 0.2% of genetic diseases. Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3′ DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events. PMID:28049704

Three-Dimensional Ultrasound-Guided Real-Time Midline Epidural Needle Placement with Epiguide: A Prospective Feasibility Study.

PubMed

Beigi, Parmida; Malenfant, Paul; Rasoulian, Abtin; Rohling, Robert; Dube, Alison; Gunka, Vit

2017-01-01

Current 2-D ultrasound technology is unable to perform a midline neuraxial needle insertion under real-time ultrasound guidance using a standard needle and without an assistant. The aim of the work described here was to determine the feasibility of a new technology providing such capability, starting with a study evaluating the selected puncture site. A novel 3-D ultrasound imaging technique was designed using thick-slice rendering in conjunction with a custom needle guide (3DUS + Epiguide). A clinical feasibility study evaluated the ability of 3DUS + Epiguide to identify the epidural needle puncture site for a midline insertion in the lumbar spine. We hypothesized that (i) the puncture site identified by 3DUS + Epiguide was within a 5-mm radius from the site chosen by standard palpation, and (ii) the difference between the two puncture sites was not correlated to the patient characteristics age, weight, height, body mass index and gestational age. The mean (±standard deviation) distances between puncture sites determined by 3DUS + Epiguide and palpation were 3.1 (±1.7) mm and 2.8 (±1.3) mm, for the L2-3 and L3-4 interspaces of 20 patients, respectively. Distances were comparable to intra-observer variability, indicating the potential for a thick-slice rendering of 3-D ultrasound along the Epiguide trajectory to select the puncture site of a midline neuraxial needle insertion. The long-term potential benefits of this system include increased efficiency and use of anesthesia, and a reduction in the frequency and severity of the complications from incorrect needle insertions. Epidural success in the most difficult cases (e.g., the obese) will be the focus of future work. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics

PubMed Central

Brough, Rachel; Papanastasiou, Antigoni M; Porter, Andrew CG

2007-01-01

Background The ability to regulate transgene expression has many applications, mostly concerning the analysis of gene function. Desirable induction characteristics, such as low un-induced expression, high induced expression and limited cellular heterogeneity, can be seriously impaired by chromosomal position effects at the site of transgene integration. Many clones may therefore need to be screened before one with optimal induction characteristics is identified. Furthermore, such screens must be repeated for each new transgene investigated, and comparisons between clones with different transgenes is complicated by their different integration sites. Results To circumvent these problems we have developed a "screen and insert" strategy in which clones carrying a transgene for a fluorescent reporter are first screened for those with optimal induction characteristics. Site-specific recombination (SSR) is then be used repeatedly to insert any new transgene at the reporter transgene locus of such clones so that optimal induction characteristics are conferred upon it. Here we have tested in a human fibrosarcoma cell line (HT1080) two of many possible implementations of this approach. Clones (e.g. Rht14-10) in which a GFP reporter gene is very stringently regulated by the tetracycline (tet) transactivator (tTA) protein were first identified flow-cytometrically. Transgenes encoding luciferase, I-SceI endonuclease or Rad52 were then inserted by SSR at a LoxP site adjacent to the GFP gene resulting stringent tet-regulated transgene expression. In clone Rht14-10, increases in expression from essentially background levels (+tet) to more than 104-fold above background (-tet) were reproducibly detected after Cre-mediated insertion of either the luciferase or the I-SceI transgenes. Conclusion Although previous methods have made use of SSR to integrate transgenes at defined sites, none has effectively combined this with a pre-selection step to identify integration sites that support optimal regulatory characteristics. Rht14-10 and similar HT1080-derived clones can now be used in conjunction with a convenient delivery vector (pIN2-neoMCS), in a simple 3-step protocol leading to stringent and reproducible transgene regulation. This approach will be particularly useful for transgenes whose products are very active at low concentrations and/or for comparisons of multiple related transgenes. PMID:17493262

Ty1-copia elements reveal diverse insertion sites linked to polymorphisms among flax (Linum usitatissimum L.) accessions.

PubMed

Galindo-González, Leonardo; Mhiri, Corinne; Grandbastien, Marie-Angèle; Deyholos, Michael K

2016-12-07

Initial characterization of the flax genome showed that Ty1-copia retrotransposons are abundant, with several members being recently inserted, and in close association with genes. Recent insertions indicate a potential for ongoing transpositional activity that can create genomic diversity among accessions, cultivars or varieties. The polymorphisms generated constitute a good source of molecular markers that may be associated with phenotype if the insertions alter gene activity. Flax, where accessions are bred mainly for seed nutritional properties or for fibers, constitutes a good model for studying the relationship of transpositional activity with diversification and breeding. In this study, we estimated copy number and used a type of transposon display known as Sequence-Specific Amplification Polymorphisms (SSAPs), to characterize six families of Ty1-copia elements across 14 flax accessions. Polymorphic insertion sites were sequenced to find insertions that could potentially alter gene expression, and a preliminary test was performed with selected genes bearing transposable element (TE) insertions. Quantification of six families of Ty1-copia elements indicated different abundances among TE families and between flax accessions, which suggested diverse transpositional histories. SSAPs showed a high level of polymorphism in most of the evaluated retrotransposon families, with a trend towards higher levels of polymorphism in low-copy number families. Ty1-copia insertion polymorphisms among cultivars allowed a general distinction between oil and fiber types, and between spring and winter types, demonstrating their utility in diversity studies. Characterization of polymorphic insertions revealed an overwhelming association with genes, with insertions disrupting exons, introns or within 1 kb of coding regions. A preliminary test on the potential transcriptional disruption by TEs of four selected genes evaluated in three different tissues, showed one case of significant impact of the insertion on gene expression. We demonstrated that specific Ty1-copia families have been active since breeding commenced in flax. The retrotransposon-derived polymorphism can be used to separate flax types, and the close association of many insertions with genes defines a good source of potential mutations that could be associated with phenotypic changes, resulting in diversification processes.

The enthesis organ concept and its relevance to the spondyloarthropathies.

PubMed

Benjamin, Michael; McGonagle, Dennis

2009-01-01

A characteristic feature of the spondyloarthropathies is inflammation at tendon or ligament attachment sites. This has traditionally been viewed as a focal abnormality, even though the inflammatory reaction intrinsic to enthesitis may be quite extensive. We argue that the diffuse nature of the pathology is best understood in the context of an 'enthesis organ concept'. This highlights the fact that stress concentration at an insertion site involves not only the enthesis itself, but neighbouring tissues as well. The archetypal enthesis organ is that of the Achilles tendon where intermittent contact between tendon and bone immediately proximal to the enthesis leads to the formation of fibrocartilages on the deep surface of the tendon and on the opposing calcaneal tuberosity, but similar functional modifications are widespread throughout the skeleton. Many entheses have bursae and fat near the insertion site and both of these serve to promote frictionless movement. Collectively, the fibrocartilages, bursa, fat pad and the enthesis itself constitute the enthesis organ. However, it also includes both the immediately adjacent trabecular bone networks and in some cases deep fascia. The concept of a synovio-entheseal complex (SEC) and of a 'functional enthesis' are complimentary to that of an enthesis organ and also have important implications for understanding spondyloarthropathy. The SEC concept emphasizes the interdependence between synovial membrane and entheses within enthesis organs. It draws attention to the fact that one component (the enthesis) is prone to microdamage and the other (the synovium) to inflammation. If an enthesis is damaged, any ensuing inflammatory reaction is likely to occur in the synovium. The concept of a 'functional enthesis' serves to emphasise anatomical, biomechanical and pathological features that are shared between true fibrocartilaginous entheses and regions proximal to the attachment sites themselves where tendons or ligaments wrap around bony pulleys. Such'wrap-around regions' are well documented sites of pathology in SpA-for tenosynovitis is a recognized feature. Stress concentration at the enthesis itself is dissipated at many sites by fibrous connections between one tendon or ligament and another, close to the insertion site. At a microscopic level, enthesis fibrocartilage is of paramount importance in ensuring that fibre bending of the tendon or ligament is not focused at the hard tissue interface. Normal enthesis organs are avascular in their fibrocartilaginous regions, but tissue microdamage to entheses is common and appears to be associated with tissue repair responses and vessel ingrowth. This makes the enthesis organ a site where adjuvant molecules derived from bacteria may be preferentially deposited. This microdamage and propensity for bacterial molecule deposition in the context of genetic factors such as HLA-B27 appears to lead to the characteristic inflammatory changes of AS. Understanding the enthesis organ concept helps to explain synovitis and osteitis in spondyloarthropathy. An appreciation of the complex anatomy of 'articular enthesis organs' (e.g., that associated with the distal interphalangeal joints) is helpful in understanding disease patterns in psoriatic arthritis. In this chapter, we review the extent and types ofenthesis organs and show how a patho-anatomic appreciation of these structures leads to a new platform for understanding the pathogenesis of SpA.

Peripherally inserted central catheters. Guidewire versus nonguidewire use: a comparative study.

PubMed

Loughran, S C; Edwards, S; McClure, S

1992-01-01

To date, no research articles have been published that explore the practice of using guidewires for placement of peripherally inserted central catheters. The literature contains speculations regarding the pros and cons of guidewire use. However, no studies to date have compared patient outcomes when peripherally inserted central catheter lines are inserted with and without guidewires. To examine the use of guidewires for peripherally inserted central lines, a comparative study was conducted at two acute care facilities, one using guidewires for insertion and one inserting peripherally inserted central catheter lines without guidewires. 109 catheters were studied between January 1, 1990 and January 1, 1991. The primary focus of this study was to examine whether guidewire use places patients at higher risk for catheter-related complications, particularly phlebitis. No significant differences in phlebitis rates between the two study sites were found. Other catheter-related and noncatheter-related complications were similar between the two facilities. The results of this study do not support the belief that guidewire use increases complication rates.

Defects in ion-implanted hcp-titanium: A first-principles study of electronic structures

NASA Astrophysics Data System (ADS)

Raji, Abdulrafiu T.; Mazzarello, Riccardo; Scandolo, Sandro; Nsengiyumva, Schadrack; Härting, Margit; Britton, David T.

2011-12-01

The electronic structures of hexagonal closed-packed (h.c.p) titanium containing a vacancy and krypton impurity atoms at various insertion sites are calculated by first-principles methods in the framework of the density-functional theory (DFT). The density of states (DOS) for titanium containing a vacancy defect shows resonance-like features. Also, the bulk electron density decreases from ˜0.15/Å 3 to ˜0.05/Å 3 at the vacancy centre. Electronic structure calculations have been performed to investigate what underlies the krypton site preference in titanium. The DOS of the nearest-neighbour (NN) titanium atoms to the octahedral krypton appears to be less distorted (relative to pure titanium) when compared to the NN titanium atoms to the tetrahedral krypton. The electronic density deformation maps show that polarization of the titanium atoms is stronger when the krypton atom is located at the tetrahedral site. Since krypton is a closed-shell atom, thus precluding any bonding with the titanium atoms, we may conclude that the polarization of the electrons in the vicinity of the inserted krypton atoms and the distortion of the DOS of the NN titanium atoms to the krypton serve to indicate which defect site is preferred when a krypton atom is inserted into titanium. Based on these considerations, we conclude that the substitutional site is the most favourable one, and the octahedral is the preferred interstitial site, in agreement with recent DFT calculations of the energetics of krypton impurity sites.

Ultrasonic implant site preparation using piezosurgery: a multicenter case series study analyzing 3,579 implants with a 1- to 3-year follow-up.

PubMed

Vercellotti, Tomaso; Stacchi, Claudio; Russo, Crescenzo; Rebaudi, Alberto; Vincenzi, Giampaolo; Pratella, Umberto; Baldi, Domenico; Mozzati, Marco; Monagheddu, Chiara; Sentineri, Rosario; Cuneo, Tommaso; Di Alberti, Luca; Carossa, Stefano; Schierano, Gianmario

2014-01-01

This multicenter case series introduces an innovative ultrasonic implant site preparation (UISP) technique as an alternative to the use of traditional rotary instruments. A total of 3,579 implants were inserted in 1,885 subjects, and the sites were prepared using a specific ultrasonic device with a 1- to 3-year follow-up. No surgical complications related to the UISP protocol were reported for any of the implant sites. Seventy-eight implants (59 maxillary, 19 mandibular) failed within 5 months of insertion, for an overall osseointegration percentage of 97.82% (97.14% maxilla, 98.75% mandible). Three maxillary implants failed after 3 years of loading, with an overall implant survival rate of 97.74% (96.99% maxilla, 98.75% mandible).

Skin microdialysis coupled with laser speckle contrast imaging to assess microvascular reactivity.

PubMed

Cracowski, J L; Gaillard-Bigot, F; Cracowski, C; Roustit, M; Millet, C

2011-11-01

Laser speckle contrast imaging (LSCI) can be used to assess real-time responses of skin microcirculation to pharmacological interventions. The main objective of this study was to determine whether intradermal or subdermal microdialysis fiber insertion, coupled with skin flux recording using LSCI, can be used to assess baseline cutaneous flux and the post-occlusive reactive hyperemic response. The microdialysis sites were compared to control area without microdialysis fibers. One dermal and two subdermal microdialysis fibers were randomly inserted in the right forearm skin of six healthy volunteers. We performed consecutively tests of post-occlusive hyperemia, infusion of 29 mM sodium nitroprusside (SNP), local thermal hyperemia at 43°C and a second 29 mM SNP infusion at the end of the experiment. Two hours after fiber insertion, cutaneous vascular conductances (CVC) at the subdermal fiber sites were not different from their respective control regions of interest, while at the dermal site CVC remained higher (0.48+/-0.15 versus 0.37+/-0.1 PU.mm Hg(-1), P=0.003). The peak CVC and area under the curve observed during post-occlusive reactive hyperemia were similar at all fiber sites and their respective controls. We observed a similar increase in CVC using 29 mM SNP infusion, 40 min local heating at 43°C, and their combination. Finally, physiological and pharmacological responses of the subdermal sites were reproducible in terms of amplitude, whether expressed as raw CVC or as % CVCmax. We showed that studying skin microvascular physiological or pharmacological responses using inserted subdermal microdialysis fibers coupled with LSCI is feasible and reproducible, and provides two-dimensional information. This technique will be useful for future mechanistic studies of skin microcirculation. Copyright © 2011 Elsevier Inc. All rights reserved.

Miyake-Apple view of inner side of sclerotomy during microincision vitrectomy surgery.

PubMed

Inoue, Makoto; Ota, Ichiro; Taniuchi, Shutaro; Nagamoto, Toshiyuki; Miyake, Kensaku; Hirakata, Akito

2011-08-01

To examine the inner surface of the sclerotomy during microincision vitrectomy surgery by Miyake-Apple view. The anterior half of porcine eyes was attached to a transparent acrylic plate with cyanoacrylate glue. Then, either a 23-gauge or a 25-gauge trocar-cannula was inserted through the sclera obliquely. The inner surface of the entrance site was observed posteriorly by Miyake-Apple view. These images were compared with the endoscopic view of two patients who underwent vitreous surgery for an epiretinal membrane. When the trocar-cannula was inserted obliquely, the Miyake-Apple view showed that the ciliary epithelium at the sclerotomy site was stretched. When the trocar-cannula was inserted vertically, the ciliary epithelium was folded, and the folds remained even after the trocar was removed. Vitreous strands were seen incarcerated into the sclerotomy site. In human eyes, a folding of the ciliary epithelium was not clearly seen with the endoscopic view but the incarcerated vitreous was seen. The Miyake-Apple view provided a precise, in vivo, observation of the inner surface of the entry site. It disclosed the morphological stress on the ciliary epithelium by the sclerotomy. © 2011 The Authors. Acta Ophthalmologica © 2011 Acta Ophthalmologica Scandinavica Foundation.

Percutaneous insertion of peritoneal dialysis catheters using ultrasound and fluoroscopic guidance: A single centre experience and review of literature.

PubMed

De Boo, Diederick W; Mott, Nigel; Tregaskis, Peter; Quach, Trung; Menahem, Solomon; Walker, Rowan G; Koukounaras, Jim

2015-12-01

Various methods of peritoneal dialysis (PD) catheter insertion are available. The purpose of this study was to evaluate a percutaneous insertion technique using ultrasound (US) and fluoroscopy performed under conscious sedation and as day case procedure. Data of 87 percutaneous inserted dialysis catheters were prospectively collected, including patients' age, gender, body mass index, history of previous abdominal surgery and cause of end stage renal failure. Length of hospital stay, early complications and time to first use were also recorded. Institutional review board approval was obtained. A 100% technical success rate was observed. Early complications included bleeding (n = 3), catheter dysfunction (n = 6), exit site infection (n = 1) and exit site leakage (n = 1). All cases of catheter dysfunction and one case of bleeding required surgical revision. Median time of follow-up was 18 months (range 3-35), and median time from insertion to first use was days 14 (1-47). Of the 82 patients who started dialysis, 20 (23%) ceased PD at some stage during follow-up. Most frequently encountered reasons include deteriorating patient cognitive or functional status (n = 5), successful transplant kidney (n = 4) and pleuro-peritoneal fistula (n = 4). Sixty-two (71%) PD catheter insertions were performed as day case. The remaining insertions were performed on patients already admitted to the hospital. Percutaneous insertion of dialysis catheter using US and fluoroscopy is not only safe but can be performed as day case procedure in most patients, even with a medical history of abdominal surgery and/or obesity. © 2015 The Royal Australian and New Zealand College of Radiologists.

[Reducing the Incidence of Phlebitis Related to Intravenous Injection in Pediatric Patients].

PubMed

Cho, Yen-Hua; Yen, Li-Ling; Yu, Kai-Ling; Chang, Chun-Chu; Chen, Hsuen-Ling

2015-06-01

Peripheral venous catheter (PVC) is commonly used to provide nutrition and medicine to pediatric inpatients. Phlebitis is a common side effect of PVC insertion. Over 90% of pediatric patients in the paedi-atric medical ward at the Chang Gung Memorial Hospital (CGMH) receive PVC insertion, with an incident rate of phlebitis of 5.07%. Common cause factors of phlebitis are: insufficient sterilization time, inappropriate methods used to fix the PVC, the use of fixtures that loosen easily, high re-fix rates, and inadequate wound care after catheter removal. The purpose of this project was to reduce the incidence rate of PVC-insertion-related phlebitis in children from 5.07% to 2.5%. A one-week clinical observation identified the re-inserting / re-fixing of existing PVCs as the principal cause of phlebitis in the CGMH paediatric ward. Therefore, the researchers modified the catheter care bundle based on a review of the literature and the suggestions of clinical pediatric experts. Modifications included applying 2% chlorhexidine to sterilize the insertion site; using a new, non-woven fabric splint to fix the PVC site; providing cartoon-themed waterproof dressings for the first bath after the removal of the PVC; and setting standard operating procedures (SOPs) for PVC insertion and catheter removal. After applying these modifications, the incident rate of phlebitis in children with PVC insertions decreased from 5.07% to 2.08%. The application of 2% chlorhexidine reduces the waiting time for sterilization; the purpose-designed splint strengthens the fixation of the PVC; and the development of the SOPs for PVC insertion and post-removal catheter care reduces the risk of phlebitis. The combination of these strategies effectively reduces the incidence of phlebitis and improves the nursing care quality.

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe.

PubMed

Chen, Bo-Ruei; Hale, Devin C; Ciolek, Peter J; Runge, Kurt W

2012-05-03

Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.

Improving patient safety during insertion of peripheral venous catheters: an observational intervention study.

PubMed

Kampf, Günter; Reise, Gesche; James, Claudia; Gittelbauer, Kirsten; Gosch, Jutta; Alpers, Birgit

2013-01-01

Peripheral venous catheters are frequently used in hospitalized patients but increase the risk of nosocomial bloodstream infection. Evidence-based guidelines describe specific steps that are known to reduce infection risk. However, the degree of guideline implementation in clinical practice is not known. The aim of this study was to determine the use of specific steps for insertion of peripheral venous catheters in clinical practice and to implement a multimodal intervention aimed at improving both compliance and the optimum order of the steps. The study was conducted at University Hospital Hamburg. An optimum procedure for inserting a peripheral venous catheter was defined based on three evidence-based guidelines (WHO, CDC, RKI) including five steps with 1A or 1B level of evidence: hand disinfection before patient contact, skin antisepsis of the puncture site, no palpation of treated puncture site, hand disinfection before aseptic procedure, and sterile dressing on the puncture site. A research nurse observed and recorded procedures for peripheral venous catheter insertion for healthcare workers in four different departments (endoscopy, central emergency admissions, pediatrics, and dermatology). A multimodal intervention with 5 elements was established (teaching session, dummy training, e-learning tool, tablet and poster, and direct feedback), followed by a second observation period. During the last observation week, participants evaluated the intervention. In the control period, 207 insertions were observed, and 202 in the intervention period. Compliance improved significantly for four of five steps (e.g., from 11.6% to 57.9% for hand disinfection before patient contact; p<0.001, chi-square test). Compliance with skin antisepsis of the puncture site was high before and after intervention (99.5% before and 99.0% after). Performance of specific steps in the correct order also improved (e.g., from 7.7% to 68.6% when three of five steps were done; p<0.001). The intervention was described as helpful by 46.8% of the participants, as neutral by 46.8%, and as disruptive by 6.4%. A multimodal strategy to improve both compliance with safety steps for peripheral venous catheter insertion and performance of an optimum procedure was effective and was regarded helpful by healthcare workers.

Photograph of moon after transearth insertion

NASA Image and Video Library

1969-05-24

AS10-27-3956 (24 May 1969) --- This photograph of the moon was taken after trans-Earth insertion when the Apollo 10 spacecraft was high above the lunar equator near 27 degrees east longitude. North is about 20 degrees left of the top of the photograph. Apollo Landing Site 3 is on the lighted side of the terminator in a dark area just north of the equator. Apollo Landing Site 2 is near the lower left margin of the Sea of Tranquility (Mare Tranquillitatis), which is the large, dark area near the center of the photograph.

A Novel Locomotion-based Validation Assay for Candidate Drugs Using Drosophila DYT1 Disease Model

DTIC Science & Technology

2014-06-01

rescue the locomotion defects of Drosophila larvae caused by the expression of human torsinAΔE. These results demonstrated that human torsinA can... Drosophila dtorsin∆D transgenic lines dtorsin∆E and dtorsin∆D cDNA constructs were made from the wild type dtorsin cDNA using QuikChange II XL Site...After confirming mutated sequences , the insert was again cut out with EcoRI and NotI and inserted between EcoRI and NotI sites of pUAST [2] to produce

The Relationship of Foot Shape and Sensitivity to Comfort of Shoe-Inserts

DTIC Science & Technology

1998-07-30

tension of the plantar fascia have been implicated by several authors as a cause of plan tar fasciitis which results in symptomatic heel pain and...Pressure distribution Relationship Between Foot Sensitivity and Plantar Pressure RESULTS AND DISCUSSION The shoe and insert factors Factor Sl...sensitivity of the plantar surface of the foot). For activities which are typical for army personal the choice of an appropriate shoe is essential

Key diffusion mechanisms involved in regulating bidirectional water permeation across E. coli outer membrane lectin

PubMed Central

Sachdeva, Shivangi; Kolimi, Narendar; Nair, Sanjana Anilkumar; Rathinavelan, Thenmalarchelvi

2016-01-01

Capsular polysaccharides (CPSs) are major bacterial virulent determinants that facilitate host immune evasion. E. coli group1 K30CPS is noncovalently attached to bacterial surface by Wzi, a lectin. Intriguingly, structure based phylogenetic analysis indicates that Wzi falls into porin superfamily. Molecular dynamics (MD) simulations further shed light on dual role of Wzi as it also functions as a bidirectional passive water specific porin. Such a functional role of Wzi was not realized earlier, due to the occluded pore. While five water specific entry points distributed across extracellular & periplasmic faces regulate the water diffusion involving different mechanisms, a luminal hydrophobic plug governs water permeation across the channel. Coincidently, MD observed open state structure of “YQF” triad is seen in sugar-binding site of sodium-galactose cotransporters, implicating its involvement in K30CPS surface anchorage. Importance of Loop 5 (L5) in membrane insertion is yet another highlight. Change in water diffusion pattern of periplasmic substitution mutants suggests Wzi’s role in osmoregulation by aiding in K30CPS hydration, corroborating earlier functional studies. Water molecules located inside β-barrel of Wzi crystal structure further strengthens the role of Wzi in osmoregulation. Thus, interrupting water diffusion or L5 insertion may reduce bacterial virulence. PMID:27320406

Development of genetic tools for in vivo virulence analysis of Streptococcus sanguinis.

PubMed

Turner, Lauren Senty; Das, Sankar; Kanamoto, Taisei; Munro, Cindy L; Kitten, Todd

2009-08-01

Completion of the genome sequence of Streptococcus sanguinis SK36 necessitates tools for further characterization of this species. It is often desirable to insert antibiotic resistance markers and other exogenous genes into the chromosome; therefore, we sought to identify a chromosomal site for ectopic expression of foreign genes, and to verify that insertion into this site did not affect important cellular phenotypes. We designed three plasmid constructs for insertion of erm, aad9 or tetM resistance determinants into a genomic region encoding only a small (65 aa) hypothetical protein. To determine whether this insertion affected important cellular properties, SK36 and its erythromycin-resistant derivative, JFP36, were compared for: (i) growth in vitro, (ii) genetic competence, (iii) biofilm formation and (iv) virulence for endocarditis in the rabbit model of infective endocarditis (IE). The spectinomycin-resistant strain, JFP56, and tetracycline-resistant strain, JFP76, were also tested for virulence in vivo. Insertion of erm did not affect growth, competence or biofilm development of JFP36. Recovery of bacteria from heart valves of co-inoculated rabbits was similar to wild-type for JFP36, JFP56 and JFP76, indicating that IE virulence was not significantly affected. The capacity for mutant complementation in vivo was explored in an avirulent ssaB mutant background. Expression of ssaB from its predicted promoter in the target region restored IE virulence. Thus, the chromosomal site utilized is a good candidate for further manipulations of S. sanguinis. In addition, the resistant strains developed may be further applied as controls to facilitate screening for virulence factors in vivo.

Development of genetic tools for in vivo virulence analysis of Streptococcus sanguinis

PubMed Central

Senty Turner, Lauren; Das, Sankar; Kanamoto, Taisei; Munro, Cindy L.; Kitten, Todd

2009-01-01

Completion of the genome sequence of Streptococcus sanguinis SK36 necessitates tools for further characterization of this species. It is often desirable to insert antibiotic resistance markers and other exogenous genes into the chromosome; therefore, we sought to identify a chromosomal site for ectopic expression of foreign genes, and to verify that insertion into this site did not affect important cellular phenotypes. We designed three plasmid constructs for insertion of erm, aad9 or tetM resistance determinants into a genomic region encoding only a small (65 aa) hypothetical protein. To determine whether this insertion affected important cellular properties, SK36 and its erythromycin-resistant derivative, JFP36, were compared for: (i) growth in vitro, (ii) genetic competence, (iii) biofilm formation and (iv) virulence for endocarditis in the rabbit model of infective endocarditis (IE). The spectinomycin-resistant strain, JFP56, and tetracycline-resistant strain, JFP76, were also tested for virulence in vivo. Insertion of erm did not affect growth, competence or biofilm development of JFP36. Recovery of bacteria from heart valves of co-inoculated rabbits was similar to wild-type for JFP36, JFP56 and JFP76, indicating that IE virulence was not significantly affected. The capacity for mutant complementation in vivo was explored in an avirulent ssaB mutant background. Expression of ssaB from its predicted promoter in the target region restored IE virulence. Thus, the chromosomal site utilized is a good candidate for further manipulations of S. sanguinis. In addition, the resistant strains developed may be further applied as controls to facilitate screening for virulence factors in vivo. PMID:19423626

Local Structural Differences in Homologous Proteins: Specificities in Different SCOP Classes

PubMed Central

Joseph, Agnel Praveen; Valadié, Hélène; Srinivasan, Narayanaswamy; de Brevern, Alexandre G.

2012-01-01

The constant increase in the number of solved protein structures is of great help in understanding the basic principles behind protein folding and evolution. 3-D structural knowledge is valuable in designing and developing methods for comparison, modelling and prediction of protein structures. These approaches for structure analysis can be directly implicated in studying protein function and for drug design. The backbone of a protein structure favours certain local conformations which include α-helices, β-strands and turns. Libraries of limited number of local conformations (Structural Alphabets) were developed in the past to obtain a useful categorization of backbone conformation. Protein Block (PB) is one such Structural Alphabet that gave a reasonable structure approximation of 0.42 Å. In this study, we use PB description of local structures to analyse conformations that are preferred sites for structural variations and insertions, among group of related folds. This knowledge can be utilized in improving tools for structure comparison that work by analysing local structure similarities. Conformational differences between homologous proteins are known to occur often in the regions comprising turns and loops. Interestingly, these differences are found to have specific preferences depending upon the structural classes of proteins. Such class-specific preferences are mainly seen in the all-β class with changes involving short helical conformations and hairpin turns. A test carried out on a benchmark dataset also indicates that the use of knowledge on the class specific variations can improve the performance of a PB based structure comparison approach. The preference for the indel sites also seem to be confined to a few backbone conformations involving β-turns and helix C-caps. These are mainly associated with short loops joining the regular secondary structures that mediate a reversal in the chain direction. Rare β-turns of type I’ and II’ are also identified as preferred sites for insertions. PMID:22745680

Spliceosomal Intron Insertions in Genome Compacted Ray-Finned Fishes as Evident from Phylogeny of MC Receptors, Also Supported by a Few Other GPCRs

PubMed Central

Sinha, Rahul; Goyal, Pankaj; Grapputo, Alessandro

2011-01-01

Background Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution. Methodology/Principal Findings We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes. Conclusions/Significance The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality. PMID:21850219

In and out of the rRNA genes: characterization of Pokey elements in the sequenced Daphnia genome

PubMed Central

2013-01-01

Background Only a few transposable elements are known to exhibit site-specific insertion patterns, including the well-studied R-element retrotransposons that insert into specific sites within the multigene rDNA. The only known rDNA-specific DNA transposon, Pokey (superfamily: piggyBac) is found in the freshwater microcrustacean, Daphnia pulex. Here, we present a genome-wide analysis of Pokey based on the recently completed whole genome sequencing project for D. pulex. Results Phylogenetic analysis of Pokey elements recovered from the genome sequence revealed the presence of four lineages corresponding to two divergent autonomous families and two related lineages of non-autonomous miniature inverted repeat transposable elements (MITEs). The MITEs are also found at the same 28S rRNA gene insertion site as the Pokey elements, and appear to have arisen as deletion derivatives of autonomous elements. Several copies of the full-length Pokey elements may be capable of producing an active transposase. Surprisingly, both families of Pokey possess a series of 200 bp repeats upstream of the transposase that is derived from the rDNA intergenic spacer (IGS). The IGS sequences within the Pokey elements appear to be evolving in concert with the rDNA units. Finally, analysis of the insertion sites of Pokey elements outside of rDNA showed a target preference for sites similar to the specific sequence that is targeted within rDNA. Conclusions Based on the target site preference of Pokey elements and the concerted evolution of a segment of the element with the rDNA unit, we propose an evolutionary path by which the ancestors of Pokey elements have invaded the rDNA niche. We discuss how specificity for the rDNA unit may have evolved and how this specificity has played a role in the long-term survival of these elements in the subgenus Daphnia. PMID:24059783

Topology of the membrane protein LamB by epitope tagging and a comparison with the X-ray model.

PubMed

Newton, S M; Klebba, P E; Michel, V; Hofnung, M; Charbit, A

1996-06-01

We previously developed a genetic approach to study, with a single antibody, the topology of the outer membrane protein LamB, an Escherichia coli porin with specificity towards maltodextrins and a receptor for bacteriophage lambda. Our initial procedure consisted of inserting at random the same reporter epitope (the C3 neutralization epitope from poliovirus) into permissive sites of LamB (i.e., sites which tolerate insertions without deleterious effects on the protein activities or the cell). A specific monoclonal antibody was then used to examine the position of the inserted epitope with respect to the protein and the membrane. In the present work, we set up a site-directed procedure to insert the C3 epitope at new sites in order to distinguish between two-dimensional folding models. This allowed us to identify two new surface loops of LamB and to predict another periplasmic exposed region. The results obtained by random and directed epitope tagging are analyzed in light of the recently published X-ray structure of the LamB protein. Study of 23 hybrid LamB-C3 proteins led to the direct identification of five of the nine external loops (L4, L5, L6, L7, and L9) and led to the prediction of four periplasmic loops (I1, I4, I5, and I8) of LamB. Nine of the hybrid proteins did not lead to topological conclusions, and none led to the wrong predictions or conclusions. The comparison indicates that parts of models based on secondary structure predictions alone are not reliable and points to the importance of experimental data in the establishment of outer membrane protein topological models. The advantages and limitations of genetic foreign epitope insertion for the study of integral membrane proteins are discussed.

Changes in implant stability using different site preparation techniques: twist drills versus piezosurgery. A single-blinded, randomized, controlled clinical trial.

PubMed

Stacchi, Claudio; Vercellotti, Tomaso; Torelli, Lucio; Furlan, Fabio; Di Lenarda, Roberto

2013-04-01

The objective of the present investigation was to longitudinally monitor stability changes of implants inserted using traditional rotary instruments or piezoelectric inserts, and to follow their variations during the first 90 days of healing. A randomized, controlled trial was conducted on 20 patients. Each patient received two identical, adjacent implants in the upper premolar area: the test site was prepared with piezosurgery, and the control site was prepared using twist drills. Resonance frequency analysis measurements were taken by a blinded operator on the day of surgery and after 7, 14, 21, 28, 42, 56, and 90 days. At 90 days, 39 out of 40 implants were osseointegrated (one failure in the control group). Both groups showed an initial decrease in mean implant stability quotient (ISQ) values: a shift in implant stability to increasing ISQ values occurred after 14 days in the test group and after 21 days in the control group. The lowest mean ISQ value was recorded at 14 days for test implants (97.3% of the primary stability) and at 21 days for the control implants (90.8% of the primary stability). ISQ variations with respect to primary stability differed significantly between the two groups during the entire period of observation: from day 14 to day 42, in particular, the differences were extremely significant (p < .0001). All 39 implants were in function successfully at the visit scheduled 1 year after insertion. The findings from this study suggest that ultrasonic implant site preparation results in a limited decrease of ISQ values and in an earlier shifting from a decreasing to an increasing stability pattern, when compared with the traditional drilling technique. From a clinical point of view, implants inserted with the piezoelectric technique demonstrated a short-term clinical success similar to those inserted using twist drills. © 2011 Wiley Periodicals, Inc.

Primary implant stability in augmented sinuslift-sites after completed bone regeneration: a randomized controlled clinical study comparing four subantrally inserted biomaterials

PubMed Central

Troedhan, Angelo; Schlichting, Izabela; Kurrek, Andreas; Wainwright, Marcel

2014-01-01

Implant-Insertion-Torque-Value (ITV) proved to be a significant clinical parameter to predict long term implant success-rates and to decide upon immediate loading. The study evaluated ITVs, when four different and commonly used biomaterials were used in sinuslift-procedures compared to natural subantral bone in two-stage-implant-procedures. The tHUCSL-INTRALIFT-method was chosen for sinuslifting in 155 sinuslift-sites for its minimal invasive transcrestal approach and scalable augmentation volume. Four different biomaterials were inserted randomly (easy-graft CRYSTAL n = 38, easy-graft CLASSIC n = 41, NanoBone n = 42, BioOss n = 34), 2 ccm in each case. After a mean healing period of 8,92 months uniform tapered screw Q2-implants were inserted and Drill-Torque-Values (DTV) and ITV were recorded and compared to a group of 36 subantral sites without need of sinuslifting. DTV/ITV were processed for statistics by ANOVA-tests. Mean DTV/ITV obtained in Ncm were: Control Group 10,2/22,2, Bio-Oss 12,7/26,2, NanoBone 17,5/33,3, easy-graft CLASSIC 20,3/45,9, easy-graft CRYSTAL 23,8/56,6 Ncm, significance-level of differences throughout p < 0,05. Within the limits of this study the results suggest self-hardening solid-block-like bone-graft-materials to achieve significantly better DTV/ITV than loose granulate biomaterials for its suspected improvement of vascularization and mineralization of the subantral scaffold by full immobilization of the augmentation site towards pressure changes in the human sinus at normal breathing. PMID:25073446

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

PubMed

Christensen, Shawn M; Ye, Junqiang; Eickbush, Thomas H

2006-11-21

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

Survey of the enthesopathy of X-linked hypophosphatemia and its characterization in Hyp mice.

PubMed

Liang, Guoying; Katz, Lee D; Insogna, Karl L; Carpenter, Thomas O; Macica, Carolyn M

2009-09-01

X-linked hypophosphatemia (XLH) is characterized by rickets and osteomalacia as a result of an inactivating mutation of the PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) gene. PHEX encodes an endopeptidase that, when inactivated, results in elevated circulating levels of FGF-23, a novel phosphate-regulating hormone (a phosphatonin), thereby resulting in increased phosphate excretion and impaired bone mineralization. A generalized and severe mineralizing enthesopathy in patients with XLH was first reported in 1985; we likewise report a survey in which we found evidence of enthesopathy in fibrocartilaginous insertion sites, as well as osteophyte formation, in the majority of patients. Nonetheless, there has been very little focus on the progression and pathogenesis underlying the paradoxical heterotopic calcification of tendon and ligament insertion sites. Such studies have been hampered by lack of a model of mineralizing enthesopathy. We therefore characterized the involvement of the most frequently targeted fibrocartilaginous tendon insertion sites in Hyp mice, a murine model of the XLH mutation that phenocopies the human syndrome in every detail including hypophosphatemia and elevated FGF-23. Histological examination of the affected entheses revealed that mineralizing insertion sites, while thought to involve bone spur formation, were not due to bone-forming osteoblasts but instead to a significant expansion of mineralizing fibrocartilage. Our finding that enthesis fibrocartilage cells specifically express fibroblast growth factor receptor 3 (FGFR3)/Klotho suggests that the high circulating levels of FGF-23, characteristic of XLH and Hyp mice, may be part of the biochemical milieu that underlies the expansion of mineralizing enthesis fibrocartilage.

Primary implant stability in augmented sinuslift-sites after completed bone regeneration: a randomized controlled clinical study comparing four subantrally inserted biomaterials.

PubMed

Troedhan, Angelo; Schlichting, Izabela; Kurrek, Andreas; Wainwright, Marcel

2014-07-30

Implant-Insertion-Torque-Value (ITV) proved to be a significant clinical parameter to predict long term implant success-rates and to decide upon immediate loading. The study evaluated ITVs, when four different and commonly used biomaterials were used in sinuslift-procedures compared to natural subantral bone in two-stage-implant-procedures. The tHUCSL-INTRALIFT-method was chosen for sinuslifting in 155 sinuslift-sites for its minimal invasive transcrestal approach and scalable augmentation volume. Four different biomaterials were inserted randomly (easy-graft CRYSTAL n = 38, easy-graft CLASSIC n = 41, NanoBone n = 42, BioOss n = 34), 2 ccm in each case. After a mean healing period of 8,92 months uniform tapered screw Q2-implants were inserted and Drill-Torque-Values (DTV) and ITV were recorded and compared to a group of 36 subantral sites without need of sinuslifting. DTV/ITV were processed for statistics by ANOVA-tests. Mean DTV/ITV obtained in Ncm were: Control Group 10,2/22,2, Bio-Oss 12,7/26,2, NanoBone 17,5/33,3, easy-graft CLASSIC 20,3/45,9, easy-graft CRYSTAL 23,8/56,6 Ncm, significance-level of differences throughout p < 0,05. Within the limits of this study the results suggest self-hardening solid-block-like bone-graft-materials to achieve significantly better DTV/ITV than loose granulate biomaterials for its suspected improvement of vascularization and mineralization of the subantral scaffold by full immobilization of the augmentation site towards pressure changes in the human sinus at normal breathing.

Retrovirus Integration Database (RID): a public database for retroviral insertion sites into host genomes.

PubMed

Shao, Wei; Shan, Jigui; Kearney, Mary F; Wu, Xiaolin; Maldarelli, Frank; Mellors, John W; Luke, Brian; Coffin, John M; Hughes, Stephen H

2016-07-04

The NCI Retrovirus Integration Database is a MySql-based relational database created for storing and retrieving comprehensive information about retroviral integration sites, primarily, but not exclusively, HIV-1. The database is accessible to the public for submission or extraction of data originating from experiments aimed at collecting information related to retroviral integration sites including: the site of integration into the host genome, the virus family and subtype, the origin of the sample, gene exons/introns associated with integration, and proviral orientation. Information about the references from which the data were collected is also stored in the database. Tools are built into the website that can be used to map the integration sites to UCSC genome browser, to plot the integration site patterns on a chromosome, and to display provirus LTRs in their inserted genome sequence. The website is robust, user friendly, and allows users to query the database and analyze the data dynamically. https://rid.ncifcrf.gov ; or http://home.ncifcrf.gov/hivdrp/resources.htm .

The Tgm9-induced indexed insertional mutant collection to conduct community-based reverse genetics studies in soybean

USDA-ARS?s Scientific Manuscript database

Until now, functional analyses of soybean genes have been very arduous because of the lack of a rapid transformation procedure. Recently identified the active endogenous type II transposable element, Tgm9, excises from insertion sites and restores wild-type phenotypes. Thus, this element provides a ...

Transposon Insertions of magellan-4 That Impair Social Gliding Motility in Myxococcus xanthus

PubMed Central

Youderian, Philip; Hartzell, Patricia L.

2006-01-01

Myxococcus xanthus has two different mechanisms of motility, adventurous (A) motility, which permits individual cells to glide over solid surfaces, and social (S) motility, which permits groups of cells to glide. To identify the genes involved in S-gliding motility, we mutagenized a ΔaglU (A−) strain with the defective transposon, magellan-4, and screened for S− mutants that form nonmotile colonies. Sequence analysis of the sites of the magellan-4 insertions in these mutants and the alignment of these sites with the M. xanthus genome sequence show that two-thirds of these insertions lie within 27 of the 37 nonessential genes known to be required for social motility, including those necessary for the biogenesis of type IV pili, exopolysaccharide, and lipopolysaccharide. The remaining insertions also identify 31 new, nonessential genes predicted to encode both structural and regulatory determinants of S motility. These include three tetratricopeptide repeat proteins, several regulators of transcription that may control the expression of genes involved in pilus extension and retraction, and additional enzymes involved in polysaccharide metabolism. Three insertions that abolish S motility lie within genes predicted to encode glycolytic enzymes, suggesting that the signal for pilus retraction may be a simple product of exopolysaccharide catabolism. PMID:16299386

Construction of Signature-tagged Mutant Library in Mesorhizobium loti as a Powerful Tool for Functional Genomics

PubMed Central

Shimoda, Yoshikazu; Mitsui, Hisayuki; Kamimatsuse, Hiroko; Minamisawa, Kiwamu; Nishiyama, Eri; Ohtsubo, Yoshiyuki; Nagata, Yuji; Tsuda, Masataka; Shinpo, Sayaka; Watanabe, Akiko; Kohara, Mitsuyo; Yamada, Manabu; Nakamura, Yasukazu; Tabata, Satoshi; Sato, Shusei

2008-01-01

Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology. PMID:18658183

MMTV insertional mutagenesis identifies genes, gene families and pathways involved in mammary cancer.

PubMed

Theodorou, Vassiliki; Kimm, Melanie A; Boer, Mandy; Wessels, Lodewyk; Theelen, Wendy; Jonkers, Jos; Hilkens, John

2007-06-01

We performed a high-throughput retroviral insertional mutagenesis screen in mouse mammary tumor virus (MMTV)-induced mammary tumors and identified 33 common insertion sites, of which 17 genes were previously not known to be associated with mammary cancer and 13 had not previously been linked to cancer in general. Although members of the Wnt and fibroblast growth factors (Fgf) families were frequently tagged, our exhaustive screening for MMTV insertion sites uncovered a new repertoire of candidate breast cancer oncogenes. We validated one of these genes, Rspo3, as an oncogene by overexpression in a p53-deficient mammary epithelial cell line. The human orthologs of the candidate oncogenes were frequently deregulated in human breast cancers and associated with several tumor parameters. Computational analysis of all MMTV-tagged genes uncovered specific gene families not previously associated with cancer and showed a significant overrepresentation of protein domains and signaling pathways mainly associated with development and growth factor signaling. Comparison of all tagged genes in MMTV and Moloney murine leukemia virus-induced malignancies showed that both viruses target mostly different genes that act predominantly in distinct pathways.

Cross-sectional evaluation of the prevalence and factors associated with soft tissue scarring after the removal of miniscrews.

PubMed

Jung, Sung-ah; Choi, Yoon Jeong; Lee, Dong-Won; Kim, Kyung-Ho; Chung, Chooryung J

2015-05-01

To investigate the prevalence of distinguishable soft tissue scarring after the removal of temporary anchorage devices (TADs) such as orthodontic miniscrews and to analyze the factors associated with scar formation. The prevalence of soft tissue scarring in 66 patients (202 miniscrew removal sites) was clinically investigated at least 1 year after miniscrew removal. To determine the clinical factors associated with soft tissue scar formation, miniscrew stability; host factors including age, gender, and gingival biotype; and miniscrew-related factors such as insertion site, vertical position, and insertion period were evaluated. The prevalence of a distinguishable scar remaining at least 1 year after miniscrew removal was 44.6%. Patients with flat gingiva showed a significantly higher prevalence of soft tissue scar formation than did those with pronounced scalloped gingiva (P < .05). Maxillary buccal removal sites showed a significantly higher prevalence of soft tissue scar formation than did those in the mandible or palatal slope (P < .05). Miniscrew sites at the alveolar mucosa showed a significantly lower prevalence of soft tissue scar formation than did those in the mucogingival junction or the attached gingiva (P < .01). The prevalence of distinguishable scarring after miniscrew removal was fairly high. On the basis of our results, patients with flat gingiva and buccal interdental gingival insertion sites are more susceptible to scar formation.

Arginine insertion and loss of N-linked glycosylation site in HIV-1 envelope V3 region confer CXCR4-tropism

PubMed Central

Tsuchiya, Kiyoto; Ode, Hirotaka; Hayashida, Tsunefusa; Kakizawa, Junko; Sato, Hironori; Oka, Shinichi; Gatanaga, Hiroyuki

2013-01-01

The third variable region (V3) of HIV-1 envelope glycoprotein gp120 plays a key role in determination of viral coreceptor usage (tropism). However, which combinations of mutations in V3 confer a tropism shift is still unclear. A unique pattern of mutations in antiretroviral therapy-naive HIV-1 patient was observed associated with the HIV-1 tropism shift CCR5 to CXCR4. The insertion of arginine at position 11 and the loss of the N-linked glycosylation site were indispensable for acquiring pure CXCR4-tropism, which were confirmed by cell-cell fusion assay and phenotype analysis of recombinant HIV-1 variants. The same pattern of mutations in V3 and the associated tropism shift were identified in two of 53 other patients (3.8%) with CD4+ cell count <200/mm3. The combination of arginine insertion and loss of N-linked glycosylation site usually confers CXCR4-tropism. Awareness of this rule will help to confirm the tropism prediction from V3 sequences by conventional rules. PMID:23925152

Human lamina cribrosa insertion and age.

PubMed

Sigal, Ian A; Flanagan, John G; Lathrop, Kira L; Tertinegg, Inka; Bilonick, Richard

2012-10-03

To test the hypothesis that in healthy human eyes the lamina cribrosa (LC) insertion into the pia mater increases with age. The optic nerve heads (ONHs) of donor eyes fixed at either 5 or 50 mm Hg of IOP were sectioned, stained, and imaged under bright- and dark-field conditions. A 3-dimensional (3D) model of each ONH was reconstructed. From the 3D models we measured the area of LC insertion into the peripapillary scleral flange and into the pia, and computed the total area of insertion and fraction of LC inserting into the pia. Linear mixed effect models were used to determine if the measurements were associated with age or IOP. We analyzed 21 eyes from 11 individuals between 47 and 91 years old. The LC inserted into the pia in all eyes. The fraction of LC inserting into the pia (2.2%-29.6%) had a significant decrease with age (P = 0.049), which resulted from a nonsignificant increase in the total area of LC insertion (P = 0.41) and a nonsignificant decrease in the area of LC insertion into the pia (P = 0.55). None of the measures was associated with fixation IOP (P values 0.44-0.81). Differences between fellow eyes were smaller than differences between unrelated eyes. The LC insertion into the pia mater is common in middle-aged and older eyes, and does not increase with age. The biomechanical and vascular implications of the LC insertion into the pia mater are not well understood and should be investigated further.

Human Lamina Cribrosa Insertion and Age

PubMed Central

Sigal, Ian A.; Flanagan, John G.; Lathrop, Kira L.; Tertinegg, Inka; Bilonick, Richard

2012-01-01

Purpose. To test the hypothesis that in healthy human eyes the lamina cribrosa (LC) insertion into the pia mater increases with age. Methods. The optic nerve heads (ONHs) of donor eyes fixed at either 5 or 50 mm Hg of IOP were sectioned, stained, and imaged under bright- and dark-field conditions. A 3-dimensional (3D) model of each ONH was reconstructed. From the 3D models we measured the area of LC insertion into the peripapillary scleral flange and into the pia, and computed the total area of insertion and fraction of LC inserting into the pia. Linear mixed effect models were used to determine if the measurements were associated with age or IOP. Results. We analyzed 21 eyes from 11 individuals between 47 and 91 years old. The LC inserted into the pia in all eyes. The fraction of LC inserting into the pia (2.2%–29.6%) had a significant decrease with age (P = 0.049), which resulted from a nonsignificant increase in the total area of LC insertion (P = 0.41) and a nonsignificant decrease in the area of LC insertion into the pia (P = 0.55). None of the measures was associated with fixation IOP (P values 0.44–0.81). Differences between fellow eyes were smaller than differences between unrelated eyes. Conclusions. The LC insertion into the pia mater is common in middle-aged and older eyes, and does not increase with age. The biomechanical and vascular implications of the LC insertion into the pia mater are not well understood and should be investigated further. PMID:22956611

Heat generation during implant placement in low-density bone: effect of surgical technique, insertion torque and implant macro design.

PubMed

Marković, Aleksa; Mišić, Tijana; Miličić, Biljana; Calvo-Guirado, Jose Luis; Aleksić, Zoran; Ðinić, Ana

2013-07-01

The study aimed to investigate the effect of surgical technique, implant macrodesign and insertion torque on bone temperature changes during implant placement. In the in vitro study, 144 self-tapping (blueSKY(®) 4 × 10 mm; Bredent) and 144 non-self-tapping (Standard implant(®) 4.1 × 10 mm; Straumann) were placed in osteotomies prepared in pig ribs by lateral bone condensing or bone drilling techniques. The maximum insertion torque values of 30, 35 and 40 Ncm were used. Real-time bone temperature measurement during implant placement was performed by three thermocouples positioned vertically, in tripod configuration around every osteotomy, at a distance of 5 mm from it and at depths of 1, 5 and 10 mm. Data were analysed using Kruskal-Wallis, Mann-Whitney U-tests and Regression analysis. Significant predictor of bone temperature at the osteotomy depth of 1 mm was insertion torque (P = 0.003) and at the depth of 10-mm implant macrodesign (P = 0.029), while no significant predictor at depth of 5 mm was identified (P > 0.05). Higher insertion torque values as well as non-self-tapping implant macrodesign were related to higher temperatures. Implant placement in sites prepared by bone drilling induced significantly higher temperature increase (P = 0.021) compared with bone condensing sites at the depth of 5 mm, while no significant difference was recorded at other depths. Compared with 30 Ncm, insertion torque values of 35 and 40 Ncm produced significantly higher temperature increase (P = 0.005; P = 0.003, respectively) at the depth of 1 mm. There was no significant difference in temperature change induced by 35 and 40 Ncm, neither by implant macrodesign at all investigated depths (P > 0.05). Placement of self-tapping implants with low insertion torque into sites prepared by lateral bone condensing technique might be advantageous in terms of thermal effect on bone. © 2012 John Wiley & Sons A/S.

Solution structure of a DNA decamer duplex containing the stable 3′ T⋅G base pair of the pyrimidine(6–4)pyrimidone photoproduct [(6–4) adduct]: Implications for the highly specific 3′ T → C transition of the (6–4) adduct

PubMed Central

Lee, Joon-Hwa; Hwang, Geum-Sook; Choi, Byong-Seok

1999-01-01

The pyrimidine(6–4)pyrimidone photoproduct [(6–4) adduct] is one of the major photoproducts induced by UV irradiation of DNA and occurs at TpT sites. The (6–4) adduct is highly mutagenic and leads most often to a 3′ T → C transition with 85% replicating error frequency [LeClerc, J. E., Borden, A. & Lawrence, C. W. (1991) Proc. Natl. Acad. Sci. USA 88, 9685–9689]. To determine the origin of the specific 3′ T → C transition of the (6–4) adduct, we have used experimental NMR restraints and molecular dynamics to determine the solution structure of a (6–4)-lesion DNA decamer duplex that contains a mismatched base pair between the 3′ T residue and an opposed G residue. Normal Watson–Crick-type hydrogen bonding is retained at the 5′ T of the lesion site. The O2 carbonyl of the 3′ T residue forms hydrogen bonds with the imino and amino protons of the opposed G residue. This potential hydrogen bonding stabilizes the overall helix and restores the highly distorted conformation of the (6–4) adduct to the typical B-form-like DNA structure. This structural feature can explain the marked preference for the insertion of an A residue opposite the 5′ T and a G residue opposite the 3′ T of the (6–4) lesion during trans-lesion synthesis. Thus these insertions yield the predominant 3′ T → C transition. PMID:10359763

Rupture of the anterior tibial tendon: three clinical cases, anatomical study, and literature review.

PubMed

Anagnostakos, Konstantinos; Bachelier, Felix; Fürst, Oliver Alexander; Kelm, Jens

2006-05-01

We report three cases of anterior tibial tendon ruptures and the results of an anatomical study in regard to the tendon's insertion site and a literature review. Three patients were referred to our hospital with anterior tibial tendon ruptures. In the anatomical study, 53 feet were dissected, looking in particular for variants of the bony insertion of the tendon. Two patients had surgical treatment (one primary repair and one semimembranosus tendon graft) and one conservative treatment. After a mean followup of 14 weeks all patients had satisfactory outcomes. In the anatomical study, we noted three different insertion sites: in 36 feet the tendon inserted into the medial side of the cuneiform and the base of the first metatarsal bone and in 13 feet only into the medial side of the cuneiform bone. In the remaining four feet the tendon inserted into the cuneiform and the first metatarsal bone, but an additional tendon was noted taking its origin from the anterior tibial tendon near its insertion into the medial cuneiform and attaching to the proximal part of the first metatarsal. According to literature, surgical repair is the treatment of choice for acute ruptures and for patients with high activity levels. For chronic ruptures and patients with low demands, conservative management may lead to an equally good outcome. Knowledge of the anatomy in this region may be helpful for diagnosis and for the interpretation of intraoperative findings and choosing the most appropriate surgical procedure.

Correlation of Fractal Dimension Values with Implant Insertion Torque and Resonance Frequency Values at Implant Recipient Sites.

PubMed

Suer, Berkay Tolga; Yaman, Zekai; Buyuksarac, Bora

2016-01-01

Fractal analysis is a mathematical method used to describe the internal architecture of complex structures such as trabecular bone. Fractal analysis of panoramic radiographs of implant recipient sites could help to predict the quality of the bone prior to implant placement. This study investigated the correlations between the fractal dimension values obtained from panoramic radiographs and the insertion torque and resonance frequency values of mandibular implants. Thirty patients who received a total of 55 implants of the same brand, diameter, and length in the mandibular premolar and molar regions were included in the study. The same surgical procedures were applied to each patient, and the insertion torque and resonance frequency values were recorded for each implant at the time of placement. The radiographic fractal dimensions of the alveolar bone in the implant recipient area were calculated from preoperative panoramic radiographs using a box-counting algorithm. The insertion torque and resonance frequency values were compared with the fractal dimension values using the Spearman test. All implants were successful, and none were lost during the follow-up period. Linear correlations were observed between the fractal dimension and resonance frequency, between the fractal dimension and insertion torque, and between resonance frequency and insertion torque. These results suggest that the noninvasive measurement of the fractal dimension from panoramic radiographs might help to predict the bone quality, and thus the primary stability of dental implants, before implant surgery.

Formation of cis-diamminedichloroplatinum(II) 1,2-intrastrand cross-links on DNA is flanking-sequence independent.

PubMed

Burstyn, J N; Heiger-Bernays, W J; Cohen, S M; Lippard, S J

2000-11-01

Mapping of cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) DNA adducts over >3000 nucleotides was carried out using a replication blockage assay. The sites of inhibition of modified T4 DNA polymerase, also referred to as stop sites, were analyzed to determine the effects of local sequence context on the distribution of intrastrand cisplatin cross-links. In a 3120 base fragment from replicative form M13mp18 DNA containing 24.6% guanine, 25.5% thymine, 26.9% adenine and 23.0% cytosine, 166 individual stop sites were observed at a bound platinum/nucleotide ratio of 1-2 per thousand. The majority of stop sites (90%) occurred at G(n>2) sequences and the remainder were located at sites containing an AG dinucleotide. For all of the GG sites present in the mapped sequences, including those with Gn(>)2, 89% blocked replication, whereas for the AG sites only 17% blocked replication. These blockage sites were independent of flanking nucleotides in a sequence of N(1)G*G*N(2) where N(1), N(2) = A, C, G, T and G*G* indicates a 1,2-intrastrand platinum cross-link. The absence of long-range sequence dependence was confirmed by monitoring the reaction of cisplatin with a plasmid containing an 800 bp insert of the human telomere repeat sequence (TTAGGG)(n). Platination reactions monitored at several formal platinum/nucleotide ratios or as a function of time reveal that the telomere insert was not preferentially damaged by cisplatin. Both replication blockage and telomere-insert plasmid platination experiments indicate that cisplatin 1,2-intrastrand adducts do not form preferentially at G-rich sequences in vitro.

Comparison of Ultra-Conserved Elements in Drosophilids and Vertebrates

PubMed Central

Makunin, Igor V.; Shloma, Viktor V.; Stephen, Stuart J.; Pheasant, Michael; Belyakin, Stepan N.

2013-01-01

Metazoan genomes contain many ultra-conserved elements (UCEs), long sequences identical between distant species. In this study we identified UCEs in drosophilid and vertebrate species with a similar level of phylogenetic divergence measured at protein-coding regions, and demonstrated that both the length and number of UCEs are larger in vertebrates. The proportion of non-exonic UCEs declines in distant drosophilids whilst an opposite trend was observed in vertebrates. We generated a set of 2,126 Sophophora UCEs by merging elements identified in several drosophila species and compared these to the eutherian UCEs identified in placental mammals. In contrast to vertebrates, the Sophophora UCEs are depleted around transcription start sites. Analysis of 52,954 P-element, piggyBac and Minos insertions in the D. melanogaster genome revealed depletion of the P-element and piggyBac insertions in and around the Sophophora UCEs. We examined eleven fly strains with transposon insertions into the intergenic UCEs and identified associated phenotypes in five strains. Four insertions behave as recessive lethals, and in one case we observed a suppression of the marker gene within the transgene, presumably by silenced chromatin around the integration site. To confirm the lethality is caused by integration of transposons we performed a phenotype rescue experiment for two stocks and demonstrated that the excision of the transposons from the intergenic UCEs restores viability. Sequencing of DNA after the transposon excision in one fly strain with the restored viability revealed a 47 bp insertion at the original transposon integration site suggesting that the nature of the mutation is important for the appearance of the phenotype. Our results suggest that the UCEs in flies and vertebrates have both common and distinct features, and demonstrate that a significant proportion of intergenic drosophila UCEs are sensitive to disruption. PMID:24349264

The Influence of Primary and Secondary DNA Structure in Deletion and Duplication between Direct Repeats in Escherichia Coli

PubMed Central

Trinh, T. Q.; Sinden, R. R.

1993-01-01

We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events. PMID:8325478

Read count-based method for high-throughput allelic genotyping of transposable elements and structural variants.

PubMed

Kuhn, Alexandre; Ong, Yao Min; Quake, Stephen R; Burkholder, William F

2015-07-08

Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed. We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate. This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

48 CFR 970.5223-3 - Agreement regarding Workplace Substance Abuse Programs at DOE sites.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Workplace Substance Abuse Programs at DOE sites. 970.5223-3 Section 970.5223-3 Federal Acquisition... Agreement regarding Workplace Substance Abuse Programs at DOE sites. As prescribed in 970.2305-4(a), the contracting officer shall insert the following provision: Agreement Regarding Workplace Substance Abuse...

48 CFR 970.5223-3 - Agreement regarding Workplace Substance Abuse Programs at DOE sites.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Workplace Substance Abuse Programs at DOE sites. 970.5223-3 Section 970.5223-3 Federal Acquisition... Agreement regarding Workplace Substance Abuse Programs at DOE sites. As prescribed in 970.2305-4(a), the contracting officer shall insert the following provision: Agreement Regarding Workplace Substance Abuse...

48 CFR 970.5223-3 - Agreement regarding Workplace Substance Abuse Programs at DOE sites.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Workplace Substance Abuse Programs at DOE sites. 970.5223-3 Section 970.5223-3 Federal Acquisition... Agreement regarding Workplace Substance Abuse Programs at DOE sites. As prescribed in 970.2305-4(a), the contracting officer shall insert the following provision: Agreement Regarding Workplace Substance Abuse...

48 CFR 970.5223-3 - Agreement regarding Workplace Substance Abuse Programs at DOE sites.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Workplace Substance Abuse Programs at DOE sites. 970.5223-3 Section 970.5223-3 Federal Acquisition... Agreement regarding Workplace Substance Abuse Programs at DOE sites. As prescribed in 970.2305-4(a), the contracting officer shall insert the following provision: Agreement Regarding Workplace Substance Abuse...

48 CFR 970.5223-3 - Agreement regarding Workplace Substance Abuse Programs at DOE sites.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Workplace Substance Abuse Programs at DOE sites. 970.5223-3 Section 970.5223-3 Federal Acquisition... Agreement regarding Workplace Substance Abuse Programs at DOE sites. As prescribed in 970.2305-4(a), the contracting officer shall insert the following provision: Agreement Regarding Workplace Substance Abuse...

Gene and enhancer trap tagging of vascular-expressed genes in poplar trees

Treesearch

Andrew Groover; Joseph R. Fontana; Gayle Dupper; Caiping Ma; Robert Martienssen; Steven Strauss; Richard Meilan

2004-01-01

We report a gene discovery system for poplar trees based on gene and enhancer traps. Gene and enhancer trap vectors carrying the β-glucuronidase (GUS) reporter gene were inserted into the poplar genome via Agrobacterium tumefaciens transformation, where they reveal the expression pattern of genes at or near the insertion sites. Because GUS...

Methods and compositions for controlling gene expression by RNA processing

DOEpatents

Doudna, Jennifer A.; Qi, Lei S.; Haurwitz, Rachel E.; Arkin, Adam P.

2017-08-29

The present disclosure provides nucleic acids encoding an RNA recognition sequence positioned proximal to an insertion site for the insertion of a sequence of interest; and host cells genetically modified with the nucleic acids. The present disclosure also provides methods of modifying the activity of a target RNA, and kits and compositions for carrying out the methods.

Anatomical Footprint of the Tibialis Anterior Tendon: Surgical Implications for Foot and Ankle Reconstructions.

PubMed

Willegger, Madeleine; Seyidova, Nargiz; Schuh, Reinhard; Windhager, Reinhard; Hirtler, Lena

2017-01-01

This study aimed to analyze precisely the dimensions, shapes, and variations of the insertional footprints of the tibialis anterior tendon (TAT) at the medial cuneiform (MC) and first metatarsal (MT1) base. Forty-one formalin-fixed human cadaveric specimens were dissected. After preparation of the TAT footprint, standardized photographs were made and the following parameters were evaluated: the footprint length, width, area of insertion, dorsoplantar location, shape, and additional tendon slips. Twenty feet (48.8%) showed an equal insertion at the MC and MT1, another 20 feet (48.8%) had a wide insertion at the MC and a narrow insertion at the MT1, and 1 foot (2.4%) demonstrated a narrow insertion at the MC and a wide insertion at the MT1. Additional tendon slips inserting at the metatarsal shaft were found in two feet (4.8%). Regarding the dorsoplantar orientation, the footprints were located medial in 29 feet (70.7%) and medioplantar in 12 feet (29.3%). The most common shape at the MT1 base was the crescent type (75.6%) and the oval type at the MC (58.5%). The present study provided more detailed data on the dimensions and morphologic types of the tibialis anterior tendon footprint. The established anatomical data may allow for a safer surgical preparation and a more anatomical reconstruction.

Treatment and disease management of multiple sclerosis patients: A review for nurse practitioners.

PubMed

Roman, Cortnee; Menning, Kara

2017-10-01

This review discusses the role of the nurse practitioner (NP) in evaluating the clinical effects, potential side effects, and monitoring requirements for treatment options in multiple sclerosis (MS) and provides guidance on how to help patients understand these issues. A literature search was conducted on PubMed to identify publications on monitoring and disease management of MS patients. Additional resources included drug information web sites and package inserts. NPs play an active role in the management of MS patients via effective monitoring and communication throughout the patient's treatment regimen and disease course. In the shared decision-making model of MS treatment, NPs ensure that patients understand the implications of their disease-modifying therapies (DMTs). As patients move through treatments during the course of their disease, the importance of this role increases, and it is critical that NPs follow the guidelines in each medication's product label and take into account any potential lingering effects of prior medications. It is critical for NPs to promote patient adherence, to ensure that patients understand treatment side effects and monitoring requirements, and to take sequencing and reversibility implications of DMTs into account when making clinical decisions. ©2017 American Association of Nurse Practitioners.

Arthroscopic fixation of the clavicle shaft fracture.

PubMed

Kim, Yang-Soo; Lee, Hyo-Jin; Kim, Jong-Ick; Yang, Hyo; Jin, Hong-Ki; Patel, Hiren Kirtibhai; Kim, Jong-Ho; Park, In

2017-01-01

This article describes an arthroscopic technique for the fixation of clavicle shaft fractures. A viewing portal is made 2 cm anterior to the fracture site, and a working portal is made 2 cm lateral to the fracture site. The guide wire for a 4.0-mm cannulated screw is inserted through the fracture site to the medial fracture fragment under arthroscopic guidance. Through the medial fragment, the guide wire is delivered through the skin anteriorly. The fracture is reduced, and then, the guide wire is drilled back across the fracture site to the lateral fracture fragment. After confirming the reduction under arthroscopy, the appropriately sized cannulated screw is inserted after reaming. This arthroscopic technique would be useful for the precise reduction and minimal invasive fixation of clavicle shaft fractures. Preliminary results are encouraging, and further studies with long-term follow-up are needed to determine the precise indications and limitations of the procedure.

Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramirez, Jean Marie; Houzet, Laurent; Koller, Richard

2004-12-20

Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associatedmore » with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.« less

Evidence for a Retroviral Insertion in TRPM1 as the Cause of Congenital Stationary Night Blindness and Leopard Complex Spotting in the Horse

PubMed Central

Bellone, Rebecca R.; Holl, Heather; Setaluri, Vijayasaradhi; Devi, Sulochana; Maddodi, Nityanand; Archer, Sheila; Sandmeyer, Lynne; Ludwig, Arne; Foerster, Daniel; Pruvost, Melanie; Reissmann, Monika; Bortfeldt, Ralf; Adelson, David L.; Lim, Sim Lin; Nelson, Janelle; Haase, Bianca; Engensteiner, Martina; Leeb, Tosso; Forsyth, George; Mienaltowski, Michael J.; Mahadevan, Padmanabhan; Hofreiter, Michael; Paijmans, Johanna L. A.; Gonzalez-Fortes, Gloria; Grahn, Bruce; Brooks, Samantha A.

2013-01-01

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ2=1022.00, p<<0.0005), and CSNB, testing 43 horses (χ2=43, p<<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder. PMID:24167615

Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse.

PubMed

Bellone, Rebecca R; Holl, Heather; Setaluri, Vijayasaradhi; Devi, Sulochana; Maddodi, Nityanand; Archer, Sheila; Sandmeyer, Lynne; Ludwig, Arne; Foerster, Daniel; Pruvost, Melanie; Reissmann, Monika; Bortfeldt, Ralf; Adelson, David L; Lim, Sim Lin; Nelson, Janelle; Haase, Bianca; Engensteiner, Martina; Leeb, Tosso; Forsyth, George; Mienaltowski, Michael J; Mahadevan, Padmanabhan; Hofreiter, Michael; Paijmans, Johanna L A; Gonzalez-Fortes, Gloria; Grahn, Bruce; Brooks, Samantha A

2013-01-01

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ(2)=1022.00, p<0.0005), and CSNB, testing 43 horses (χ(2)=43, p<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.

Risk Management of New Microelectronics for NASA: Radiation Knowledge-base

NASA Technical Reports Server (NTRS)

LaBel, Kenneth A.

2004-01-01

Contents include the following: NASA Missions - implications to reliability and radiation constraints. Approach to Insertion of New Technologies Technology Knowledge-base development. Technology model/tool development and validation. Summary comments.

Improving patient safety during insertion of peripheral venous catheters: an observational intervention study

PubMed Central

Kampf, Günter; Reise, Gesche; James, Claudia; Gittelbauer, Kirsten; Gosch, Jutta; Alpers, Birgit

2013-01-01

Background: Peripheral venous catheters are frequently used in hospitalized patients but increase the risk of nosocomial bloodstream infection. Evidence-based guidelines describe specific steps that are known to reduce infection risk. However, the degree of guideline implementation in clinical practice is not known. The aim of this study was to determine the use of specific steps for insertion of peripheral venous catheters in clinical practice and to implement a multimodal intervention aimed at improving both compliance and the optimum order of the steps. Methods: The study was conducted at University Hospital Hamburg. An optimum procedure for inserting a peripheral venous catheter was defined based on three evidence-based guidelines (WHO, CDC, RKI) including five steps with 1A or 1B level of evidence: hand disinfection before patient contact, skin antisepsis of the puncture site, no palpation of treated puncture site, hand disinfection before aseptic procedure, and sterile dressing on the puncture site. A research nurse observed and recorded procedures for peripheral venous catheter insertion for healthcare workers in four different departments (endoscopy, central emergency admissions, pediatrics, and dermatology). A multimodal intervention with 5 elements was established (teaching session, dummy training, e-learning tool, tablet and poster, and direct feedback), followed by a second observation period. During the last observation week, participants evaluated the intervention. Results: In the control period, 207 insertions were observed, and 202 in the intervention period. Compliance improved significantly for four of five steps (e.g., from 11.6% to 57.9% for hand disinfection before patient contact; p<0.001, chi-square test). Compliance with skin antisepsis of the puncture site was high before and after intervention (99.5% before and 99.0% after). Performance of specific steps in the correct order also improved (e.g., from 7.7% to 68.6% when three of five steps were done; p<0.001). The intervention was described as helpful by 46.8% of the participants, as neutral by 46.8%, and as disruptive by 6.4%. Conclusions: A multimodal strategy to improve both compliance with safety steps for peripheral venous catheter insertion and performance of an optimum procedure was effective and was regarded helpful by healthcare workers. PMID:24327944

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

PubMed Central

2012-01-01

Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches. PMID:22554201

Evidence for Watson-Crick and not Hoogsteen or wobble base pairing in the selection of nucleotides for insertion opposite pyrimidines and a thymine dimer by yeast DNA pol eta.

PubMed

Hwang, Hanshin; Taylor, John-Stephen

2005-03-29

We have recently reported that pyrene nucleotide is preferentially inserted opposite an abasic site, the 3'-T of a thymine dimer, and most undamaged bases by yeast DNA polymerase eta (pol eta). Because pyrene is a nonpolar molecule with no H-bonding ability, the unusually high efficiencies of dPMP insertion are ascribed to its superior base stacking ability, and underscore the importance of base stacking in the selection of nucleotides by pol eta. To investigate the role of H-bonding and base pair geometry in the selection of nucleotides by pol eta, we determined the insertion efficiencies of the base-modified nucleotides 2,6-diaminopurine, 2-aminopurine, 6-chloropurine, and inosine which would make a different number of H-bonds with the template base depending on base pair geometry. Watson-Crick base pairing appears to play an important role in the selection of nucleotide analogues for insertion opposite C and T as evidenced by the decrease in the relative insertion efficiencies with a decrease in the number of Watson-Crick H-bonds and an increase in the number of donor-donor and acceptor-acceptor interactions. The selectivity of nucleotide insertion is greater opposite the 5'-T than the 3'-T of the thymine dimer, in accord with previous work suggesting that the 5'-T is held more rigidly than the 3'-T. Furthermore, insertion of A opposite both Ts of the dimer appears to be mediated by Watson-Crick base pairing and not by Hoogsteen base pairing based on the almost identical insertion efficiencies of A and 7-deaza-A, the latter of which lacks H-bonding capability at N7. The relative efficiencies for insertion of nucleotides that can form Watson-Crick base pairs parallel those for the Klenow fragment, whereas the Klenow fragment more strongly discriminates against mismatches, in accord with its greater shape selectivity. These results underscore the importance of H-bonding and Watson-Crick base pair geometry in the selection of nucleotides by both pol eta and the Klenow fragment, and the lesser role of shape selection in insertion by pol eta due to its more open and less constrained active site.

Implant Evaluation of an Insertable Cardiac Monitor Outside the Electrophysiology Lab Setting

PubMed Central

Pachulski, Roman; Cockrell, James; Solomon, Hemant; Yang, Fang; Rogers, John

2013-01-01

Background To date, insertable cardiac monitors (ICM) have been implanted in the hospital without critical evaluation of other potential settings. Providing alternatives to in-hospital insertion may increase access to ICM, decrease waiting times for patients awaiting diagnosis, and reduce hospital resources. Methods This was a prospective, non-randomized, clinical trial involving nine clinical sites throughout the United States designed to assess the feasibility of ICM implants in a non-hospital setting. Other than the Reveal® ICM, implant supplies and techniques were left to physician discretion in patients who met indications. Patients were followed up to 90 days post-implant. The primary objective was to characterize the number of procedure-related adverse events that required surgical intervention within 90 days. Results Sixty-five patients were implanted at nine out-of-hospital sites. The insertion procedure was well tolerated by all patients. There were no deaths, systemic infections or endocarditis. There were two (3%) procedure-related adverse events requiring device explant and four (6%) adverse events not requiring explant. ICM use led to 16 diagnoses (24.6%) with 9 patients proceeding to alternate cardiac device implants during the course of the 90-day follow up. Conclusion Out-of-hospital ICM insertion can be accomplished with comparable procedural safety and represents a reasonable alternative to the in-hospital setting. Clinicaltrials.gov registration number: NCT01168427 PMID:23977071

Evaluation of noise barriers.

DOT National Transportation Integrated Search

1979-01-01

Noise measurements were taken at six barrier sites: two wooden, two metal, and one concrete barrier were studied; the sixth site had no barrier and was studied to determine the ground effect. The approach was to determine insertion losses by taking s...

HIV genetic information and clonal growth

Cancer.gov

Based on an analysis of blood cells from five HIV-infected individuals, NCI researchers have identified more than 2,400 HIV DNA insertion sites. Analysis of these sites showed that there is extensive clonal expansion (growth) of HIV infected cells.

Vulvar Pain-Associations Between First-Time Vaginal Intercourse, Tampon Insertion, and Later Experiences of Pain.

PubMed

Elmerstig, Eva; Thomtén, Johanna

2016-11-16

This study examines associations between the first experience of vaginal intercourse/tampon insertion and later experiences of vulvar pain. The study is based on questionnaire data from 1,259 Swedish female senior high-school students, aged 18 to 22 years old. Of these, 592 women reported present vulvar pain. Present vulvar pain was associated with first-time experiences of vaginal intercourse (pain, negative experience, against will) and with pain at tampon insertion. First-time experiences were also related to temporal aspects of present vulvar pain during vaginal intercourse (at the beginning, after a while during, and after). Implications of first-time experiences of vaginal intercourse for future symptoms of vulvar pain are discussed.

Templated sequence insertion polymorphisms in the human genome

NASA Astrophysics Data System (ADS)

Onozawa, Masahiro; Aplan, Peter

2016-11-01

Templated Sequence Insertion Polymorphism (TSIP) is a recently described form of polymorphism recognized in the human genome, in which a sequence that is templated from a distant genomic region is inserted into the genome, seemingly at random. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; Class 1 TSIPs show features of insertions that are mediated via the LINE-1 ORF2 protein, including 1) target-site duplication (TSD), 2) polyadenylation 10-30 nucleotides downstream of a “cryptic” polyadenylation signal, and 3) preference for insertion at a 5’-TTTT/A-3’ sequence. In contrast, class 2 TSIPs show features consistent with repair of a DNA double-strand break via insertion of a DNA “patch” that is derived from a distant genomic region. Survey of a large number of normal human volunteers demonstrates that most individuals have 25-30 TSIPs, and that these TSIPs track with specific geographic regions. Similar to other forms of human polymorphism, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases.

Human Alu insertion polymorphisms in North African populations.

PubMed

Cherni, Loth; Frigi, Sabeh; Ennafaa, Hajer; Mtiraoui, Nabil; Mahjoub, Touhami; Benammar-Elgaaied, Amel

2011-10-01

Several features make Alu insertions a powerful tool used in population genetic studies: the polymorphic nature of many Alu insertions, the stability of an Alu insertion event and, furthermore, the ancestral state of an Alu insertion is known to be the absence of the Alu element at a particular locus and the presence of an Alu insertion at the site that forward mutational change. This study analyses seven Alu insertion polymorphisms in a sample of 297 individuals from the autochthonous population of Tunisia (Thala, Smar, Zarzis, and Bou Salem) and Libya with the aim of studying their genetic structure with respect to the populations of North Africa, Western, Eastern and Central Europe. The comparative analyses carried out using the MDS and AMOVA methods reveal the existence of spatial heterogeneity, and identify four population groups. Study populations (Libya, Smar, Zarzis, and Bou Salem) are closest to North African populations whereas Thala is isolated and is closest to Western European populations. In conclusion, Results of the present study support the important role that migratory movements have played in the North African gene pool, at least since the Neolithic period.

Indel PDB: a database of structural insertions and deletions derived from sequence alignments of closely related proteins.

PubMed

Hsing, Michael; Cherkasov, Artem

2008-06-25

Insertions and deletions (indels) represent a common type of sequence variations, which are less studied and pose many important biological questions. Recent research has shown that the presence of sizable indels in protein sequences may be indicative of protein essentiality and their role in protein interaction networks. Examples of utilization of indels for structure-based drug design have also been recently demonstrated. Nonetheless many structural and functional characteristics of indels remain less researched or unknown. We have created a web-based resource, Indel PDB, representing a structural database of insertions/deletions identified from the sequence alignments of highly similar proteins found in the Protein Data Bank (PDB). Indel PDB utilized large amounts of available structural information to characterize 1-, 2- and 3-dimensional features of indel sites. Indel PDB contains 117,266 non-redundant indel sites extracted from 11,294 indel-containing proteins. Unlike loop databases, Indel PDB features more indel sequences with secondary structures including alpha-helices and beta-sheets in addition to loops. The insertion fragments have been characterized by their sequences, lengths, locations, secondary structure composition, solvent accessibility, protein domain association and three dimensional structures. By utilizing the data available in Indel PDB, we have studied and presented here several sequence and structural features of indels. We anticipate that Indel PDB will not only enable future functional studies of indels, but will also assist protein modeling efforts and identification of indel-directed drug binding sites.

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae.

PubMed

Chung, K-R; Ehrenshaft, M; Wetzel, D K; Daub, M E

2003-11-01

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.

48 CFR 52.236-3 - Site Investigation and Conditions Affecting the Work.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Conditions Affecting the Work. 52.236-3 Section 52.236-3 Federal Acquisition Regulations System FEDERAL... Provisions and Clauses 52.236-3 Site Investigation and Conditions Affecting the Work. As prescribed in 36.503, insert the following clause: Site Investigation and Conditions Affecting the Work (APR 1984) (a) The...

A Novel Vesicoscopic Bladder Wall Suture Fixation Technique to Aid Endoscopic Vesicostomy Button Insertion

PubMed Central

Adam, Ahmed; Sookram, Jayveer

2018-01-01

Background To describe a novel bladder fixation technique for use during endoscopic vesicostomy button insertion. Methods After standard cystoscopic visualization of the bladder, a standard 18 G intravenous cannula was inserted into the bladder. A non-absorbable suture thread was placed through this intravenous cannula under cystoscopic vision. The proximal end of the suture was then removed using standard ureteroscopic grasping forceps (3 Fr) through another needle (15 G) inserted next to the initial puncture site (following a path at 30 degrees from the initial puncture tract) into the bladder. The suture ends were brought out of the bladder and tied at the skin level, 2 cm from the intended vesicostomy site. Sutures were removed on the second postoperative day. Results This fixation technique allows for adequate fixation of the bladder dome to the anterior abdominal wall. These sutures also have less potential for cutaneous scarring and pain. No complications were reported. Conclusion This simple fixation technique is easily performed using materials found in every urology suite. It also avoids the skills required with other previously reported fixation suture techniques, and can also be utilized for bladder fixation in cases of vesicoscopic laparoscopic or robotic assisted laparoscopic procedures. PMID:29692696

Preoperative Ultrasound Prediction of Essential Landmarks for Successful Fetoscopic Laser Treatment of Twin-Twin Transfusion Syndrome.

PubMed

Miller, Jena L; Block-Abraham, Dana M; Blakemore, Karin J; Baschat, Ahmet A

2018-06-06

The insertion site of the fetoscope for laser occlusion (FLOC) treatment of twin-twin transfusion syndrome (TTTS) determines the likelihood of treatment success. We assessed a standardized preoperative ultrasound approach for its ability to identify critical landmarks for successful FLOC. Three surgeons independently performed preoperative ultrasound and deduced the likely orientation of the intertwin membrane (ITM) and vascular equator (VE) based on the sites of the cord insertion, the lie of the donor, and the size discordance between twins. At FLOC, these landmarks were visually verified and compared to preoperative assessments. Fifty consecutive FLOC surgeries had 127 preoperative assessments. Basic ITM and VE orientation were accurately predicted in 115 (90.6%), 109 (85.8%), and 105 (82.7%) assessments. Predictions were anatomically correct in 96 (75.6%), 70 (55.1%), and 58 (45.7%) assessments with no differences in accuracy between operators of different training level. The ITM/VE relationship was most poorly predicted in stage-3 TTTS (χ2, p = 0.016). In TTTS, preoperative ultrasound identification of placental cord insertion sites, lie of the donor twin, and size discordance enables preoperative prediction of key landmarks for successful FLOC. © 2018 S. Karger AG, Basel.

Sequence-specific DNA cleavage by Fe2+-mediated fenton reactions has possible biological implications.

PubMed

Henle, E S; Han, Z; Tang, N; Rai, P; Luo, Y; Linn, S

1999-01-08

Preferential cleavage sites have been determined for Fe2+/H2O2-mediated oxidations of DNA. In 50 mM H2O2, preferential cleavages occurred at the nucleoside 5' to each of the dG moieties in the sequence RGGG, a sequence found in a majority of telomere repeats. Within a plasmid containing a (TTAGGG)81 human telomere insert, 7-fold more strand breakage occurred in the restriction fragment with the insert than in a similar-sized control fragment. This result implies that telomeric DNA could protect coding DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and aging. In micromolar H2O2, preferential cleavage occurred at the thymidine within the sequence RTGR, a sequence frequently found to be required in promoters for normal responses of many procaryotic and eucaryotic genes to iron or oxygen stress. Computer modeling of the interaction of Fe2+ with RTGR in B-DNA suggests that due to steric hindrance with the thymine methyl, Fe2+ associates in a specific manner with the thymine flipped out from the base stack so as to allow an octahedrally-oriented coordination of the Fe2+ with the three purine N7 residues. Fe2+-dependent changes in NMR spectra of duplex oligonucleotides containing ATGA versus those containing AUGA or A5mCGA were consistent with this model.

Occurrence of Phlebitis: A Systematic Review and Meta-analysis.

PubMed

Chang, Wen P; Peng, Yu X

Peripheral venous catheters (PVCs) are commonly used in clinical practice. However, varying degrees of phlebitis often occur in patients receiving intravenous injections. The relevant literature suggests that phlebitis occurrence is highly associated with the catheter gauge, insertion site, and catheterization duration. Nevertheless, no meta-analysis has been performed on the influence of these three factors on the occurrence of phlebitis. The objective of this study was to determine whether any significant differences exist in the occurrence of phlebitis between catheters of 20 gauge or smaller and those larger than 20 gauge, between catheters inserted in the antecubital fossa and those inserted in other locations on the upper limbs, or between catheters inserted for more than 96 hours and those inserted for 96 hours or less. Using a systematic approach, we searched for literature published between 2006 and 2017 in the Cumulative Index to Nursing and Allied Health Literature (CINAHL), PubMed, ProQuest, and Cochrane Library databases. We used Comprehensive Meta-analysis Version 2 to perform our meta-analysis. After the screening and review processes, we identified 17 studies that met our selection conditions. Among these studies, 14 contained complete data for meta-analysis. These studies involved 4,343 patients and 5,846 PVCs. Regarding the overall effect size in the meta-analysis, the results of the forest plot comparing catheters of 20 gauge or smaller and those larger than 20 gauge presented a risk ratio (RR) of 0.88 (95% confidence interval [0.67, 1.17], p = .380), indicating no statistically significant difference in the occurrence of phlebitis between catheters of the aforementioned gauges. The results of the forest plot comparing catheters inserted in the antecubital fossa and those inserted in other locations on the upper limbs presented an RR of 1.05 (95% confidence interval [0.82, 1.34], p = .696), indicating no statistically significant difference in the occurrence of phlebitis between catheters inserted in the aforementioned locations. The results of the forest plot comparing catheters inserted for more than 96 hours and those inserted for 96 hours or less presented an RR of 1.13 (95% confidence interval [0.49, 2.61], p = .779), indicating no statistically significant difference in the occurrence of phlebitis between catheters inserted for the aforementioned durations. The empirical results of this meta-analysis can serve as a reference for hospital management for selecting the PVC gauge, insertion site, and catheterization duration. In addition to the three factors that we analyzed, whether any other factors influence the occurrence of phlebitis in patients with catheter implantation is worth investigating in future research.

Factors affecting colonoscope insertion time in patients with or without a colostomy after left-sided colorectal resection.

PubMed

Jang, Hui Won; Kim, Yoon Nam; Nam, Chung Mo; Lee, Hyun Jung; Park, Soo Jung; Hong, Sung Pil; Kim, Tae Il; Kim, Won Ho; Cheon, Jae Hee

2012-12-01

We examined whether the insertion time for colonoscopies performed after left-sided resection was different in patients with a colostomy from that in patients without a colostomy and identified factors that could impact colonoscopy performance. We included consecutive patients who underwent colonoscopy between July 2005 and March 2011 after left-sided colorectal resection for colorectal cancer. We classified surgical methods according to the presence or absence of a colostomy and evaluated colonoscope insertion time retrospectively. Furthermore, we analyzed factors that might affect insertion time. A total of 1,041 patients underwent colonoscopy after left-sided colorectal resection during the study period. The colonoscopy completion rate was 98.6 %, and the mean insertion time was 6.1 ± 4.6 min (median 4.7 min, range 0.3-35.8 min). A shorter resection length of colon, the presence of a colostomy, and a lower endoscopist case volume were found to be independent factors associated with prolonged insertion time in patients with left-sided colorectal resection. Among experienced colonoscopists, no colonoscopy-associated or clinical factors were found to affect insertion time. However, a shorter resection length of colon, the presence of a colostomy, and poor bowel preparation were associated with prolonged insertion time among inexperienced endoscopists. We identified three factors that affect colonoscope insertion time after left-sided colorectal resection, including the presence of a colostomy. Inexperienced endoscopists were much more affected by the presence of a colostomy after left-sided colorectal resection. These findings have implications for the practice and teaching of colonoscopy after left-sided colorectal resection.

Effect of clinic related factors on continuation rates of IUDs.

PubMed

Reading, A E; Goldstuck, N D

1982-01-01

This paper concerns factors relating to the clinic and the way in which the insertion of IUDs is managed from a psychological and technical standpoint. 3 aspects of clinical service are examined: 1) the influence of the psychological state at time of insertion, 2) individual differences in relation to side effects amongst users and the way in which those are influenced by managment techniques, and 3) technical aspects of the insertion of the device. Insertion pain and discomfort may influence subsequent tolerance of the IUD in 3 ways: 1) patient compliance may be enhanced by trouble-free insertion, 2) an unpleasant experience may influence attitudes towards the IUD, and 3) the experience of pain at insertion may reduce subsequent tolerance of discomfort. IUDs increase the amount of menstrual flow which, along with pain, constitute frequent requests for removal. Removal rates for pain and bleeding vary from 0-16.8/100 users. Removal rates for bleeding are also influenced by cultural attitudes. Insertion techniques influence acceptability of IUDs. Also, the incidence of expulsion, pregnancy, and removal declines with increasing age and parity. Discontinuation rates after 24 months were 91% for women below 2nd parity and only 37% for women of 6 or more parity. In the youngest age group the discontinuation rate was 76% compared with 35% in the oldest age group. The timing of IUD insertion and positioning of the IUD influence continuation rates. An implication of these concerns is that IUD insertion procedures may have to become more complex to achieve compatibility between IUD-uterine configurations.

Insertion and deletion polymorphisms of the ancient AluS family in the human genome.

PubMed

Kryatova, Maria S; Steranka, Jared P; Burns, Kathleen H; Payer, Lindsay M

2017-01-01

Polymorphic Alu elements account for 17% of structural variants in the human genome. The majority of these belong to the youngest AluY subfamilies, and most structural variant discovery efforts have focused on identifying Alu polymorphisms from these currently retrotranspositionally active subfamilies. In this report we analyze polymorphisms from the evolutionarily older AluS subfamily, whose peak activity was tens of millions of years ago. We annotate the AluS polymorphisms, assess their likely mechanism of origin, and evaluate their contribution to structural variation in the human genome. Of 52 previously reported polymorphic AluS elements ascertained for this study, 48 were confirmed to belong to the AluS subfamily using high stringency subfamily classification criteria. Of these, the majority (77%, 37/48) appear to be deletion polymorphisms. Two polymorphic AluS elements (4%) have features of non-classical Alu insertions and one polymorphic AluS element (2%) likely inserted by a mechanism involving internal priming. Seven AluS polymorphisms (15%) appear to have arisen by the classical target-primed reverse transcription (TPRT) retrotransposition mechanism. These seven TPRT products are 3' intact with 3' poly-A tails, and are flanked by target site duplications; L1 ORF2p endonuclease cleavage sites were also observed, providing additional evidence that these are L1 ORF2p endonuclease-mediated TPRT insertions. Further sequence analysis showed strong conservation of both the RNA polymerase III promoter and SRP9/14 binding sites, important for mediating transcription and interaction with retrotransposition machinery, respectively. This conservation of functional features implies that some of these are fairly recent insertions since they have not diverged significantly from their respective retrotranspositionally competent source elements. Of the polymorphic AluS elements evaluated in this report, 15% (7/48) have features consistent with TPRT-mediated insertion, thus suggesting that some AluS elements have been more active recently than previously thought, or that fixation of AluS insertion alleles remains incomplete. These data expand the potential significance of polymorphic AluS elements in contributing to structural variation in the human genome. Future discovery efforts focusing on polymorphic AluS elements are likely to identify more such polymorphisms, and approaches tailored to identify deletion alleles may be warranted.

Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

PubMed

Kawabe, Yoshinori; Shimomura, Takuya; Huang, Shuohao; Imanishi, Suguru; Ito, Akira; Kamihira, Masamichi

2016-07-01

Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre-based cell engineering. Biotechnol. Bioeng. 2016;113: 1600-1610. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

New Insights into the Classification and Integration Specificity of Streptococcus Integrative Conjugative Elements through Extensive Genome Exploration

PubMed Central

Ambroset, Chloé; Coluzzi, Charles; Guédon, Gérard; Devignes, Marie-Dominique; Loux, Valentin; Lacroix, Thomas; Payot, Sophie; Leblond-Bourget, Nathalie

2016-01-01

Recent genome analyses suggest that integrative and conjugative elements (ICEs) are widespread in bacterial genomes and therefore play an essential role in horizontal transfer. However, only a few of these elements are precisely characterized and correctly delineated within sequenced bacterial genomes. Even though previous analysis showed the presence of ICEs in some species of Streptococci, the global prevalence and diversity of ICEs was not analyzed in this genus. In this study, we searched for ICEs in the completely sequenced genomes of 124 strains belonging to 27 streptococcal species. These exhaustive analyses revealed 105 putative ICEs and 26 slightly decayed elements whose limits were assessed and whose insertion site was identified. These ICEs were grouped in seven distinct unrelated or distantly related families, according to their conjugation modules. Integration of these streptococcal ICEs is catalyzed either by a site-specific tyrosine integrase, a low-specificity tyrosine integrase, a site-specific single serine integrase, a triplet of site-specific serine integrases or a DDE transposase. Analysis of their integration site led to the detection of 18 target-genes for streptococcal ICE insertion including eight that had not been identified previously (ftsK, guaA, lysS, mutT, rpmG, rpsI, traG, and ebfC). It also suggests that all specificities have evolved to minimize the impact of the insertion on the host. This overall analysis of streptococcal ICEs emphasizes their prevalence and diversity and demonstrates that exchanges or acquisitions of conjugation and recombination modules are frequent. PMID:26779141

New Insights into the Classification and Integration Specificity of Streptococcus Integrative Conjugative Elements through Extensive Genome Exploration.

PubMed

Ambroset, Chloé; Coluzzi, Charles; Guédon, Gérard; Devignes, Marie-Dominique; Loux, Valentin; Lacroix, Thomas; Payot, Sophie; Leblond-Bourget, Nathalie

2015-01-01

Recent genome analyses suggest that integrative and conjugative elements (ICEs) are widespread in bacterial genomes and therefore play an essential role in horizontal transfer. However, only a few of these elements are precisely characterized and correctly delineated within sequenced bacterial genomes. Even though previous analysis showed the presence of ICEs in some species of Streptococci, the global prevalence and diversity of ICEs was not analyzed in this genus. In this study, we searched for ICEs in the completely sequenced genomes of 124 strains belonging to 27 streptococcal species. These exhaustive analyses revealed 105 putative ICEs and 26 slightly decayed elements whose limits were assessed and whose insertion site was identified. These ICEs were grouped in seven distinct unrelated or distantly related families, according to their conjugation modules. Integration of these streptococcal ICEs is catalyzed either by a site-specific tyrosine integrase, a low-specificity tyrosine integrase, a site-specific single serine integrase, a triplet of site-specific serine integrases or a DDE transposase. Analysis of their integration site led to the detection of 18 target-genes for streptococcal ICE insertion including eight that had not been identified previously (ftsK, guaA, lysS, mutT, rpmG, rpsI, traG, and ebfC). It also suggests that all specificities have evolved to minimize the impact of the insertion on the host. This overall analysis of streptococcal ICEs emphasizes their prevalence and diversity and demonstrates that exchanges or acquisitions of conjugation and recombination modules are frequent.

GROUTING TECHNIQUES IN BOTTOM SEALING OF HAZARDOUS WASTE SITES

EPA Science Inventory

Bottom sealing of hazardous waste sites involves the injection or insertion of an inert impermeable and continuous horizontal barrier in soil below the source of contamination. This type of containment strategy could be used in conjunction with other technology such as slurry wal...

Interactions between the cytomegalovirus promoter and the estrogen response element: implications for design of estrogen-responsive reporter plasmids.

PubMed

Derecka, K; Wang, C K; Flint, A P F

2006-07-01

We aimed to produce an estrogen-responsive reporter plasmid that would permit monitoring of estrogen receptor function in the uterus in vivo. The plasmid pBL-tk-CAT(+)ERE was induced by estrogen in bovine endometrial stromal cells. When the CAT gene was replaced by the secreted alkaline phosphatase SeAP, the resulting construct pBL-tk-SeAP(+)ERE remained estrogen responsive. However when the tk promoter was replaced by the cytomegalovirus (cmv) promoter, the resulting plasmid (pBL-cmv-SeAP(+)ERE) was not estrogen responsive. Inhibition of ERE function was not due to an effect in trans or due to lack of estrogen receptor. It was not due to an interaction between the cmv promoter and the SeAP gene. cmv promoter function was dependent on NF-kappaB, and mutagenesis in the NF-kappaB sites reduced basal reporter expression without imparting responsiveness to estrogen. A mutation in the TATA box also failed to impart estrogen responsiveness. Modeling of DNA accessibility indicated the ERE was inserted at a site accessible to transcription factors. We conclude that the cmv promoter inhibits ERE function in cis when the two sequences are located in the same construct, and that this effect does not involve an interaction between cmv and reporter gene, NF-kappaB sites or the TATA box, or DNA inaccessibility.

Bilingual Crosscultural Education in Western Europe: An Overview.

ERIC Educational Resources Information Center

Blanche, Parick

1992-01-01

A European perspective on multicultural education is presented that focuses on demographic/geopolitical, public policy, and sociolinguistic dimensions, and the intercultural hypothesis. "Positive discrimination" and sociological insertion are also discussed, along with implications for America. (29 references) (LB)

A pair of new BAC and BIBAC vectors that facilitate BAC/BIBAC library construction and intact large genomic DNA insert exchange.

PubMed

Shi, Xue; Zeng, Haiyang; Xue, Yadong; Luo, Meizhong

2011-10-11

Large-insert BAC and BIBAC libraries are important tools for structural and functional genomics studies of eukaryotic genomes. To facilitate the construction of BAC and BIBAC libraries and the transfer of complete large BAC inserts into BIBAC vectors, which is desired in positional cloning, we developed a pair of new BAC and BIBAC vectors. The new BAC vector pIndigoBAC536-S and the new BIBAC vector BIBAC-S have the following features: 1) both contain two 18-bp non-palindromic I-SceI sites in an inverted orientation at positions that flank an identical DNA fragment containing the lacZ selection marker and the cloning site. Large DNA inserts can be excised from the vectors as single fragments by cutting with I-SceI, allowing the inserts to be easily sized. More importantly, because the two vectors contain different antibiotic resistance genes for transformant selection and produce the same non-complementary 3' protruding ATAA ends by I-SceI that suppress self- and inter-ligations, the exchange of intact large genomic DNA inserts between the BAC and BIBAC vectors is straightforward; 2) both were constructed as high-copy composite vectors. Reliable linearized and dephosphorylated original low-copy pIndigoBAC536-S and BIBAC-S vectors that are ready for library construction can be prepared from the high-copy composite vectors pHZAUBAC1 and pHZAUBIBAC1, respectively, without the need for additional preparation steps or special reagents, thus simplifying the construction of BAC and BIBAC libraries. BIBAC clones constructed with the new BIBAC-S vector are stable in both E. coli and Agrobacterium. The vectors can be accessed through our website http://GResource.hzau.edu.cn. The two new vectors and their respective high-copy composite vectors can largely facilitate the construction and characterization of BAC and BIBAC libraries. The transfer of complete large genomic DNA inserts from one vector to the other is made straightforward.

Risk factors for upper extremity venous thrombosis associated with peripherally inserted central venous catheters.

PubMed

Marnejon, Thomas; Angelo, Debra; Abu Abdou, Ahmed; Gemmel, David

2012-01-01

To identify clinically important risk factors associated with upper extremity venous thrombosis following peripherally inserted central venous catheters (PICC). A retrospective case control study of 400 consecutive patients with and without upper extremity venous thrombosis post-PICC insertion was performed. Patient data included demographics, body mass index (BMI), ethnicity, site of insertion, size and lumen of catheter, internal length, infusate, and co-morbidities, such as diabetes mellitus, congestive heart failure, and renal failure. Additional risk factors analyzed were active cancer, any history of cancer, recent trauma, smoking, a history of prior deep vein thrombosis, and recent surgery, defined as surgery within three months prior to PICC insertion. The prevalence of trauma, renal failure, and infusion with antibiotics and total parenteral nutrition (TPN) was higher among patients exhibiting upper extremity venous thrombosis (UEVT), when compared to controls. Patients developing UEVT were also more likely to have PICC line placement in a basilic vein and less likely to have brachial vein placement (P<.001). Left-sided PICC line sites also posed a greater risk (P=.026). The rate of standard DVT prophylaxis with low molecular weight heparin and unfractionated heparin and the use of warfarin was similar in both groups. Average length of hospital stay was almost double among patients developing UEVT, 19.5 days, when compared to patients undergoing PICC line insertion without thrombosis, 10.8 days (t=6.98, P<.001). In multivariate analysis, trauma, renal failure, left-sided catheters, basilic placement, TPN, and infusion with antibiotics, specifically vancomycin, were significant risk factors for UEVT associated with PICC insertion. Prophylaxis with low molecular weight heparin, unfractionated heparin or use of warfarin did not prevent the development of venous thrombosis in patients with PICCs. Length of hospital stay and cost are markedly increased in patients who develop PICC-associated upper extremity venous thrombosis.

Cloning of Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa, into the pBD214 vector of Bacillus subtilis.

PubMed Central

Putterman, D G; Gryczan, T J; Dubnau, D; Day, L A

1983-01-01

The genome of Pf3, a filamentous single-stranded DNA bacteriophage of Pseudomonas aeruginosa (a gram-negative organism) was cloned into pBD214, a plasmid cloning vector of Bacillus subtilis (a gram-positive organism). Cloning in the gram-positive organism was done to avoid anticipated lethal effects. The entire Pf3 genome was inserted in each orientation at a unique Bc/I site within a thymidylate synthetase gene (from B. subtilis phage beta 22) on the plasmid. Additional clones were made by inserting EcoRI fragments of Pf3 DNA into a unique EcoRI site within this gene. Images PMID:6306273

Impact of targeted counseling on reported vaginal hygiene practices and bacterial vaginosis: the HIV Prevention Trials Network 035 study.

PubMed

Kasaro, Margaret P; Husnik, Marla J; Chi, Benjamin H; Reid, Cheri; Magure, Tsitsi; Makanani, Bonus; Tembo, Tchangani; Ramjee, Gita; Maslankowski, Lisa; Rabe, Lorna; Brad Guffey, M

2017-04-01

The objective of this study was to describe the impact of intense counseling to reduce vaginal hygiene practices and its effect on bacterial vaginosis. A secondary data analysis of the HIV Prevention Trials Network 035 study was undertaken, focusing on HIV-negative, nonpregnant women who were at least 18 years old, in seven African sites and one US site. At enrollment and during follow-up quarterly visits, vaginal hygiene practices were determined by face-to-face administration of a behavioral assessment questionnaire. Vaginal hygiene practices were categorized as insertion into the vagina of (1) nothing, (2) water only, and (3) other substances with or without water. Each practice was quantified by frequency and type/combination of inserted substances. At quarterly visits, diagnosis of bacterial vaginosis was made using the Nugent score. Trends for vaginal hygiene practices and bacterial vaginosis were evaluated using generalized estimating equation models. A total of 3087 participants from the HIV Prevention Trials Network 035 study were eligible for this analysis. At enrollment, 1859 (60%) reported recent vaginal hygiene practices. By one year, this figure had decreased to 1019 (33%) with counseling. However, bacterial vaginosis prevalence remained consistent across the study observation period, with 36%-38% of women testing positive for the condition ( p for trend = 0.27). Overall, those who reported douching with water only (AOR = 1.03, 95%CI: 0.94-1.13) and those who reported inserting other substances (AOR= 0.98, 95%CI: 0.88-1.09) in the past quarter were not more likely to have bacterial vaginosis compared to those who reported no insertions. However, in South Africa, an increase in bacterial vaginosis was seen among those who reported inserting other substances (AOR: 1.48, 95%CI: 1.17, 1.88). In conclusion, targeted counseling against vaginal hygiene practices resulted in change in self-reported behavior but did not have an impact on bacterial vaginosis diagnosis in all but one site.

Direction of catheter insertion and the incidence of paresthesia during continuous epidural anesthesia in the elderly patients

PubMed Central

Kim, Jong-Hak; Lee, Jun Seop

2013-01-01

Background Continuous epidural anesthesia is useful for endoscopic urologic surgery, as mostly performed in the elderly patients. In such a case, it is necessary to obtain successful sacral anesthesia, and the insertion of epidural catheter in the caudad direction may be needed. However, continuous epidural catherization has been related to paresthesias. This study aimed to evaluate the effects of the direction of the catheter insertion on the incidence of paresthesias in the elderly patients. Methods Two hundred elderly patients scheduled for endoscopic urologic surgery were enrolled. The epidural catheter was inserted at L2-3, L3-4, and L4-5 using the Tuohy needle. In Group I (n = 100), the Tuohy needle with the bevel directed the cephalad during the catheter insertion. In Group II (n = 100), it directed the caudad. During the catheter insertion, an anesthesiologist evaluated the presence of paresthesias and the ease or difficulty during the catheter insertion. Results In Group I (n = 97), 15.5% of the patients had paresthesias versus 18.4% in Group II (n = 98), and there was no significant difference between the two groups. In paresthesia depending on the insertion site and the ease or difficulty during the catheter insertion, there were no significant differences between the two groups. Conclusions Our results concluded that the direction of epidural catheter insertion did not significantly influence the incidence of paresthesias in the elderly patients. PMID:23741568

Copy number determination of genetically-modified hematopoietic stem cells.

PubMed

Schuesler, Todd; Reeves, Lilith; Kalle, Christof von; Grassman, Elke

2009-01-01

Human gene transfer with gammaretroviral, murine leukemia virus (MLV) based vectors has been shown to effectively insert and express transgene sequences at a level of therapeutic benefit. However, there are numerous reports of disruption of the normal cellular processes caused by the viral insertion, even of replication deficient gammaretroviral vectors. Current gammaretroviral and lentiviral vectors do not control the site of insertion into the genome, hence, the possibility of disruption of the target cell genome. Risk related to viral insertions is linked to the number of insertions of the transgene into the cellular DNA, as has been demonstrated for replication competent and replication deficient retroviruses in experiments. At high number of insertions per cell, cell transformation due to vector induced activation of proto-oncogenes is more likely to occur, in particular since more than one transforming event is needed for oncogenesis. Thus, determination of the vector copy number in bulk transduced populations, individual colony forming units, and tissue from the recipient of the transduced cells is an increasingly important safety assay and has become a standard, though not straightforward assay, since the inception of quantitative PCR.

Elucidating the mechanism of posterior reversible encephalopathy syndrome: a case of transient blindness after central venous catheterization.

PubMed

Rao, Neal M; Raychev, Radoslav; Kim, Doojin; Liebeskind, David S

2012-11-01

Posterior reversible encephalopathy syndrome (PRES) is a condition characterized by reversible symptoms including headache, visual disturbances, focal neurological deficits, altered mentation, and seizures. It has been associated with circumstances that may affect the cerebrovascular system, such as hypertension, eclampsia, and immunosuppression with calcineurin inhibitors. The underlying etiology of PRES has remained unclear; however, cerebrovascular autoregulatory dysfunction, hyperperfusion, and endothelial activation have been implicated. We describe a case of a young patient with lung transplant, who presented with headache, acute binocular blindness, and seizure immediately after infusion of saline through a peripherally inserted central catheter line, which inadvertently terminated cephalad in the left internal jugular vein, near the jugular foramen. Subsequent brain magnetic resonance imaging revealed vasogenic edematous lesions in a pattern consistent with PRES--a diagnosis supported by his constellation of symptoms, history of lung transplantation on tacrolimus immunosuppression, and relative hypertension. This is the first reported case describing the development of PRES after the insertion of a peripherally inserted central catheter line. The development of PRES in a typical high-risk patient immediately after cerebral venous outflow obstruction implicates the role of the cerebral venous system and provides potential insight into the mechanism of this disorder that remains of unclear pathogenesis.

A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data.

PubMed

Park, Doori; Park, Su-Hyun; Ban, Yong Wook; Kim, Youn Shic; Park, Kyoung-Cheul; Kim, Nam-Soo; Kim, Ju-Kon; Choi, Ik-Young

2017-08-15

Genetically modified crops (GM crops) have been developed to improve the agricultural traits of modern crop cultivars. Safety assessments of GM crops are of paramount importance in research at developmental stages and before releasing transgenic plants into the marketplace. Sequencing technology is developing rapidly, with higher output and labor efficiencies, and will eventually replace existing methods for the molecular characterization of genetically modified organisms. To detect the transgenic insertion locations in the three GM rice gnomes, Illumina sequencing reads are mapped and classified to the rice genome and plasmid sequence. The both mapped reads are classified to characterize the junction site between plant and transgene sequence by sequence alignment. Herein, we present a next generation sequencing (NGS)-based molecular characterization method, using transgenic rice plants SNU-Bt9-5, SNU-Bt9-30, and SNU-Bt9-109. Specifically, using bioinformatics tools, we detected the precise insertion locations and copy numbers of transfer DNA, genetic rearrangements, and the absence of backbone sequences, which were equivalent to results obtained from Southern blot analyses. NGS methods have been suggested as an effective means of characterizing and detecting transgenic insertion locations in genomes. Our results demonstrate the use of a combination of NGS technology and bioinformatics approaches that offers cost- and time-effective methods for assessing the safety of transgenic plants.

Enhancing the GABI-Kat Arabidopsis thaliana T-DNA Insertion Mutant Database by Incorporating Araport11 Annotation.

PubMed

Kleinboelting, Nils; Huep, Gunnar; Weisshaar, Bernd

2017-01-01

SimpleSearch provides access to a database containing information about T-DNA insertion lines of the GABI-Kat collection of Arabidopsis thaliana mutants. These mutants are an important tool for reverse genetics, and GABI-Kat is the second largest collection of such T-DNA insertion mutants. Insertion sites were deduced from flanking sequence tags (FSTs), and the database contains information about mutant plant lines as well as insertion alleles. Here, we describe improvements within the interface (available at http://www.gabi-kat.de/db/genehits.php) and with regard to the database content that have been realized in the last five years. These improvements include the integration of the Araport11 genome sequence annotation data containing the recently updated A. thaliana structural gene descriptions, an updated visualization component that displays groups of insertions with very similar insertion positions, mapped confirmation sequences, and primers. The visualization component provides a quick way to identify insertions of interest, and access to improved data about the exact structure of confirmed insertion alleles. In addition, the database content has been extended by incorporating additional insertion alleles that were detected during the confirmation process, as well as by adding new FSTs that have been produced during continued efforts to complement gaps in FST availability. Finally, the current database content regarding predicted and confirmed insertion alleles as well as primer sequences has been made available as downloadable flat files. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Evaluating the patient experience after implantation of a 0.4 mg sustained release dexamethasone intracanalicular insert (Dextenza™): results of a qualitative survey.

PubMed

Gira, Joseph P; Sampson, Reginald; Silverstein, Steven M; Walters, Thomas R; Metzinger, Jamie Lynne; Talamo, Jonathan H

2017-01-01

The purpose of this study is to evaluate the patient experience of sustained release dexamethasone intracanalicular insert (Dextenza™) following cataract surgery as part of a Phase III clinical trial program. This cross-sectional, qualitative evaluation involved individual interviews lasting approximately 45 minutes. Patients from four US investigational study sites who had previously received an insert were enrolled. There were no predesignated end points; this was a qualitative survey seeking a deeper understanding of patient experience. Twenty-five patients were interviewed. Most patients (92%) reported the highest level of satisfaction grade with regard to overall product satisfaction. All patients described the insert as comfortable. Most patients (96%) described their overall experience with the insert as very convenient or extremely convenient. Twenty-two of 23 (96%) participants rated their experience with the insert as "very" or "extremely convenient", compared to previous topical therapy, and 88% of patients stated that if they were to undergo cataract surgery again, they would request the insert. When asked if they would recommend the insert to family members or friends, 92% stated they would. The survey found that 84% of participants would be willing to pay more for the insert than for eye drop therapy. The dexamethasone insert was found by patients to be highly favorable with regard to overall satisfaction, convenience, and comfort. The insert was well received and largely preferred over topical therapy alternatives following surgery. More extensive evaluation of the patient experience is warranted, and future studies should help inform design of the next generation of sustained release drug delivery systems.

Circular permutant GFP insertion folding reporters

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2008-06-24

Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

Circular permutant GFP insertion folding reporters

DOEpatents

Waldo, Geoffrey S; Cabantous, Stephanie

2013-02-12

Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

Circular permutant GFP insertion folding reporters

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2011-06-14

Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

Circular permutant GFP insertion folding reporters

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2013-04-16

Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

INS studies of Cobalt-Copper Catalyst for the Conversion of Syngas to Higher Oxygenates

NASA Astrophysics Data System (ADS)

Sprunger, Phillip; Wang, Zi; Patterson, Matthew; Kurtz, Richard; Spivey, James

Cobalt-copper catalysts have been proposed for the synthesis of ethanol and higher oxygenates as a substitute of Rh and other high-cost noble metal catalysts. Two types of sites with atomic proximity are needed to form higher oxygenates: one to dissociate CO and a second to insert CO to the intermediates to form the CHxCO intermediate. Metallic cobalt is responsible for CO dissociation, while the nature of the site for CO insertion is still under study. We have utilized inelastic neutron scattering (INS) at the VISION beamline at SNS to probe intermediate surface species of this cobalt-copper catalyst. This unique technique allows for elucidation of mechanistic details of the CO insertion and subsequent CHxCO intermediate formation on the metal surfaces (Co0, Co2C and/or Cu0) . In addition to XRD and EXAFS which show a unique surface Co-C carbide formation, a combination of both INS and computational modeling indicate that the active site for CHxCO intermediates. Sponsored through the Louisiana Consortium for Neutron Scattering, DOE No. DE-SC0012432 with additional support from the LA BOR; also ORNL's Spallation Neutron Source (VISION Beamline), DOE-BES under Contract No. DE-AC0500OR22725.

Ribosomal Binding Site Switching: An Effective Strategy for High-Throughput Cloning Constructions

PubMed Central

Li, Yunlong; Zhang, Yong; Lu, Pei; Rayner, Simon; Chen, Shiyun

2012-01-01

Direct cloning of PCR fragments by TA cloning or blunt end ligation are two simple methods which would greatly benefit high-throughput (HTP) cloning constructions if the efficiency can be improved. In this study, we have developed a ribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon and is essential for gene expression. Change from A/G to T/C in the RBS blocks its activity and thereby abolishes gene expression. Based on this property, we introduced an inactive RBS upstream of a selectable marker gene, and designed a fragment insertion site within this inactive RBS. Forward and reverse insertions of specifically tailed fragments will respectively form an active and inactive RBS, thus all background from vector self-ligation and fragment reverse insertions will be eliminated due to the non-expression of the marker gene. The effectiveness of our strategy for TA cloning and blunt end ligation are confirmed. Application of this strategy to gene over-expression, a bacterial two-hybrid system, a bacterial one-hybrid system, and promoter bank construction are also verified. The advantages of this simple procedure, together with its low cost and high efficiency, makes our strategy extremely useful in HTP cloning constructions. PMID:23185557

Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FlEx gene traps

PubMed Central

Schebelle, Laura; Wolf, Claudia; Stribl, Carola; Javaheri, Tahereh; Schnütgen, Frank; Ettinger, Andreas; Ivics, Zoltán; Hansen, Jens; Ruiz, Patricia; von Melchner, Harald; Wurst, Wolfgang; Floss, Thomas

2010-01-01

Recombinase-mediated cassette exchange (RMCE) exploits the possibility to unidirectionally exchange any genetic material flanked by heterotypic recombinase recognition sites (RRS) with target sites in the genome. Due to a limited number of available pre-fabricated target sites, RMCE in mouse embryonic stem (ES) cells has not been tapped to its full potential to date. Here, we introduce a universal system, which allows the targeted insertion of any given transcriptional unit into 85 742 previously annotated retroviral conditional gene trap insertions, representing 7013 independent genes in mouse ES cells, by RMCE. This system can be used to express any given cDNA under the control of endogenous trapped promoters in vivo, as well as for the generation of transposon ‘launch pads’ for chromosomal region-specific ‘Sleeping Beauty’ insertional mutagenesis. Moreover, transcription of the gene-of-interest is only activated upon Cre-recombinase activity, a feature that adds conditionality to this expression system, which is demonstrated in vivo. The use of the RMCE system presented in this work requires one single-cloning step followed by one overnight gateway clonase reaction and subsequent cassette exchange in ES cells with efficiencies of 40% in average. PMID:20139417

The Effect of Platelet-rich Fibrin Matrix on Rotator Cuff Healing in a Rat Model.

PubMed

Hasan, S; Weinberg, M; Khatib, O; Jazrawi, L; Strauss, E J

2016-01-01

The purpose of the current study was to determine if the application of platelet-rich fibrin matrix could improve regeneration of the tendon-bone insertion site in a rat rotator cuff repair model. 25 Lewis syngeneic rats underwent bilateral tenotomy and repair of the supraspinatus tendon. 10 separate rats were used for PRFM harvest. All left (control) shoulders underwent transosseous rotator cuff repair, while all right (treatment) shoulders were repaired similarly with PRFM augmentation. 9 rats were sacrificed at 2-weeks and ten at 4-weeks for biomechanical testing. 3 separate rats were sacrificed at 2-weeks and 4-weeks each for histologic analysis of the insertion site. At 2 weeks, the experimental group repairs were significantly stronger in ultimate load to failure (P=0.01), stress (P=0.03), and stiffness (P=0.03). Differences in biomechanical testing were not found between the groups at 4 weeks. Histological analysis revealed less collagen organization and cartilage formation at the insertion site in the experimental group. Semiquantitative histologic analysis confirmed our qualitative assessment of the specimens. PRFM does not recapitulate the native enthesis, but rather induces an exuberant and disordered healing response that is characterized by fibrovascular scar tissue. © Georg Thieme Verlag KG Stuttgart · New York.

Effects of Surface Oxygen on the Performance of Carbon as an Anode in Lithium-Ion Batteries

NASA Technical Reports Server (NTRS)

Hung, Ching-Cheh; Clark, Gregory W.

2001-01-01

Carbon materials with similar bulk structure but different surface oxygen were compared for their performance as anodes in lithium-ion battery. The bulk structure was such that the graphene planes were perpendicular to the surface. Three types of surfaces were examined: surface containing C=O type oxygen. surface containing -O-C type oxygen, and surface containing high concentration of active sites. The test involved cycles of lithium insertion into and release from the carbon materials, which was in the half cells of carbon/saturated LiI-50/50 (vol %) EC and DMC/lithium. During the first cycle of lithium insertion, the presence of adsorbed oxygen, -O-C type oxygen, active carbon sites, and C=O type oxygen resulted in the formation of solid-electrolyte interface (SEI) when the carbon's voltage relative to lithium metal was >1.35, 1 to 1.35, 0.5 to 1, and 0.67 to 0.7 V, respectively. An optimum -O-C type oxygen and a minimum C=O type oxygen was found to increase the reversible and decrease the irreversible capacity of carbon. Active sites on the carbon surface result in a large irreversible capacity and a second lithium insertion-release mechanism. However, this new mechanism has a short cycle life.

Structural effects in the interstitial solid solution system (La,Ce)(Fe,Si)13Cx-H: Correlation with hydrogenation kinetics

NASA Astrophysics Data System (ADS)

Hai, X.; Porcher, F.; Mayer, C.; Miraglia, S.

2018-02-01

Steady state and in-situ neutron powder diffraction on selected compositions of the magneto-caloric (La,Ce)(Fe,Si)13CxHy compounds has been used to locate the sites accommodated by the interstitial species and to reveal the structural modifications (breathing) that occur upon metal substitution and/or interstitial insertion. The latter type of measurement in which the sequential filling of interstitial sites is followed allows one to extract some useful hydrogenation kinetics data. This structural investigation has allowed to precise the deformations undergone by the complex metallic alloys La(Fe,Si)13 when subjected to light interstitial insertion or rare earth substitution at the cation site of the NaZn13-structure type. We attempt to correlate hydrogenation kinetics variations (depression or enhancement of the hydrogen absorption rate) with a particular inhomogeneous cell variation (breathing) and bonding of the NaZn13 structure-type.

Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.

PubMed

Bressan, Fabiana Fernandes; Dos Santos Miranda, Moyses; Perecin, Felipe; De Bem, Tiago Henrique; Pereira, Flavia Thomaz Verechia; Russo-Carbolante, Elisa Maria; Alves, Daiani; Strauss, Bryan; Bajgelman, Marcio; Krieger, José Eduardo; Binelli, Mario; Meirelles, Flavio Vieira

2011-02-01

Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

PubMed

Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

PubMed Central

Rivera-Torres, Natalia; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex. PMID:28052104

Lithiation Thermodynamics and Kinetics of the TiO 2 (B) Nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Xiao; Liu, Zheng; Fischer, Michael G.

TiO2 (B) has attracted a lot of attention in recent years because it exhibits the largest capacity among all studied titania polymorphs with high rate performance for Li intercalation achieved when this material is nanostructured. However, due to the complex nature of its lithiation mechanism and practical challenges in probing Li local environments in nanostructured materials, a definitive understanding of the lithiation thermodynamics has yet to be established. A comprehensive mechanistic investigation of the TiO2 (B) nanoparticles is therefore presented using a combination of in situ / operando X-ray pair distribution function (PDF) and electrochemical techniques. The discharge begins withmore » surface reactions involving surface hydroxyl groups. Such reactions contribute to the capacity loss and take place in parallel with Li insertion into the near-surface region of the nanoparticles. The Li bulk insertion starts with a single-phase reaction into the A2 site, a position adjacent to the b channel. A change of the Li diffusion pathway from that along this open channel to that along the c-direction is likely to occur at the composition of Li0.25TiO2 until Li0.5TiO2 is attained, leading to a two-step A2-site incorporation with one step kinetically distinct from the other. Subsequent Li insertion involves C’ site, a position situated inside the channel, and follows a rapid two-phase reaction to form Li0.75TiO2. Due to the high diffusion barrier associated with the further lithiation, Li insertion into the A1 site, another position adjacent to the channel neighboring the A2 sites, is kinetically restricted. It can be promoted by either nanostructuring or raising the operating temperature, the latter however triggering concurrent electrolyte decomposition giving rise to additional capacity loss. This study not only provides compelling experimental evidence for the unresolved reaction thermodynamics of nanoparticulate TiO2 (B), but also serves as a strong reference for future studies of the bulk phase, and for future calculations to study the kinetic properties of TiO2 (B).« less

Target sites for the transposition of rat long interspersed repeated DNA elements (LINEs) are not random.

PubMed Central

Furano, A V; Somerville, C C; Tsichlis, P N; D'Ambrosio, E

1986-01-01

The long interspersed repeated DNA family of rats (LINE or L1Rn family) contains about 40,000 6.7-kilobase (kb) long members (1). LINE members may be currently mobile since their presence or absence causes allelic variation at three single copy loci (2, 3): insulin 1, Moloney leukemia virus integration 2 (Mlvi-2) (4), and immunoglobulin heavy chain (Igh). To characterize target sites for LINE insertion, we compared the DNA sequences of the unoccupied Mlvi-2 target site, its LINE-containing allele, and several other LINE-containing sites. Although not homologous overall, the target sites share three characteristics: First, depending on the site, they are from 68% to 86% (A+T) compared to 58% (A+T) for total rat DNA (5). Depending on the site, a 7- to 15-bp target site sequence becomes duplicated and flanks the inserted LINE member. The second is a version (0 or 1 mismatch) of the hexanucleotide, TACTCA, which is also present in the LINE member, in a highly conserved region located just before the A-rich right end of the LINE member. The third is a stretch of alternating purine/pyrimidine (PQ). The A-rich right ends of different LINE members vary in length and composition, and the sequence of a particularly long one suggests that it contains the A-rich target site from a previous transposition. PMID:3012480

Active role of a human genomic insert in replication of a yeast artificial chromosome.

PubMed

van Brabant, A J; Fangman, W L; Brewer, B J

1999-06-01

Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.

Recombination Can Initiate and Terminate at a Large Number of Sites within the Rosy Locus of Drosophila Melanogaster

PubMed Central

Clark, S. H.; Hilliker, A. J.; Chovnick, A.

1988-01-01

This report presents the results of a recombination experiment designed to question the existence of special sites for the initiation or termination of a recombination heteroduplex within the region of the rosy locus. Intragenic recombination events were monitored between two physically separated rosy mutant alleles ry(301) and ry(2) utilizing DNA restriction site polymorphisms as genetic markers. Both ry(301) and ry(2) are known from previous studies to be associated with gene conversion frequencies an order of magnitude lower than single site mutations. The mutations are associated with large, well defined insertions located as internal sites within the locus in prior intragenic mapping studies. On the molecular map, they represent large insertions approximately 2.7 kb apart in the second and third exons, respectively, of the XDH coding region. The present study monitors intragenic recombination in a mutant heterozygous genotype in which DNA homology is disrupted by these large discontinuities, greater than the region of DNA homology and flanking both sides of the locus. If initiation/or termination requires separate sites at either end of the locus, then intragenic recombination within the rosy locus of the heterozygote should be eliminated. Contrary to expectation, significant recombination between these sites is seen. PMID:2834266

Reversible magnesium and aluminium ions insertion in cation-deficient anatase TiO2

NASA Astrophysics Data System (ADS)

Koketsu, Toshinari; Ma, Jiwei; Morgan, Benjamin J.; Body, Monique; Legein, Christophe; Dachraoui, Walid; Giannini, Mattia; Demortière, Arnaud; Salanne, Mathieu; Dardoize, François; Groult, Henri; Borkiewicz, Olaf J.; Chapman, Karena W.; Strasser, Peter; Dambournet, Damien

2017-11-01

In contrast to monovalent lithium or sodium ions, the reversible insertion of multivalent ions such as Mg2+ and Al3+ into electrode materials remains an elusive goal. Here, we demonstrate a new strategy to achieve reversible Mg2+ and Al3+ insertion in anatase TiO2, achieved through aliovalent doping, to introduce a large number of titanium vacancies that act as intercalation sites. We present a broad range of experimental and theoretical characterizations that show a preferential insertion of multivalent ions into titanium vacancies, allowing a much greater capacity to be obtained compared to pure TiO2. This result highlights the possibility to use the chemistry of defects to unlock the electrochemical activity of known materials, providing a new strategy for the chemical design of materials for practical multivalent batteries.

48 CFR 452.236-73 - Archaeological or Historic Sites.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Archaeological or Historic... CLAUSES AND FORMS SOLICITATION PROVISIONS AND CONTRACT CLAUSES Texts of Provisions and Clauses 452.236-73 Archaeological or Historic Sites. As prescribed in 436.573, insert the following clause: Archaeological or...

Tn5099, a xylE promoter probe transposon for Streptomyces spp.

PubMed Central

Hahn, D R; Solenberg, P J; Baltz, R H

1991-01-01

Tn5099, a promoter probe transposon for Streptomyces spp., was constructed by inserting a promoterless xylE gene and a hygromycin resistance gene into IS493. Tn5099 transposed into different sites in the Streptomyces griseofuscus genome, and the xylE reporter gene was expressed in some of the transposition mutants. Strains containing Tn5099 insertions that gave regulated expression of the xylE gene were identified. Images PMID:1653213

A randomized, crossover comparison of injected buffered lidocaine, lidocaine cream, and no analgesia for peripheral intravenous cannula insertion.

PubMed

McNaughton, Candace; Zhou, Chuan; Robert, Linda; Storrow, Alan; Kennedy, Robert

2009-08-01

We compare pain and anxiety associated with peripheral intravenous (IV) cannula insertion after pretreatment with no local anesthesia, 4% lidocaine cream, or subcutaneously injected, buffered 1% lidocaine. In a randomized, crossover design, 3 peripheral IVs were inserted in each of 70 medical students or nurses. In random order, insertion sites were pretreated with nothing, lidocaine cream, or injected, buffered lidocaine. After each IV insertion, subjects recorded pain, anxiety, and preference (as patient and provider) for each technique on a 10-point numeric rating scale. Higher scores indicated greater pain, anxiety, and preference. Median pain scores (interquartile range [IQR]) were 7 (4 to 8) without local anesthesia, 3 (2 to 5) with lidocaine cream, and 1 (1 to 2) with injected, buffered lidocaine. Median anxiety scores (IQR) were 4 (2 to 7) without local anesthesia, 2 (1 to 4) with lidocaine cream, and 2 (1 to 3) with injected, buffered lidocaine. There was no detectable difference in anxiety scores between lidocaine cream and injected, buffered lidocaine. Most IV placement attempts were successful, regardless of technique. Seventy percent of subjects indicated they would "always" request buffered lidocaine for peripheral IV insertion. In adult health care providers, pain and anxiety associated with peripheral IV insertion is significantly reduced by using topical lidocaine cream or injected, buffered lidocaine. Injected, buffered lidocaine reduces IV insertion pain more than lidocaine cream, without affecting success. Adults desire the use of local anesthetic techniques for IV insertion for themselves and for their patients.

Molecular Dynamics Simulations and Structural Analysis to Decipher Functional Impact of a Twenty Residue Insert in the Ternary Complex of Mus musculus TdT Isoform

PubMed Central

Mutt, Eshita; Sowdhamini, Ramanathan

2016-01-01

Insertions/deletions are common evolutionary tools employed to alter the structural and functional repertoire of protein domains. An insert situated proximal to the active site or ligand binding site frequently impacts protein function; however, the effect of distal indels on protein activity and/or stability are often not studied. In this paper, we have investigated a distal insert, which influences the function and stability of a unique DNA polymerase, called terminal deoxynucleotidyl transferase (TdT). TdT (EC:2.7.7.31) is a monomeric 58 kDa protein belonging to family X of eukaryotic DNA polymerases and known for its role in V(D)J recombination as well as in non-homologous end-joining (NHEJ) pathways. Two murine isoforms of TdT, with a length difference of twenty residues and having different biochemical properties, have been studied. All-atom molecular dynamics simulations at different temperatures and interaction network analyses were performed on the short and long-length isoforms. We observed conformational changes in the regions distal to the insert position (thumb subdomain) in the longer isoform, which indirectly affects the activity and stability of the enzyme through a mediating loop (Loop1). A structural rationale could be provided to explain the reduced polymerization rate as well as increased thermosensitivity of the longer isoform caused by peripherally located length variations within a DNA polymerase. These observations increase our understanding of the roles of length variants in introducing functional diversity in protein families in general. PMID:27311013

Anatomic and isometric points on femoral attachment site of popliteus muscle-tendon complex for the posterolateral corner reconstruction.

PubMed

Yang, Jae-Hyuk; Lim, Hong Chul; Bae, Ji Hoon; Fernandez, Harry; Bae, Tae Soo; Wang, Joon Ho

2011-10-01

Descriptive laboratory study. The femoral anatomic insertion site and the optimal isometric point of popliteus tendon for posterolateral reconstruction are not well known. Purpose of this study was to determine the relative relationship between the femoral anatomic insertion and isometric point of popliteus muscle-tendon complex with the lateral epicondyle of femur. Thirty unpaired cadaveric knees were dissected to determine the anatomic femoral insertion of the popliteus tendon. The distance and the angle from the lateral epicondyle of femur to the center of the anatomic insertion of the popliteus tendon were measured using digital caliper and goniometer. Eight unpaired fresh cadaveric knees were examined to determine the optimal isometric point of femoral insertion of popliteus tendon using computer-controlled motion capture analysis system (Motion Analysis, CA, USA). Distances from targeted tibial tunnel for popliteus tendon reconstruction to the 35 points gained on the lateral surface of femur were recorded at 0, 30, 60, 90, and 120° knee flexion. A point with the least excursion (<2.0 mm) was determined as the isometric point. The center of anatomic insertion points and the optimal isometric point for the main fibers of popliteus tendon were found to be posterior and distal to the lateral epicondyle of femur. The distance from the lateral epicondyle of femur to the center of anatomic femoral insertion of popliteus tendon was 11.3 ± 1.2 mm (mean ± SD). The angle between long axis of femur and the line from lateral epicondyle of femur to anatomic femoral insertion of popliteus tendon was 31.4 ± 5.3°. The isometric points for the femoral insertion of popliteus muscle-tendon complex were situated posterior and distal to the lateral epicondyle in all 8 knees. The distance between the least excursion point and the lateral epicondyle was calculated as 10.4 ± 1.7 mm. The angle between the long axis of femur and the line from lateral epicondyle of femur to optimum isometric point of popliteus tendon was calculated as 41.3 ± 14.9°. The optimal isometric point for the femoral insertion of popliteus muscle-tendon complex is situated posterior and distal to the lateral epicondyle of femur. Femoral tunnel for "posterolateral corner sling procedure" should be placed at this point to achieve least amount of graft excursion during knee motion.

Landscape of Insertion Polymorphisms in the Human Genome

PubMed Central

Onozawa, Masahiro; Goldberg, Liat; Aplan, Peter D.

2015-01-01

Nucleotide substitutions, small (<50 bp) insertions or deletions (indels), and large (>50 bp) deletions are well-known causes of genetic variation within the human genome. We recently reported a previously unrecognized form of polymorphic insertions, termed templated sequence insertion polymorphism (TSIP), in which the inserted sequence was templated from a distant genomic region, and was inserted in the genome through reverse transcription of an RNA intermediate. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; class 1 TSIPs show target site duplication, polyadenylation, and preference for insertion at a 5′-TTTT/A-3′ sequence, suggesting a LINE-1 based insertion mechanism, whereas class 2 TSIPs show features consistent with repair of a DNA double strand break by nonhomologous end joining. To gain a more complete picture of TSIPs throughout the human population, we evaluated whole-genome sequence from 52 individuals, and identified 171 TSIPs. Most individuals had 25–30 TSIPs, and common (present in >20% of individuals) TSIPs were found in individuals throughout the world, whereas rare TSIPs tended to cluster in specific geographic regions. The number of rare TSIPs was greater than the number of common TSIPs, suggesting that TSIP generation is an ongoing process. Intriguingly, mitochondrial sequences were a frequent template for class 2 insertions, used more commonly than any nuclear chromosome. Similar to single nucleotide polymorphisms and indels, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases, and can be useful in tracking historical migration of populations. PMID:25745018

Conductivity Probe Inserted in Martian Soil, Sol 46

NASA Technical Reports Server (NTRS)

2008-01-01

This image taken by the Surface Stereo Imager on NASA's Phoenix Mars Lander shows the lander's Thermal and Electrical Conductivity Probe (TECP), at the end of the Robotic Arm, on the 46th Martian day, or sol, of the mission (July 11, 2008).

The TECP is inserted at a site called Vestri, which was monitored several times over the course of the mission. The probe's measurements at this site yielded evidence that water was exchanged, daily and seasonally, between the soil and atmosphere.

The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

Efficient Mutagenesis Independent of Ligation (EMILI).

PubMed

Füzik, Tibor; Ulbrich, Pavel; Ruml, Tomáš

2014-11-01

Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealing of the PCR product's sticky ends, which are generated by exonuclease digestion. This method is fast, extremely efficient and cost-effective. It can be used to introduce large insertions and deletions, but also for multiple point mutations in a single step. To show the principle and to prove the efficiency of the method, we present a series of basic mutations (insertions, deletions, point mutations) on pUC19 plasmid DNA. Copyright © 2014 Elsevier B.V. All rights reserved.

Insertion of inter-domain linkers improves expression and bioactivity of Zygote arrest (Zar) fusion proteins.

PubMed

Cook, Jonathan M; Charlesworth, Amanda

2017-04-01

Developmentally important proteins that are crucial for fertilization and embryogenesis are synthesized through highly regulated translation of maternal mRNA. The Zygote arrest proteins, Zar1 and Zar2, are crucial for embryogenesis and have been implicated in binding mRNA and repressing mRNA translation. To investigate Zar1 and Zar2, the full-length proteins had been fused to glutathione-S-transferase (GST) or MS2 protein tags with minimal inter-domain linkers derived from multiple cloning sites; however, these fusion proteins expressed poorly and/or lacked robust function. Here, we tested the effect of inserting additional linkers between the fusion domains. Three linkers were tested, each 17 amino acids long with different physical and chemical properties: flexible hydrophilic, rigid extended or rigid helical. In the presence of any of the three linkers, GST-Zar1 and GST-Zar2 had fewer breakdown products. Moreover, in the presence of any of the linkers, MS2-Zar1 was expressed to higher levels, and in dual luciferase tethered assays, both MS2-Zar1 and MS2-Zar2 repressed luciferase translation to a greater extent. These data suggest that for Zar fusion proteins, increasing the length of linkers, regardless of their physical or chemical properties, improves stability, expression and bioactivity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Antibacterial nanosilver coated orthodontic bands with potential implications in dentistry.

PubMed

Prabha, Rahul Damodaran; Kandasamy, Rajasigamani; Sivaraman, U Sajeev; Nandkumar, Maya A; Nair, Prabha D

2016-10-01

Fixed orthodontic treatment, an indispensable procedure in orthodontics, necessitates insertion of dental bands. Insertion of band material could also introduce a site of plaque retention. It was hypothesized that band materials with slow-release antimicrobial properties could help in sustained infection control, prevention of dental plaque formation and further associated health risks. Considering the known antimicrobial proprieties of silver, a coating of silver nanoparticle (SNP) onto the stainless steel bands was done and characterized for its beneficial properties in the prevention of plaque accumulation. Coatings of SNPs on conventional stainless steel dental bands were prepared using thermal evaporation technology. The coated dental bands were characterized for their physicochemical properties and evaluated for antimicrobial activity and biocompatibility. The physiochemical characterization of band material both coated and uncoated was carried out using scanning electron microscope, energy dispersive spectroscopy, atomic force microscopyand contact angle test. Biocompatibility tests for coated band material were carried using L929 mouse fibroblast cell culture and MTT [3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. Antimicrobial activity of coated band material against Gram-positive bacteria was tested. A stable and uniform coating of SNPs was obtained. The coated band materials were biocompatible as well as possessed distinct antimicrobial activity. The SNP coated dental bands could be potential antimicrobial dental bands for future clinical use. Further studies need to be done to validate the efficiency of coated band materials in oral environments.

Detailed anatomy of the abducens nerve in the lateral rectus muscle.

PubMed

Nam, Yong Seok; Kim, In-Beom; Shin, Sun Young

2017-10-01

The aims of this study were to elucidate the detailed anatomy of the abducens nerve in the lateral rectus muscle (LRM) and the intramuscular innervation pattern using Sihler staining. In this cohort study, 32 eyes of 16 cadavers were assessed. Dissection was performed from the LRM origin to its insertion. The following distances were measured: from LRM insertion to the bifurcation point of the abducens nerve, from LRM insertion to the entry site of the superior branch or inferior branch, from the upper border of the LRM to the entry site of the superior branch, from the lower border of LRM to the entry site of inferior branch, and the widths of the main trunk and superior and inferior branches. The single trunk of the abducens nerve divided into two branches 37 mm from insertion of the LRM, and 22 of 32 (68.8%) orbits showed only two superior and inferior branches with no subdivision. The superior branch entered the LRM more anteriorly (P = 0.037) and the superior branch was thinner than the inferior branch (P = 0.040). The most distally located intramuscular nerve ending was observed at 52.9 ± 3.5% of the length of each muscle. Non-overlap between the superior and inferior intramuscular arborization of the nerve was detected in 27 of 32 cases (84.4%). Five cases (15.6%) showed definite overlap of the superior and inferior zones. This study revealed the detailed anatomy of the abducens nerve in the LRM and provides helpful information to understand abducens nerve palsy. Clin. Anat. 30:873-877, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Hepatitis B Virus Core Gene Mutations Which Block Nucleocapsid Envelopment

PubMed Central

Koschel, Matthias; Oed, Daniela; Gerelsaikhan, Tudevdagwa; Thomssen, Reiner; Bruss, Volker

2000-01-01

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment. PMID:10590084

Protective effects of biodegradable collagen implants on thinned sclera after strabismus surgery: a paired-eye study.

PubMed

Yoo, Tae Keun; Han, Sueng-Han; Han, Jinu

2017-12-01

To determine the efficacy of a biodegradable Ologen (Aeon Astron Europe BV, Leiden, The Netherlands) collagen matrix in reducing the blue color change due to exposed thinned sclera after strabismus surgery. Fourteen patients with intermittent exotropia undergoing symmetric bilateral lateral rectus recession surgery were included in this prospective, randomized, paired-eye controlled study. In each patient, Ologen was implanted at the original rectus insertion site in one randomly selected eye; the other eye underwent conventional surgery. Ologen was inserted under the conjunctiva without suturing, covering the muscle insertion site. Conjunctival color change was analyzed using computer-based image analysis immediately and 1 week, 1 month, and 3 months postoperatively. Slit-lamp photographs of each eye were evaluated using contrast limited adaptive histogram equalization (CLAHE), Canny edge, and the RGB (red-green-blue) model. Secondary outcomes were conjunctival and sclera thickness 3 months postoperatively determined by anterior segment optical coherence tomography. Immediately and 1 week postoperatively all color models showed no significant differences between Ologen-implanted and control eyes. Three months postoperatively, Ologen-implanted eyes exhibited significantly lower CLAHE (P = 0.041) and RGB model blue color (P = 0.008) values than control eyes. Canny edge (P = 0.061) and RGB model red color (P = 0.152) values did not differ between eyes. Conjunctival stroma and episcleral complex thickness was greater in Ologen-implanted eyes than in controls (P = 0.001). Blue color change was significantly less noticeable in Ologen-implanted eyes than in controls. Thus, Ologen implantation helps prevent visible blue sclera at the original rectus insertion site after lateral rectus recession. Copyright © 2017 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.

Single-row versus double-row capsulolabral repair: a comparative evaluation of contact pressure and surface area in the capsulolabral complex-glenoid bone interface.

PubMed

Kim, Doo-Sup; Yoon, Yeo-Seung; Chung, Hoi-Jeong

2011-07-01

Despite the attention that has been paid to restoration of the capsulolabral complex anatomic insertion onto the glenoid, studies comparing the pressurized contact area and mean interface pressure at the anatomic insertion site between a single-row repair and a double-row labral repair have been uncommon. The purpose of our study was to compare the mean interface pressure and pressurized contact area at the anatomic insertion site of the capsulolabral complex between a single-row repair and a double-row repair technique. Controlled laboratory study. Thirty fresh-frozen cadaveric shoulders (mean age, 61 ± 8 years; range, 48-71 years) were used for this study. Two types of repair were performed on each specimen: (1) a single-row repair and (2) a double-row repair. Using pressure-sensitive films, we examined the interface contact area and contact pressure. The mean interface pressure was greater for the double-row repair technique (0.29 ± 0.04 MPa) when compared with the single-row repair technique (0.21 ± 0.03 MPa) (P = .003). The mean pressurized contact area was also significantly greater for the double-row repair technique (211.8 ± 18.6 mm(2), 78.4% footprint) compared with the single-row repair technique (106.4 ± 16.8 mm(2), 39.4% footprint) (P = .001). The double-row repair has significantly greater mean interface pressure and pressurized contact area at the insertion site of the capsulolabral complex than the single-row repair. The double-row repair may be advantageous compared with the single-row repair in restoring the native footprint area of the capsulolabral complex.

Robot-assisted transnasal laryngoplasty in cadaveric models: Quantifying forces and identifying challenges.

PubMed

Groom, Kelly; Wang, Long; Simaan, Nabil; Netterville, James

2015-05-01

We expanded our prior work with transnasal robotic surgery (TNRS) in this study with the following aims: 1) use a cadaveric model to evaluate the feasibility of laryngoplasty with TNRS, 2) measure robot insertion times and forces, and 3) identify operational challenges to further guide the development of a flexible robotic system. Cadaveric study. A 5-mm robot was guided to the larynx via a transnasal approach. Insertion times and forces using TNRS and a 4-mm flexible fiberoptic laryngoscope (FFL) were measured. Target sites on the true vocal cords were marked, and the TNRS was telemanipulated to perform injection laryngoplasty. Insertion times averaged 5.05 seconds (range, 3.8-10.4 seconds) for the TNRS and 7.97 seconds (range, 6.2-11.6 seconds) for the FFL. Insertion forces averaged 2.06 newtons (range, 1.56-5.55 newtons) for the TNRS and 0.43 newtons (range, 0.157-1.138 newtons) for the FFL. The unpaired t test between times and forces revealed P values of .0024 and .0000658, respectively. Seven target injection sites on three vocal cords in two cadaveric larynxes were successfully injected. In two out of nine sites marked, we were unable to access the vocal cord due tongue base collapse that obscured the posterior airway. TNRS is able to effectively access the larynx, although in a supine model may be limited by tongue base collapse. Forces with TNRS were significantly higher than with the FFL, albeit within the same scale. Despite increased forces, there was no evidence of tissue trauma using TNRS. NA Laryngoscope, 125:1166-1168, 2015. © 2015 The American Laryngological, Rhinological and Otological Society, Inc.

Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

PubMed Central

Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

2011-01-01

Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

Evaluating the patient experience after implantation of a 0.4 mg sustained release dexamethasone intracanalicular insert (Dextenza™): results of a qualitative survey

PubMed Central

Gira, Joseph P; Sampson, Reginald; Silverstein, Steven M; Walters, Thomas R; Metzinger, Jamie Lynne; Talamo, Jonathan H

2017-01-01

Purpose The purpose of this study is to evaluate the patient experience of sustained release dexamethasone intracanalicular insert (Dextenza™) following cataract surgery as part of a Phase III clinical trial program. Methods This cross-sectional, qualitative evaluation involved individual interviews lasting approximately 45 minutes. Patients from four US investigational study sites who had previously received an insert were enrolled. There were no predesignated end points; this was a qualitative survey seeking a deeper understanding of patient experience. Results Twenty-five patients were interviewed. Most patients (92%) reported the highest level of satisfaction grade with regard to overall product satisfaction. All patients described the insert as comfortable. Most patients (96%) described their overall experience with the insert as very convenient or extremely convenient. Twenty-two of 23 (96%) participants rated their experience with the insert as “very” or “extremely convenient”, compared to previous topical therapy, and 88% of patients stated that if they were to undergo cataract surgery again, they would request the insert. When asked if they would recommend the insert to family members or friends, 92% stated they would. The survey found that 84% of participants would be willing to pay more for the insert than for eye drop therapy. Conclusion The dexamethasone insert was found by patients to be highly favorable with regard to overall satisfaction, convenience, and comfort. The insert was well received and largely preferred over topical therapy alternatives following surgery. More extensive evaluation of the patient experience is warranted, and future studies should help inform design of the next generation of sustained release drug delivery systems. PMID:28331295

A multicenter randomized clinical trial of one-rod etonogestrel and two-rod levonorgestrel contraceptive implants with nonrandomized copper-IUD controls: methodology and insertion data.

PubMed

Meirik, Olav; Brache, Vivian; Orawan, Kiriwat; Habib, Ndema Abu; Schmidt, Johannes; Ortayli, Nuriye; Culwell, Kelly; Jackson, Emily; Ali, Moazzam

2013-01-01

Comparative data on etonogestrel and two-rod levonorgestrel contraceptive implants are lacking. A multicenter, open, parallel-group trial with random allocation of implants was performed. For every second implant user, an age-matched woman choosing an intrauterine device (IUD) (TCu380A) was admitted. Methods and data on implant/IUD insertion and 6-week follow-up are reported. A total of 2008 women were randomized to an implant, and 974 women were enrolled in the IUD group. Results from 997 etonogestrel implant users, 997 levonorgestrel implant users and 971 IUD users were analyzed. In the etonogestrel and levonorgestrel groups, respectively, mean insertion durations were 51 (SD 50.2) s and 88 (SD 60.8) s; complication rates at insertion were 0.8% and 0.2%; and at follow-up, 27.2% and 26.7% of women, respectively, had signs or symptoms at the insertion site. At follow-up within 6 weeks after insertion, all implants were in situ, while 2.1% of IUDs were expelled. Performance of etonogestrel and levonorgestrel implants at insertion and within the first 6 weeks is similar. Short-term (6 weeks) continuation rates appear higher for implants than TCu380A. Copyright © 2013 Elsevier Inc. All rights reserved.

Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.

PubMed

Jia, Xianbo; Lin, Xinjian; Chen, Jichen

2017-11-02

Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.

Targeted gene insertion for molecular medicine.

PubMed

Voigt, Katrin; Izsvák, Zsuzsanna; Ivics, Zoltán

2008-11-01

Genomic insertion of a functional gene together with suitable transcriptional regulatory elements is often required for long-term therapeutical benefit in gene therapy for several genetic diseases. A variety of integrating vectors for gene delivery exist. Some of them exhibit random genomic integration, whereas others have integration preferences based on attributes of the targeted site, such as primary DNA sequence and physical structure of the DNA, or through tethering to certain DNA sequences by host-encoded cellular factors. Uncontrolled genomic insertion bears the risk of the transgene being silenced due to chromosomal position effects, and can lead to genotoxic effects due to mutagenesis of cellular genes. None of the vector systems currently used in either preclinical experiments or clinical trials displays sufficient preferences for target DNA sequences that would ensure appropriate and reliable expression of the transgene and simultaneously prevent hazardous side effects. We review in this paper the advantages and disadvantages of both viral and non-viral gene delivery technologies, discuss mechanisms of target site selection of integrating genetic elements (viruses and transposons), and suggest distinct molecular strategies for targeted gene delivery.

Structure and Expression of Hybrid Dysgenesis-Induced Alleles of the Ovarian Tumor (Otu) Gene in Drosophila Melanogaster

PubMed Central

Sass, G. L.; Mohler, J. D.; Walsh, R. C.; Kalfayan, L. J.; Searles, L. L.

1993-01-01

Mutations at the ovarian tumor (otu) gene of Drosophila melanogaster cause female sterility and generate a range of ovarian phenotypes. Quiescent (QUI) mutants exhibit reduced germ cell proliferation; in oncogenic (ONC) mutants germ cells undergo uncontrolled proliferation generating excessive numbers of undifferentiated cells; the egg chambers of differentiated (DIF) mutants differentiate to variable degrees but fail to complete oogenesis. We have examined mutations caused by insertion and deletion of P elements at the otu gene. The P element insertion sites are upstream of the major otu transcription start sites. In deletion derivatives, the P element, regulatory regions and/or protein coding sequences have been removed. In both insertion and deletion mutants, the level of otu expression correlates directly with the severity of the phenotype: the absence of otu function produces the most severe QUI phenotype while the ONC mutants express lower levels of otu than those which are DIF. The results of this study demonstrate that the diverse mutant phenotypes of otu are the consequence of different levels of otu function. PMID:8436274

Method for introducing unidirectional nested deletions

DOEpatents

Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

2001-01-01

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment in the context of a cloning vector which contains an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment. Also disclosed is a method for producing single-stranded DNA probes utilizing the same cloning vector. An optimal vector, PZIP is described. Methods for introducing unidirectional deletions into a terminal location of a cloned DNA sequence which is inserted into the vector of the present invention are also disclosed. These methods are useful for introducing deletions into either or both ends of a cloned DNA insert, for high throughput sequencing of any DNA of interest.

Structural changes in the S 3 state of the oxygen evolving complex in photosystem II

DOE PAGES

Hatakeyama, Makoto; Ogata, Koji; Fujii, Katsushi; ...

2016-03-19

The S 3 state of the Mn 4CaO 5-cluster in photosystem II was investigated by DFT calculations and compared with EXAFS data. Considering previously proposed mechanism; a water molecule is inserted into an open coordination site of Mn upon S 2 to S 3 transition that becomes a substrate water, we examined if the water insertion is essential for the S 3 formation, or if one cannot eliminate other possible routes that do not require a water insertion at the S 3 stage. The novel S 3 state structure consisting of only short 2.7–2.8 Å MnMn distances was discussed.

Structural changes in the S 3 state of the oxygen evolving complex in photosystem II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatakeyama, Makoto; Ogata, Koji; Fujii, Katsushi

The S 3 state of the Mn 4CaO 5-cluster in photosystem II was investigated by DFT calculations and compared with EXAFS data. Considering previously proposed mechanism; a water molecule is inserted into an open coordination site of Mn upon S 2 to S 3 transition that becomes a substrate water, we examined if the water insertion is essential for the S 3 formation, or if one cannot eliminate other possible routes that do not require a water insertion at the S 3 stage. The novel S 3 state structure consisting of only short 2.7–2.8 Å MnMn distances was discussed.

Peripherally inserted central catheters in the neonatal period.

PubMed

Uygun, Ibrahim; Okur, Mehmet Hanifi; Otcu, Selcuk; Ozturk, Hayrettin

2011-10-01

Peripherally inserted central catheters (PICC) have been extensively used in neonates. However, insertion of these thinnest catheters is a very delicate procedure associated with a high failure rate. In our Neonatal Surgical Intensive Care Unit, we developed a very easy new PICC insertion and evaluated the neonates treated with PICCs which were inserted by using our technique as well as catheter features such as success rate, number of insertion attempts, reason for removal and complications. Information was retrospectively collected on all 40 PICCs inserted at Kutahya Evliya Celebi Goverment Hospital and Dicle University Hospital during a 6-years period from September 2004 to September 2010. A total of 40 PICCs were inserted in 37 patients (26, 70% males, 11, 30% females) by using new technique. The median age of patients was 8.3 days (range 1 to 66 days) and the median weight of patients was 2365 g (range 600 to 5000 g). The vein most commonly accessed was long saphenous vein (85%). The length of PICCs in the body was 19.6 cm (range 5 cm to 30 cm). The tip was located in a central vein in all patients. Surgical abdomen was the most common cause for PICC insertion (38%). Duration of catheterization was 7.7±5.6 days (1-F 5.5 days, 2-F 8.6 days). Almost all of the PICCs were inserted successfully (40/42, success rate 95%) and in the first venipucture (36/42, 86%). Completion of therapy and removed after death were achieved with 87% of PICCs. Three minor complications were noted. Minor bleeding in the insertion site which was stopped via compression occurred in two neonates. Major complication was not seen. No deaths were directly attributed to PICCs use. The new insertion technique of the neonatal peripherally inserted central catheters may be one of the easiest and safest techniques, in comparison to previous techniques reported in the literature.

In-office insertion of a miniaturized insertable cardiac monitor: Results from the Reveal LINQ In-Office 2 randomized study.

PubMed

Rogers, John D; Sanders, Prashanthan; Piorkowski, Christopher; Sohail, M Rizwan; Anand, Rishi; Crossen, Karl; Khairallah, Farhat S; Kaplon, Rachelle E; Stromberg, Kurt; Kowal, Robert C

2017-02-01

Recent miniaturization of an insertable cardiac monitor (ICM) may make it possible to move device insertion from a hospital to office setting. However, the safety of this strategy is unknown. The primary objective was to compare the safety of inserting the Reveal LINQ ICM in an office vs a hospital environment. Ancillary objectives included summarizing device- and procedure-related adverse events and responses to a physician questionnaire. Five hundred twenty-one patients indicated for an ICM were randomized (1:1 ratio) to undergo ICM insertion in a hospital or office environment at 26 centers in the United States in the Reveal LINQ In-Office 2 study (ClinicalTrials.gov identifier NCT02395536). Patients were followed for 90 days. ICM insertion was successful in all 482 attempted patients (office: 251; hospital: 231). The untoward event rate (composite of unsuccessful insertion and ICM- or insertion-related complications) was 0.8% (2 of 244) in the office and 0.9% (2 of 227) in the hospital (95% confidence interval, -3.0% to 2.9%; 5% noninferiority: P < .001). In addition, adverse events occurred during 2.5% (6 of 244) of office and 4.4% (10 of 227) of hospital insertions (95% confidence interval [office minus inhospital rates], -5.8% to 1.9%; 5% noninferiority: P < .001). Physicians indicated that for procedures performed in an office vs a hospital, there were fewer delays >15 minutes (16% vs 35%; P < .001) and patient response was more often "very positive." Physicians considered the office location "very convenient" more frequently than the hospital location (85% vs 27%; P < .001). The safety profile for the insertion of the Reveal LINQ ICM is excellent irrespective of insertion environment. These results may expand site of service options for LINQ insertion. Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus.

PubMed

Barutcu, A Rasim; Maass, Philipp G; Lewandowski, Jordan P; Weiner, Catherine L; Rinn, John L

2018-04-13

The binding of the transcriptional regulator CTCF to the genome has been implicated in the formation of topologically associated domains (TADs). However, the general mechanisms of folding the genome into TADs are not fully understood. Here we test the effects of deleting a CTCF-rich locus on TAD boundary formation. Using genome-wide chromosome conformation capture (Hi-C), we focus on one TAD boundary on chromosome X harboring ~ 15 CTCF binding sites and located at the long non-coding RNA (lncRNA) locus Firre. Specifically, this TAD boundary is invariant across evolution, tissues, and temporal dynamics of X-chromosome inactivation. We demonstrate that neither the deletion of this locus nor the ectopic insertion of Firre cDNA or its ectopic expression are sufficient to alter TADs in a sex-specific or allele-specific manner. In contrast, Firre's deletion disrupts the chromatin super-loop formation of the inactive X-chromosome. Collectively, our findings suggest that apart from CTCF binding, additional mechanisms may play roles in establishing TAD boundary formation.

Social Class, Identity, and Migrant Students

ERIC Educational Resources Information Center

Darvin, Ron; Norton, Bonny

2014-01-01

A necessary component of the neoliberal mechanisms of globalization, migration addresses the economic and labor needs of postindustrial countries while producing new modes of social fragmentation and inequality (Crompton, 2008). As migrant students insert themselves into segmented spaces, their countries of origin are themselves implicated in a…

Transfemoral transcatheter aortic valve insertion-related intraoperative morbidity: Implications of the minimalist approach.

PubMed

Greason, Kevin L; Pochettino, Alberto; Sandhu, Gurpreet S; King, Katherine S; Holmes, David R

2016-04-01

Transfemoral transcatheter aortic valve insertion may be performed in a catheterization laboratory (ie, the minimalist approach). It seems reasonable when considering this approach to avoid it in patients at risk for intraoperative morbidity that would require surgical intervention. We hypothesized that it would be possible to associate baseline characteristics with such morbidity, which would help heart teams select patients for the minimalist approach. We reviewed the records of 215 consecutive patients who underwent transfemoral transcatheter aortic valve insertion with a current commercially available device from November 2008 through July 2015. Demographic characteristics of the patients included a mean age of 78.9 ± 10.6 years, female sex in 73 patients (34.0%), and a mean Society of Thoracic Surgeons predicted risk of mortality of 8.7% ± 5.4%. Valve prostheses were balloon-expandable in 126 patients (58.6%) and self-expanding in 89 patients (41.4%). Significant intraoperative morbidity occurred in 22 patients (10.2%) and included major vascular injury in 12 patients (5.6%), hemodynamic compromise requiring cardiopulmonary bypass support in 4 patients (1.9%), cardiac tamponade requiring intervention in 3 patients (1.4%), ventricular valve embolization in 2 patients (0.9%), and inability to obtain percutaneous access requiring open vascular access in 1 patient (0.5%). Intraoperative morbidity was similarly distributed across all valve types (P = .556) and sheath sizes (P = .369). There were no baseline patient characteristics predictive of intraoperative morbidity. Patient and valve characteristics are not predictive of significant intraoperative morbidity during transfemoral transcatheter aortic valve insertion. The finding has implications for patient selection for the minimalist approach. Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Related factors with extravasation of non-cytostatic agents in peripheral vein catheters.

PubMed

Fernández-García, Cristina; Mata-Peón, Esther; Avanzas-Fernández, Sara

To know the independent variables related to the occurrence of extravasation in patients with peripheral vein catheters (PVC). Retrospective study carried out in 6 longitudinal cuts between July 2013 an January 2014. A total of 1,442 PVC were reviewed, of which 730 met the inclusion criteria, and were divided into 2 groups: extravasation and not extravasation, with 365 cases each. The variables of age, gender, admission unit, catheter gauge, insertion site, previous insertion into the same limb, hospital unit where the insertion took place, communication difficulties, personal health history and analyzed parenterally drug administered were considered. Risk factors to develop extravasation were: female gender, with previous insertion in the same limb, <72h PVC of insertion, communication difficulties, personal health history of neoplasia and KCl, gentamicin or beta lactam treatment. Our study allows to know the variables that are related to the emergence of extravasations in patients with non-cancer treatments (gender, medical service of admission, catheter gauge, elapsed time since the insertion, patient communication difficulties, personal health history, and intravenous treatments), as well as the factors that may be considered protective. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

The LENR-CANR.ORG Website, its Past and Future

NASA Astrophysics Data System (ADS)

Rothwell, J.; Storms, E.

2005-12-01

The LENR-CANR.org web site has proven to be a popular source of information about cold fusion. This site has distributed more full text papers about LENR than any other source. In addition, it contains many features that allow easy search and insertion of the discovered references into a document.

Tissue reaction of deproteinized bovine bone matrix grafting in ectopic site: histological study on sheep.

PubMed

Grossi, João Ricardo Almeida; Bonacin, Rodrigo; Crivelaro, Viviane Rozeira; Giovanini, Allan Fernando; Zielak, João César; Deliberador, Tatiana Miranda

2016-12-01

The aim of this paper was to evaluate through histological analysis of the tissue reaction of deproteinized bovine bone matrix (DBBM) when inserted into the site of intramuscular ectopic sheep. In this study, 16 sheep received 3 groups and these were divided into 2 experimental times: Group 1-sham group, Group 2-particulate autogenous bone and Group 3-DBBM granules. All animals underwent surgical procedures for insertion of materials in an ectopic site (muscles of the lower back and after 3 and 6 months postoperatively, the samples were evaluated by histological analysis. The results indicated that the Sham group showed dense collagen fibers and thin, characterizing fibrosis at 3 and 6 months. In the autograft group there was a significant amount of collagen deposition and decreased inflammation at 6 months postoperatively. Group of DBBM, it was noted the presence of dense connective tissue and surrounding remaining particles was observed the formation of with osteoid characteristic tissue. The DBBM demonstrated biocompatibility, osteoconductivity and small osteogenesis capacity on ectopic site.

Get3 is a holdase chaperone and moves to deposition sites for aggregated proteins when membrane targeting is blocked

PubMed Central

Powis, Katie; Schrul, Bianca; Tienson, Heather; Gostimskaya, Irina; Breker, Michal; High, Stephen; Schuldiner, Maya; Jakob, Ursula; Schwappach, Blanche

2013-01-01

Summary The endomembrane system of yeast contains different tail-anchored proteins that are post-translationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment could be hazardous in the cytosol if membrane insertion fails, resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum, where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function: promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis. PMID:23203805

IS1598 (IsPg4) distributed to abscess-forming strains of Porphyromonas gingivalis may enhance virulence through upregulation of nrdD-like gene expression.

PubMed

Sonoi, Norihiro; Maeda, Hiroshi; Murauchi, Toshimitsu; Yamamoto, Tadashi; Omori, Kazuhiro; Kokeguchi, Susumu; Naruishi, Koji; Takashiba, Shogo

2018-01-01

An insertion sequence, IS1598 (IsPg4) has been found in virulent strains of Porphyromonas gingivalis in a murine abscess model. The present study was performed to investigate the effects of genetic rearrangements by IS1598 on the phenotypic characteristics of the virulent strains. For this purpose, we searched for a common insertion site of IS1598 among the virulent strains. Through cloning and database search, a common insertion site was identified beside an nrdD-like gene in the virulent FDC 381, W83 and W50 strains. In this region, predicted promoters of the nrdD-like gene and IS1598 are located in tandem, and accumulation of nrdD-like gene mRNA was 5-fold higher in virulent strains (W83, W50, FDC 381) than avirulent strains (ATCC33277, SU63, SUNY1021, ESO59 without IS1598). The role of the nrdD-like gene in virulence of P. gingivalis was investigated by constructing a nrdD-deficient mutant. In the murine abscess model, the parental W83 strain produced necrotic abscesses, while the nrdD-deficient mutant had almost lost this ability. Insertion of IS1598 into the nrdD-like gene promoter region may be related to the phenotypic differences in virulence among P. gingivalis strains through upregulation of the expression of this gene.

Reversible magnesium and aluminium ions insertion in cation-deficient anatase TiO 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koketsu, Toshinari; Ma, Jiwei; Morgan, Benjamin J.

In contrast to monovalent lithium or sodium ions, the reversible insertion of multivalent ions such as Mg 2+ and Al 3+ into electrode materials remains an elusive goal. In this work, we demonstrate a new strategy to achieve reversible Mg 2+ and Al 3+ insertion in anatase TiO 2, achieved through aliovalent doping, to introduce a large number of titanium vacancies that act as intercalation sites. We present a broad range of experimental and theoretical characterizations that show a preferential insertion of multivalent ions into titanium vacancies, allowing a much greater capacity to be obtained compared to pure TiO 2.more » In conclusion, this result highlights the possibility to use the chemistry of defects to unlock the electrochemical activity of known materials providing a new strategy for the chemical design of materials for practical multivalent batteries.« less

Reversible magnesium and aluminium ions insertion in cation-deficient anatase TiO 2

DOE PAGES

Koketsu, Toshinari; Ma, Jiwei; Morgan, Benjamin J.; ...

2017-09-18

In contrast to monovalent lithium or sodium ions, the reversible insertion of multivalent ions such as Mg 2+ and Al 3+ into electrode materials remains an elusive goal. In this work, we demonstrate a new strategy to achieve reversible Mg 2+ and Al 3+ insertion in anatase TiO 2, achieved through aliovalent doping, to introduce a large number of titanium vacancies that act as intercalation sites. We present a broad range of experimental and theoretical characterizations that show a preferential insertion of multivalent ions into titanium vacancies, allowing a much greater capacity to be obtained compared to pure TiO 2.more » In conclusion, this result highlights the possibility to use the chemistry of defects to unlock the electrochemical activity of known materials providing a new strategy for the chemical design of materials for practical multivalent batteries.« less

How Y-Family DNA polymerase IV is more accurate than Dpo4 at dCTP insertion opposite an N2-dG adduct of benzo[a]pyrene.

PubMed

Sholder, Gabriel; Creech, Amanda; Loechler, Edward L

2015-11-01

To bypass DNA damage, cells have Y-Family DNA polymerases (DNAPs). One Y-Family-class includes DNAP κ and DNAP IV, which accurately insert dCTP opposite N(2)-dG adducts, including from the carcinogen benzo[a]pyrene (BP). Another class includes DNAP η and DNAP V, which insert accurately opposite UV-damage, but inaccurately opposite BP-N(2)-dG. To investigate structural differences between Y-Family-classes, regions are swapped between DNAP IV (a κ/IV-class-member) and Dpo4 (a η/V-class-member); the kinetic consequences are evaluated via primer-extension studies with a BP-N(2)-dG-containing template. Four key structural elements are revealed. (1) Y-Family DNAPs have discreet non-covalent contacts between their little finger-domain (LF-Domain) and their catalytic core-domain (CC-Domain), which we call "non-covalent bridges" (NCBs). Arg37 and Arg38 in DNAP IV's CC-Domain near the active site form a non-covalent bridge (AS-NCB) by interacting with Glu251 and Asp252, respectively, in DNAP IV's LF-Domain. Without these interactions dATP/dGTP/dTTP misinsertions increase. DNAP IV's AS-NCB suppresses misinsertions better than Dpo4's equivalent AS-NCB. (2) DNAP IV also suppresses dATP/dGTP/dTTP misinsertions via a second non-covalent bridge, which is ∼8Å from the active site (Distal-NCB). Dpo4 has no Distal-NCB, rendering it inferior at dATP/dGTP/dTTP suppression. (3) dCTP insertion is facilitated by the larger minor groove opening near the active site in DNAP IV versus Dpo4, which is sensible given that Watson/Crick-like [dCTP:BP-N(2)-dG] pairing requires the BP-moiety to be in the minor groove. (4) Compared to Dpo4, DNAP IV has a smaller major groove opening, which suppresses dGTP misinsertion, implying BP-N(2)-dG bulk in the major groove during Hoogsteen syn-adduct-dG:dGTP pairing. In summary, DNAP IV has a large minor groove opening to enhance dCTP insertion, a plugged major groove opening to suppress dGTP misinsertion, and two non-covalent bridges (near and distal to the active site) to suppress dATP/dGTP/dTTP misinsertions; collectively these four structural features enhance DNAP IV's dNTP insertion fidelity opposite a BP-N(2)-dG adduct compared to Dpo4. Copyright © 2015 Elsevier B.V. All rights reserved.

Sex and the community: the implications of neighbourhoods and social networks for sexual risk behaviours among urban gay men.

PubMed

Kelly, Brian C; Carpiano, Richard M; Easterbrook, Adam; Parsons, Jeffrey T

2012-09-01

Gay neighbourhoods have historically served as vital places for gay socialising, and gay social networks are important sources of social support. Yet, few studies have examined the influence of these forms of community on sexual health. Informed by theoretical frameworks on neighbourhoods and networks, we employ multi-level modelling to test hypotheses concerning whether gay neighbourhoods and social network factors are associated with five sexual risk behaviours: receptive and insertive unprotected anal intercourse (UAI), barebacking identity, recent internet use for finding sexual partners, and 'Party and Play' (PnP). Our analyses of a community-based sample of gay men in New York City reveal little evidence for the direct effect of gay enclaves on sexual risk with the exception of PnP, which was more likely among gay enclave residents. Having a network composed predominantly of other gay men was associated with insertive UAI, PnP, and internet use for meeting sexual partners. This network type also mediated the association between gay neighbourhoods and higher odds of insertive UAI as well as PnP. Our findings highlight the sexual health implications of two important facets of gay community and, in doing so, indicate the need to better contextualise the sexual health risks faced by gay men. © 2012 The Authors. Sociology of Health & Illness © 2012 Foundation for the Sociology of Health & Illness/Blackwell Publishing Ltd.

Plasmid partition system of the P1par family from the pWR100 virulence plasmid of Shigella flexneri.

PubMed

Sergueev, Kirill; Dabrazhynetskaya, Alena; Austin, Stuart

2005-05-01

P1par family members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by a parS partition site. The large virulence plasmid pWR100 of Shigella flexneri contains a new P1par family member: pWR100par. Although typical parA and parB genes are present, the putative pWR100parS site is atypical in sequence and organization. However, pWR100parS promoted accurate plasmid partition in Escherichia coli when the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs within parS define species specificities among previously described family members. Although substantially different from P1parS from the P1 plasmid prophage of E. coli, pWR100parS has the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parS and P1parS is the presence of a 21-bp insert at the center of the pWR100parS site. Deletion of this insert left much of the parS activity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.

Lentiviral-Mediated Gene Therapy in Fanconi Anemia-A Mice Reveals Long-Term Engraftment and Continuous Turnover of Corrected HSCs.

PubMed

Molina-Estevez, F Javier; Nowrouzi, Ali; Lozano, M Luz; Galy, Anne; Charrier, Sabine; von Kalle, Christof; Guenechea, Guillermo; Bueren, Juan A; Schmidt, Manfred

2015-01-01

Fanconi anemia is a DNA repair-deficiency syndrome mainly characterized by cancer predisposition and bone marrow failure. Trying to restore the hematopoietic function in these patients, lentiviral vector-mediated gene therapy trials have recently been proposed. However, because no insertional oncogenesis studies have been conducted so far in DNA repair-deficiency syndromes such as Fanconi anemia, we have carried out a genome-wide screening of lentiviral insertion sites after the gene correction of Fanca(-/-) hematopoietic stem cells (HSCs), using LAM-PCR and 454-pyrosequencing. Our studies first demonstrated that transduction of Fanca(-/-) HSCs with a lentiviral vector designed for clinical application efficiently corrects the phenotype of Fanconi anemia repopulating cells without any sign of toxicity. The identification of more than 6,500 insertion sites in primary and secondary recipients showed a polyclonal pattern of reconstitution, as well as a continuous turnover of corrected Fanca(-/-) HSC clones, without evidences of selection towards specific common integration sites. Taken together our data show, for the first time in a DNA repair-deficiency syndrome, that lentiviral vector-mediated gene therapy efficiently corrects the phenotype of affected HSCs and promotes a healthy pattern of clonal turnover in vivo. These studies will have a particular impact in the development of new gene therapy trials in patients affected by DNA repair syndromes, particularly in Fanconi anemia.

Fluorophore Labeled Kinase Detects Ligands That Bind within the MAPK Insert of p38α Kinase

PubMed Central

Termathe, Martin; Grütter, Christian; Rabiller, Matthias; van Otterlo, Willem A. L.; Rauh, Daniel

2012-01-01

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway. PMID:22768308

Drosophila mini-white model system: new insights into positive position effects and the role of transcriptional terminators and gypsy insulator in transgene shielding

PubMed Central

Silicheva, Margarita; Golovnin, Anton; Pomerantseva, Ekaterina; Parshikov, Aleksander; Georgiev, Pavel; Maksimenko, Oksana

2010-01-01

The white gene, which is responsible for eye pigmentation, is widely used to study position effects in Drosophila. As a result of insertion of P-element vectors containing mini-white without enhancers into random chromosomal sites, flies with different eye color phenotypes appear, which is usually explained by the influence of positive/negative regulatory elements located around the insertion site. We found that, in more than 70% of cases when mini-white expression was subject to positive position effects, deletion of the white promoter had no effect on eye pigmentation; in these cases, the transposon was inserted into the transcribed regions of genes. Therefore, transcription through the mini-white gene could be responsible for high levels of its expression in most of chromosomal sites. Consistently with this conclusion, transcriptional terminators proved to be efficient in protecting mini-white expression from positive position effects. On the other hand, the best characterized Drosophila gypsy insulator was poorly effective in terminating transcription and, as a consequence, only partially protected mini-white expression from these effects. Thus, to ensure maximum protection of a transgene from position effects, a perfect boundary/insulator element should combine three activities: to block enhancers, to provide a barrier between active and repressed chromatin, and to terminate transcription. PMID:19854952

Central Venous Catheter Insertion Site and Colonization in Pediatric Cardiac Surgery

ClinicalTrials.gov

2017-11-04

Central Line-associated Bloodstream Infection (CLABSI); Central Venous Catheter Associated Bloodstream Infection; Heart; Surgery, Heart, Functional Disturbance as Result; Congenital Heart Disease; Newborn; Infection

[Construction of a general AAV vector regulated by minimal and artificial hypoxic-responsive element].

PubMed

Nie, Xiao-wei; Sun, Li-jun; Hao, Yue-wen; Yang, Guang-xiao; Wang, Quan-ying

2011-03-01

To synthesize the minimal and artificial HRE, and to insert it into the anterior extremity of CMV promoter of a AAV plasmid, and then to construct the AAV regulated by hypoxic-responsive element which was introduced into 293 cell by method of Ca3(PO4)2 using three plasmids. Thus obtaining the adenoassociated virus vector regulated by hypoxic-responsive element was possibly used for gene therapy in ischemia angiocardiopathy and cerebrovascular disease. Artificially synthesize the 36 bp nucleotide sequences of four connection in series HIF-binding sites A/GCGTG(4×HBS)and a 35 bp nucleotide sequences spacing inserted into anterior extremity of CMV promoter TATA Box, then amplified by PCR. The cDNA fragment was confirmed to be right by DNA sequencing. Molecular biology routine method was used to construct a AAV vector regulated by minimal hypoxic-responsive element after the normal CMV promoter in AAV vector was replaced by the CMV promoter included minimal hypoxic-responsive element. Then, NT4-6His-PR39 fusogenic peptide was inserted into MCS of the plasmid, the recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells, in which we can also investigate the expression of 6×His using immunochemistry in hypoxia environment. Artificial HRE was inserted into anterior extremity of CMV promoter and there was a correct spacing between the HRE and the TATA-box. The DNA sequencing and restriction enzyme digestion results indicated that the AAV regulated by hypoxic-responsive element was successfully constructed. Compared to the control group, the expressions of 6×His was significantly increased in the experimental groups in hypoxia environment, which confirmed that the AAV effectually regulated by the minimal HRE was inserted into anterior extremity of CMV promoter. The HRE is inserted into anterior extremity of CMV promoter to lack incision enzyme recognition site by PCR. And eukaryotic expression vector regulated by hypoxic-responsive is constructed. The AAV effectually regulated by the minimal HRE inserted into anterior extremity of CMV promoter. The vector is successfully constructed and it has important theoretical and practical value in the synteresis and therapy of ischemia angiocardiopathy and cerebrovascular disease.

Fluoroscopically guided nose tube drainage of mediastinal abscesses in post-operative gastro-oesophageal anastomotic leakage.

PubMed

Xu, Q Y; Yin, G W; Chen, S X; Jiang, F; Bai, X J; Wu, J D

2012-11-01

The aim of this study was to retrospectively evaluate the technical success rates and clinical effectiveness of fluoroscopically guided nose tube drainage of mediastinal abscesses and a nasojejunum feeding tube in post-operative gastro-oesophageal anastomotic leakage (GEAL). From January 2006 to June 2011, 18 cases of post-operative GEAL with mediastinal abscesses after oesophagectomy with intrathoracic oesophagogastric anastomotic procedures for oesophageal and cardiac carcinoma were treated by insertion of a nose drainage tube and nasojejunum feeding tube under fluoroscopic guidance. We evaluated the feasibility of two-tube insertion to facilitate leakage site closure and complete resolution of the abscess, and the patients' nutritional benefit was also evaluated by checking the serum albumin level between pre- and post-enteral feeding via the feeding tube. The two tubes were placed successfully under fluoroscopic guidance in 18 patients (100%). The procedure time for two-tube insertion ranged from 20 to 40 min (mean 30 min). 17 patients (94%) achieved leakage site closure after two-tube insertion and had a good tolerance of two tubes in the nasal cavity. The serum albumin level was significant, increased from pre-enteral feeding (2.49 ± 0.42 g dl(-1)) to the post-enteral feeding (3.58 ± 0.47 g dl(-1)) via the feeding tube (p<0.001). The duration of follow-up ranged from 1 to 49 months (mean 19 months). The insertion of nose tube drainage and a nasojejunum feeding tube under fluoroscopic guidance is safe, and it provides effective relief from mediastinal abscesses in GEAL after oesophagectomy. Moreover, our findings indicate that two-tube insertion may be used as a selective procedure to treat mediastinal abscesses in post-operative GEAL. Advances in knowledge Directive drainage of mediastinal abscesses in post-operative GEAL may be an effective treatment.

X-Linked Congenital Hypertrichosis Syndrome Is Associated with Interchromosomal Insertions Mediated by a Human-Specific Palindrome near SOX3

PubMed Central

Zhu, Hongwen; Shang, Dandan; Sun, Miao; Choi, Sunju; Liu, Qing; Hao, Jiajie; Figuera, Luis E.; Zhang, Feng; Choy, Kwong Wai; Ao, Yang; Liu, Yang; Zhang, Xiao-Lin; Yue, Fengzhen; Wang, Ming-Rong; Jin, Li; Patel, Pragna I.; Jing, Tao; Zhang, Xue

2011-01-01

X-linked congenital generalized hypertrichosis (CGH), an extremely rare condition characterized by universal overgrowth of terminal hair, was first mapped to chromosome Xq24-q27.1 in a Mexican family. However, the underlying genetic defect remains unknown. We ascertained a large Chinese family with an X-linked congenital hypertrichosis syndrome combining CGH, scoliosis, and spina bifida and mapped the disease locus to a 5.6 Mb critical region within the interval defined by the previously reported Mexican family. Through the combination of a high-resolution copy-number variation (CNV) scan and targeted genomic sequencing, we identified an interchromosomal insertion at Xq27.1 of a 125,577 bp intragenic fragment of COL23A1 on 5q35.3, with one X breakpoint within and the other very close to a human-specific short palindromic sequence located 82 kb downstream of SOX3. In the Mexican family, we found an interchromosomal insertion at the same Xq27.1 site of a 300,036 bp genomic fragment on 4q31.2, encompassing PRMT10 and TMEM184C and involving parts of ARHGAP10 and EDNRA. Notably, both of the two X breakpoints were within the short palindrome. The two palindrome-mediated insertions fully segregate with the CGH phenotype in each of the families, and the CNV gains of the respective autosomal genomic segments are not present in the public database and were not found in 1274 control individuals. Analysis of control individuals revealed deletions ranging from 173 bp to 9104 bp at the site of the insertions with no phenotypic consequence. Taken together, our results strongly support the pathogenicity of the identified insertions and establish X-linked congenital hypertrichosis syndrome as a genomic disorder. PMID:21636067

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.

PubMed

Schouten, Henk J; Vande Geest, Henri; Papadimitriou, Sofia; Bemer, Marian; Schaart, Jan G; Smulders, Marinus J M; Perez, Gabino Sanchez; Schijlen, Elio

2017-03-01

Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

Deep electrode insertion and sound coding in cochlear implants.

PubMed

Hochmair, Ingeborg; Hochmair, Erwin; Nopp, Peter; Waller, Melissa; Jolly, Claude

2015-04-01

Present-day cochlear implants demonstrate remarkable speech understanding performance despite the use of non-optimized coding strategies concerning the transmission of tonal information. Most systems rely on place pitch information despite possibly large deviations from correct tonotopic placement of stimulation sites. Low frequency information is limited as well because of the constant pulse rate stimulation generally used and, being even more restrictive, of the limited insertion depth of the electrodes. This results in a compromised perception of music and tonal languages. Newly available flexible long straight electrodes permit deep insertion reaching the apical region with little or no insertion trauma. This article discusses the potential benefits of deep insertion which are obtained using pitch-locked temporal stimulation patterns. Besides the access to low frequency information, further advantages of deeply inserted long electrodes are the possibility to better approximate the correct tonotopic location of contacts, the coverage of a wider range of cochlear locations, and the somewhat reduced channel interaction due to the wider contact separation for a given number of channels. A newly developed set of strategies has been shown to improve speech understanding in noise and to enhance sound quality by providing a more "natural" impression, which especially becomes obvious when listening to music. The benefits of deep insertion should not, however, be compromised by structural damage during insertion. The small cross section and the high flexibility of the new electrodes can help to ensure less traumatic insertions as demonstrated by patients' hearing preservation rate. This article is part of a Special Issue entitled . Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Family of pH-Low-Insertion-Peptides (pHLIPs)

NASA Astrophysics Data System (ADS)

Weerakkody, Dhammika; Moshnikova, Anna; Moshnikova, Valentina; Thakur, Mak; Rossi, Bethany; Engelman, Donald; Andreev, Oleg; Reshetnyak, Yana

2012-02-01

pHLIP (pH (Low) Insertion Peptide) is a novel delivery system for targeting of acidic diseased tissue such as solid tumors, sites of inflammation, arthritis and other pathological states. The molecular mechanism of pHLIP action is based on pH-dependent insertion and folding of pHLIP in membrane. We performed sequence variation and investigated 16 pHLIP variants with main goals of understanding the main principles of peptide-lipid interactions and tune delivery capability of pHLIP. The biophysical studies including thermodynamics and kinetics of the peptides interaction with a lipid bilayer of liposomes and cellular membranes were carried out. We found that peptides association to membrane at neutral and low pH could be modulated by 3-4 times. The apparent pK of transition from surface bound to membrane-inserted state could be tuned from 6.5 to 4.5. The rate of peptide's insertion across a bilayer could be enhanced 100 times compared to parent pHLIP. As a result, blood clearance and tumor targeting were modulated in a significant degree. The work is supported by NIH grants CA133890 to OAA, DME, YRK.

Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solera, J.; Magallon, M.; Martin-Villar, J.

1992-02-01

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends ofmore » the deleted DNA fragment.« less

A Novel Retrotransposon Inserted in the Dominant Vrn-B1 Allele Confers Spring Growth Habit in Tetraploid Wheat (Triticum turgidum L.).

PubMed

Chu, C-G; Tan, C T; Yu, G-T; Zhong, S; Xu, S S; Yan, L

2011-12-01

Vernalization genes determine winter/spring growth habit in temperate cereals and play important roles in plant development and environmental adaptation. In wheat (Triticum L. sp.), it was previously shown that allelic variation in the vernalization gene VRN1 was due to deletions or insertions either in the promoter or in the first intron. Here, we report a novel Vrn-B1 allele that has a retrotransposon in its promoter conferring spring growth habit. The VRN-B1 gene was mapped in a doubled haploid population that segregated for winter-spring growth habit but was derived from two spring tetraploid wheat genotypes, the durum wheat (T. turgidum subsp. durum) variety 'Lebsock' and T. turgidum subsp. carthlicum accession PI 94749. Genetic analysis revealed that Lebsock carried the dominant Vrn-A1 and recessive vrn-B1 alleles, whereas PI 94749 had the recessive vrn-A1 and dominant Vrn-B1 alleles. The Vrn-A1 allele in Lebsock was the same as the Vrn-A1c allele previously reported in hexaploid wheat. No differences existed between the vrn-B1 and Vrn-B1 alleles, except that a 5463-bp insertion was detected in the 5'-UTR region of the Vrn-B1 allele. This insertion was a novel retrotransposon (designated as retrotrans_VRN), which was flanked by a 5-bp target site duplication and contained primer binding site and polypurine tract motifs, a 325-bp long terminal repeat, and an open reading frame encoding 1231 amino acids. The insertion of retrotrans_VRN resulted in expression of Vrn-B1 without vernalization. Retrotrans_VRN is prevalent among T. turgidum subsp. carthlicum accessions, less prevalent among T. turgidum subsp. dicoccum accessions, and rarely found in other tetraploid wheat subspecies.

The Role(s) of Heparan Sulfate Proteoglycan(s) in the wnt-1 Signaling Pathway

DTIC Science & Technology

1998-08-01

First , the sequence of the cDNA, when compared to the genomic site of insertion of the P-element, revealed that the P-element is inserted 686 bp...stages 8 to 13 (Yoffe et al. 1995). We first examined whether ectopic expression of Wgts effectively restores the naked cuticle as it does in wg and...by Kjell~n and Lindahl, 1991) . HS/heparin N-deacetylase/N-sulfotransferase catalyzes N-deacetylation and N-sulfation that is the first and key step

Effects of Hematopoietic Lineage and Precursor Age on CML Disease Progression

DTIC Science & Technology

2007-03-01

ABL gene was inserted is bi-cistronic and contains the gene encoding green fluorescence protein (GFP) in addition to BCR-ABL, we were able to assess...ribosome entry site (IRES) enhanced green fluorescent protein (EGFP) or 5’ LTR-driven IRES EGFP for 24-36 hours; We received the BCR-ABL construct...from our collaborator, Dr. Owen Witte. This gene was then inserted into a murine retrovirus. We then prepared stocks of retrovirus to be used for

Quality assurance: recommended guidelines for safe heating by capacitive-type heating technique to treat patients with metallic implants.

PubMed

Kato, Hirokazu; Kondo, Motoharu; Imada, Hajime; Kuroda, Masahiro; Kamimura, Yoshitsugu; Saito, Kazuyuki; Kuroda, Kagayaki; Ito, Koichi; Takahashi, Hideaki; Matsuki, Hidetoshi

2013-05-01

This article is a redissemination of the previous Japanese Quality Assurance Guide guidelines. Specific absorption rate and temperature distribution were investigated with respect to various aspects including metallic implant size and shape, insertion site, insertion direction, blood flow and heating power, and simulated results were compared with adverse reactions of patients treated by radio frequency capacitive-type heating. Recommended guidelines for safe heating methods for patients with metallic implants are presented based on our findings.

Retrotransposon Tf1 is targeted to pol II promoters by transcription activators

PubMed Central

Leem, Young-Eun; Ripmaster, Tracy; Kelly, Felice; Ebina, Hirotaka; Heincelman, Marc; Zhang, Ke; Grewal, Shiv I. S.; Hoffman, Charles S.; Levin, Henry L.

2008-01-01

SUMMARY The LTR-retrotransposon Tf1 preserves the coding capacity of its host Schizosaccharomyces pombe by integrating upstream of open reading frames (ORFs). To determine which features of the target sites were recognized by the transposon, we introduced plasmids containing candidate insertion sites into S. pombe and mapped the positions of integration. We found that Tf1 was targeted specifically to the promoters of pol II transcribed genes. A detailed analysis of integration in plasmids that contained either ade6 or fbp1 revealed insertions occurred in the promoters at positions where transcription factors bound. Further experiments revealed that the activator Atf1p and its binding site were required for directing integration to the promoter of fbp1. An interaction between Tf1 integrase and Atf1p was observed indicating that integration at fbp1 was mediated by the activator bound to its promoter. Surprisingly we found Tf1 contained sequences that activated transcription and these substituted for elements of the ade6 promoter disrupted by integration. PMID:18406330

Retrotransposon Tf1 is targeted to Pol II promoters by transcription activators.

PubMed

Leem, Young-Eun; Ripmaster, Tracy L; Kelly, Felice D; Ebina, Hirotaka; Heincelman, Marc E; Zhang, Ke; Grewal, Shiv I S; Hoffman, Charles S; Levin, Henry L

2008-04-11

The LTR-retrotransposon Tf1 preserves the coding capacity of its host Schizosaccharomyces pombe by integrating upstream of open reading frames (ORFs). To determine which features of the target sites were recognized by the transposon, we introduced plasmids containing candidate insertion sites into S. pombe and mapped the positions of integration. We found that Tf1 was targeted specifically to the promoters of Pol II-transcribed genes. A detailed analysis of integration in plasmids that contained either ade6 or fbp1 revealed insertions occurred in the promoters at positions where transcription factors bound. Further experiments revealed that the activator Atf1p and its binding site were required for directing integration to the promoter of fbp1. An interaction between Tf1 integrase and Atf1p was observed, indicating that integration at fbp1 was mediated by the activator bound to its promoter. Surprisingly, we found Tf1 contained sequences that activated transcription, and these substituted for elements of the ade6 promoter disrupted by integration.

Effects of insertion speed and trocar stiffness on the accuracy of needle position for brachytherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

McGill, Carl S.; Schwartz, Jonathon A.; Moore, Jason Z.

2012-04-15

Purpose: In prostate brachytherapy, accurate positioning of the needle tip to place radioactive seeds at its target site is critical for successful radiation treatment. During the procedure, needle deflection leads to seed misplacement and suboptimal radiation dose to cancerous cells. In practice, radiation oncologists commonly use high-speed hand needle insertion to minimize displacement of the prostate as well as the needle deflection. Effects of speed during needle insertion and stiffness of trocar (a solid rod inside the hollow cannula) on needle deflection are studied. Methods: Needle insertion experiments into phantom were performed using a 2{sup 2} factorial design (2 parametersmore » at 2 levels), with each condition having replicates. Analysis of the deflection data included calculating the average, standard deviation, and analysis of variance (ANOVA) to find significant single and two-way interaction factors. Results: The stiffer tungsten carbide trocar is effective in reducing the average and standard deviation of needle deflection. The fast insertion speed together with the stiffer trocar generated the smallest average and standard deviation for needle deflection for almost all cases. Conclusions: The combination of stiff tungsten carbide trocar and fast needle insertion speed are important to decreasing needle deflection. The knowledge gained from this study can be used to improve the accuracy of needle insertion during brachytherapy procedures.« less

48 CFR 1511.011-74 - Work assignments.

Code of Federal Regulations, 2010 CFR

2010-10-01

... insert the contract clause at 1552.211-74, Work Assignments, in cost-reimbursement type term form... conflict of interest certificates (e.g., Site Specific contracts, the Contract Laboratory Program (CLP...

Suboptimal Doses of Raltegravir Cause Aberrant HIV Integrations | Center for Cancer Research

Cancer.gov

When a cell is infected with HIV, a DNA copy of the HIV genome is inserted into that cell’s chromosomal DNA. This insertion reaction is carried out by the viral enzyme integrase (IN) and involves two distinct steps: removal of two nucleotides from each 3’ end of the viral DNA, followed by the strand transfer reaction, in which the viral DNA ends are inserted into the host chromosomal DNA. Integration is essential for viral replication, making it an important target for antiviral therapy. Raltegravir, and the other approved integrase inhibitor, Elvitegravir, are called integrase strand transfer inhibitors (INSTIs), because they bind to the active site of IN and block the strand transfer reaction.      

RNA detection using peptide-inserted Renilla luciferase.

PubMed

Andou, Takashi; Endoh, Tamaki; Mie, Masayasu; Kobatake, Eiry

2009-01-01

A novel complementation system with short peptide-inserted-Renilla luciferase (PI-Rluc) and split-RNA probes was constructed for noninvasive RNA detection. The RNA binding peptides HIV-1 Rev and BIV Tat were used as inserted peptides. They display induced fit conformational changes upon binding to specific RNAs and trigger complementation or discomplementation of Rluc. Split-RNA probes were designed to reform the peptide binding site upon hybridization with arbitrarily selected target RNA. This set of recombinant protein and split-RNA probes enabled a high degree of sensitivity in RNA detection. In this study, we show that the Rluc system is comparable to Fluc, but that its detection limit for arbitrarily selected RNA (at least 100 pM) exceeds that of Fluc by approximately two orders of magnitude.

Detection of possible restriction sites for type II restriction enzymes in DNA sequences.

PubMed

Gagniuc, P; Cimponeriu, D; Ionescu-Tîrgovişte, C; Mihai, Andrada; Stavarachi, Monica; Mihai, T; Gavrilă, L

2011-01-01

In order to make a step forward in the knowledge of the mechanism operating in complex polygenic disorders such as diabetes and obesity, this paper proposes a new algorithm (PRSD -possible restriction site detection) and its implementation in Applied Genetics software. This software can be used for in silico detection of potential (hidden) recognition sites for endonucleases and for nucleotide repeats identification. The recognition sites for endonucleases may result from hidden sequences through deletion or insertion of a specific number of nucleotides. Tests were conducted on DNA sequences downloaded from NCBI servers using specific recognition sites for common type II restriction enzymes introduced in the software database (n = 126). Each possible recognition site indicated by the PRSD algorithm implemented in Applied Genetics was checked and confirmed by NEBcutter V2.0 and Webcutter 2.0 software. In the sequence NG_008724.1 (which includes 63632 nucleotides) we found a high number of potential restriction sites for ECO R1 that may be produced by deletion (n = 43 sites) or insertion (n = 591 sites) of one nucleotide. The second module of Applied Genetics has been designed to find simple repeats sizes with a real future in understanding the role of SNPs (Single Nucleotide Polymorphisms) in the pathogenesis of the complex metabolic disorders. We have tested the presence of simple repetitive sequences in five DNA sequence. The software indicated exact position of each repeats detected in the tested sequences. Future development of Applied Genetics can provide an alternative for powerful tools used to search for restriction sites or repetitive sequences or to improve genotyping methods.

Mechanisms by which herpes simplex virus DNA polymerase limits translesion synthesis through abasic sites.

PubMed

Zhu, Yali; Song, Liping; Stroud, Jason; Parris, Deborah S

2008-01-01

Results suggest a high probability that abasic (AP) sites occur at least once per herpes simplex virus type 1 (HSV-1) genome. The parameters that control the ability of HSV-1 DNA polymerase (pol) to engage in AP translesion synthesis (TLS) were examined because AP lesions could influence the completion and fidelity of viral DNA synthesis. Pre-steady-state kinetic experiments demonstrated that wildtype (WT) and exonuclease-deficient (exo-) pol could incorporate opposite an AP lesion, but full TLS required absence of exo function. Virtually all of the WT pol was bound at the exo site to AP-containing primer-templates (P/Ts) at equilibrium, and the pre-steady-state rate of excision by WT pol was higher on AP-containing than on matched DNA. However, several factors influencing polymerization work synergistically with exo activity to prevent HSV-1 pol from engaging in TLS. Although the pre-steady-state catalytic rate constant for insertion of dATP opposite a T or AP site was similar, ground-state-binding affinity of dATP for insertion opposite an AP site was reduced 3-9-fold. Single-turnover running-start experiments demonstrated a reduced proportion of P/Ts extended to the AP site compared to the preceding site during processive synthesis by WT or exo- pol. Only the exo- pol engaged in TLS, though inefficiently and without burst kinetics, suggesting a much slower rate-limiting step for extension beyond the AP site.

Localization of PKA Phosphorylation Site, Serine-2030, in the Three-Dimensional Structure of Cardiac Ryanodine Receptor

PubMed Central

Jones, Peter P.; Meng, Xing; Xiao, Bailong; Cai, Shitian; Bolstad, Jeff; Wagenknecht, Terence; Liu, Zheng; Chen, S. R. Wayne

2009-01-01

Protein kinase A (PKA)-dependent phosphorylation of the cardiac Ca2+ release channel/ryanodine receptor (RyR2) is believed to directly dissociate FKBP12.6 from the channel, causing abnormal channel activation and Ca2+ release. To gain insight into the structural basis of the regulation of RyR2 by PKA, we determined the three-dimensional location of the PKA site S2030. Green fluorescent protein (GFP) was inserted into the wild type (wt) RyR2 and RyR2 mutant, A4860G, after T2023. The resultant GFP-RyR2 fusion proteins, RyR2T2023-GFP and RyR2(A4860G)T2023-GFP, were expressed in HEK293 cells and functionally characterized. Ca2+ release assays revealed that both GFP-RyR2 fusion proteins formed caffeine- and ryanodine-sensitive Ca2+ release channels. Further analyses using [3H]ryanodine binding demonstrated that the insertion of GFP into RyR2 wt after T2023 reduced the sensitivity of the channel to activation by Ca2+ or caffeine. RyR2(A4860G)T2023-GFP was found to be structurally more stable than RyR2T2023-GFP and was subsequently used as a basis for three-dimensional reconstruction. Cryo-electron microscopy and single particle image processing of the purified RyR2(A4860G)T2023-GFP protein revealed the location of the inserted GFP, and hence the S2030 PKA site in domain 4, a region that may be involved in signal transduction between the transmembrane and cytoplasmic domains. Like the S2808 PKA site reported previously, the S2030 site is not located close to the FKBP12.6 binding site mapped previously, indicating that neither of these PKA sites is directly involved in FKBP12.6 binding. Based on the three-dimensional localizations of a number of residues or regions, a model for the subunit organization in the structure of RyR2 is proposed. PMID:17967164

Recombination of an intrachromosomal paracentric insertion of chromosome 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Best, R.G.; Burnett, W.J.; Brock, J.K.

1994-09-01

Cytogenetic studies were initiated on a newborn female due to multiple congenital anomalies including microcephaly, clinodactyly, abnormal positioning of hands, left facial palsy, heart defect, sacral dimple, and facial dysmorphic features. Facial features were described as low set rotated ears, nystagmus, and a small, flattened nose. A structural rearrangement of the long arm of chromosome 3 was observed with a complex banding pattern. Study of parental chromosomes revealed a normal male pattern for the father, and an intrachromosomal insertion on the long arm of chromosome 3 for the mother described as 46,XX,dir ins(3)(q21q23q26.2). Further characterization of the proband`s structurally abnormalmore » chromosome 3 revealed a karyotype best described as: 46,XX,rec(3),dupq23{r_arrow}q26.2::q21{r_arrow}q23,dir ins(3)(q21q23q26.2), which is a partial duplication of both the inserted segment as well as the intervening segment between the inserted segment and the insertion site. This would appear to be the result of a three-strand double cross-over within the insertion loop. Molecular cytogenetic studies are presently underway to further elucidate chromosome structure of the proband and her mother.« less

Evaluation of manhole inserts as structural barriers to mosquito entry into belowground stormwater systems using a simulated treatment device.

PubMed

Harbison, Justin E; Metzger, Marco E; Allen, Vaikko; Hu, Renjie

2009-09-01

Belowground proprietary stormwater treatment devices can produce mosquitoes, including vectors of West Nile virus. Elimination of vertical entry points such as pick holes in manhole covers may reduce the number of mosquitoes entering and reproducing in these structures. Plastic manhole dish inserts were evaluated as structural barriers against mosquito entry through pick holes in a simulated stormwater treatment device. Inserts were 100% effective at preventing mosquito entry through covers when no other openings existed. In devices configured with an open lateral conveyance pipe, the addition of an insert under the cover reduced mosquito oviposition significantly. Subsequent trials to further elucidate mosquito entry through manhole covers found a significant positive correlation between increasing number of pick holes and mosquito oviposition. Results of the study suggest the potential for manhole dish inserts to decrease the number of mosquitoes entering belowground structures. The different available stormwater treatment systems and site-specific installations may, however, provide a much greater variety of possible alternate entry points for mosquitoes than was addressed in the current study. Further work is needed in field installations to quantify the significance of pick holes to mosquito entry and determine under what conditions, if any, manhole dish inserts would be most effective and appropriate.

Mechanism for DNA transposons to generate introns on genomic scales

PubMed Central

Huff, Jason T.; Zilberman, Daniel; Roy, Scott W.

2017-01-01

Discovered four decades ago, the existence of introns was one of the most unexpected findings in molecular biology1. Introns are sequences interrupting genes that must be removed as part of mRNA production. Genome sequencing projects have documented that most eukaryotic genes contain at least one and frequently many introns2,3. Comparison of these genomes reveals a history of long evolutionary periods with little intron gain punctuated by episodes of rapid, extensive gain2,3. However, no detailed mechanism for such episodic intron generation has been empirically supported on a sufficient scale, despite several proposals4–8. Here we show how short non-autonomous DNA transposons independently generated hundreds to thousands of introns in the prasinophyte Micromonas pusilla and the pelagophyte Aureococcus anophagefferens. Each transposon carries one splice site. The other splice site is co-opted from gene sequence duplicated upon transposon insertion, allowing perfect splicing out of RNA. The distributions of sequences that can be co-opted are biased with respect to codons, and phasing of transposon-generated introns is similarly biased. These transposons insert between preexisting nucleosomes, so that multiple nearby insertions generate nucleosome-sized intervening segments. Thus, transposon insertion and sequence co-option may explain the intron phase biases2 and prevalence of nucleosome-sized exons9 observed in eukaryotes. Overall, the two independent examples of proliferating elements illustrate a general DNA transposon mechanism plausibly accounting for episodes of rapid, extensive intron gain during eukaryotic evolution2,3. PMID:27760113

Crystal Structure of the Extracellular Cholinesterase-Like Domain from Neuroligin-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koehnke,J.; Jin, X.; Budreck, E.

Neuroligins (NLs) are catalytically inactive members of a family of cholinesterase-like transmembrane proteins that mediate cell adhesion at neuronal synapses. Postsynaptic neuroligins engage in Ca2+-dependent transsynaptic interactions via their extracellular cholinesterase domain with presynaptic neurexins (NRXs). These interactions may be regulated by two short splice insertions (termed A and B) in the NL cholinesterase domain. Here, we present the 3.3- Angstroms crystal structure of the ectodomain from NL2 containing splice insertion A (NL2A). The overall structure of NL2A resembles that of cholinesterases, but several structural features are unique to the NL proteins. First, structural elements surrounding the esterase active-site regionmore » differ significantly between active esterases and NL2A. On the opposite surface of the NL2A molecule, the positions of the A and B splice insertions identify a candidate NRX interaction site of the NL protein. Finally, sequence comparisons of NL isoforms allow for mapping the location of residues of previously identified mutations in NL3 and NL4 found in patients with autism spectrum disorders. Overall, the NL2 structure promises to provide a valuable model for dissecting NL isoform- and synapse-specific functions.« less

The imaging features of the meniscal roots on isotropic 3D MRI in young asymptomatic volunteers.

PubMed

Wang, Ping; Zhang, Cheng-Zhou; Zhang, Di; Liu, Quan-Yuan; Zhong, Xiao-Fei; Yin, Zhi-Jie; Wang, Bin

2018-05-01

This study aimed to describe clearly the normal imaging features of the meniscal roots on the magnetic resonance imaging (MRI) with a 3-dimensional (3D) proton density-weighted (PDW) sequence at 3T. A total of 60 knees of 31 young asymptomatic volunteers were examined using a 3D MRI. The insertion patterns, constitution patterns, and MR signals of the meniscal roots were recorded. The anterior root of the medial meniscus (ARMM), the anterior root of the lateral meniscus (ARLM), and the posterior root of the medial meniscus (PRMM) had 1 insertion site, whereas the posterior root of the lateral meniscus (PRLM) can be divided into major and minor insertion sites. The ARLM and the PRMM usually consisted of multiple fiber bundles (≥3), whereas the ARMM and the PRLM often consisted of a single fiber bundle. The ARMM and the PRLM usually appeared as hypointense, whereas the ARLM and the PRMM typically exhibited mixed signals. The meniscal roots can be complex and diverse, and certain characteristics of them were observed on 3D MRI. Understanding the normal imaging features of the meniscal roots is extremely beneficial for further diagnosis of root tears.

Development of a Robotic Colonoscopic Manipulation System, Using Haptic Feedback Algorithm.

PubMed

Woo, Jaehong; Choi, Jae Hyuk; Seo, Jong Tae; Kim, Tae Il; Yi, Byung Ju

2017-01-01

Colonoscopy is one of the most effective diagnostic and therapeutic tools for colorectal diseases. We aim to propose a master-slave robotic colonoscopy that is controllable in remote site using conventional colonoscopy. The master and slave robot were developed to use conventional flexible colonoscopy. The robotic colonoscopic procedure was performed using a colonoscope training model by one expert endoscopist and two unexperienced engineers. To provide the haptic sensation, the insertion force and the rotating torque were measured and sent to the master robot. A slave robot was developed to hold the colonoscopy and its knob, and perform insertion, rotation, and two tilting motions of colonoscope. A master robot was designed to teach motions of the slave robot. These measured force and torque were scaled down by one tenth to provide the operator with some reflection force and torque at the haptic device. The haptic sensation and feedback system was successful and helpful to feel the constrained force or torque in colon. The insertion time using robotic system decreased with repeated procedures. This work proposed a robotic approach for colonoscopy using haptic feedback algorithm, and this robotic device would effectively perform colonoscopy with reduced burden and comparable safety for patients in remote site.

Crystal structure of the extracellular cholinesterase-like domain from neuroligin-2

PubMed Central

Koehnke, Jesko; Jin, Xiangshu; Budreck, Elaine C.; Posy, Shoshana; Scheiffele, Peter; Honig, Barry; Shapiro, Lawrence

2008-01-01

Neuroligins (NLs) are catalytically inactive members of a family of cholinesterase-like transmembrane proteins that mediate cell adhesion at neuronal synapses. Postsynaptic neuroligins engage in Ca2+-dependent transsynaptic interactions via their extracellular cholinesterase domain with presynaptic neurexins (NRXs). These interactions may be regulated by two short splice insertions (termed A and B) in the NL cholinesterase domain. Here, we present the 3.3-Å crystal structure of the ectodomain from NL2 containing splice insertion A (NL2A). The overall structure of NL2A resembles that of cholinesterases, but several structural features are unique to the NL proteins. First, structural elements surrounding the esterase active-site region differ significantly between active esterases and NL2A. On the opposite surface of the NL2A molecule, the positions of the A and B splice insertions identify a candidate NRX interaction site of the NL protein. Finally, sequence comparisons of NL isoforms allow for mapping the location of residues of previously identified mutations in NL3 and NL4 found in patients with autism spectrum disorders. Overall, the NL2 structure promises to provide a valuable model for dissecting NL isoform- and synapse-specific functions. PMID:18250328

SfiI genomic cleavage map of Escherichia coli K-12 strain MG1655.

PubMed Central

Perkins, J D; Heath, J D; Sharma, B R; Weinstock, G M

1992-01-01

An SfiI restriction map of Escherichia coli K-12 strain MG1655 is presented. The map contains thirty-one cleavage sites separating fragments ranging in size from 407 kb to 3.7 kb. Several techniques were used in the construction of this map, including CHEF pulsed field gel electrophoresis; physical analysis of a set of twenty-six auxotrophic transposon insertions; correlation with the restriction map of Kohara and coworkers using the commercially available E. coli Gene Mapping Membranes; analysis of publicly available sequence information; and correlation of the above data with the combined genetic and physical map developed by Rudd, et al. The combination of these techniques has yielded a map in which all but one site can be localized within a range of +/- 2 kb, and over half the sites can be localized precisely by sequence data. Two sites present in the EcoSeq5 sequence database are not cleaved in MG1655 and four sites are noted to be sensitive to methylation by the dcm methylase. This map, combined with the NotI physical map of MG1655, can aid in the rapid, precise mapping of several different types of genetic alterations, including transposon mediated mutations and other insertions, inversions, deletions and duplications. Images PMID:1312707

A Modular Vaccine Development Platform Based on Sortase-Mediated Site-Specific Tagging of Antigens onto Virus-Like Particles

PubMed Central

Tang, Shubing; Xuan, Baoqin; Ye, Xiaohua; Huang, Zhong; Qian, Zhikang

2016-01-01

Virus-like particles (VLPs) can be used as powerful nanoscale weapons to fight against virus infection. In addition to direct use as vaccines, VLPs have been extensively exploited as platforms on which to display foreign antigens for prophylactic vaccination and immunotherapeutic treatment. Unfortunately, fabrication of new chimeric VLP vaccines in a versatile, site-specific and highly efficient manner is beyond the capability of traditional VLP vaccine design approaches, genetic insertion and chemical conjugation. In this study, we described a greatly improved VLP display strategy by chemoenzymatic site-specific tailoring antigens on VLPs surface with high efficiency. Through the transpeptidation mediated by sortase A, one protein and two epitopes containing N-terminal oligoglycine were conjugated to the LPET motif on the surface of hepatitis B virus core protein (HBc) VLPs with high density. All of the new chimeric VLPs induced strong specific IgG responses. Furthermore, the chimeric VLPs with sortase A tagged enterovirus 71 (EV71) SP70 epitope could elicit effective antibodies against EV71 lethal challenging as well as the genetic insertion chimeric VLPs. The sortase A mediated chemoenzymatic site-specific tailoring of the HBc VLP approach shows great potential in new VLP vaccine design for its simplicity, site specificity, high efficiency, and versatility. PMID:27170066

Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

PubMed

Luo, Ming; Gilbert, Brian; Ayliffe, Michael

2016-07-01

Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.

An interferon regulatory factor binding site in the U5 region of the bovine leukemia virus long terminal repeat stimulates Tax-independent gene expression.

PubMed

Kiermer, V; Van Lint, C; Briclet, D; Vanhulle, C; Kettmann, R; Verdin, E; Burny, A; Droogmans, L

1998-07-01

Bovine leukemia virus (BLV) replication is controlled by both cis- and trans-acting elements. The virus-encoded transactivator, Tax, is necessary for efficient transcription from the BLV promoter, although it is not present during the early stages of infection. Therefore, sequences that control Tax-independent transcription must play an important role in the initiation of viral gene expression. This study demonstrates that the R-U5 sequence of BLV stimulates Tax-independent reporter gene expression directed by the BLV promoter. R-U5 was also stimulatory when inserted immediately downstream from the transcription initiation site of a heterologous promoter. Progressive deletion analysis of this region revealed that a 46-bp element corresponding to the 5' half of U5 is principally responsible for the stimulation. This element exhibited enhancer activity when inserted upstream or downstream from the herpes simplex virus thymidine kinase promoter. This enhancer contains a binding site for the interferon regulatory factors IRF-1 and IRF-2. A 3-bp mutation that destroys the IRF recognition site caused a twofold decrease in Tax-independent BLV long terminal repeat-driven gene expression. These observations suggest that the IRF binding site in the U5 region of BLV plays a role in the initiation of virus replication.

Genome Engineering in Bacillus anthracis Using Cre Recombinase

PubMed Central

Pomerantsev, Andrei P.; Sitaraman, Ramakrishnan; Galloway, Craig R.; Kivovich, Violetta; Leppla, Stephen H.

2006-01-01

Genome engineering is a powerful method for the study of bacterial virulence. With the availability of the complete genomic sequence of Bacillus anthracis, it is now possible to inactivate or delete selected genes of interest. However, many current methods for disrupting or deleting more than one gene require use of multiple antibiotic resistance determinants. In this report we used an approach that temporarily inserts an antibiotic resistance marker into a selected region of the genome and subsequently removes it, leaving the target region (a single gene or a larger genomic segment) permanently mutated. For this purpose, a spectinomycin resistance cassette flanked by bacteriophage P1 loxP sites oriented as direct repeats was inserted within a selected gene. After identification of strains having the spectinomycin cassette inserted by a double-crossover event, a thermo-sensitive plasmid expressing Cre recombinase was introduced at the permissive temperature. Cre recombinase action at the loxP sites excised the spectinomycin marker, leaving a single loxP site within the targeted gene or genomic segment. The Cre-expressing plasmid was then removed by growth at the restrictive temperature. The procedure could then be repeated to mutate additional genes. In this way, we sequentially mutated two pairs of genes: pepM and spo0A, and mcrB and mrr. Furthermore, loxP sites introduced at distant genes could be recombined by Cre recombinase to cause deletion of large intervening regions. In this way, we deleted the capBCAD region of the pXO2 plasmid and the entire 30 kb of chromosomal DNA between the mcrB and mrr genes, and in the latter case we found that the 32 intervening open reading frames were not essential to growth. PMID:16369025

Probing the Li Insertion Mechanism of ZnFe 2O 4 in Li-Ion Batteries: A Combined X-Ray Diffraction, Extended X-Ray Absorption Fine Structure, and Density Functional Theory Study [Probing the Li insertion mechanism of ZnFe 2O 4 in Li ion batteries: A combined XRD, EXAFS, and DFT study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yiman; Pelliccione, Christopher J.; Brady, Alexander B.

Here, we report an extensive study on fundamental properties that determine the functional electrochemistry of ZnFe 2O 4 spinel (theoretical capacity of 1000 mAh/g). For the first time, the reduction mechanism is followed through a combination of in situ X-ray diffraction data, synchrotron based powder diffraction, and ex-situ extended X-ray absorption fine structure allowing complete visualization of reduction products irrespective of their crystallinity. The first 0.5 electron equivalents (ee) do not significantly change the starting crystal structure. Subsequent lithiation results in migration of Zn 2+ ions from 8a tetrahedral sites into vacant 16c sites. Density functional theory shows that Limore » + ions insert into 16c site initially and then 8a site with further lithiation. Fe metal is formed over the next eight ee of reduction with no evidence of concurrent Zn 2+ reduction to Zn metal. Despite the expected formation of LiZn alloy from the electron count, we find no evidence for this phase under the tested conditions. Additionally, upon oxidation to 3 V, we observe an FeO phase with no evidence of Fe 2O 3. Electrochemistry data show higher electron equivalent transfer than can be accounted for solely based on ZnFe 2O 4 reduction indicating excess capacity ascribed to carbon reduction or surface electrolyte interphase formation.« less

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

PubMed

Bishop, Kathleen A; Harrington, Anne; Kouranova, Evguenia; Weinstein, Edward J; Rosen, Clifford J; Cui, Xiaoxia; Liaw, Lucy

2016-07-07

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Copyright © 2016 Bishop et al.

Probing the Li Insertion Mechanism of ZnFe 2O 4 in Li-Ion Batteries: A Combined X-Ray Diffraction, Extended X-Ray Absorption Fine Structure, and Density Functional Theory Study [Probing the Li insertion mechanism of ZnFe 2O 4 in Li ion batteries: A combined XRD, EXAFS, and DFT study

DOE PAGES

Zhang, Yiman; Pelliccione, Christopher J.; Brady, Alexander B.; ...

2017-04-24

Here, we report an extensive study on fundamental properties that determine the functional electrochemistry of ZnFe 2O 4 spinel (theoretical capacity of 1000 mAh/g). For the first time, the reduction mechanism is followed through a combination of in situ X-ray diffraction data, synchrotron based powder diffraction, and ex-situ extended X-ray absorption fine structure allowing complete visualization of reduction products irrespective of their crystallinity. The first 0.5 electron equivalents (ee) do not significantly change the starting crystal structure. Subsequent lithiation results in migration of Zn 2+ ions from 8a tetrahedral sites into vacant 16c sites. Density functional theory shows that Limore » + ions insert into 16c site initially and then 8a site with further lithiation. Fe metal is formed over the next eight ee of reduction with no evidence of concurrent Zn 2+ reduction to Zn metal. Despite the expected formation of LiZn alloy from the electron count, we find no evidence for this phase under the tested conditions. Additionally, upon oxidation to 3 V, we observe an FeO phase with no evidence of Fe 2O 3. Electrochemistry data show higher electron equivalent transfer than can be accounted for solely based on ZnFe 2O 4 reduction indicating excess capacity ascribed to carbon reduction or surface electrolyte interphase formation.« less

Nurse education: a feminist approach.

PubMed

Chapman, E

1997-06-01

Nursing is predominantly a female profession. This paper seeks to explore the implications of this for curriculum design and suggests that insights from feminist theory should be applied to curricula. To insert the 'subject' of feminism into the curriculum is different from allowing its theories to affect the design of the curriculum itself. The paper seeks to justify such a change and asks what the resulting characteristics would be. Would such a curriculum change succeed and what would be its limitations? The paper concludes by highlighting the implications for nurse education.

Transferable green fluorescence-tagged pEI2 in Edwardsiella ictaluri

USDA-ARS?s Scientific Manuscript database

The pEI2 plasmid of Edwardsiella ictaluri isolate, I49, was tagged using a Tn10-GFP-kan cassette to create the green fluorescence-expressing derivative I49-gfp. The Tn10-GFP-kan insertion site was mapped by plasmid sequencing to 663 bp upstream of orf2 and appeared to be at a neutral site in the pla...

Peripherally inserted central catheter - dressing change

MedlinePlus

... chlorhexidine) in a single-use small applicator Special sponges or wipes that contain a cleaning agent, such ... Clean your skin around the site with the sponge and cleaning solution for 30 seconds. Let the ...

Cas9-Guide RNA Directed Genome Editing in Soybean[OPEN

PubMed Central

Li, Zhongsen; Liu, Zhan-Bin; Xing, Aiqiu; Moon, Bryan P.; Koellhoffer, Jessica P.; Huang, Lingxia; Ward, R. Timothy; Clifton, Elizabeth; Falco, S. Carl; Cigan, A. Mark

2015-01-01

Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing. PMID:26294043

Foldback intercoil DNA and the mechanism of DNA transposition.

PubMed

Kim, Byung-Dong

2014-09-01

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as 180° and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.

Evaluation of human recession defects treated with coronally advanced flaps and either purified recombinant human platelet-derived growth factor-BB with beta tricalcium phosphate or connective tissue: a histologic and microcomputed tomographic examination.

PubMed

McGuire, Michael K; Scheyer, Todd; Nevins, Myron; Schupbach, Peter

2009-02-01

The current study examined the histologic and microcomputed tomographic (micro CT) outcomes of the treatment of gingival recession defects with either a subepithelial connective tissue graft (CTG) or 0.3 mg/mL recombinant human platelet-derived growth factor (rhPDGF-BB) on a beta tricalcium phosphate (beta-TCP) matrix. Gingival recession defects were surgically created in six premolar teeth with no more than 3 mm of keratinized marginal tissue, an osseous crest 2 to 3 mm apical to the newly created gingival margin, and recession depth of at least 3 mm. The defects were left untouched for 2 months; then, four defects were grafted with rhPDGF-BB + beta-TCP + a wound healing dressing, and two defects received CTGs. A coronally advanced flap covered each grafted site. Nine months later, sections were obtained for examination. All four sites treated with rhPDGF-BB + beta-TCP showed connective tissue fibers (Sharpey fibers) perpendicularly inserting into newly formed cementum and alveolar bone. In the two sites treated with CTGs, a long junctional epithelium was seen coronal to the osseous crest and connective tissue fibers ran parallel to the adjacent root surfaces, with no evidence of insertion into cementum or bone. There was no evidence of regeneration of cementum, inserting connective tissue fibers, or supporting alveolar bone. Regeneration of the periodontium in gingival recession defects is possible through growth factor-mediated therapy.

Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells.

PubMed

Chang, Chia-Wei; Lai, Yi-Shin; Pawlik, Kevin M; Liu, Kaimao; Sun, Chiao-Wang; Li, Chao; Schoeb, Trenton R; Townes, Tim M

2009-05-01

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3' long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic "hit and run" vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells.

Large exon size does not limit splicing in vivo.

PubMed

Chen, I T; Chasin, L A

1994-03-01

Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases. It has been proposed that this limitation may derive from the exon definition model of splice site recognition. In this model, a downstream donor site enhances splicing at the upstream acceptor site of the same exon. This enhancement may require contact between factors bound to each end of the exon; an exon size limitation would promote such contact. To test the idea that proximity was required for exon definition, we inserted random DNA fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase minigene and tested whether the expanded exons were efficiently spliced. DNA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus. PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides. A parallel analysis of the RNA from this population by reverse transcription followed by PCR showed a similar size distribution. Central exons as large as 1,400 bases could be spliced into mRNA. We also tested individual plasmid clones containing exon inserts of defined sizes. The largest exon included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the survey of naturally occurring exons. We conclude that a limitation in exon size is not part of the exon definition mechanism.

Structural and mechanistic studies of polymerase η bypass of phenanthriplatin DNA damage.

PubMed

Gregory, Mark T; Park, Ga Young; Johnstone, Timothy C; Lee, Young-Sam; Yang, Wei; Lippard, Stephen J

2014-06-24

Platinum drugs are a mainstay of anticancer chemotherapy. Nevertheless, tumors often display inherent or acquired resistance to platinum-based treatments, prompting the search for new compounds that do not exhibit cross-resistance with current therapies. Phenanthriplatin, cis-diamminephenanthridinechloroplatinum(II), is a potent monofunctional platinum complex that displays a spectrum of activity distinct from those of the clinically approved platinum drugs. Inhibition of RNA polymerases by phenanthriplatin lesions has been implicated in its mechanism of action. The present study evaluates the ability of phenanthriplatin lesions to inhibit DNA replication, a function disrupted by traditional platinum drugs. Phenanthriplatin lesions effectively inhibit DNA polymerases ν, ζ, and κ and the Klenow fragment. In contrast to results obtained with DNA damaged by cisplatin, all of these polymerases were capable of inserting a base opposite a phenanthriplatin lesion, but only Pol η, an enzyme efficient in translesion synthesis, was able to fully bypass the adduct, albeit with low efficiency. X-ray structural characterization of Pol η complexed with site-specifically platinated DNA at both the insertion and +1 extension steps reveals that phenanthriplatin on DNA interacts with and inhibits Pol η in a manner distinct from that of cisplatin-DNA adducts. Unlike cisplatin and oxaliplatin, the efficacies of which are influenced by Pol η expression, phenanthriplatin is highly toxic to both Pol η+ and Pol η- cells. Given that increased expression of Pol η is a known mechanism by which cells resist cisplatin treatment, phenanthriplatin may be valuable in the treatment of cancers that are, or can easily become, resistant to cisplatin.

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli

PubMed Central

Díez-Villaseñor, César; Guzmán, Noemí M.; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J.M.

2013-01-01

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism. PMID:23445770

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli.

PubMed

Díez-Villaseñor, César; Guzmán, Noemí M; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J M

2013-05-01

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.

Histological evidence for muscle insertion in extant amniote femora: implications for muscle reconstruction in fossils.

PubMed

Petermann, Holger; Sander, Martin

2013-04-01

Since the 19th century, identification of muscle attachment sites on bones has been important for muscle reconstructions, especially in fossil tetrapods, and therefore has been the subject of numerous biological and paleontological studies. At the microscopic level, in histological thin sections, the only features that can be used reliably for identifying tendon-bone or muscle-tendon-bone interactions are Sharpey's fibers. Muscles, however, do not only attach to the bone indirectly with tendons, but also directly. Previous studies failed to provide new indicators for muscle attachment, or to address the question of whether muscles with direct attachment can be identified histologically. However, histological identification of direct muscle attachments is important because these attachments do not leave visible marks (e.g. scars and rugosities) on the bone surface. We dissected the right hind limb and mapped the muscle attachment sites on the femur of one rabbit (Oryctolagus cuniculus), one Alligator mississippiensis, and one turkey (Meleagris cuniculus). We then extracted the femur and prepared four histological thin sections for the rabbit and the turkey and five histological thin sections for the alligator. Sharpey's fibers, vascular canal orientation, and a frayed periosteal margin can be indicators for indirect but also direct muscle attachment. Sharpey's fibers can be oriented to the cutting plane of the thin section at high angles, and two Sharpey's fibers orientations can occur in one area, possibly indicating a secondary force axis. However, only about 60% of mapped muscle attachment sites could be detected in thin sections, and frequently histological features suggestive of muscle attachment occurred outside mapped sites. While these insights should improve our ability to successfully identify and reconstruct muscles in extinct species, they also show the limitations of this approach. © 2013 The Authors Journal of Anatomy © 2013 Anatomical Society.

Histological evidence for muscle insertion in extant amniote femora: implications for muscle reconstruction in fossils

PubMed Central

Petermann, Holger; Sander, Martin

2013-01-01

Since the 19th century, identification of muscle attachment sites on bones has been important for muscle reconstructions, especially in fossil tetrapods, and therefore has been the subject of numerous biological and paleontological studies. At the microscopic level, in histological thin sections, the only features that can be used reliably for identifying tendon–bone or muscle–tendon-bone interactions are Sharpey's fibers. Muscles, however, do not only attach to the bone indirectly with tendons, but also directly. Previous studies failed to provide new indicators for muscle attachment, or to address the question of whether muscles with direct attachment can be identified histologically. However, histological identification of direct muscle attachments is important because these attachments do not leave visible marks (e.g. scars and rugosities) on the bone surface. We dissected the right hind limb and mapped the muscle attachment sites on the femur of one rabbit (Oryctolagus cuniculus), one Alligator mississippiensis, and one turkey (Meleagris cuniculus). We then extracted the femur and prepared four histological thin sections for the rabbit and the turkey and five histological thin sections for the alligator. Sharpey's fibers, vascular canal orientation, and a frayed periosteal margin can be indicators for indirect but also direct muscle attachment. Sharpey's fibers can be oriented to the cutting plane of the thin section at high angles, and two Sharpey's fibers orientations can occur in one area, possibly indicating a secondary force axis. However, only about 60% of mapped muscle attachment sites could be detected in thin sections, and frequently histological features suggestive of muscle attachment occurred outside mapped sites. While these insights should improve our ability to successfully identify and reconstruct muscles in extinct species, they also show the limitations of this approach. PMID:23439026

The anatomy and ontogeny of the head, neck, pectoral, and upper limb muscles of Lemur catta and Propithecus coquereli (primates): discussion on the parallelism between ontogeny and phylogeny and implications for evolutionary and developmental biology.

PubMed

Diogo, Rui; Molnar, Julia L; Smith, Timothy D

2014-08-01

Most anatomical studies of primates focus on skeletal tissues, but muscular anatomy can provide valuable information about phylogeny, functional specializations, and evolution. Herein, we present the first detailed description of the head, neck, pectoral, and upper limb muscles of the fetal lemuriforms Lemur catta (Lemuridae) and Propithecus coquereli (Indriidae). These two species belong to the suborder Strepsirrhini, which is often presumed to possess some plesiomorphic anatomical features within primates. We compare the muscular anatomy of the fetuses with that of infants and adults and discuss the evolutionary and developmental implications. The fetal anatomy reflects a phylogenetically more plesiomorphic condition in nine of the muscles we studied and a more derived condition in only two, supporting a parallel between ontogeny and phylogeny. The derived exceptions concern muscles with additional insertions in the fetus which are lost in adults of the same species, that is, flexor carpi radialis inserts on metacarpal III and levator claviculae inserts on the clavicle. Interestingly, these two muscles are involved in movements of the pectoral girdle and upper limb, which are mainly important for activities in later stages of life, such as locomotion and prey capture, rather than activities in fetal life. Accordingly, our findings suggest that some exceptions to the "ontogeny parallels phylogeny" rule are probably driven more by ontogenetic constraints than by adaptive plasticity. © 2014 Wiley Periodicals, Inc.

Climate matching: implications for the biological control of hemlock woolly adelgid

Treesearch

R. Talbot III Trotter

2008-01-01

Classical biological control programs are faced with a daunting challenge: inserting a new species into an existing ecological system. In order for the newly introduced biological control species to survive and reproduce, the recipient ecosystem must provide the required biotic and abiotic requirements. The Adelgid Biological Control simulator (ABCs), a simulation...

Biomechanical and histologic basis of osseodensification drilling for endosteal implant placement in low density bone. An experimental study in sheep.

PubMed

Lahens, Bradley; Neiva, Rodrigo; Tovar, Nick; Alifarag, Adham M; Jimbo, Ryo; Bonfante, Estevam A; Bowers, Michelle M; Cuppini, Marla; Freitas, Helora; Witek, Lukasz; Coelho, Paulo G

2016-10-01

A bone drilling concept, namely osseodensification, has been introduced for the placement of endosteal implants to increase primary stability through densification of the osteotomy walls. This study investigated the effect of osseodensification on the initial stability and early osseointegration of conical and parallel walled endosteal implants in low density bone. Five male sheep were used. Three implants were inserted in the ilium, bilaterally, totaling 30 implants (n=15 conical, and n=15 parallel). Each animal received 3 implants of each type, inserted into bone sites prepared as follows: (i) regular-drilling (R: 2mm pilot, 3.2mm, and 3.8mm twist drills), (ii) clockwise osseodensification (CW), and (iii) counterclockwise (CCW) osseodensification drilling with Densah Bur (Versah, Jackson, MI, USA): 2.0mm pilot, 2.8mm, and 3.8mm multi-fluted burs. Insertion torque as a function of implant type and drilling technique, revealed higher values for osseodensification relative to R-drilling, regardless of implant macrogeometry. A significantly higher bone-to-implant contact (BIC) for both osseodensification techniques (p<0.05) was observed compared to R-drilling. There was no statistical difference in BIC as a function of implant type (p=0.58), nor in bone-area-fraction occupancy (BAFO) as a function of drilling technique (p=0.22), but there were higher levels of BAFO for parallel than conic implants (p=0.001). Six weeks after surgery, new bone formation along with remodeling sites was observed for all groups. Bone chips in proximity with the implants were seldom observed in the R-drilling group, but commonly observed in the CW, and more frequently under the CCW osseodensification technique. In low-density bone, endosteal implants present higher insertion torque levels when placed in osseodensification drilling sites, with no osseointegration impairment compared to standard subtractive drilling methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Trinucleotide Insertions, Deletions, and Point Mutations in Glucose Transporters Confer K+ Uptake in Saccharomyces cerevisiae

PubMed Central

Liang, Hong; Ko, Christopher H.; Herman, Todd; Gaber, Richard F.

1998-01-01

Deletion of TRK1 and TRK2 abolishes high-affinity K+ uptake in Saccharomyces cerevisiae, resulting in the inability to grow on typical synthetic growth medium unless it is supplemented with very high concentrations of potassium. Selection for spontaneous suppressors that restored growth of trk1Δ trk2Δ cells on K+-limiting medium led to the isolation of cells with unusual gain-of-function mutations in the glucose transporter genes HXT1 and HXT3 and the glucose/galactose transporter gene GAL2. 86Rb uptake assays demonstrated that the suppressor mutations conferred increased uptake of the ion. In addition to K+, the mutant hexose transporters also conferred permeation of other cations, including Na+. Because the selection strategy required such gain of function, mutations that disrupted transporter maturation or localization to the plasma membrane were avoided. Thus, the importance of specific sites in glucose transport could be independently assessed by testing for the ability of the mutant transporter to restore glucose-dependent growth to cells containing null alleles of all of the known functional glucose transporter genes. Twelve sites, most of which are conserved among eukaryotic hexose transporters, were revealed to be essential for glucose transport. Four of these have previously been shown to be essential for glucose transport by animal or plant transporters. Eight represented sites not previously known to be crucial for glucose uptake. Each suppressor mutant harbored a single mutation that altered an amino acid(s) within or immediately adjacent to a putative transmembrane domain of the transporter. Seven of 38 independent suppressor mutations consisted of in-frame insertions or deletions. The nature of the insertions and deletions revealed a striking DNA template dependency: each insertion generated a trinucleotide repeat, and each deletion involved the removal of a repeated nucleotide sequence. PMID:9447989

Perforation of the urinary bladder caused by transurethral insertion of a pencil for the purpose of masturbation in a 29-year-old female.

PubMed

Bantis, Athanasios; Sountoulides, Petros; Kalaitzis, Christos; Giannakopoulos, Stelios; Agelonidou, Eleni; Foutzitzi, Soultana; Touloupidis, Stavros

2010-01-01

The urethra is a usual site of introduction of foreign bodies for autoerotic stimulation. We present an unusual case of bladder perforation caused by foreign body that was self-inserted in the urethra and consequently slipped inside the bladder in a 29-year-old female patient with psychiatric disease. The patient was referred to our department for macroscopic hematuria and abdominal pain. Imaging studies revealed the presence of a foreign body in the pelvic area which had perforated the left lateral wall of the bladder. The foreign body was removed via open cystotomy. In psychiatric patients hematuria and pelvic pain may result from insertion of a foreign body in the bladder usually during masturbation.

Perforation of the Urinary Bladder Caused by Transurethral Insertion of a Pencil for the Purpose of Masturbation in a 29-Year-Old Female

PubMed Central

Bantis, Athanasios; Sountoulides, Petros; Kalaitzis, Christos; Giannakopoulos, Stelios; Agelonidou, Eleni; Foutzitzi, Soultana; Touloupidis, Stavros

2010-01-01

The urethra is a usual site of introduction of foreign bodies for autoerotic stimulation. We present an unusual case of bladder perforation caused by foreign body that was self-inserted in the urethra and consequently slipped inside the bladder in a 29-year-old female patient with psychiatric disease. The patient was referred to our department for macroscopic hematuria and abdominal pain. Imaging studies revealed the presence of a foreign body in the pelvic area which had perforated the left lateral wall of the bladder. The foreign body was removed via open cystotomy. In psychiatric patients hematuria and pelvic pain may result from insertion of a foreign body in the bladder usually during masturbation. PMID:20862362

High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

PubMed

Inagaki, Soichi; Henry, Isabelle M; Lieberman, Meric C; Comai, Luca

2015-01-01

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

Tissue Engineering Strategies for the Tendon/ligament-to-bone insertion

PubMed Central

Smith, Lester; Xia, Younan; Galatz, Leesa M.; Genin, Guy M.; Thomopoulos, Stavros

2012-01-01

Injuries to connective tissues are painful and disabling and result in costly medical expenses. These injuries often require re-attachment of an unmineralized connective tissue to bone. The uninjured tendon/ligament-to-bone insertion (enthesis) is a functionally graded material that exhibits a gradual transition from soft tissue (i.e., tendon or ligament) to hard tissue (i.e., mineralized bone) through a fibrocartilaginous transition region. This transition is believed to facilitate force transmission between the two dissimilar tissues by ameliorating potentially damaging interfacial stress concentrations. The transition region is impaired or lost upon tendon/ligament injury and is not regenerated following surgical repair or natural healing, exposing the tissue to risk of re-injury. The need to regenerate a robust tendon-to-bone insertion has led a number of tissue engineering repair strategies. This review treats the tendon-to-bone insertion site as a tissue structure whose primary role is mechanical and discusses current and emerging strategies for engineering the tendon/ligament-to-bone insertion in this context. The focus lies on strategies for producing mechanical structures that can guide and subsequently sustain a graded tissue structure and the associated cell populations. PMID:22185608

An evaluation of the urban stormwater pollutant removal efficiency of catch basin inserts.

PubMed

Morgan, Robert A; Edwards, Findlay G; Brye, Kristofor R; Burian, Stephen J

2005-01-01

In a storm sewer system, the catch basin is the interface between surface runoff and the sewer. Responding to the need to improve the quality of stormwater from urban areas and transportation facilities, and spurred by Phase I and II Stormwater Rules from the U.S. Environmental Protection Agency, several companies market catch basin inserts as best management practices for urban water quality management. However, little data have been collected under controlled tests that indicate the pollutant removal efficiency of these inserts when the inflow is near what can be expected to occur in the field. A stormwater simulator was constructed to test inserts under controlled and replicable conditions. The inserts were tested for removal efficiency of total suspended solids (TSS) and total petroleum hydrocarbons (TPH) at an inflow rate of 757 to 814 L/min, with influent pollutant concentrations of 225 mg/L TSS and 30 mg/L TPH. These conditions are similar to stormwater runoff from small commercial sites in the southeastern United States. Results from the tests indicate that at the test flowrate and pollutant concentration, average TSS removal efficiencies ranged from 11 to 42% and, for TPH, the removal efficiency ranged from 10 to 19%.

Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from Tobacco

PubMed Central

Mazier, Marianne; Botton, Emmanuel; Flamain, Fabrice; Bouchet, Jean-Paul; Courtial, Béatrice; Chupeau, Marie-Christine; Chupeau, Yves; Maisonneuve, Brigitte; Lucas, Hélène

2007-01-01

The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3β-hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome. PMID:17351058

Tissue-engineering strategies for the tendon/ligament-to-bone insertion.

PubMed

Smith, Lester; Xia, Younan; Galatz, Leesa M; Genin, Guy M; Thomopoulos, Stavros

2012-01-01

Injuries to connective tissues are painful and disabling and result in costly medical expenses. These injuries often require reattachment of an unmineralized connective tissue to bone. The uninjured tendon/ligament-to-bone insertion (enthesis) is a functionally graded material that exhibits a gradual transition from soft tissue (i.e., tendon or ligament) to hard tissue (i.e., mineralized bone) through a fibrocartilaginous transition region. This transition is believed to facilitate force transmission between the two dissimilar tissues by ameliorating potentially damaging interfacial stress concentrations. The transition region is impaired or lost upon tendon/ligament injury and is not regenerated following surgical repair or natural healing, exposing the tissue to risk of reinjury. The need to regenerate a robust tendon-to-bone insertion has led a number of tissue engineering repair strategies. This review treats the tendon-to-bone insertion site as a tissue structure whose primary role is mechanical and discusses current and emerging strategies for engineering the tendon/ligament-to-bone insertion in this context. The focus lies on strategies for producing mechanical structures that can guide and subsequently sustain a graded tissue structure and the associated cell populations.

The Tc1/mariner transposable element family shapes genetic variation and gene expression in the protist Trichomonas vaginalis

PubMed Central

2014-01-01

Background Trichomonas vaginalis is the most prevalent non-viral sexually transmitted parasite. Although the protist is presumed to reproduce asexually, 60% of its haploid genome contains transposable elements (TEs), known contributors to genome variability. The availability of a draft genome sequence and our collection of >200 global isolates of T. vaginalis facilitate the study and analysis of TE population dynamics and their contribution to genomic variability in this protist. Results We present here a pilot study of a subset of class II Tc1/mariner TEs that belong to the T. vaginalis Tvmar1 family. We report the genetic structure of 19 Tvmar1 loci, their ability to encode a full-length transposase protein, and their insertion frequencies in 94 global isolates from seven regions of the world. While most of the Tvmar1 elements studied exhibited low insertion frequencies, two of the 19 loci (locus 1 and locus 9) show high insertion frequencies of 1.00 and 0.96, respectively. The genetic structuring of the global populations identified by principal component analysis (PCA) of the Tvmar1 loci is in general agreement with published data based on genotyping, showing that Tvmar1 polymorphisms are a robust indicator of T. vaginalis genetic history. Analysis of expression of 22 genes flanking 13 Tvmar1 loci indicated significantly altered expression of six of the genes next to five Tvmar1 insertions, suggesting that the insertions have functional implications for T. vaginalis gene expression. Conclusions Our study is the first in T. vaginalis to describe Tvmar1 population dynamics and its contribution to genetic variability of the parasite. We show that a majority of our studied Tvmar1 insertion loci exist at very low frequencies in the global population, and insertions are variable between geographical isolates. In addition, we observe that low frequency insertion is related to reduced or abolished expression of flanking genes. While low insertion frequencies might be expected, we identified two Tvmar1 insertion loci that are fixed across global populations. This observation indicates that Tvmar1 insertion may have differing impacts and fitness costs in the host genome and may play varying roles in the adaptive evolution of T. vaginalis. PMID:24834134

Viewing Pornography Depicting Unprotected Anal Intercourse: Are There Implications for HIV Prevention among Men Who Have Sex with Men?

PubMed Central

Stein, Dylan J; Silvera, Richard J; Hagerty, Robert; Marmor, Michael

2011-01-01

We used an Internet-based questionnaire to investigate whether viewing pornography depicting unprotected anal intercourse (UAI) was associated with engaging in UAI in a sample of 821 non-monogamous men who have sex with men (MSM). In the three months prior to interview, 77.2% viewed pornography depicting UAI, 42.6% engaged in insertive UAI, and 38.9% engaged in receptive UAI. Polytomous logistic regression of the 751 subjects who provided data on pornography viewing showed significantly elevated odds ratios for having engaged in receptive UAI, insertive UAI, and both receptive and insertive UAI associated with increasing percentage of pornography viewed that depicted UAI. We also found independently significant associations of engaging in UAI with age, use of inhalant nitrites, and HIV status. Although the data cannot establish causality, our findings indicate that viewing pornography depicting UAI and engaging in UAI are correlated. Further research is needed to determine if this observation may have utility for HIV prevention. PMID:21755381

78 FR 58300 - Texas Eastern Transmission, LP; Notice of Availability of the Environmental Assessment for the...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-23

... Mississippi. \\2\\ A ``pig'' is a tool that is inserted into and moves through the pipeline, and is used for... Web site ( www.ferc.gov ) using the eLibrary link. A limited number of copies of the EA are available... using the eComment feature on the Commission's Web site ( www.ferc.gov ) under the link to Documents and...

Intraosseous Infusion Rates under High Pressure: A Cadaveric Comparison of Anatomic Sites

DTIC Science & Technology

2014-01-01

compartment syndrome, growth plate disruption, hematoma formation, fat embolization , and tissue necrosis [34-37]. These complications can not only be... fat embolism . Pediatr Crit Care Med 2001; 2(2):133-8. 37. Simmons CM, Johnson NE, Perkin RM, van Stralen D. Intraosseous extravasation complication...myeloproliferative malignancy, fracture of targeted bone, previous orthopedic procedures near insertion site, recent IO placement, prosthetic limb or

Recombinase-Mediated Cassette Exchange Using Adenoviral Vectors.

PubMed

Kolb, Andreas F; Knowles, Christopher; Pultinevicius, Patrikas; Harbottle, Jennifer A; Petrie, Linda; Robinson, Claire; Sorrell, David A

2017-01-01

Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

Surface expression of an immunodominant malaria protein B cell epitope by yellow fever virus.

PubMed

Bonaldo, Myrna C; Garratt, Richard C; Caufour, Philippe S; Freire, Marcos S; Rodrigues, Mauricio M; Nussenzweig, Ruth S; Galler, Ricardo

2002-01-25

The yellow fever 17D virus (YF17D) has several characteristics that are desirable for the development of new, live attenuated vaccines. We approached its development as a vector for heterologous antigens by studying the expression of a humoral epitope at the surface of the E protein based on the results of modelling its three-dimensional structure. This model indicated that the most promising insertion site is between beta-strands f and g, a site that is exposed at the external surface of the virus. The large deletion of six residues from the fg loop of the E protein from yellow fever virus, compared to tick-born encephalitis virus, leaves space at the dimer interface for a large insertion without creating steric hindrance. We have tested this hypothesis by inserting a model humoral epitope from the circumsporozoite protein of Plasmodium falciparum consisting of triple NANP repeats. Recombinant virus (17D/8) expressing this insertion flanked by two glycine residues at each end, is specifically neutralized by a monoclonal antibody to the model epitope. Furthermore, mouse antibodies raised to the recombinant virus recognize the parasite protein in an ELISA assay. Serial passage analysis confirmed the genetic stability of the insertion made in the viral genome and the resulting 17D/8 virus is significantly more attenuated in mouse neurovirulence tests than the 17DD vaccine. The fg loop belongs to the dimerization domain of the E protein and lies at the interface between monomers. This domain undergoes a low pH transition, which is related to the fusion of the viral envelope to the endosome membrane. It is conceivable that a slower rate of fusion, resulting from the insertion close to the dimer interface, may delay the onset of virus production and thereby lead to a milder infection of the host. This would account for the more attenuated phenotype of the recombinant virus in the mouse model and lower extent of replication in cultured cells. The vectorial capacity of the yellow fever virus is being further explored for the expression and presentation of other epitopes, including those mediating T-cell responses. Copyright 2002 Academic Press.

Identification of two integration sites in favor of transgene expression in Trichoderma reesei.

PubMed

Qin, Lina; Jiang, Xianzhang; Dong, Zhiyang; Huang, Jianzhong; Chen, Xiuzhen

2018-01-01

The ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei . Currently, only the locus of exocellobiohydrolases I encoding gene ( cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized. To search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified. We have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei . Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions.

Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism.

PubMed

Zhang, Zhenyu; Zhao, Wei; Li, Deshan; Yang, Jinlong; Zsak, Laszlo; Yu, Qingzhong

2015-08-01

In the present study, we developed a novel approach for foreign gene expression by Newcastle disease virus (NDV) from a second ORF through an internal ribosomal entry site (IRES). Six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene behind the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) or large polymerase (L) gene ORF were generated using reverse genetics technology. The insertion of the second ORF slightly attenuated virus pathogenicity, but did not affect ability of the virus to grow. Quantitative measurements of RFP expression in virus-infected DF-1 cells revealed that the abundance of viral mRNAs and red fluorescence intensity were positively correlated with the gene order of NDV, 3'-NP-P-M-F-HN-L-5', proving the sequential transcription mechanism for NDV. The results herein suggest that the level of foreign gene expression could be regulated by selecting the second ORF insertion site to maximize the efficacy of vaccine and gene therapy.

Escherichia coli ArgR mutants defective in cer/Xer recombination, but not in DNA binding.

PubMed

Sénéchal, Hélène; Delesques, Jérémy; Szatmari, George

2010-04-01

The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls the expression of the arginine biosynthetic genes and is required as an accessory factor for Xer site-specific recombination at cer and related recombination sites in plasmids. We used the technique of pentapeptide scanning mutagenesis to isolate a series of ArgR mutants that were considerably reduced in cer recombination, but were still able to repress an argA::lacZ fusion. DNA sequence analysis showed that all of the mutants mapped to the same nucleotide, resulting in a five amino acid insertion between residues 149 and 150 of ArgR, corresponding to the end of the alpha6 helix. A truncated ArgR containing a stop codon at residue 150 displayed the same phenotype as the protein with the five amino acid insertion, and both mutants displayed sequence-specific DNA-binding activity that was L-arginine dependent. These results show that the C-terminus of ArgR is more important in cer/Xer site-specific recombination than in DNA binding.

Analysis of the 227 bp short interspersed nuclear element (SINE) insertion of the promoter of the myostatin (MSTN) gene in different horse breeds.

PubMed

Dall'Olio, Stefania; Scotti, Emilio; Fontanesi, Luca; Tassinari, Marco

2014-01-01

The myostatin (MSTN) gene encodes a protein known to be a negative regulator of muscle mass in mammalian species. Different polymorphisms of the horse (Equus caballus) MSTN gene have been identified, including single nucleotide polymorphisms and a short interspersed nuclear element (SINE) insertion of 227 bp within the promoter of the gene. The SINE insertion has been associated with performance traits in Thoroughbred racehorses and it was proposed as a predictor of optimum racing distance. The aims of this study were to perform in silico analysis to identify putative gains or abrogation of transcription-factor binding sites (TFBSs) generated by the SINE allele of the promoter and to analyse the frequency of the SINE insertion in horses used for racing (gallop and trot) and other purposes. The SINE insertion was genotyped in 227 horses from 10 breeds belonging to different morphological types (brachimorphic, mesomorphic, meso-dolichomorphic and dolichomorphic). The presence of the insertion was confirmed in the Quarter Horse (SINE allele frequency of 0.81) and in the Thoroughbred (0.51), whereas the SINE allele did not segregate in any of the other analysed breeds. As the SINE MSTN gene polymorphism may be population or breed specific, it is not a useful marker for association studies in all breeds.

Cancer gene discovery: exploiting insertional mutagenesis

PubMed Central

Ranzani, Marco; Annunziato, Stefano; Adams, David J.; Montini, Eugenio

2013-01-01

Insertional mutagenesis has been utilized as a functional forward genetics screen for the identification of novel genes involved in the pathogenesis of human cancers. Different insertional mutagens have been successfully used to reveal new cancer genes. For example, retroviruses (RVs) are integrating viruses with the capacity to induce the deregulation of genes in the neighborhood of the insertion site. RVs have been employed for more than 30 years to identify cancer genes in the hematopoietic system and mammary gland. Similarly, another tool that has revolutionized cancer gene discovery is the cut-and-paste transposons. These DNA elements have been engineered to contain strong promoters and stop cassettes that may function to perturb gene expression upon integration proximal to genes. In addition, complex mouse models characterized by tissue-restricted activity of transposons have been developed to identify oncogenes and tumor suppressor genes that control the development of a wide range of solid tumor types, extending beyond those tissues accessible using RV-based approaches. Most recently, lentiviral vectors (LVs) have appeared on the scene for use in cancer gene screens. LVs are replication defective integrating vectors that have the advantage of being able to infect non-dividing cells, in a wide range of cell types and tissues. In this review, we describe the various insertional mutagens focusing on their advantages/limitations and we discuss the new and promising tools that will improve the insertional mutagenesis screens of the future. PMID:23928056

Staphylococcus aureus infections following knee and hip prosthesis insertion procedures.

PubMed

Arduino, Jean Marie; Kaye, Keith S; Reed, Shelby D; Peter, Senaka A; Sexton, Daniel J; Chen, Luke F; Hardy, N Chantelle; Tong, Steven Yc; Smugar, Steven S; Fowler, Vance G; Anderson, Deverick J

2015-01-01

Staphylococcus aureus is the most common and most important pathogen following knee and hip arthroplasty procedures. Understanding the epidemiology of invasive S. aureus infections is important to quantify this serious complication. This nested retrospective cohort analysis included adult patients who had undergone insertion of knee or hip prostheses with clean or clean-contaminated wound class at 11 hospitals between 2003-2006. Invasive S. aureus infections, non-superficial incisional surgical site infections (SSIs) and blood stream infections (BSIs), were prospectively identified following each procedure. Prevalence rates, per 100 procedures, were estimated. 13,719 prosthetic knee (62%) and hip (38%) insertion procedures were performed. Of 92 invasive S. aureus infections identified, SSIs were more common (80%) than SSI and BSI (10%) or BSI alone (10%). The rate of invasive S. aureus infection/100 procedures was 0.57 [95% CI: 0.43-0.73] for knee insertion and 0.83 [95% CI: 0.61-1.08] for hip insertion. More than half (53%) were methicillin-resistant. Median time-to-onset of infection was 34 and 26 days for knee and hip insertion, respectively. Infection was associated with higher National Healthcare Safety Network risk index (p ≤ 0.0001). Post-operative invasive S. aureus infections were rare, but difficult-to-treat methicillin-resistant infections were relatively common. Optimizing preventative efforts may greatly reduce the healthcare burden associated with S. aureus infections.

Deregulation of EIF4E: a novel mechanism for autism.

PubMed

Neves-Pereira, M; Müller, B; Massie, D; Williams, J H G; O'Brien, P C M; Hughes, A; Shen, S-B; Clair, David St; Miedzybrodzka, Z

2009-11-01

Autism is a common childhood onset neurodevelopmental disorder, characterised by severe and sustained impairment of social interaction and social communication, as well as a notably restricted repertoire of activities and interests. Its aetiology is multifactorial with a strong genetic basis. EIF4E is the rate limiting component of eukaryotic translation initiation, and plays a key role in learning and memory through its control of translation within the synapse. EIF4E mediated translation is the final common process modulated by the mammalian target of rapamycin (mTOR), PTEN and fragile X mental retardation protein (FMRP) pathways, which are implicated in autism. Linkage of autism to the EIF4E region on chromosome 4q has been found in genome wide linkage studies. The authors present evidence that directly implicates EIF4E in autism. In a boy with classic autism, the authors observed a de novo chromosome translocation between 4q and 5q and mapped the breakpoint site to within a proposed alternative transcript of EIF4E. They then screened 120 autism families for mutations and found two unrelated families where in each case both autistic siblings and one of the parents harboured the same single nucleotide insertion at position -25 in the basal element of the EIF4E promoter. Electrophoretic mobility shift assays and reporter gene studies show that this mutation enhances binding of a nuclear factor and EIF4E promoter activity. These observations implicate EIF4E, and more specifically control of EIF4E activity, directly in autism. The findings raise the exciting possibility that pharmacological manipulation of EIF4E may provide therapeutic benefit for those with autism caused by disturbance of the converging pathways controlling EIF4E activity.

Determination of Surface-Exposed, Functional Domains of Gonococcal Transferrin-Binding Protein A

PubMed Central

Yost-Daljev, Mary Kate; Cornelissen, Cynthia Nau

2004-01-01

The gonococcal transferrin receptor is composed of two distinct proteins, TbpA and TbpB. TbpA is a member of the TonB-dependent family of integral outer membrane transporters, while TbpB is lipid modified and thought to be peripherally surface exposed. We previously proposed a hypothetical topology model for gonococcal TbpA that was based upon computer predictions and similarity with other TonB-dependent transporters for which crystal structures have been determined. In the present study, the hemagglutinin epitope was inserted into TbpA to probe the surface topology of this protein and secondarily to test the functional impacts of site-specific mutagenesis. Twelve epitope insertion mutants were constructed, five of which allowed us to confirm the surface exposure of loops 2, 3, 5, 7, and 10. In contrast to the predictions set forth by the hypothetical model, insertion into the plug region resulted in an epitope that was surface accessible, while epitope insertions into two putative loops (9 and 11) were not surface accessible. Insertions into putative loop 3 and β strand 9 abolished transferrin binding and utilization, and the plug insertion mutant exhibited decreased transferrin-binding affinity concomitant with an inability to utilize it. Insertion into putative β strand 16 generated a mutant that was able to bind transferrin normally but that was unable to mediate utilization. Mutants with insertions into putative loops 2, 9, and 11 maintained wild-type binding affinity but could utilize only transferrin in the presence of TbpB. This is the first demonstration of the ability of TbpB to compensate for a mutation in TbpA. PMID:14977987

Insertion of a solo LTR retrotransposon associates with spur mutations in 'Red Delicious' apple (Malus × domestica).

PubMed

Han, Mengxue; Sun, Qibao; Zhou, Junyong; Qiu, Huarong; Guo, Jing; Lu, Lijuan; Mu, Wenlei; Sun, Jun

2017-09-01

Insertion of a solo LTR, which possesses strong bidirectional, stem-specific promoter activities, is associated with the evolution of a dwarfing apple spur mutation. Spur mutations in apple scions revolutionized global apple production. Since long terminal repeat (LTR) retrotransposons are tightly related to natural mutations, inter-retrotransposon-amplified polymorphism technique and genome walking were used to find sequences in the apple genome based on these LTRs. In 'Red Delicious' spur mutants, a novel, 2190-bp insertion was identified as a spur-specific, solo LTR (sLTR) located at the 1038th nucleotide of another sLTR, which was 1536 bp in length. This insertion-within-an-insertion was localized within a preexisting Gypsy-50 retrotransposon at position 3,762,767 on chromosome 4. The analysis of transcriptional activity of the two sLTRs (the 2190- and 1536-bp inserts) indicated that the 2190-bp sLTR is a promoter, capable of bidirectional transcription. GUS expression in the 2190-bp-sense and 2190-bp-antisense transgenic lines was prominent in stems. In contrast, no promoter activity from either the sense or the antisense strand of the 1536-bp sLTR was detected. From ~150 kb of DNA on each side of the 2190 bp, sLTR insertion site, corresponding to 300 kb of the 'Golden Delicious' genome, 23 genes were predicted. Ten genes had predicted functions that could affect shoot development. This first report, of a sLTR insertion associated with the evolution of apple spur mutation, will facilitate apple breeding, cloning of spur-related genes, and discovery of mechanisms behind dwarf habit.

Lunar Orbit Insertion Targeting and Associated Outbound Mission Design for Lunar Sortie Missions

NASA Technical Reports Server (NTRS)

Condon, Gerald L.

2007-01-01

This report details the Lunar Orbit Insertion (LOI) arrival targeting and associated mission design philosophy for Lunar sortie missions with up to a 7-day surface stay and with global Lunar landing site access. It also documents the assumptions, methodology, and requirements validated by TDS-04-013, Integrated Transit Nominal and Abort Characterization and Sensitivity Study. This report examines the generation of the Lunar arrival parking orbit inclination and Longitude of the Ascending Node (LAN) targets supporting surface missions with global Lunar landing site access. These targets support the Constellation Program requirement for anytime abort (early return) by providing for a minimized worst-case wedge angle [and an associated minimum plane change delta-velocity (V) cost] between the Crew Exploration Vehicle (CEV) and the Lunar Surface Access Module (LSAM) for an LSAM launch anytime during the Lunar surface stay.

Sites of intermolecular crosslinking of fatty acyl chains in phospholipids carrying a photoactivable carbene precursor

PubMed Central

Gupta, Chhitar M.; Costello, Catherine E.; Khorana, H. Gobind

1979-01-01

Sonicated vesicles of 1-fatty acyl-2-ω-(2-diazo-3,3,3-trifluoropropionoxy) fatty acyl sn-glycero-3-phosphoryl-cholines were shown recently to form intermolecular crosslinks by insertion of the photogenerated carbene into a C—H bond of a neighboring hydrocarbon chain. We now report that photolysis of multilamellar dispersions gives a second series of products in which carbene insertion is accompanied by elimination of a molecule of hydrogen fluoride. The sites of crosslinking in the latter compounds have been studied by mass spectrometry using phospholipids with varying chain lengths of the fatty acyl groups carrying the carbene precursor. The patterns observed show that the point of maximum crosslinking is consistent with the recent conclusion that in phospholipids the sn-2 fatty acyl chain trails the sn-1 chain by 2-4 atoms. Images PMID:16592675

Technical note: assessment of recovery site of mobile nylon bags for measuring ileal digestibility of starch in dairy cows.

PubMed

Norberg, E; Volden, H; Harstad, O M

2007-01-01

The objective of this study was to evaluate recovery site of mobile nylon bags for measuring ileal digestibility of ruminally undegraded starch in dairy cows. Eight feed samples of untreated and treated concentrates were examined. Three lactating cows equipped with rumen fistula and duodenal and ileal cannulas were used in the experiment. The mobile nylon bags containing intact feeds or residues after a 12-h ruminal incubation were pretreated using a 2-step procedure to simulate abomasal digestion before insertion through the duodenal cannula. To assess the effect of hindgut fermentation on starch digestibility, approximately half of the bags were collected from the ileum and half from the feces. The results indicate that feed samples should be preincubated in rumen before insertion into duodenum, and that samples with relatively high fractions of rumen-undigestible starch should be collected from the ileum instead of from feces.

Reactions of Persistent Carbenes with Hydrogen-Terminated Silicon Surfaces.

PubMed

Zhukhovitskiy, Aleksandr V; Mavros, Michael G; Queeney, K T; Wu, Tony; Voorhis, Troy Van; Johnson, Jeremiah A

2016-07-13

Surface passivation has enabled the development of silicon-based solar cells and microelectronics. However, a number of emerging applications require a paradigm shift from passivation to functionalization, wherein surface functionality is installed proximal to the silicon surface. To address this need, we report here the use of persistent aminocarbenes to functionalize hydrogen-terminated silicon surfaces via Si-H insertion reactions. Through the use of model compounds (H-Si(TMS)3 and H-Si(OTMS)3), nanoparticles (H-SiNPs), and planar Si(111) wafers (H-Si(111)), we demonstrate that among different classes of persistent carbenes, the more electrophilic and nucleophilic ones, in particular, a cyclic (alkyl)(amino)carbene (CAAC) and an acyclic diaminocarbene (ADAC), are able to undergo insertion into Si-H bonds at the silicon surface, forming persistent C-Si linkages and simultaneously installing amine or aminal functionality in proximity to the surface. The CAAC (6) is particularly notable for its clean insertion reactivity under mild conditions that produces monolayers with 21 ± 3% coverage of Si(111) atop sites, commensurate with the expected maximum of ∼20%. Atomic force and transmission electron microscopy, nuclear magnetic resonance, X-ray photoelectron, and infrared spectroscopy, and time-of-flight secondary ion mass spectrometry provided evidence for the surface Si-H insertion process. Furthermore, computational studies shed light on the reaction energetics and indicated that CAAC 6 should be particularly effective at binding to silicon dihydride, trihydride, and coupled monohyride motifs, as well as oxidized surface sites. Our results pave the way for the further development of persistent carbenes as universal ligands for silicon and potentially other nonmetallic substrates.

Structures of the Signal Recognition Particle Receptor from the Archaeon Pyrococcus furiosus: Implications for the Targeting Step at the Membrane

PubMed Central

Egea, Pascal F.; Tsuruta, Hiro; de Leon, Gladys P.; Napetschnig, Johanna; Walter, Peter; Stroud, Robert M.

2008-01-01

In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP•magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP•SR targeting complexes. PMID:18978942

Novel splice site mutation in the growth hormone receptor gene in Turkish patients with Laron-type dwarfism.

PubMed

Arman, Ahmet; Ozon, Alev; Isguven, Pinar S; Coker, Ajda; Peker, Ismail; Yordam, Nursen

2008-01-01

Growth hormone (GH) is involved in growth, and fat and carbohydrate metabolism. Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of insulin-like growth factor-I (IGF-I) which mediates GH actions. Mutations in the GHR cause severe postnatal growth failure; the disorder is an autosomal recessive genetic disease resulting in GH insensitivity, called Laron syndrome. It is characterized by dwarfism with elevated serum GH and low levels of IGF-I. We analyzed the GHR gene for mutations and polymorphisms in eight patients with Laron-type dwarfism from six families. We found three missense mutations (S40L, V125A, I526L), one nonsense mutation (W157X), and one splice site mutation in the extracellular domain of GHR. Furthermore, G168G and exon 3 deletion polymorphisms were detected in patients with Laron syndrome. The splice site mutation, which is a novel mutation, was located at the donor splice site of exon 2/ intron 2 within GHR. Although this mutation changed the highly conserved donor splice site consensus sequence GT to GGT by insertion of a G residue, the intron splicing between exon 2 and exon 3 was detected in the patient. These results imply that the splicing occurs arthe GT site in intron 2, leaving the extra inserted G residue at the end of exon 2, thus changing the open reading frame of GHR resulting in a premature termination codon in exon 3.

Presence of multiple sites containing polar material in spherical Escherichia coli cells that lack MreB.

PubMed

Nilsen, Trine; Yan, Arthur W; Gale, Gregory; Goldberg, Marcia B

2005-09-01

In rod-shaped bacteria, certain proteins are specifically localized to the cell poles. The nature of the positional information that leads to the proper localization of these proteins is unclear. In a screen for factors required for the localization of the Shigella sp. actin assembly protein IcsA to the bacterial pole, a mutant carrying a transposon insertion in mreB displayed altered targeting of IcsA. The phenotype of cells containing a transposon insertion in mreB was indistinguishable from that of cells containing a nonpolar mutation in mreB or that of wild-type cells treated with the MreB inhibitor A22. In cells lacking MreB, a green fluorescent protein (GFP) fusion to a cytoplasmic derivative of IcsA localized to multiple sites. Secreted full-length native IcsA was present in multiple faint patches on the surfaces of these cells in a pattern similar to that seen for the cytoplasmic IcsA-GFP fusion. EpsM, the polar Vibrio cholerae inner membrane protein, also localized to multiple sites in mreB cells and colocalized with IcsA, indicating that localization to multiple sites is not unique to IcsA. Our results are consistent with the requirement, either direct or indirect, for MreB in the restriction of certain polar material to defined sites within the cell and, in the absence of MreB, with the formation of ectopic sites containing polar material.

piggyBac Transposon-Mediated Transgenesis in the Apicomplexan Parasite Eimeria tenella

PubMed Central

Su, Huali; Liu, Xianyong; Yan, Wenchao; Shi, Tuanyuan; Zhao, Xinxin; Blake, Damer P.; Tomley, Fiona M.; Suo, Xun

2012-01-01

piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5′ and 3′ ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac. PMID:22768223

Development of a Robotic Colonoscopic Manipulation System, Using Haptic Feedback Algorithm

PubMed Central

Woo, Jaehong; Choi, Jae Hyuk; Seo, Jong Tae

2017-01-01

Purpose Colonoscopy is one of the most effective diagnostic and therapeutic tools for colorectal diseases. We aim to propose a master-slave robotic colonoscopy that is controllable in remote site using conventional colonoscopy. Materials and Methods The master and slave robot were developed to use conventional flexible colonoscopy. The robotic colonoscopic procedure was performed using a colonoscope training model by one expert endoscopist and two unexperienced engineers. To provide the haptic sensation, the insertion force and the rotating torque were measured and sent to the master robot. Results A slave robot was developed to hold the colonoscopy and its knob, and perform insertion, rotation, and two tilting motions of colonoscope. A master robot was designed to teach motions of the slave robot. These measured force and torque were scaled down by one tenth to provide the operator with some reflection force and torque at the haptic device. The haptic sensation and feedback system was successful and helpful to feel the constrained force or torque in colon. The insertion time using robotic system decreased with repeated procedures. Conclusion This work proposed a robotic approach for colonoscopy using haptic feedback algorithm, and this robotic device would effectively perform colonoscopy with reduced burden and comparable safety for patients in remote site. PMID:27873506

Synthesis of Defect Perovskites (He 2–x⟂ x)(CaZr)F 6 by Inserting Helium into the Negative Thermal Expansion Material CaZrF 6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hester, Brett R.; dos Santos, António M.; Molaison, Jamie J.

Defect perovskites (He 2–x⟂ x)(CaZr)F 6 can be prepared by inserting helium into CaZrF 6 at high pressure. They can be recovered to ambient pressure at low temperature. There are no prior examples of perovskites with noble gases on the A-sites. The insertion of helium gas into CaZrF 6 both elastically stiffens the material and reduces the magnitude of its negative thermal expansion. It also suppresses the onset of structural disorder, which is seen on compression in other media. Measurements of the gas released on warming to room temperature and Rietveld analyses of neutron diffraction data at low temperature indicatemore » that exposure to helium gas at 500 MPa leads to a stoichiometry close to (He 1⟂ 1)(CaZr)F 6. Helium has a much higher solubility in CaZrF 6 than silica glass or crystobalite. An analogue with composition (H 2) 2(CaZr)F 6 would have a volumetric hydrogen storage capacity greater than current US DOE targets. We anticipate that other hybrid perovskites with small neutral molecules on the A-site can also be prepared and that they will display a rich structural chemistry.« less

High-throughput analysis of T-DNA location and structure using sequence capture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously,more » using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less

High-throughput analysis of T-DNA location and structure using sequence capture

DOE PAGES

Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.; ...

2015-10-07

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously,more » using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less

Long-term effect of tetracycline fibers on recurrent lesions in periodontal maintenance patients.

PubMed

Corsair, A

1994-01-01

Thirty-one patients with chronic adult periodontitis participated in this long-term private practice study. Sixty-one sites with moderate (4-6 mm, n = 21) or deep (> or = 7 mm, n = 40) periodontal pockets and bleeding on probing were treated with an antibiotic-releasing fiber, Actisite (Periodontal Therapeutic System) (tetracycline hydrochloride). Scaling/root planing (SRP) was performed on all teeth 3 weeks before start of the study, and on most teeth immediately before fiber insertion. Monolithic fibers loaded with 25% tetracycline hydrochloride were inserted in periodontal pockets, where they were retained for a mean of 6.7 days, at which point they were removed. Patients were evaluated at 1, 3, and 6 months after treatment; subgroups were also evaluated at normalized 12-month (10 patients, 20 sites) and 24-month (13 patients, 20 sites) time points. Sites showed a mean probing depth reduction of 2.5 mm at 6 months, and 2.2 mm at 24 months, with the deepest sites showing the greatest reduction. Bleeding on probing was 100% at baseline, 34% at 3 months, and 50% at 6 months. Attachment gains in 18 patients (25 sites) were 2.4 mm at 1 month, 3.0 mm at 3 months, and 2.5 mm at 6 months. Fibers were well tolerated; no adverse effects from treatment were noted. These results indicate that use of tetracycline fiber plus SRP clearly reduced the clinical signs of periodontal disease and maintained or improved attachment for up to 24 months in sites previously refractory to treatment.

The role of lipid composition for insertion and stabilization of amino acids in membranes

NASA Astrophysics Data System (ADS)

Johansson, Anna C. V.; Lindahl, Erik

2009-05-01

While most membrane protein helices are clearly hydrophobic, recent experiments have indicated that it is possible to insert marginally hydrophobic helices into bilayers and have suggested apparent in vivo free energies of insertion for charged residues that are low, e.g., a few kcals for arginine. In contrast, a number of biophysical simulation studies have predicted that the bilayer interior is close to a pure hydrophobic environment with large penalties for hydrophilic amino acids—and yet the experimental scales do significantly better at predicting actual membrane proteins from sequence. Here, we have systematically studied the dependence of the free energy profiles on lipid properties, including tail length, saturation, headgroup hydrogen bond strength, and charge, both to see to whether the in vivo insertion can be explained in whole or part from lipid composition of the endoplasmic reticulum (ER) membranes, and if the solvation properties can help interpret how protein function depends on the lipids. We find that lipid charge is important to stabilize charged amino acids inside the bilayer (with implications, e.g., for ion channels), that thicker bilayers have higher solvation costs for hydrophilic side chains, and that headgroup hydrogen bond strength determines how adaptive the lipids are as a hydrophobic/hydrophilic solvent. None of the different free energy profiles are even close to the low apparent in vivo insertion cost, which suggests that regardless of the specific ER membrane composition the current experimental results cannot be explained by normal lipid-type variation.

Molecular landscape of the interaction between the urease accessory proteins UreE and UreG.

PubMed

Merloni, Anna; Dobrovolska, Olena; Zambelli, Barbara; Agostini, Federico; Bazzani, Micaela; Musiani, Francesco; Ciurli, Stefano

2014-09-01

Urease, the most efficient enzyme so far discovered, depends on the presence of nickel ions in the catalytic site for its activity. The transformation of inactive apo-urease into active holo-urease requires the insertion of two Ni(II) ions in the substrate binding site, a process that involves the interaction of four accessory proteins named UreD, UreF, UreG and UreE. This study, carried out using calorimetric and NMR-based structural analysis, is focused on the interaction between UreE and UreG from Sporosarcina pasteurii, a highly ureolytic bacterium. Isothermal calorimetric protein-protein titrations revealed the occurrence of a binding event between SpUreE and SpUreG, entailing two independent steps with positive cooperativity (Kd1=42±9μM; Kd2=1.7±0.3μM). This was interpreted as indicating the formation of the (UreE)2(UreG)2 hetero-oligomer upon binding of two UreG monomers onto the pre-formed UreE dimer. The molecular details of this interaction were elucidated using high-resolution NMR spectroscopy. The occurrence of SpUreE chemical shift perturbations upon addition of SpUreG was investigated and analyzed to establish the protein-protein interaction site. The latter appears to involve the Ni(II) binding site as well as mobile portions on the C-terminal and the N-terminal domains. Docking calculations based on the information obtained from NMR provided a structural basis for the protein-protein contact site. The high sequence and structural similarity within these protein classes suggests a generality of the interaction mode among homologous proteins. The implications of these results on the molecular details of the urease activation process are considered and analyzed. Copyright © 2014 Elsevier B.V. All rights reserved.

Reverse genetics of Newcastle disease virus

USDA-ARS?s Scientific Manuscript database

Reverse genetics allows the generation of recombinant viruses or vectors used in functional studies, vaccine development, and gene therapy. This technique allows genetic manipulation and cloning of viral genomes, mutation through site-directed mutagenesis, and gene insertion or deletion, among othe...

40 CFR 725.455 - Information to be included in the Tier II exemption request.

Code of Federal Regulations, 2011 CFR

2011-07-01

... identification. (2) Type of genetic modification and the function of the introduced genetic material. (3) Site of insertion. (4) Certification of compliance with the introduced genetic material criteria described in § 725...

40 CFR 725.455 - Information to be included in the Tier II exemption request.

Code of Federal Regulations, 2012 CFR

2012-07-01

... identification. (2) Type of genetic modification and the function of the introduced genetic material. (3) Site of insertion. (4) Certification of compliance with the introduced genetic material criteria described in § 725...

40 CFR 725.455 - Information to be included in the Tier II exemption request.

Code of Federal Regulations, 2013 CFR

2013-07-01

... identification. (2) Type of genetic modification and the function of the introduced genetic material. (3) Site of insertion. (4) Certification of compliance with the introduced genetic material criteria described in § 725...

40 CFR 725.455 - Information to be included in the Tier II exemption request.

Code of Federal Regulations, 2014 CFR

2014-07-01

... identification. (2) Type of genetic modification and the function of the introduced genetic material. (3) Site of insertion. (4) Certification of compliance with the introduced genetic material criteria described in § 725...

48 CFR 903.1004 - Contract clauses.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Contract clauses. (b)(2)(ii) Insert the DOE Web site address http://ig.energy.gov/hotline.htm in paragraph (b)(3) of the 48 CFR 52.203-14 clause, Display of Hotline Poster(s). [76 FR 7690, Feb. 11, 2011] ...

48 CFR 903.1004 - Contract clauses.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Contract clauses. (b)(2)(ii) Insert the DOE Web site address http://ig.energy.gov/hotline.htm in paragraph (b)(3) of the 48 CFR 52.203-14 clause, Display of Hotline Poster(s). [76 FR 7690, Feb. 11, 2011] ...

48 CFR 903.1004 - Contract clauses.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Contract clauses. (b)(2)(ii) Insert the DOE Web site address http://ig.energy.gov/hotline.htm in paragraph (b)(3) of the 48 CFR 52.203-14 clause, Display of Hotline Poster(s). [76 FR 7690, Feb. 11, 2011] ...

48 CFR 903.1004 - Contract clauses.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Contract clauses. (b)(2)(ii) Insert the DOE Web site address http://ig.energy.gov/hotline.htm in paragraph (b)(3) of the 48 CFR 52.203-14 clause, Display of Hotline Poster(s). [76 FR 7690, Feb. 11, 2011] ...

Influence of underpreparation on primary stability of implants inserted in poor quality bone sites: an in vitro study.

PubMed

Degidi, Marco; Daprile, Giuseppe; Piattelli, Adriano

2015-06-01

The purpose of this present study was to investigate the relation between implant site underpreparation and primary stability in the presence of poor-quality bone. A study was performed on fresh humid bovine bone; samples presented no cortical layer with a cancellous structure inside and were obtained from the hip. The bones were firmly attached to a base device. Sixty sites were prepared according to the protocol provided by the manufacturer: a 2-mm pilot drill was introduced to the proper depth and then twist drills of 3 and 3.4 mm were used. After site preparation, 20 3.4- × 11-mm (standard protocol group), 20 3.8- × 11-mm (10% undersized group), and 20 4.5- × 11-mm (25% undersized group) implants were inserted at a calibrated maximum torque of 70 N-cm at the predetermined speed of 30 rpm. After implant insertion, variable torque work (VTW), maximum insertion torque (peak IT), and resonance frequency analysis (RFA) values were recorded. The standard protocol group showed a mean VTW of 565.77 ± 219.12 N-cm, a peak IT of 11.3 ± 4.44 N-cm, and an RFA of 69.35 ± 7.35 implant stability quotient (ISQ). The 10% undersized group showed a mean VTW of 1,240.24 ± 407.78 N-cm, a peak IT of 20.26 ± 7.03 N-cm, and an RFA of 73.40 ± 2.33 ISQ. The 25% undersized group showed a mean VTW of 1,254.96 ± 727.49 N-cm, a peak IT of 17.15 ± 10.39 N-cm, and an RFA of 72.30 ± 6.70 ISQ. For VTW, the difference between the standard and undersized protocol values was statistically significant; for peak IT, the difference between the standard and 10% undersized protocol values was statistically significant; no other statistical differences were found between mean values. In the presence of poor-bone quality, a 10% undersized protocol is sufficient to improve the primary stability of the implant; additional decreases do not seem to enhance primary stability values. Copyright © 2015 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Molecular mechanisms of retroviral integration site selection

PubMed Central

Kvaratskhelia, Mamuka; Sharma, Amit; Larue, Ross C.; Serrao, Erik; Engelman, Alan

2014-01-01

Retroviral replication proceeds through an obligate integrated DNA provirus, making retroviral vectors attractive vehicles for human gene-therapy. Though most of the host cell genome is available for integration, the process of integration site selection is not random. Retroviruses differ in their choice of chromatin-associated features and also prefer particular nucleotide sequences at the point of insertion. Lentiviruses including HIV-1 preferentially integrate within the bodies of active genes, whereas the prototypical gammaretrovirus Moloney murine leukemia virus (MoMLV) favors strong enhancers and active gene promoter regions. Integration is catalyzed by the viral integrase protein, and recent research has demonstrated that HIV-1 and MoMLV targeting preferences are in large part guided by integrase-interacting host factors (LEDGF/p75 for HIV-1 and BET proteins for MoMLV) that tether viral intasomes to chromatin. In each case, the selectivity of epigenetic marks on histones recognized by the protein tether helps to determine the integration distribution. In contrast, nucleotide preferences at integration sites seem to be governed by the ability for the integrase protein to locally bend the DNA duplex for pairwise insertion of the viral DNA ends. We discuss approaches to alter integration site selection that could potentially improve the safety of retroviral vectors in the clinic. PMID:25147212

A training program for novice paramedics provides initial laryngeal mask airway insertion skill and improves skill retention at 6 months.

PubMed

Hein, Cindy; Owen, Harry; Plummer, John

2010-02-01

Major resuscitation councils endorse the use of the laryngeal mask airway (LMA) by paramedics for lifesaving airway interventions. Learning and maintaining adequate skill level is important for patient safety. The aim of this project was to develop a training program that provides student paramedics with initial knowledge and experience in LMA insertion skills but equally important to provide ongoing skill retention. After ethics approval and informed consent, 55 first year Paramedic degree students watched a manufacturer's LMA instruction video and practiced insertion in three different part task trainers. Six months later, subjects were randomized to an intervention (reviewing the video and 10 minutes unsupervised practice) or control group before participating in a high-fidelity simulated clinical scenario. For equity of training, the control group received the intervention after the scenario. Main outcomes measured were time to insertion; success rate; and LMA skill retention (sum of LMA orientation; cuff inflation; bite block; securing; patient positioning; and overall subject performance). Fifty subjects completed the study. Those in the intervention group displayed significantly shorter insertion times (P = 0.029), fewer attempts to achieve success (P = 0.033), and had significantly higher LMA skill performance levels (P = 0.019) at 6 months. We devised a short intervention based on our training program using a video and practice in part task trainers. In an assessment using high-fidelity simulation, we demonstrated significant improvements in maintenance of LMA insertion skills in student paramedics at 6 months. Our model of just-in-time assessment and reinforcement of training prevents skill decay and has implications for healthcare skills training in general.

Adapted preparation technique for screw-type implants: explorative in vitro pilot study in a porcine bone model.

PubMed

Beer, Andreas; Gahleitner, André; Holm, Anders; Birkfellner, Wolfgang; Homolka, Peter

2007-02-01

The aim of this study was to quantify the effect of adapted preparation on the insertion torque of self-tapping implants in cancellous bone. In adapted preparation, bone condensation - and thus, insertion torque - is controlled by changing the diameter of the drilling. After preparation of cancellous porcine vertebral bone with drills of 2.85, 3, 3.15 or 3.35 mm final diameters, Brånemark sytem Mk III implants (3.75 x 11.5 mm) were inserted in 141 sites. During implantation, the insertion torque was recorded. Prior to implant insertion, bone mineralization (bone mineral density (BMD)) was measured with dental quantative computed tomography. The BMD values measured at the implant position were correlated with insertion torque for varying bone condensation. Based on the average torque recorded during implant insertion into the pre-drilled canals with a diameter of 3 mm, torque increased by approximately 17% on reducing the diameter of the drill by 5% (to 2.85 mm). On increasing the diameter of the osteotomy to 3.15 mm (5%) or 3.35 mm (12%), torque values decreased by approximately 21% and 50%, respectively. The results demonstrate a correlation between primary stability (average insertion torque) and the diameter of the implant bed on using a screw-shaped implant. Thus, using an individualized bone mineralization-dependent drilling technique, optimized torque values could be achieved in all tested bone qualities with BMDs ranging from 330 to 500 mg/cm(3). The results indicate that using a bone-dependent drilling technique, higher torque values can also be achieved in poor bone using an individualized drilling resulting in higher bone condensation. As immediate function is dependent on primary stability (high insertion torque), this indicates that primary stability can be increased using a modified drilling technique in lesser mineralized bone.

Refoldable Foldamers: Global Conformational Switching by Deletion or Insertion of a Single Hydrogen Bond

PubMed Central

Le Bailly, Bryden A. F.; Byrne, Liam

2016-01-01

Abstract Small changes in the structure of a foldamer may lead to gross changes in conformational preference. We show that the simple insertion or deletion of a single hydrogen bond by changes in pH or by photochemical deprotection is sufficient to refold a helical oligomer, interconverting M and P screw‐sense preference. As a consequence of the switch, information may be transmitted to a remote catalytic site, selectively directing the formation of either of two enantiomeric products by a reaction involving 1,22‐remote intermolecular asymmetric induction. PMID:26762559

Disulfide-Bridged (Mo3S11) Cluster Polymer: Molecular Dynamics and Application as Electrode Material for a Rechargeable Magnesium Battery.

PubMed

Truong, Quang Duc; Kempaiah Devaraju, Murukanahally; Nguyen, Duc N; Gambe, Yoshiyuki; Nayuki, Keiichiro; Sasaki, Yoshikazu; Tran, Phong D; Honma, Itaru

2016-09-14

Exploring novel electrode materials is critical for the development of a next-generation rechargeable magnesium battery with high volumetric capacity. Here, we showed that a distinct amorphous molybdenum sulfide, being a coordination polymer of disulfide-bridged (Mo3S11) clusters, has great potential as a rechargeable magnesium battery cathode. This material provided good reversible capacity, attributed to its unique structure with high flexibility and capability of deformation upon Mg insertion. Free-terminal disulfide moiety may act as the active site for reversible insertion and extraction of magnesium.

Tendon entheses of the human masticatory muscles.

PubMed

Hems, T; Tillmann, B

2000-09-01

Tendons attach to the limb skeleton via chondral-apophysary or periosteal-diaphysary entheses. It was the aim of the present study to investigate the tendon entheses of the temporal, the masseter, as well as the medial and lateral pterygoid muscles, considering the biomechanics and the mode of osteogenesis at the attachment sites. The origin and insertion zones of the four masticatory muscles were studied histologically and by polarization light microscopy in six halves of human heads. Contrary to the limb skeleton no causal relationship between the histological structure of the tendon entheses and the osteogenic mode of the bone areas involved was observed in the masticatory muscles that were studied. Based on the histological findings, a purely structural classification of the tendon attachments irrespective of the osteogenesis is therefore proposed that is applicable to the entire skeleton. It is possible to distinguish between tendon entheses inserting into periosteum, into bone or into fibrocartilage. Tendon attachments with periosteal insertion are found at the temporal plane, the retromolar triangle, zygomatic arch, lateral pterygoid plate, in the caudal zone of the pterygoid fovea of the neck of mandible as well as major portions of the ramus and angle of the mandible. The attachment zones in which collagen fibrils of tendons insert into the bone via the periosteum correspond in their structure to plane periosteal-diaphysary insertions into the diaphyses of long bones. Attachment zones to the bone are present at the inferior temporal line, the base of the coronoid process, the caudal surface of the zygomatic arch, the cranial zones of the pterygoid fovea of the neck of the mandible as well as at circumscribed areas of the ramus and angle of the mandible. In these zones the collagen fibers of the tendon insert immediately into the bone without any mediation of other tissues. The entheses resemble those of circumscribed periosteal-diaphysary attachments to long bones. Fibrocartilaginous entheses occur at the coronoid process, the cranialmost portions of the pterygoid fovea of the neck of the mandible as well as in circumscribed areas of the medial and lateral facets of the angle of the mandible. The structures of these attachment sites are comparable to chondral-apophysary tendon attachments. As for masticatory muscles, the described forms of tendon entheses occur at the same time in the majority of the attachment sites. From the structure of the three types of tendon entheses it is possible to conclude that they fulfill a biomechanical function similar to that of the limb skeleton, namely adapting the different elasticity moduli of bone and tendon tissues. From a technical perspective they can be considered to act as an "angle and stretching brake".

Rapid discrimination of sequences flanking and within T-DNA insertions in the Arabidopsis genome.

PubMed

Ponce, M R; Quesada, V; Micol, J L

1998-05-01

An improvement to previous methods for recovering Arabidopsis thaliana genomic DNA flanking T-DNA insertions is presented that allows for the avoidance of some of the cloning difficulties caused by the concatameric nature of T-DNA inserts. The principle of the procedure is to categorize by size restriction fragments of mutant DNA, produced in separate digestions with NdeI and Bst1107I. Given that the sites for these two enzymes are contiguous within the pGV3850:1003 T-DNA construct, the restriction fragments obtained fall into two categories: those showing identical size in both digestions, which correspond to sequences internal to T-DNA concatamers; and those of different sizes, that contain the junctions between plant DNA and the T-DNA insert. Such a criterion makes it possible to easily distinguish the digestion products corresponding to internal T-DNA parts, which do not deserve further attention, and those which presumably include a segment of the locus of interest. Discrimination between restriction fragments of genomic mutant DNA can be made on rescued plasmids, inverse PCR amplification products or bands in a genomic blot.

[Isolation and function of genes regulating aphB expression in Vibrio cholerae].

PubMed

Chen, Haili; Zhu, Zhaoqin; Zhong, Zengtao; Zhu, Jun; Kan, Biao

2012-02-04

We identified genes that regulate the expression of aphB, the gene encoding a key virulence regulator in Vibrio cholerae O1 E1 Tor C6706(-). We constructed a transposon library in V. cholerae C6706 strain containing a P(aphB)-luxCDABE and P(aphB)-lacZ transcriptional reporter plasmids. Using a chemiluminescence imager system, we rapidly detected aphB promoter expression level at a large scale. We then sequenced the transposon insertion sites by arbitrary PCR and sequencing analysis. We obtained two candidate mutants T1 and T2 which displayed reduced aphB expression from approximately 40,000 transposon insertion mutants. Sequencing analysis shows that Tn inserted in vc1585 reading frame in the T1 mutant and Tn inserted in the end of coding sequence of vc1602 in the T2 mutant. By using a genetic screen, we identified two potential genes that may involve in regulation of the expression of the key virulence regulator AphB. This study sheds light on our further investigation to fully understand V. cholerae virulence gene regulatory cascades.

Familial retinoblastoma due to intronic LINE-1 insertion causes aberrant and noncanonical mRNA splicing of the RB1 gene.

PubMed

Rodríguez-Martín, Carlos; Cidre, Florencia; Fernández-Teijeiro, Ana; Gómez-Mariano, Gema; de la Vega, Leticia; Ramos, Patricia; Zaballos, Ángel; Monzón, Sara; Alonso, Javier

2016-05-01

Retinoblastoma (RB, MIM 180200) is the paradigm of hereditary cancer. Individuals harboring a constitutional mutation in one allele of the RB1 gene have a high predisposition to develop RB. Here, we present the first case of familial RB caused by a de novo insertion of a full-length long interspersed element-1 (LINE-1) into intron 14 of the RB1 gene that caused a highly heterogeneous splicing pattern of RB1 mRNA. LINE-1 insertion was inferred by mRNA studies and full-length sequenced by massive parallel sequencing. Some of the aberrant mRNAs were produced by noncanonical acceptor splice sites, a new finding that up to date has not been described to occur upon LINE-1 retrotransposition. Our results clearly show that RNA-based strategies have the potential to detect disease-causing transposon insertions. It also confirms that the incorporation of new genetic approaches, such as massive parallel sequencing, contributes to characterize at the sequence level these unique and exceptional genetic alterations.

Influence of insertion site of the avian influenza virus haemagglutinin (HA) gene within the Newcastle disease virus genome on HA expression.

PubMed

Ramp, Kristina; Skiba, Martin; Karger, Axel; Mettenleiter, Thomas C; Römer-Oberdörfer, Angela

2011-02-01

Members of the order Mononegavirales express their genes in a transcription gradient from 3' to 5'. To assess how this impacts on expression of a foreign transgene, the haemagglutinin (HA) of highly pathogenic avian influenza virus (HPAIV) A/chicken/Vietnam/P41/05 (subtype H5N1) was inserted between the phosphoprotein (P) and matrix protein (M), M and fusion protein (F), or F and haemagglutinin-neuraminidase protein (HN) genes of attenuated Newcastle disease virus (NDV) Clone 30. In addition, the gene encoding the neuraminidase of HPAIV A/duck/Vietnam/TG24-01/05 (subtype H5N1) was inserted into the NDV genome either alone or in combination with the HA gene. All recombinants replicated well in embryonated chicken eggs. The expression levels of HA-specific mRNA and protein were quantified by Northern blot analysis and mass spectrometry, with good correlation. HA expression levels differed only moderately and were highest in the recombinant carrying the HA insertion between the F and HN genes of NDV.

Sound tuning of amygdala plasticity in auditory fear conditioning

PubMed Central

Park, Sungmo; Lee, Junuk; Park, Kyungjoon; Kim, Jeongyeon; Song, Beomjong; Hong, Ingie; Kim, Jieun; Lee, Sukwon; Choi, Sukwoo

2016-01-01

Various auditory tones have been used as conditioned stimuli (CS) for fear conditioning, but researchers have largely neglected the effect that different types of auditory tones may have on fear memory processing. Here, we report that at lateral amygdala (LA) synapses (a storage site for fear memory), conditioning with different types of auditory CSs (2.8 kHz tone, white noise, FM tone) recruits distinct forms of long-term potentiation (LTP) and inserts calcium permeable AMPA receptor (CP-AMPAR) for variable periods. White noise or FM tone conditioning produced brief insertion (<6 hr after conditioning) of CP-AMPARs, whereas 2.8 kHz tone conditioning induced more persistent insertion (≥6 hr). Consistently, conditioned fear to 2.8 kHz tone but not to white noise or FM tones was erased by reconsolidation-update (which depends on the insertion of CP-AMPARs at LA synapses) when it was performed 6 hr after conditioning. Our data suggest that conditioning with different auditory CSs recruits distinct forms of LA synaptic plasticity, resulting in more malleable fear memory to some tones than to others. PMID:27488731

Effects of porous insert on flame dynamics in a lean premixed swirl-stabilized combustor

NASA Astrophysics Data System (ADS)

Brown, Marcus; Agrawal, Ajay; Allen, James; Kornegay, John

2016-11-01

In this study, we investigated different methods of determining the effect a porous insert has on flame dynamics during lean premixed combustion. A metallic porous insert is used to mitigate instabilities in a swirl-stabilized combustor. Thermoacoustic instabilities are seen as negative consequences of lean premixed combustion and eliminating them is the motivation for our research. Three different diagnostics techniques with high-speed Photron SA5 cameras were used to monitor flame characteristics. Particle image velocimetry (PIV) was used to observe vortical structures and recirculation zones within the combustor. Using planar laser induced fluorescence (PLIF), we were able to observe changes in the reaction zones during instabilities. Finally, utilizing a color high-speed camera, visual images depicting a flame's oscillations during the instability were captured. Using these monitoring techniques, we are able to support the claims made in previous studies stating that the porous insert in the combustor significantly reduces the thermoacoustic instability. Funding for this research was provided by the NSF REU site Grant EEC 1358991 and NASA Grant NNX13AN14A.

Enhancers Are Major Targets for Murine Leukemia Virus Vector Integration

PubMed Central

De Ravin, Suk See; Su, Ling; Theobald, Narda; Choi, Uimook; Macpherson, Janet L.; Poidinger, Michael; Symonds, Geoff; Pond, Susan M.; Ferris, Andrea L.; Hughes, Stephen H.

2014-01-01

ABSTRACT Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (<15%) occur in this region. To resolve this apparent discrepancy, we created a high-resolution genome-wide integration map of more than one million integration sites from CD34+ hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ∼60,000 tight clusters. These clusters comprise ∼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors. PMID:24501411

The effect of undersizing and tapping on bone to implant contact and implant primary stability: A histomorphometric study on bovine ribs.

PubMed

Di Stefano, Danilo Alessio; Perrotti, Vittoria; Greco, Gian Battista; Cappucci, Claudia; Arosio, Paolo; Piattelli, Adriano; Iezzi, Giovanna

2018-06-01

Implant site preparation may be adjusted to achieve the maximum possible primary stability. The aim of this investigation was to study the relation among bone-to-implant contact at insertion, bone density, and implant primary stability intra-operatively measured by a torque-measuring implant motor, when implant sites were undersized or tapped. Undersized (n=14), standard (n=13), and tapped (n=13) implant sites were prepared on 9 segments of bovine ribs. After measuring bone density using the implant motor, 40 implants were placed, and their primary stability assessed by measuring the integral of the torque-depth insertion curve. Bovine ribs were then processed histologically, the bone-to-implant contact measured and statistically correlated to bone density and the integral. Bone-to-implant contact and the integral of the torque-depth curve were significantly greater for undersized sites than tapped sites. Moreover, a correlation between bone to implant contact, the integral and bone density was found under all preparation conditions. The slope of the bone-to-implant/density and integral/density lines was significantly greater for undersized sites, while those corresponding to standard prepared and tapped sites did not differ significantly. The integral of the torque-depth curve provided reliable information about bone-to-implant contact and primary implant stability even in tapped or undersized sites. The linear relations found among the parameters suggests a connection between extent and modality of undersizing and the corresponding increase of the integral and, consequently, of primary stability. These results might help the physician determine the extent of undersizing needed to achieve the proper implant primary stability, according to the planned loading protocol.

Lambda Red Mediated Gap Repair Utilizes a Novel Replicative Intermediate in Escherichia coli

PubMed Central

Reddy, Thimma R.; Fevat, Léna M. S.; Munson, Sarah E.; Stewart, A. Francis; Cowley, Shaun M.

2015-01-01

The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination. PMID:25803509

Method of inactivation of an end product of energy metabolism in Zymomonas mobilis

DOEpatents

Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Lakewood, CO

2008-05-20

The present invention briefly provides a method of site-specific insertion in Zymomonas, comprising, providing a Zymomonas gene fragment, interrupting a DNA sequence the fragment, and transforming the Zymomonas through homologous recombination with the interrupted fragment.

Anthrax Toxin Receptor 1 / Tumor Endothelial Marker 8: Mutation of Conserved Inserted Domain Residues Overrides Cytosolic Control of Protective Antigen Binding†

PubMed Central

Ramey, Jordan D.; Villareal, Valerie A.; Ng, Charles; Ward, Sabrina; Xiong, Jian-Ping; Clubb, Robert T.; Bradley, Kenneth A.

2010-01-01

Anthrax toxin receptor 1 (ANTXR1) / tumor endothelial marker 8 (TEM8) is one of two known proteinaceous cell surface anthrax toxin receptors. A metal ion dependent adhesion site (MIDAS) present in the integrin-like inserted (I) domain of ANTXR1 mediates the binding of the anthrax toxin subunit, protective antigen (PA). Here we provide evidence that single point mutations in the I domain can override regulation of ANTXR1 ligand-binding activity mediated by intracellular signals. A previously reported MIDAS-mutant of ANTXR1 (T118A) was found to retain normal metal ion binding and secondary structure but failed to bind PA, consistent with a locked inactive state. Conversely, mutation of a conserved I domain phenylalanine residue to a tryptophan (F205W) increased the proportion of cell-surface ANTXR1 that bound PA, consistent with a locked active state. Interestingly, the KD and total amount of PA bound by the isolated ANTXR1 I domain was not affected by the F205W mutation, indicating that ANTXR1 is preferentially found in the active state in the absence of inside-out signaling. Circular dichroism (CD) spectroscopy and 1H-15N heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) revealed that structural changes between T118A, F205W and WT I domains were minor despite a greater than 103-fold difference in their abilities to bind toxin. Regulation of toxin binding has important implications for the design of toxin inhibitors and for the targeting of ANTXR1 for anti-tumor therapies. PMID:20690680

Novel cytochrome P450 (CYP6D1) and voltage sensitive sodium channel (Vssc) alleles of the house fly (Musca domestica) and their roles in pyrethroid resistance.

PubMed

Pan, Jing; Yang, Chan; Liu, Yan; Gao, Qi; Li, Mei; Qiu, Xinghui

2018-04-01

The house fly Musca domestica is an important disease vector. Point mutation-mediated target-site insensitivity of the voltage sensitive sodium channel (VSSC) and increased detoxification mediated by cytochrome P450 (CYP6D1) overexpression have been characterized as two major mechanisms of pyrethroid resistance. In this study, genetic mutations in the Vssc and CYP6D1 genes and their contribution to pyrethroid resistance were investigated. Twelve lines of house flies homozygous for four genotypes were established. House flies carrying the VSSC 1014F mutation and overexpressing CYP6D1 had higher resistance to pyrethroids than those carrying 1014F alone. The presence of the 15-bp insert in the promoter region of the CYP6D1 gene did not necessarily result in a significant increase in CYP6D1 mRNA and pyrethroid resistance levels. A novel Vssc allele carrying two mutations (G1924D and G2004S) in combination with the classic 1014F and a novel CYP6D1 allele that is very similar to CYP6D1v1 were identified in Chinese house flies. This work demonstrates the effect of genetic mutations in CYP6D1 and Vssc on the susceptibility of house flies to pyrethroids, and verifies that 15-bp insert-containing CYP6D1 alleles have a single origin. These findings offer insights into the evolution of insecticide resistance and have implications for house fly control. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Discerning the catalytic mechanism of Staphylococcus aureus sortase A with QM/MM free energy calculations.

PubMed

Shrestha, Pooja; Wereszczynski, Jeff

2016-06-01

Sortases are key virulence factors in Gram-positive bacteria. These enzymes embed surface proteins in the cell wall through a transpeptidation reaction that involves recognizing a penta-peptide "sorting signal" in a target protein, cleaving it, and covalently attaching it to a second substrate that is later inserted into the cell wall. Although well studied, several aspects of the mechanism by which sortases perform these functions remains unclear. In particular, experiments have revealed two potential sorting signal binding motifs: a "Threonine-Out" (Thr-Out) structure in which the catalytically critical threonine residues protrudes into solution, and a "Threonine-In" (Thr-In) configuration in which this residue inserts into the binding site. To determine which of these is the biologically relevant state, we have performed a series of conventional and hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of the Staphylococcus aureus sortase A (SrtA) enzyme bound to a sorting signal substrate. Through the use of multi-dimensional metadynamics, our simulations were able to both map the acylation mechanism of SrtA in the Thr-In and Thr-Out states, as well as determine the free energy minima and barriers along these reactions. Results indicate that in both states the catalytic mechanisms are similar, however the free energy barriers are lower in the Thr-In configuration, suggesting that Thr-In is the catalytically relevant state. This has important implications for advancing our understanding of the mechanisms of sortase enzymes, as well we for future structure based drug design efforts aimed at inhibiting sortase function in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Occurence of adverse events due to continuous glucose monitoring.

PubMed

Jadviscokova, Tereza; Fajkusova, Zuzana; Pallayova, Maria; Luza, Jiri; Kuzmina, Galina

2007-12-01

Continuous glucose monitoring (CGM) using transcutaneous sensors is becoming a sophisticated method to control and regulate glucose metabolism. The transcutaneous sensor of the CGM system (CGMS Medtronic Minimed, Northridge, CA, USA) is chosen to measure glucose concentration in interstitial fluid up to three days after insertion even though its function remains stable for a longer period. The question arises, which factors really limit the period of sensor insertion without unnecessary risk. The aim of this study was to assess any adverse events occurring in the course of 9 days after the sensor insertion. In a group of 22 healthy volunteers aged 21.8+/-1.30 y (mean +/- SE) a total of 26 sensors was inserted subcutaneously in gluteal or lumbar region for 9 days. Before insertion the site was sprayed with an antiseptic (Cutasept F, Bode Chemie, Hamburg, Germany). Local adverse reactions and disturbances in general condition were examined. In the course of 184 sensor-days, there were only minor local adverse events: hypersensitivity, itching, pain, redness, burning, subcutaneous hemorrhage. Additionally, sleep disturbances, attention deficits, problems related to the CGMS monitor, to adhesive tape and/or sensor were found. None of these resulted in sensor withdrawal. In 12 volunteers (55 %) no complications were observed. The sensor function measured according to electrical signals (ISIG) failed (always on day 1-2) in 4 cases (16 %). The present FDA approved 3-day insertion period for Medtronic transcutaneous sensor does not seem to limit its use and appears to be worth a careful revision.

Nephric duct insertion is a crucial step in urinary tract maturation that is regulated by a Gata3-Raldh2-Ret molecular network in mice.

PubMed

Chia, Ian; Grote, David; Marcotte, Michael; Batourina, Ekaterina; Mendelsohn, Cathy; Bouchard, Maxime

2011-05-01

Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility.

Effect of self-deflection on a totally asymmetric simple exclusion process with functions of site assignments

NASA Astrophysics Data System (ADS)

Tsuzuki, Satori; Yanagisawa, Daichi; Nishinari, Katsuhiro

2018-04-01

This study proposes a model of a totally asymmetric simple exclusion process on a single-channel lane with functions of site assignments along the pit lane. The system model attempts to insert a new particle to the leftmost site at a certain probability by randomly selecting one of the empty sites in the pit lane, and reserving it for the particle. Thereafter, the particle is directed to stop at the site only once during its travel. Recently, the system was determined to show a self-deflection effect, in which the site usage distribution biases spontaneously toward the leftmost site, and the throughput becomes maximum when the site usage distribution is slightly biased to the rightmost site. Our exact analysis describes this deflection effect and show a good agreement with simulations.

Use of a national continuing medical education meeting to provide simulation-based training in temporary hemodialysis catheter insertion skills: a pre-test post-test study.

PubMed

Clark, Edward G; Paparello, James J; Wayne, Diane B; Edwards, Cedric; Hoar, Stephanie; McQuillan, Rory; Schachter, Michael E; Barsuk, Jeffrey H

2014-01-01

Simulation-based-mastery-learning (SBML) is an effective method to train nephrology fellows to competently insert temporary, non-tunneled hemodialysis catheters (NTHCs). Previous studies of SBML for NTHC-insertion have been conducted at a local level. Determine if SBML for NTHC-insertion can be effective when provided at a national continuing medical education (CME) meeting. Describe the correlation of demographic factors, prior experience with NTHC-insertion and procedural self-confidence with simulated performance of the procedure. Pre-test - post-test study. 2014 Canadian Society of Nephrology annual meeting. Nephrology fellows, internal medicine residents and medical students. Participants were surveyed regarding demographics, prior NTHC-insertion experience, procedural self-confidence and attitudes regarding the training they received. NTHC-insertion skills were assessed using a 28-item checklist. Participants underwent a pre-test of their NTHC-insertion skills at the internal jugular site using a realistic patient simulator and ultrasound machine. Participants then had a training session that included a didactic presentation and 2 hours of deliberate practice using the simulator. On the following day, trainees completed a post-test of their NTHC-insertion skills. All participants were required to meet or exceed a minimum passing score (MPS) previously set at 79%. Trainees who did not reach the MPS were required to perform more deliberate practice until the MPS was achieved. Twenty-two individuals participated in SBML training. None met or exceeded the MPS at baseline with a median checklist score of 20 (IQR, 7.25 to 21). Seventeen of 22 participants (77%) completed post-testing and improved their scores to a median of 27 (IQR, 26 to 28; p < 0.001). All met or exceeded the MPS on their first attempt. There were no significant correlations between demographics, prior experience or procedural self-confidence with pre-test performance. Small sample-size and self-selection of participants. Costs could limit the long-term feasibility of providing this type of training at a CME conference. Despite most participants reporting having previously inserted NTHCs in clinical practice, none met the MPS at baseline; this suggests their prior training may have been inadequate.

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

PubMed

Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D; Nystul, Todd G; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E; Murphy, Terence D; Levis, Robert W; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A; Spradling, Allan C

2007-03-01

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

Maturation of nitrogenase cofactor—the role of a class E radical SAM methyltransferase NifB

PubMed Central

Hu, Yilin; Ribbe, Markus W.

2016-01-01

Nitrogenase catalyzes the important reactions of N2-, CO- and CO2-reduction at its active cofactor site. Designated the M-cluster, this complex metallocofactor is assembled through the generation of a characteristic 8Fe-core prior to the insertion of Mo and homocitrate that completes the stoichiometry of the M-cluster. NifB catalyzes the critical step of radical SAM-dependent carbide insertion that occurs concomitant with the insertion a “9th” sulfur and the rearrangement/coupling of two 4Fe-clusters into a complete 8Fe-core of the M-cluster. Further categorization of a family of NifB proteins as a new class of radical SAM methyltransferases suggests a general function of these proteins in complex metallocofactor assembly and provides a new platform for unveiling unprecedented chemical reactions catalyzed by biological systems. PMID:26969410

Phosphine-alkene ligands as mechanistic probes in the Pauson-Khand reaction.

PubMed

Ferrer, Catalina; Benet-Buchholz, Jordi; Riera, Antoni; Verdaguer, Xavier

2010-07-26

An alkyne tetracarbonyl dicobalt complex with a chelated phosphine-alkene ligand, in which the phosphorus atom and the alkene from the ligand are attached to the same cobalt atom has been prepared, isolated, and characterized by X-ray crystallography. The complex serves as a mechanistic model for an intermediate of the Pauson-Khand (PK) reaction. Although the alkene fragment is located in an equatorial coordination site with an appropriate orientation, and, therefore, should undergo insertion, it failed to give the PK product upon either thermal or N-methylmorpholine N-oxide activation. However, a phosphine-alkene complex that contains a terminal alkene readily provided the corresponding PK product. We attribute this change in reactivity to the different ability of each olefin to undergo 1,2-insertion. These results provide further insights into the factors that govern a crucial step in the PK reaction, the olefin insertion.

Optimal effect-site concentration of remifentanil when combined with dexmedetomidine in patients undergoing cystoscopy

PubMed Central

Heo, Bongha; Kim, Minsun; Lee, Hyunjung; Park, Sanghee

2014-01-01

Background Cystoscopic procedure is a very common practice in the field of urology due to its ability to survey the bladder for a variety of indications. However, patients who undergo cystoscopy feel intense pain and discomfort. This study investigated the half maximal effective concentration (EC50) of remifentanil in preventing cystoscope insertion pain under sedation using dexmedetomidine. Methods The study was prospectively conducted on 18 male patients, aged 18 to 65. Remifentail infusion was initiated together with dexmedetomidine, and started at a dose of 2.4 ng/ml on the first patient. The effect-site concentration (Ce) of remifentanil for each subsequent patient was determined by the previous patient's response using Dixon's up-and-down method with an interval of 0.3 ng/ml. Patients received a loading dose of 1.0 µg/kg dexmedetomidine over 10 minutes, followed by a maintenance dose of 0.6 µg/kg/hr. After the patient's OAA/S score (Observer's Assessment of Alertness/Sedation scale) reached 3-4, and the Ce of remifentanil reached target concentration, the urologist was allowed to insert the cystoscope and the pain responses were observed. Results The effect-site concentration of remifentanil required to prevent cystoscope insertion pain in 50% of patients under sedation using dexmedetomidine was 1.30 ± 0.12 ng/ml by Dixon's up-and-down method. The logistic regression curve of the probability of response showed that the EC50 and EC95 values (95% confidence limits) of remifentanil were 1.33 ng/ml (1.12-1.52 ng/ml) and 1.58 ng/ml (1.44-2.48 ng/ml), respectively. Conclusions Cystoscopic procedure can be carried out successfully without any pain or adverse effects by optimal remifentanil effect-site concentration (EC50, 1.33 ng/ml; EC95, 1.58 ng/ ml) combined with sedation using dexmedetomidine. PMID:24567812

Intelligent Embedded Instruction for Computer-Aided Design (CAD) systems

DTIC Science & Technology

1988-10-01

difficulties were predicted and six lessons were prepared that were aimed at preventing error pattern formation. The lessons were programmed in AUTOLISP ...and arcs, angles of lines, layering (linetype and color), and block creation and insertion. A program written in AUTOLISP examined the values in the...One site had AutoCAD reference manuals nearby and others had no manuals . * Only one site set a schedule for the users. * The attitudes of managers

Tendon healing in a bone tunnel. Part II: Histologic analysis after biodegradable interference fit fixation in a model of anterior cruciate ligament reconstruction in sheep.

PubMed

Weiler, Andreas; Hoffmann, Reinhard F G; Bail, Hermann J; Rehm, Oliver; Südkamp, Norbert P

2002-02-01

Tendon-to-bone healing of soft-tissue grafts has been described to progress by the development of a fibrous interzone that undergoes a maturation process leading to the development of an indirect type of ligament insertion. Previous studies used extra-articular models or fixation far away from the joint line; thus, no data are available investigating tendon-to-bone healing of a soft-tissue graft fixed anatomically. Therefore, we studied the tendon-to-bone healing of the anatomic soft-tissue graft interference fit fixation in a model of anterior cruciate ligament (ACL) reconstruction in sheep. Animal study. Thirty-five mature sheep underwent ACL reconstruction with an autologous Achilles tendon split graft. Grafts were directly fixed with biodegradable poly-(D,L-lactide) interference screws. Animals were euthanized after 6, 9, 12, 24, and 52 weeks and histologic evaluations were performed. Undecalcified specimens were evaluated under normal and polarized light. Additionally, animals received a polychrome sequential labeling (tetracycline, xylenol orange, and calcein green) to determine bone growth per time under fluorescent light. Intratunnel histologic findings at 6 weeks showed a tendon-bone junction with only a partial fibrous interzone between the graft tissue and the surrounding bone. A mature intratunnel tendon-bone junction with a zone of fibrocartilage was found at 9 to 12 weeks. At the tunnel entrance site a wide regular ligamentous insertion site was seen in all specimens after 24 weeks. This insertion showed regular patterns such as the direct type of insertion of a normal ligament with a dense basophilic transition zone consisting of mineralized cartilage. A fibrous interzone between the graft tissue and the bone tunnel was only partially developed, which is in contrast to all previous studies in which nonanatomic fixation was used. Thus, it is reasonable to assume that the tendon-to-bone healing in the present study may progress partially by direct-contact healing without the development of a fibrous interzone. To our knowledge, this is the first report describing the development of a direct type of ligament insertion after ACL replacement with a soft-tissue graft. This is in contrast to previous studies reporting the development of an indirect type of insertion when using nonanatomic fixation far away from the joint line. Thus, histologic data strongly indicate that anatomic interference fit fixation is beneficial for tendon-to-bone incorporation by leading to the development of a direct type of ligament insertion.

Genome-wide analysis of short interspersed nuclear elements SINES revealed high sequence conservation, gene association and retrotranspositional activity in wheat

PubMed Central

Ben-David, Smadar; Yaakov, Beery; Kashkush, Khalil

2013-01-01

Short interspersed nuclear elements (SINEs) are non-autonomous non-LTR retroelements that are present in most eukaryotic species. While SINEs have been intensively investigated in humans and other animal systems, they are poorly studied in plants, especially in wheat (Triticum aestivum). We used quantitative PCR of various wheat species to determine the copy number of a wheat SINE family, termed Au SINE, combined with computer-assisted analyses of the publicly available 454 pyrosequencing database of T. aestivum. In addition, we utilized site-specific PCR on 57 Au SINE insertions, transposon methylation display and transposon display on newly formed wheat polyploids to assess retrotranspositional activity, epigenetic status and genetic rearrangements in Au SINE, respectively. We retrieved 3706 different insertions of Au SINE from the 454 pyrosequencing database of T. aestivum, and found that most of the elements are inserted in A/T-rich regions, while approximately 38% of the insertions are associated with transcribed regions, including known wheat genes. We observed typical retrotransposition of Au SINE in the second generation of a newly formed wheat allohexaploid, and massive hypermethylation in CCGG sites surrounding Au SINE in the third generation. Finally, we observed huge differences in the copy numbers in diploid Triticum and Aegilops species, and a significant increase in the copy numbers in natural wheat polyploids, but no significant increase in the copy number of Au SINE in the first four generations for two of three newly formed allopolyploid species used in this study. Our data indicate that SINEs may play a prominent role in the genomic evolution of wheat through stress-induced activation. PMID:23855320

Genome-wide analysis of short interspersed nuclear elements SINES revealed high sequence conservation, gene association and retrotranspositional activity in wheat.

PubMed

Ben-David, Smadar; Yaakov, Beery; Kashkush, Khalil

2013-10-01

Short interspersed nuclear elements (SINEs) are non-autonomous non-LTR retroelements that are present in most eukaryotic species. While SINEs have been intensively investigated in humans and other animal systems, they are poorly studied in plants, especially in wheat (Triticum aestivum). We used quantitative PCR of various wheat species to determine the copy number of a wheat SINE family, termed Au SINE, combined with computer-assisted analyses of the publicly available 454 pyrosequencing database of T. aestivum. In addition, we utilized site-specific PCR on 57 Au SINE insertions, transposon methylation display and transposon display on newly formed wheat polyploids to assess retrotranspositional activity, epigenetic status and genetic rearrangements in Au SINE, respectively. We retrieved 3706 different insertions of Au SINE from the 454 pyrosequencing database of T. aestivum, and found that most of the elements are inserted in A/T-rich regions, while approximately 38% of the insertions are associated with transcribed regions, including known wheat genes. We observed typical retrotransposition of Au SINE in the second generation of a newly formed wheat allohexaploid, and massive hypermethylation in CCGG sites surrounding Au SINE in the third generation. Finally, we observed huge differences in the copy numbers in diploid Triticum and Aegilops species, and a significant increase in the copy numbers in natural wheat polyploids, but no significant increase in the copy number of Au SINE in the first four generations for two of three newly formed allopolyploid species used in this study. Our data indicate that SINEs may play a prominent role in the genomic evolution of wheat through stress-induced activation. © 2013 Ben-Gurion University The Plant Journal © 2013 John Wiley & Sons Ltd.

Non-interventional 1-year follow-up study of peri-implant soft tissues following previous soft tissue augmentation and crown insertion in single-tooth gaps.

PubMed

Huber, Samuel; Zeltner, Marco; Hämmerle, Christoph H F; Jung, Ronald E; Thoma, Daniel S

2018-04-01

To assess peri-implant soft tissue dimensions at implant sites, previously augmented with a collagen matrix (VCMX) or an autogenous subepithelial connective tissue graft (SCTG), between crown insertion and 1 year. Twenty patients with single-tooth implants received soft tissue augmentation prior to abutment connection randomly using VCMX or SCTG. Following abutment connection 3 months later, final reconstructions were fabricated and inserted (baseline). Patients were recalled at 6 months (6M) and at 1 year (FU-1). Measurements included clinical data, soft tissue thickness, volumetric outcomes and patient-reported outcome measures (PROMs). The buccal soft tissue thickness showed a median decrease of -0.5 mm (-1.0;0.3) (VCMX) and 0.0 mm (-0.5;1.0) (SCTG) (p = .243) up to FU-1. The soft tissue volume demonstrated a median decrease between BL and FU-1 of -0.1 mm (-0.2;0.0) (p = .301) for VCMX and a significant decrease of -0.2 mm (-0.4; -0.1) (p = .002) for SCTG, respectively. Intergroup comparisons did not reveal any significant differences between the groups for peri-implant soft tissue dimensions and changes up to FU-1 (p > .05). PROMs did not show any significant changes over time nor differences between the groups. Between crown insertion and 1 year, the buccal peri-implant soft tissue dimensions remained stable without relevant differences between sites that had previously been grafted with VCMX or SCTG. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evolutionary genomics of miniature inverted-repeat transposable elements (MITEs) in Brassica.

PubMed

Nouroz, Faisal; Noreen, Shumaila; Heslop-Harrison, J S

2015-12-01

Miniature inverted-repeat transposable elements (MITEs) are truncated derivatives of autonomous DNA transposons, and are dispersed abundantly in most eukaryotic genomes. We aimed to characterize various MITEs families in Brassica in terms of their presence, sequence characteristics and evolutionary activity. Dot plot analyses involving comparison of homoeologous bacterial artificial chromosome (BAC) sequences allowed identification of 15 novel families of mobile MITEs. Of which, 5 were Stowaway-like with TA Target Site Duplications (TSDs), 4 Tourist-like with TAA/TTA TSDs, 5 Mutator-like with 9-10 bp TSDs and 1 novel MITE (BoXMITE1) flanked by 3 bp TSDs. Our data suggested that there are about 30,000 MITE-related sequences in Brassica rapa and B. oleracea genomes. In situ hybridization showed one abundant family was dispersed in the A-genome, while another was located near 45S rDNA sites. PCR analysis using primers flanking sequences of MITE elements detected MITE insertion polymorphisms between and within the three Brassica (AA, BB, CC) genomes, with many insertions being specific to single genomes and others showing evidence of more recent evolutionary insertions. Our BAC sequence comparison strategy enables identification of evolutionarily active MITEs with no prior knowledge of MITE sequences. The details of MITE families reported in Brassica enable their identification, characterization and annotation. Insertion polymorphisms of MITEs and their transposition activity indicated important mechanism of genome evolution and diversification. MITE families derived from known Mariner, Harbinger and Mutator DNA transposons were discovered, as well as some novel structures. The identification of Brassica MITEs will have broad applications in Brassica genomics, breeding, hybridization and phylogeny through their use as DNA markers.

Polyaxial Screws in Locked Plating of Tibial Pilon Fractures.

PubMed

Yenna, Zachary C; Bhadra, Arup K; Ojike, Nwakile I; Burden, Robert L; Voor, Michael J; Roberts, Craig S

2015-08-01

This study examined the axial and torsional stiffness of polyaxial locked plating techniques compared with fixed-angle locked plating techniques in a distal tibia pilon fracture model. The effect of using a polyaxial screw to cross the fracture site was examined to determine its ability to control relative fracture site motion. A laboratory experiment was performed to investigate the biomechanical stiffness of distal tibia fracture models repaired with 3.5-mm anterior polyaxial distal tibial plates and locking screws. Sawbones Fourth Generation Composite Tibia models (Pacific Research Laboratories, Inc, Vashon, Washington) were used to model an Orthopaedic Trauma Association 43-A1.3 distal tibia pilon fracture. The polyaxial plates were inserted with 2 central locking screws at a position perpendicular to the cortical surface of the tibia and tested for load as a function of axial displacement and torque as a function of angular displacement. The 2 screws were withdrawn and inserted at an angle 15° from perpendicular, allowing them to span the fracture and insert into the opposing fracture surface. Each tibia was tested again for axial and torsional stiffness. In medial and posterior loading, no statistically significant difference was found between tibiae plated with the polyaxial plate and the central screws placed in the neutral position compared with the central screws placed at a 15° position. In torsional loading, a statistically significant difference was noted, showing greater stiffness in tibiae plated with the polyaxial plate and the central screws placed at a 15° position compared with tibiae plated with the central screws placed at a 0° (or perpendicular) position. This study showed that variable angle constructs show similar stiffness properties between perpendicular and 15° angle insertions in axial loading. The 15° angle construct shows greater stiffness in torsional loading. Copyright 2015, SLACK Incorporated.

Poststroke upper-limb rehabilitation using 5 to 7 inserted microstimulators: implant procedure, safety, and efficacy for restoration of function.

PubMed

Davis, Ross; Sparrow, Owen; Cosendai, Gregoire; Burridge, Jane H; Wulff, Christian; Turk, Ruth; Schulman, Joseph

2008-10-01

To investigate the feasibility of implanting microstimulators to deliver programmed nerve stimulation for sequenced muscle activation to recover arm-hand functions. By using a minimally invasive procedure and local anesthesia, 5 to 7 microstimulators can be safely and comfortably implanted adjacent to targeted radial nerve branches in the arm and forearm of 7 subjects with poststroke paresis. The microstimulators' position should remain stable with no tissue infection and can be programmed to produce effective personalized functional muscle activity with no discomfort for a preliminary 12-week study. Clinical testing, before and after the study, is reported in the accompanying study. Microstimulator implantations in a sterile operating room. Seven adults, with poststroke hemiparesis of 12 months or more. Under local anesthesia, a stimulating probe was inserted to identify radial nerve branches. Microstimulators were inserted by using an introducer and were retrievable for 6 days by attached suture. Each device was powered via a radiofrequency link from 2 external cuff coils connected to a control unit. To achieve low threshold values at the target sites with minimal implant discomfort. Microstimulators and external equipment were monitored over 12 weeks of exercise. Seven subjects were implanted with 41 microstimulators, 5 to 7 per subject, taking 3.5 to 6 hours. Implantation pain levels were 20% more than anticipated. No infections or microstimulator failures occurred. Mean nerve thresholds ranged between 4.0 to 7.7 microcoulomb/cm(2)/phase over 90 days, indicating that cathodes were within 2 to 4 mm of target sites. In 1 subject, 2 additional microstimulators were inserted. Microstimulators were safely implanted with no infection or failure. The system was reliable and programmed effectively to perform exercises at home for functional restoration.

Complete replication-competent adenovirus 11p vectors with E1 or E3 insertions show improved heat stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

2016-10-15

Conventional adenovirus vectors harboring E1 or E3 deletions followed by the insertion of an exogenous gene show considerably reduced virion stability. Here, we report strategies to generate complete replication-competent Ad11p(RCAd11p) vectors that overcome the above disadvantage. A GFP cassette was successfully introduced either upstream of E1A or in the E3A region. The resulting vectors showed high expression levels of the hexon and E1genes and also strongly induced the cytopathic effect in targeted cells. When harboring oversized genomes, the RCAd11pE1 and RCAd11pE3 vectors showed significantly improved heat stability in comparison to Ad11pwt;of the three, RCAd11pE3 was the most tolerant to heatmore » treatment. Electron microscopy showed that RCAd11pE3, RCAd11pE1, Ad11pwt, and Ad11pE1 Delmanifested dominant, moderate, minimum, or no full virus particles after heat treatment at 47 °C for 5 h. Our results demonstrated that both genome size and the insertion site in the viral genome affect virion stability. -- Highlights: •Replicating adenovirus 11p GFP vectors at the E1 or E3 region were generated. •RCAd11pE3 and RCAd11pE1 vectors manifested significantly improved heat stability. •RCAd11pE3 and RCAd11pE1 showed more full viral particles than Ad11pwt after heating. •We demonstrated that both genome size and the insertion site affect virion stability.« less

Comparison between vestibular and subcutaneous insertion of deslorelin implants for oestrus induction in bitches.

PubMed

Kutzler, M; Lamb, S V; Volkmann, D

2009-07-01

Investigations using sustained-release deslorelin implants at various insertion sites have shown that this method consistently induces oestrus in anoestus bitches. However, fertility comparisons between implant insertion sites have not been performed. Anestrous beagle bitches received one 2.1 mg deslorelin implant beneath the vestibular mucosa (VM group; n = 6) or in the subcutaneous tissue between the shoulder blades (SubQ group; n = 8). Vestibular implants were removed when serum progesterone concentrations first exceeded 1.5 ng/ml. Vaginal cytologies and blood samples were collected daily and bitches were inseminated during oestrus. Serum progesterone and deslorelin concentrations were measured and pregnancy status was determined using ultrasonography. There were no differences between groups in the intervals between implant administration and the onset of proestrus, the time of the luteinizing hormone surge and the onset of cytologic diestrus. There were also no differences in the number of corpora lutea or foetuses. However, conception rate was significantly lower in the SubQ group. The pregnancy rate did not differ significantly between the VM and SubQ groups [4 out of 6 (66.7%) and 3 out of 8 (37.5%), respectively]. One bitch (16.7%) in the VM group and three bitches (37.5%) in the SubQ group suffered distinct, premature declines in serum progesterone concentrations starting 1-4 weeks after cytologic diestrus. Serum progesterone concentrations did not recover (premature luteal failure), resulting in abortion. Bitches with premature luteal failure in the SubQ group still had serum deslorelin concentrations >100 pg/ml 20 days after implant insertion, suggesting a possible association between prolonged deslorelin release and luteal failure.

Intein-modified enzymes, their production and industrial applications

DOEpatents

Apgar, James; Lessard, Philip; Raab, Michael R.; Shen, Binzhang; Lazar, Gabor; de la Vega, Humberto

2016-10-11

A method of predicting an intein insertion site in a protein that will lead to a switching phenotype is provided. The method includes identifying a plurality of C/T/S sites within the protein; selecting from the plurality of C/T/S/ sites those that are ranked 0.75 or higher by a support vector machine, within ten angstroms of the active site of the protein, and at or near a loop-.beta.-sheet junction or a loop-.alpha.-helix junction. A method of controlling protein activity and hosts including proteins with controlled activity are also provided. Also, intein modified proteins and plants containing intein modified proteins are provided.

An Assessment of Radiologically Inserted Transoral and Transgastric Gastroduodenal Stents to Treat Malignant Gastric Outlet Obstruction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, Bethany H. T., E-mail: bmiller@doctors.org.uk; Griffiths, Ewen A., E-mail: Eagriffiths@doctors.org.uk; Pursnani, Kishore G., E-mail: Kish.Pursnani@lthtr.nhs.uk

2013-12-15

IntroductionSelf-expanding metallic stents (SEMS) are used to palliate malignant gastric outlet obstruction (GOO) and are useful in patients with limited life expectancy or severe medical comorbidity, which would preclude surgery. Stenting can be performed transorally or by a percutaneous transgastric technique. Our goal was to review the outcome of patients who underwent radiological SEMS insertion performed by a single consultant interventional radiologist. Methods: Patients were identified from a prospectively collected database held by one consultant radiologist. Data were retrieved from radiological reports, multidisciplinary team meetings, and the patients' case notes. Univariate survival analysis was performed. Results: Between December 2000 andmore » January 2011, 100 patients (63 males, 37 females) had 110 gastroduodenal stenting procedures. Median age was 73 (range 39-89) years. SEMS were inserted transorally (n = 66) or transgastrically (n = 44). Site of obstruction was the stomach (n = 37), duodenum (n = 50), gastric pull-up (n = 10), or gastroenterostomy (n = 13). Seven patients required biliary stents. Technical success was 86.4 %: 83.3 % for transoral insertion, 90.9 % for transgastric insertion. Eleven patients developed complications. Median GOO severity score: 1 pre-stenting, 2 post-stenting (p = 0.0001). Median survival was 54 (range 1-624) days. Post-stenting GOO severity score was predictive of survival (p = 0.0001). Conclusions: The technical success rate for insertion of palliative SEMS is high. Insertional technique can be tailored to the individual depending on the location of the tumor and whether it is possible to access the stomach percutaneously. Patients who have successful stenting and return to eating a soft/normal diet have a statistically significant increase in survival.« less

Preventing central venous catheter-associated primary bloodstream infections: characteristics of practices among hospitals participating in the Evaluation of Processes and Indicators in Infection Control (EPIC) study.

PubMed

Braun, Barbara I; Kritchevsky, Stephen B; Wong, Edward S; Solomon, Steve L; Steele, Lynn; Richards, Cheryl L; Simmons, Bryan P

2003-12-01

To describe the conceptual framework and methodology of the Evaluation of Processes and Indicators in Infection Control (EPIC) study and present results of CVC insertion characteristics and organizational practices for preventing BSIs. The goal of the EPIC study was to evaluate relationships among processes of care, organizational characteristics, and the outcome of BSI. This was a multicenter prospective observational study of variation in hospital practices related to preventing CVC-associated BSIs. Process of care information (eg, barrier use during insertions and experience of the inserting practitioner) was collected for a random sample of approximately 5 CVC insertions per month per hospital during November 1998 to December 1999. Organization demographic and practice information (eg, surveillance activities and staff and ICU nurse staffing levels) was also collected. Medical, surgical, or medical-surgical ICUs from 55 hospitals (41 U.S. and 14 international sites). Process information was obtained for 3,320 CVC insertions with an average of 58.2 (+/- 16.1) insertions per hospital. Fifty-four hospitals provided policy and practice information. Staff spent an average of 13 hours per week in study ICU surveillance. Most patients received nontunneled, multiple lumen CVCs, of which fewer than 25% were coated with antimicrobial material. Regarding barriers, most clinicians wore masks (81.5%) and gowns (76.8%); 58.1% used large drapes. Few hospitals (18.1%) used an intravenous team to manage ICU CVCs. Substantial variation exists in CVC insertion practice and BSI prevention activities. Understanding which practices have the greatest impact on BSI rates can help hospitals better target improvement interventions.

Ocular Inserts for Sustained Release of the Angiotensin-Converting Enzyme 2 Activator, Diminazene Aceturate, to Treat Glaucoma in Rats

PubMed Central

Nogueira, José Carlos; Fulgêncio, Gustavo de Oliveira; Ribeiro, Tatiana Gomes; Castilho, Rachel Oliveira; Yoshida, Maria Irene; Fuscaldi, Leonardo Lima; Fernandes, Simone Odília Antunes; Cardoso, Valbert Nascimento; Cronemberger, Sebastião; Faraco, André Augusto Gomes; Ferreira, Anderson José

2015-01-01

The aim of this study was to develop and evaluate the effects of chitosan inserts for sustained release of the angiotensin-converting enzyme 2 (ACE2) activator, diminazene aceturate (DIZE), in experimental glaucoma. Monolayer DIZE loaded inserts (D+I) were prepared and characterized through swelling, attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and in vitro drug release. Functionally, the effects of D+I were tested in glaucomatous rats. Glaucoma was induced by weekly injections of hyaluronic acid (HA) into the anterior chamber and intraocular pressure (IOP) measurements were performed. Retinal ganglion cells (RGC) and optic nerve head cupping were evaluated in histological sections. Biodistribution of the drug was accessed by scintigraphic images and ex vivo radiation counting. We found that DIZE increased the swelling index of the inserts. Also, it was molecularly dispersed and interspersed in the polymeric matrix as a freebase. DIZE did not lose its chemical integrity and activity when loaded in the inserts. The functional evaluation demonstrated that D+I decreased the IOP and maintained the IOP lowered for up to one month (last week: 11.0±0.7 mmHg). This effect of D+I prevented the loss of RGC and degeneration of the optic nerve. No toxic effects in the eyes related to application of the inserts were observed. Moreover, biodistribution studies showed that D+I prolonged the retention of DIZE in the corneal site. We concluded that D+I provided sustained DIZE delivery in vivo, thereby evidencing the potential application of polymeric-based DIZE inserts for glaucoma management. PMID:26204514

Nursing care as a predictor of phlebitis related to insertion of a peripheral venous cannula in emergency departments: findings from a prospective study.

PubMed

Palese, A; Ambrosi, E; Fabris, F; Guarnier, A; Barelli, P; Zambiasi, P; Allegrini, E; Bazoli, L; Casson, P; Marin, M; Padovan, M; Picogna, M; Taddia, P; Salmaso, D; Chiari, P; Marognolli, O; Canzan, F; Saiani, L

2016-03-01

To date, few studies have investigated the occurrence of phlebitis related to insertion of a peripheral venous cannula (PVC) in an emergency department (ED). To describe the natural history of ED-inserted PVC site use; the occurrence and severity of PVC-related phlebitis; and associations with patient, PVC and nursing care factors. A prospective study was undertaken of 1262 patients treated as urgent cases in EDs who remained in a medical unit for at least 24h. The first PVC inserted was observed daily until its removal; phlebitis was measured using the Visual Infusion Phlebitis Scale. Data on patient, PVC, nursing care and organizational variables were collected, and a time-to-event analysis was performed. The prevalence of PVC-related phlebitis was 31%. The cumulative incidence (78/391) was almost 20% three days after insertion, and reached >50% (231/391) five days after insertion. Being in a specialized hospital [hazard ratio (HR) 0.583, 95% confidence interval (CI) 0.366-0.928] and receiving more nursing care (HR 0.988, 95% CI 0.983-0.993) were protective against PVC-related phlebitis at all time points. Missed nursing care increased the incidence of PVC-related phlebitis by approximately 4% (HR 1.038, 95% CI 1.001-1.077). Missed nursing care and expertise of the nurses caring for the patient after PVC insertion affected the incidence of phlebitis; receiving more nursing care and being in a specialized hospital were associated with lower risk of PVC-related phlebitis. These are modifiable risk factors of phlebitis, suggesting areas for intervention at both hospital and unit level. Copyright © 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Impact insertion of osteochondral grafts: Interference fit and central graft reduction affect biomechanics and cartilage damage.

PubMed

Su, Alvin W; Chen, Yunchan; Wailes, Dustin H; Wong, Van W; Cai, Shengqiang; Chen, Albert C; Bugbee, William D; Sah, Robert L

2018-01-01

An osteochondral graft (OCG) is an effective treatment for articular cartilage and osteochondral defects. Impact of an OCG during insertion into the osteochondral recipient site (OCR) can cause chondrocyte death and matrix damage. The aim of the present study was to analyze the effects of graft-host interference fit and a modified OCG geometry on OCG insertion biomechanics and cartilage damage. The effects of interference fit (radius of OCG - radius of OCR), loose (0.00 mm), moderate (0.05 mm), tight (0.10 mm), and of a tight fit with OCG geometry modification (central region of decreased radius), were analyzed for OCG cylinders and OCR blocks from adult bovine knee joints with an instrumented drop tower apparatus. An increasingly tight (OCG - OCR) interference fit led to increased taps for insertion, peak axial force, graft cartilage axial compression, cumulative and total energy delivery to cartilage, lower time of peak axial force, lesser graft advancement during each tap, higher total crack length in the cartilage surface, and lower chondrocyte viability. The modified OCG, with reduction of diameter in the central area, altered the biomechanical insertion variables and biological consequences to be similar to those of the moderate interference fit scenario. Micro-computed tomography confirmed structural interference between the OCR bone and both the proximal and distal bone segments of the OCGs, with the central regions being slightly separated for the modified OCGs. These results clarify OCG insertion biomechanics and mechanobiology, and introduce a simple modification of OCGs that facilitates insertion with reduced energy while maintaining a structural interference fit. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:377-386, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Phylogenetic inference under varying proportions of indel-induced alignment gaps

PubMed Central

Dwivedi, Bhakti; Gadagkar, Sudhindra R

2009-01-01

Background The effect of alignment gaps on phylogenetic accuracy has been the subject of numerous studies. In this study, we investigated the relationship between the total number of gapped sites and phylogenetic accuracy, when the gaps were introduced (by means of computer simulation) to reflect indel (insertion/deletion) events during the evolution of DNA sequences. The resulting (true) alignments were subjected to commonly used gap treatment and phylogenetic inference methods. Results (1) In general, there was a strong – almost deterministic – relationship between the amount of gap in the data and the level of phylogenetic accuracy when the alignments were very "gappy", (2) gaps resulting from deletions (as opposed to insertions) contributed more to the inaccuracy of phylogenetic inference, (3) the probabilistic methods (Bayesian, PhyML & "MLε, " a method implemented in DNAML in PHYLIP) performed better at most levels of gap percentage when compared to parsimony (MP) and distance (NJ) methods, with Bayesian analysis being clearly the best, (4) methods that treat gapped sites as missing data yielded less accurate trees when compared to those that attribute phylogenetic signal to the gapped sites (by coding them as binary character data – presence/absence, or as in the MLε method), and (5) in general, the accuracy of phylogenetic inference depended upon the amount of available data when the gaps resulted from mainly deletion events, and the amount of missing data when insertion events were equally likely to have caused the alignment gaps. Conclusion When gaps in an alignment are a consequence of indel events in the evolution of the sequences, the accuracy of phylogenetic analysis is likely to improve if: (1) alignment gaps are categorized as arising from insertion events or deletion events and then treated separately in the analysis, (2) the evolutionary signal provided by indels is harnessed in the phylogenetic analysis, and (3) methods that utilize the phylogenetic signal in indels are developed for distance methods too. When the true homology is known and the amount of gaps is 20 percent of the alignment length or less, the methods used in this study are likely to yield trees with 90–100 percent accuracy. PMID:19698168

Additional Surgical Method Aimed to Increase Distractive Force during Occipitocervical Stabilization : Technical Note.

PubMed

Antar, Veysel; Turk, Okan

2018-03-01

Craniovertebral junctional anomalies constitute a technical challenge. Surgical opening of atlantoaxial joint region is a complex procedure especially in patients with nuchal deformity like basilar invagination. This region has actually very complicated anatomical and functional characteristics, including multiple joints providing extension, flexion, and wide rotation. In fact, it is also a bottleneck region where bones, neural structures, and blood vessels are located. Stabilization surgery regarding this region should consider the fact that the area exposes excessive and life-long stress due to complex movements and human posture. Therefore, all options should be considered for surgical stabilization, and they could be interchanged during the surgery, if required. A 53-year-old male patient applied to outpatients' clinic with complaints of head and neck pain persisting for a long time. Physical examination was normal except increased deep tendon reflexes. The patient was on long-term corticosteroid due to an allergic disease. Magnetic resonance imaging and computed tomography findings indicated basilar invagination and atlantoaxial dislocation. The patient underwent C0-C3-C4 (lateral mass) and additional C0-C2 (translaminar) stabilization surgery. In routine practice, the sites where rods are bound to occipital plates were placed as paramedian. Instead, we inserted lateral mass screw to the sites where occipital screws were inserted on the occipital plate, thereby creating a site where extra rod could be bound. When C2 translaminar screw is inserted, screw caps remain on the median plane, which makes them difficult to bind to contralateral system. These bind directly to occipital plate without any connection from this region to the contralateral system. Advantages of this technique include easy insertion of C2 translaminar screws, presence of increased screw sizes, and exclusion of pullout forces onto the screw from neck movements. Another advantage of the technique is the median placement of the rod; i.e., thick part of the occipital bone is in alignment with axial loading. We believe that this technique, which could be easily performed as adjuvant to classical stabilization surgery with no need for special screw and rod, may improve distraction force in patients with low bone density.

Identification of Targets for Prevention of Peritoneal Catheter Tunnel and Exit-Site Infections in Low Incidence Settings.

PubMed

Santos, Clara; Pérez-Fontán, Miguel; Rodríguez-Carmona, Ana; Calvo-Rodríguez, María; López-Muñiz, Andrés; López-Calviño, Beatriz; García-Falcón, Teresa

2016-01-01

♦ Peritoneal catheter tunnel and exit-site infection (TESI) complicates the clinical course of peritoneal dialysis (PD) patients. Adherence to recommendations for catheter insertion, exit-site care, and management of Staphylococcus aureus (SAu) carriage reduces, but does not abrogate the risk of these infections. ♦ To reappraise the risk profile for TESI in an experienced center with a long-term focus on management of SAu carriage and a low incidence of these infections. ♦ Following a retrospective, observational design, we investigated 665 patients incident on PD. The main study variable was survival to the first episode of TESI. We considered selected demographic, clinical, and technical variables, applying multivariate strategies of analysis. ♦ The overall incidence of TESI was 1 episode/68.5 patient-months. Staphylococcus aureus carriage disclosed at inception of PD (but not if observed sporadically during follow-up) (hazard ratio [HR] 1.53, p = 0.009), PD started shortly after catheter insertion (HR 0.98 per day, p = 0.011), PD after kidney transplant failure (HR 2.18, p = 0.017), lower hemoglobin levels (HR 0.88 per g/dL, p = 0.013) and fast peritoneal transport rates (HR 2.92, p = 0.03) portended an increased risk of TESI. Delaying PD ≥ 30 days after catheter insertion markedly improved the probability of TESI. Carriage of methicillin-resistant SAu since the start of PD was associated with a high incidence of TESI by these bacteria. On the contrary, resistance to mupirocin did not predict such a risk, probably due to the use of an alternative regime in affected patients. ♦ Adherence to current recommendations results in a low incidence of TESI in PD patients. Interventions on specific risk subsets have a potential to bring incidence close to negligible levels. Despite systematic screening and management, SAu carriage is still a predictor of TESI. Antibiotic susceptibility patterns may help to refine stratification of the risk of TESI by these bacteria. Early insertion of the peritoneal catheter should be considered whenever possible, to reduce the risk of later TESI. Copyright © 2016 International Society for Peritoneal Dialysis.

An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease.

PubMed Central

Urban, M; Bhargava, T; Hamer, J E

1999-01-01

Cells tolerate exposure to cytotoxic compounds through the action of ATP-driven efflux pumps belonging to the ATP-binding cassette (ABC) superfamily of membrane transporters. Phytopathogenic fungi encounter toxic environments during plant invasion as a result of the plant defense response. Here we demonstrate the requirement for an ABC transporter during host infection by the fungal plant pathogen Magnaporthe grisea. The ABC1 gene was identified in an insertional mutagenesis screen for pathogenicity mutants. The ABC1 insertional mutant and a gene-replacement mutant arrest growth and die shortly after penetrating either rice or barley epidermal cells. The ABC1-encoded protein is similar to yeast ABC transporters implicated in multidrug resistance, and ABC1 gene transcripts are inducible by toxic drugs and a rice phytoalexin. However, abc1 mutants are not hypersensitive to antifungal compounds. The non-pathogenic, insertional mutation in ABC1 occurs in the promoter region and dramatically reduces transcript induction by metabolic poisons. These data strongly suggest that M.grisea requires the up-regulation of specific ABC transporters for pathogenesis; most likely to protect itself against plant defense mechanisms. PMID:9927411

Big-bubble deep anterior lamellar keratoplasty using central vs peripheral air injection: a clinical trial.

PubMed

Feizi, Sepehr; Daryabari, Seyed-Hashem; Najdi, Danial; Javadi, Mohammad Ali; Karimian, Farid

2016-06-10

To compare 2 sites of air injection to achieve Descemet membrane (DM) detachment in big-bubble deep anterior lamellar keratoplasty (DALK). In this prospective, randomized study, 48 eyes of 48 keratoconus-affected patients who underwent DALK by cornea fellows were enrolled. Each patient was randomly assigned into one of 2 groups. After trephination to approximately 80% of the corneal thickness, a 27-G needle was inserted into the stroma from the trephination site. The needle was moved radially inside the trephination site and advanced to the central or paracentral cornea in group 1. In group 2, the needle was inserted into the deep stroma from the trephination site and advanced into the peripheral cornea to approximately 1.5 mm anterior to the limbus. Air was gently injected into the deep stroma until a big bubble was formed. The rates of DM separation and complications were compared between the 2 groups. Big-bubble formation was successful in 79.2% of the eyes in the study group. A bare DM was achieved by central injection in 68.0% of group 1 and by peripheral injection in 69.6% of group 2 (p = 0.68). This rate was increased to 80.0% and 78.3% in groups 1 and 2, respectively, after the injection site was shifted when injections failed. The study groups were comparable in terms of complications including DM perforation and bubble bursting. Both injection sites were equivalent in their rates of big-bubble formation and complications. Less experienced surgeons are advised to initially inject air outside the trephination.

Structural-electrochemical relations in the aqueous copper hexacyanoferrate-zinc system examined by synchrotron X-ray diffraction

NASA Astrophysics Data System (ADS)

Renman, Viktor; Ojwang, Dickson O.; Valvo, Mario; Gómez, Cesar Pay; Gustafsson, Torbjörn; Svensson, Gunnar

2017-11-01

The storage process of Zn2+ in the Prussian blue analogue (PBA) copper hexacyanoferrate (Cu[Fe(CN)6]2/3·nH2O - CuHCF) framework structure in a context of rechargeable aqueous batteries is examined by means of in operando synchrotron X-ray diffraction. Via sequential unit-cell parameter refinements of time-resolved diffraction data, it is revealed that the step-profile of the cell output voltage curves during repeated electrochemical insertion and removal of Zn2+ in the CuHCF host structure is associated with a non-linear contraction and expansion of the unit-cell in the range 0.36 < x < 1.32 for Znx/3Cu[Fe(CN)6]2/3·nH2O. For a high insertion cation content there is no apparent change in the unit-cell contraction. Furthermore, a structural analysis with respect to the occupancies of possible Zn2+ sites suggests that the Fe(CN)6 vacancies within the CuHCF framework play an important role in the structural-electrochemical behavior of this particular system. More specifically, it is observed that Zn2+ swaps position during electrochemical cycling, hopping between cavity sites to vacant ferricyanide sites.

Optimal approach for complete liver tumor ablation using radiofrequency ablation: a simulation study.

PubMed

Givehchi, Sogol; Wong, Yin How; Yeong, Chai Hong; Abdullah, Basri Johan Jeet

2018-04-01

To investigate the effect of radiofrequency ablation (RFA) electrode trajectory on complete tumor ablation using computational simulation. The RFA of a spherical tumor of 2.0 cm diameter along with 0.5 cm clinical safety margin was simulated using Finite Element Analysis software. A total of 86 points inside one-eighth of the tumor volume along the axial, sagittal and coronal planes were selected as the target sites for electrode-tip placement. The angle of the electrode insertion in both craniocaudal and orbital planes ranged from -90° to +90° with 30° increment. The RFA electrode was simulated to pass through the target site at different angles in combination of both craniocaudal and orbital planes before being advanced to the edge of the tumor. Complete tumor ablation was observed whenever the electrode-tip penetrated through the epicenter of the tumor regardless of the angles of electrode insertion in both craniocaudal and orbital planes. Complete tumor ablation can also be achieved by placing the electrode-tip at several optimal sites and angles. Identification of the tumor epicenter on the central slice of the axial images is essential to enhance the success rate of complete tumor ablation during RFA procedures.

The Geographic Distribution of Human Y Chromosome Variation

PubMed Central

Hammer, M. F.; Spurdle, A. B.; Karafet, T.; Bonner, M. R.; Wood, E. T.; Novelletto, A.; Malaspina, P.; Mitchell, R. J.; Horai, S.; Jenkins, T.; Zegura, S. L.

1997-01-01

We examined variation on the nonrecombining portion of the human Y chromosome to investigate human evolution during the last 200,000 years. The Y-specific polymorphic sites included the Y Alu insertional polymorphism or ``YAP'' element (DYS287), the poly(A) tail associated with the YAP element, three point mutations in close association with the YAP insertion site, an A-G polymorphic transition (DYS271), and a tetranucleotide microsatellite (DYS19). Global variation at the five bi-allelic sites (DYS271, DYS287, and the three point mutations) gave rise to five ``YAP haplotypes'' in 60 populations from Africa, Europe, Asia, Australasia, and the New World (n = 1500). Combining the multi-allelic variation at the microsatellite loci (poly(A) tail and DYS19) with the YAP haplotypes resulted in a total of 27 ``combination haplotypes''. All five of the YAP haplotypes and 21 of the 27 combination haplotypes were found in African populations, which had greater haplotype diversity than did populations from other geographical locations. Only subsets of the five YAP haplotypes were found outside of Africa. Patterns of observed variation were compatible with a variety of hypotheses, including multiple human migrations and range expansions. PMID:9055088

Consistency of Continuous Ambulatory Interstitial Glucose Monitoring Sensors.

PubMed

Wu, Pei T; Segovia, David E; Lee, Cathy C; Nguyen, Kim-Lien

2018-05-16

The abdominal region is the most common location for continuous glucose monitor (CGM) sensor insertion. However, a paucity of post-marketing data is available to demonstrate intra-individual consistency of CGM readings at different abdominal insertion sites. Healthy adults (fasting glucose (FG) < 5.5 mmol/L; BMI < 30 kg/m²) were recruited and a CGM sensor was placed on each side of the abdomen. Postprandial and continuous 48-h interstitial glucose levels were analyzed. There was no significant difference in the 3-h postprandial glucose (PPG) level derived from the left versus right CGM, which remained non-significant after adjusting for waist circumference or FG. Among the glucose levels recorded over 48-h, values on the left site were greater in 3.6% of the data points ( p < 0.05). After adjusting for waist circumference, only 0.5% of the glucose values remained significantly greater on the left ( p < 0.05). When adjusted for FG, similar results were observed. For both PPG and 48-h readings, the mean absolute relative difference was not significant between the two abdominal sites. CGM-derived glucose measures were highly consistent between the left and right abdomen during both the postprandial and post-absorptive periods.

Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors.

PubMed

Vega, Juan M; Yu, Weichang; Han, Fangpu; Kato, Akio; Peters, Eric M; Zhang, Zhanyuan J; Birchler, James A

2008-04-01

The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

Bacteriophage-based vectors for site-specific insertion of DNA in the chromosome of Corynebacteria.

PubMed

Oram, Mark; Woolston, Joelle E; Jacobson, Andrew D; Holmes, Randall K; Oram, Diana M

2007-04-15

In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as beta. beta-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally encoded genes, is regulated by the DtxR protein in response to Fe(2+) levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the beta-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae DeltadtxR strain. Additionally, strains of beta-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for beta, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species.

The Tn7 transposition regulator TnsC interacts with the transposase subunit TnsB and target selector TnsD

PubMed Central

Choi, Ki Young; Spencer, Jeanelle M.; Craig, Nancy L.

2014-01-01

The excision of transposon Tn7 from a donor site and its insertion into its preferred target site, attachment site attTn7, is mediated by four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC, and TnsD. Transposition requires the assembly of a nucleoprotein complex containing all four Tns proteins and the DNA substrates, the donor site containing Tn7, and the preferred target site attTn7. TnsA and TnsB together form the heteromeric Tn7 transposase, and TnsD is a target-selecting protein that binds specifically to attTn7. TnsC is the key regulator of transposition, interacting with both the TnsAB transposase and TnsD-attTn7. We show here that TnsC interacts directly with TnsB, and identify the specific region of TnsC involved in the TnsB–TnsC interaction during transposition. We also show that a TnsC mutant defective in interaction with TnsB is defective for Tn7 transposition both in vitro and in vivo. Tn7 displays cis-acting target immunity, which blocks Tn7 insertion into a target DNA that already contains Tn7. We provide evidence that the direct TnsB–TnsC interaction that we have identified also mediates cis-acting Tn7 target immunity. We also show that TnsC interacts directly with the target selector protein TnsD. PMID:24982178

An Intraoperative Site-specific Bone Density Device: A Pilot Test Case.

PubMed

Arosio, Paolo; Moschioni, Monica; Banfi, Luca Maria; Di Stefano, Anilo Alessio

2015-08-01

This paper reports a case of all-on-four rehabilitation where bone density at implant sites was assessed both through preoperative computed tomographic (CT) scans and using a micromotor working as an intraoperative bone density measurement device. Implant-supported rehabilitation is a predictable treatment option for tooth replacement whose success depends on the clinician's experience, the implant characteristics and location and patient-related factors. Among the latter, bone density is a determinant for the achievement of primary implant stability and, eventually, for implant success. The ability to measure bone density at the placement site before implant insertion could be important in the clinical setting. A patient complaining of masticatory impairment was presented with a plan calling for extraction of all her compromised teeth, followed by implant rehabilitation. A week before surgery, she underwent CT examination, and the bone density on the CT scans was measured. When the implant osteotomies were created, the bone density was again measured with a micromotor endowed with an instantaneous torque-measuring system. The implant placement protocols were adapted for each implant, according to the intraoperative measurements, and the patient was rehabilitated following an all-on-four immediate loading protocol. The bone density device provided valuable information beyond that obtained from CT scans, allowing for site-specific, intraoperative assessment of bone density immediately before implant placement and an estimation of primary stability just after implant insertion. Measuring jaw-bone density could help clinicians to select implant-placement protocols and loading strategies based on site-specific bone features.

A novel protocol for the production of recombinant LL-37 expressed as a thioredoxin fusion protein.

PubMed

Li, Yifeng

2012-02-01

LL-37 is the only cathelicidin-derived antimicrobial peptide found in humans and it has a multifunctional role in host defense. The peptide has been shown to possess immunomodulatory functions in addition to antimicrobial activity. To provide sufficient material for biological and structural characterization of this important peptide, various systems were developed to produce recombinant LL-37 in Escherichia coli. In one previous approach, LL-37 coding sequence was cloned into vector pET-32a, allowing the peptide to be expressed as a thioredoxin fusion. The fusion protein contains two thrombin cleavage sites: a vector-encoded one that is 30-residue upstream of the insert and an engineered one that is immediately adjacent to LL-37. Cleavage at these two sites shall generate three fragments, one of which is the target peptide. However, when the fusion protein was treated with thrombin, cleavage only occurred at the remote upstream site. A plausible explanation is that the thrombin site adjacent to LL-37 is less accessible due to the peptide's aggregation tendency and cleavage at the remote site generates a fragment, which forms a large aggregate that buries the intended site. In this study, I deleted the vector-encoded thrombin site and S tag in pET-32a, and then inserted the coding sequence for LL-37 plus a thrombin site into the modified vector. Although removing the S tag did not change the oligomeric state of the fusion protein, deletion of the vector-encoded thrombin site allowed the fusion to be cleaved at the engineered site to release LL-37. The released peptide was separated from the carrier and cleavage enzyme by size-exclusion chromatography. This new approach enables a quick production of high quality active LL-37 with a decent amount. Copyright © 2011 Elsevier Inc. All rights reserved.

Influencing factors for port-site hernias after single-incision laparoscopy.

PubMed

Buckley, F P; Vassaur, H E; Jupiter, D C; Crosby, J H; Wheeless, C J; Vassaur, J L

2016-10-01

Single-incision laparoscopic surgery (SILS) has been demonstrated to be a feasible alternative to multiport laparoscopy, but concerns over port-site incisional hernias have not been well addressed. A retrospective study was performed to determine the rate of port-site hernias as well as influencing risk factors for developing this complication. A review of all consecutive patients who underwent SILS over 4 years was conducted using electronic medical records in a multi-specialty integrated healthcare system. Statistical evaluation included descriptive analysis of demographics in addition to bivariate and multivariate analyses of potential risk factors, which were age, gender, BMI, procedure, existing insertion-site hernia, wound infection, tobacco use, steroid use, and diabetes. 787 patients who underwent SILS without conversion to open were reviewed. There were 454 cholecystectomies, 189 appendectomies, 72 colectomies, 21 fundoplications, 15 transabdominal inguinal herniorrhaphies, and 36 other surgeries. Cases included 532 (67.6 %) women, and among all patients mean age was 44.65 (±19.05) years and mean BMI of 28.04 (±6). Of these, 50 (6.35 %) patients were documented as developing port-site incisional hernias by a health care provider or by incidental imaging. Of the risk factors analyzed, insertion-site hernia, age, and BMI were significant. Multivariate analysis indicated that both preexisting hernia and BMI were significant risk factors (p value = 0.00212; p value = 0.0307). Morbidly obese patients had the highest incidence of incisional hernias at 18.18 % (p value = 0.02). When selecting patients for SILS, surgeons should consider the presence of an umbilical hernia, increased age and obesity as risk factors for developing a port-site hernia.

Finite Element Analysis of Patella Alta: A Patellofemoral Instability Model.

PubMed

Watson, Nicole A; Duchman, Kyle R; Grosland, Nicole M; Bollier, Matthew J

2017-01-01

This study aims to provide biomechanical data on the effect of patella height in the setting of medial patellofemoral ligament (MPFL) reconstruction using finite element analysis. The study will also examine patellofemoral joint biomechanics using variable femoral insertion sites for MPFL reconstruction. A previously validated finite element knee model was modified to study patella alta and baja by translating the patella a given distance to achieve each patella height ratio. Additionally, the models were modified to study various femoral insertion sites of the MPFL (anatomic, anterior, proximal, and distal) for each patella height model, resulting in 32 unique scenarios available for investigation. In the setting of patella alta, the patellofemoral contact area decreased, resulting in a subsequent increase in maximum patellofemoral contact pressures as compared to the scenarios with normal patellar height. Additionally, patella alta resulted in decreased lateral restraining forces in the native knee scenario as well as following MPFL reconstruction. Changing femoral insertion sites had a variable effect on patellofemoral contact pressures; however, distal and anterior femoral tunnel malpositioning in the setting of patella alta resulted in grossly elevated maximum patellofemoral contact pressures as compared to other scenarios. Patella alta after MPFL reconstruction results in decreased lateral restraining forces and patellofemoral contact area and increased maximum patellofemoral contact pressures. When the femoral MPFL tunnel is malpositioned anteriorly or distally on the femur, the maximum patellofemoral contact pressures increase with severity of patella alta. When evaluating patients with patellofemoral instability, it is important to recognize patella alta as a potential aggravating factor. Failure to address patella alta in the setting of MPFL femoral tunnel malposition may result in even further increases in patellofemoral contact pressures, making it essential to optimize intraoperative techniques to confirm anatomic MPFL femoral tunnel positioning.

Finite Element Analysis of Patella Alta: A Patellofemoral Instability Model

PubMed Central

Duchman, Kyle R.; Grosland, Nicole M.; Bollier, Matthew J.

2017-01-01

Abstract Background: This study aims to provide biomechanical data on the effect of patella height in the setting of medial patellofemoral ligament (MPFL) reconstruction using finite element analysis. The study will also examine patellofemoral joint biomechanics using variable femoral insertion sites for MPFL reconstruction. Methods: A previously validated finite element knee model was modified to study patella alta and baja by translating the patella a given distance to achieve each patella height ratio. Additionally, the models were modified to study various femoral insertion sites of the MPFL (anatomic, anterior, proximal, and distal) for each patella height model, resulting in 32 unique scenarios available for investigation. Results: In the setting of patella alta, the patellofemoral contact area decreased, resulting in a subsequent increase in maximum patellofemoral contact pressures as compared to the scenarios with normal patellar height. Additionally, patella alta resulted in decreased lateral restraining forces in the native knee scenario as well as following MPFL reconstruction. Changing femoral insertion sites had a variable effect on patellofemoral contact pressures; however, distal and anterior femoral tunnel malpositioning in the setting of patella alta resulted in grossly elevated maximum patellofemoral contact pressures as compared to other scenarios. Conclusions: Patella alta after MPFL reconstruction results in decreased lateral restraining forces and patellofemoral contact area and increased maximum patellofemoral contact pressures. When the femoral MPFL tunnel is malpositioned anteriorly or distally on the femur, the maximum patellofemoral contact pressures increase with severity of patella alta. Clinical Relevance: When evaluating patients with patellofemoral instability, it is important to recognize patella alta as a potential aggravating factor. Failure to address patella alta in the setting of MPFL femoral tunnel malposition may result in even further increases in patellofemoral contact pressures, making it essential to optimize intraoperative techniques to confirm anatomic MPFL femoral tunnel positioning. PMID:28852343

HIV evolution in early infection: selection pressures, patterns of insertion and deletion, and the impact of APOBEC.

PubMed

Wood, Natasha; Bhattacharya, Tanmoy; Keele, Brandon F; Giorgi, Elena; Liu, Michael; Gaschen, Brian; Daniels, Marcus; Ferrari, Guido; Haynes, Barton F; McMichael, Andrew; Shaw, George M; Hahn, Beatrice H; Korber, Bette; Seoighe, Cathal

2009-05-01

The pattern of viral diversification in newly infected individuals provides information about the host environment and immune responses typically experienced by the newly transmitted virus. For example, sites that tend to evolve rapidly across multiple early-infection patients could be involved in enabling escape from common early immune responses, could represent adaptation for rapid growth in a newly infected host, or could represent reversion from less fit forms of the virus that were selected for immune escape in previous hosts. Here we investigated the diversification of HIV-1 env coding sequences in 81 very early B subtype infections previously shown to have resulted from transmission or expansion of single viruses (n = 78) or two closely related viruses (n = 3). In these cases, the sequence of the infecting virus can be estimated accurately, enabling inference of both the direction of substitutions as well as distinction between insertion and deletion events. By integrating information across multiple acutely infected hosts, we find evidence of adaptive evolution of HIV-1 env and identify a subset of codon sites that diversified more rapidly than can be explained by a model of neutral evolution. Of 24 such rapidly diversifying sites, 14 were either i) clustered and embedded in CTL epitopes that were verified experimentally or predicted based on the individual's HLA or ii) in a nucleotide context indicative of APOBEC-mediated G-to-A substitutions, despite having excluded heavily hypermutated sequences prior to the analysis. In several cases, a rapidly evolving site was embedded both in an APOBEC motif and in a CTL epitope, suggesting that APOBEC may facilitate early immune escape. Ten rapidly diversifying sites could not be explained by CTL escape or APOBEC hypermutation, including the most frequently mutated site, in the fusion peptide of gp41. We also examined the distribution, extent, and sequence context of insertions and deletions, and we provide evidence that the length variation seen in hypervariable loop regions of the envelope glycoprotein is a consequence of selection and not of mutational hotspots. Our results provide a detailed view of the process of diversification of HIV-1 following transmission, highlighting the role of CTL escape and hypermutation in shaping viral evolution during the establishment of new infections.

HIV Evolution in Early Infection: Selection Pressures, Patterns of Insertion and Deletion, and the Impact of APOBEC

PubMed Central

Wood, Natasha; Bhattacharya, Tanmoy; Keele, Brandon F.; Giorgi, Elena; Liu, Michael; Gaschen, Brian; Daniels, Marcus; Ferrari, Guido; Haynes, Barton F.; McMichael, Andrew; Shaw, George M.; Hahn, Beatrice H.; Korber, Bette; Seoighe, Cathal

2009-01-01

The pattern of viral diversification in newly infected individuals provides information about the host environment and immune responses typically experienced by the newly transmitted virus. For example, sites that tend to evolve rapidly across multiple early-infection patients could be involved in enabling escape from common early immune responses, could represent adaptation for rapid growth in a newly infected host, or could represent reversion from less fit forms of the virus that were selected for immune escape in previous hosts. Here we investigated the diversification of HIV-1 env coding sequences in 81 very early B subtype infections previously shown to have resulted from transmission or expansion of single viruses (n = 78) or two closely related viruses (n = 3). In these cases, the sequence of the infecting virus can be estimated accurately, enabling inference of both the direction of substitutions as well as distinction between insertion and deletion events. By integrating information across multiple acutely infected hosts, we find evidence of adaptive evolution of HIV-1 env and identify a subset of codon sites that diversified more rapidly than can be explained by a model of neutral evolution. Of 24 such rapidly diversifying sites, 14 were either i) clustered and embedded in CTL epitopes that were verified experimentally or predicted based on the individual's HLA or ii) in a nucleotide context indicative of APOBEC-mediated G-to-A substitutions, despite having excluded heavily hypermutated sequences prior to the analysis. In several cases, a rapidly evolving site was embedded both in an APOBEC motif and in a CTL epitope, suggesting that APOBEC may facilitate early immune escape. Ten rapidly diversifying sites could not be explained by CTL escape or APOBEC hypermutation, including the most frequently mutated site, in the fusion peptide of gp41. We also examined the distribution, extent, and sequence context of insertions and deletions, and we provide evidence that the length variation seen in hypervariable loop regions of the envelope glycoprotein is a consequence of selection and not of mutational hotspots. Our results provide a detailed view of the process of diversification of HIV-1 following transmission, highlighting the role of CTL escape and hypermutation in shaping viral evolution during the establishment of new infections. PMID:19424423

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

PubMed Central

2017-01-01

ABSTRACT Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in “enhancerless” self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required. IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy. PMID:29046446

Genome-Wide Identification by Transposon Insertion Sequencing of Escherichia coli K1 Genes Essential for In Vitro Growth, Gastrointestinal Colonizing Capacity, and Survival in Serum.

PubMed

McCarthy, Alex J; Stabler, Richard A; Taylor, Peter W

2018-04-01

Escherichia coli K1 strains are major causative agents of invasive disease of newborn infants. The age dependency of infection can be reproduced in neonatal rats. Colonization of the small intestine following oral administration of K1 bacteria leads rapidly to invasion of the blood circulation; bacteria that avoid capture by the mesenteric lymphatic system and evade antibacterial mechanisms in the blood may disseminate to cause organ-specific infections such as meningitis. Some E. coli K1 surface constituents, in particular the polysialic acid capsule, are known to contribute to invasive potential, but a comprehensive picture of the factors that determine the fully virulent phenotype has not emerged so far. We constructed a library and constituent sublibraries of ∼775,000 Tn 5 transposon mutants of E. coli K1 strain A192PP and employed transposon-directed insertion site sequencing (TraDIS) to identify genes required for fitness for infection of 2-day-old rats. Transposon insertions were lacking in 357 genes following recovery on selective agar; these genes were considered essential for growth in nutrient-replete medium. Colonization of the midsection of the small intestine was facilitated by 167 E. coli K1 gene products. Restricted bacterial translocation across epithelial barriers precluded TraDIS analysis of gut-to-blood and blood-to-brain transits; 97 genes were required for survival in human serum. This study revealed that a large number of bacterial genes, many of which were not previously associated with systemic E. coli K1 infection, are required to realize full invasive potential. IMPORTANCE Escherichia coli K1 strains cause life-threatening infections in newborn infants. They are acquired from the mother at birth and colonize the small intestine, from where they invade the blood and central nervous system. It is difficult to obtain information from acutely ill patients that sheds light on physiological and bacterial factors determining invasive disease. Key aspects of naturally occurring age-dependent human infection can be reproduced in neonatal rats. Here, we employ transposon-directed insertion site sequencing to identify genes essential for the in vitro growth of E. coli K1 and genes that contribute to the colonization of susceptible rats. The presence of bottlenecks to invasion of the blood and cerebrospinal compartments precluded insertion site sequencing analysis, but we identified genes for survival in serum. Copyright © 2018 McCarthy et al.

Genome-Wide Identification by Transposon Insertion Sequencing of Escherichia coli K1 Genes Essential for In Vitro Growth, Gastrointestinal Colonizing Capacity, and Survival in Serum

PubMed Central

McCarthy, Alex J.

2018-01-01

ABSTRACT Escherichia coli K1 strains are major causative agents of invasive disease of newborn infants. The age dependency of infection can be reproduced in neonatal rats. Colonization of the small intestine following oral administration of K1 bacteria leads rapidly to invasion of the blood circulation; bacteria that avoid capture by the mesenteric lymphatic system and evade antibacterial mechanisms in the blood may disseminate to cause organ-specific infections such as meningitis. Some E. coli K1 surface constituents, in particular the polysialic acid capsule, are known to contribute to invasive potential, but a comprehensive picture of the factors that determine the fully virulent phenotype has not emerged so far. We constructed a library and constituent sublibraries of ∼775,000 Tn5 transposon mutants of E. coli K1 strain A192PP and employed transposon-directed insertion site sequencing (TraDIS) to identify genes required for fitness for infection of 2-day-old rats. Transposon insertions were lacking in 357 genes following recovery on selective agar; these genes were considered essential for growth in nutrient-replete medium. Colonization of the midsection of the small intestine was facilitated by 167 E. coli K1 gene products. Restricted bacterial translocation across epithelial barriers precluded TraDIS analysis of gut-to-blood and blood-to-brain transits; 97 genes were required for survival in human serum. This study revealed that a large number of bacterial genes, many of which were not previously associated with systemic E. coli K1 infection, are required to realize full invasive potential. IMPORTANCE Escherichia coli K1 strains cause life-threatening infections in newborn infants. They are acquired from the mother at birth and colonize the small intestine, from where they invade the blood and central nervous system. It is difficult to obtain information from acutely ill patients that sheds light on physiological and bacterial factors determining invasive disease. Key aspects of naturally occurring age-dependent human infection can be reproduced in neonatal rats. Here, we employ transposon-directed insertion site sequencing to identify genes essential for the in vitro growth of E. coli K1 and genes that contribute to the colonization of susceptible rats. The presence of bottlenecks to invasion of the blood and cerebrospinal compartments precluded insertion site sequencing analysis, but we identified genes for survival in serum. PMID:29339415

Knotted versus knotless suture bridge repair of the achilles tendon insertion: a biomechanical study.

PubMed

Cox, Joseph T; Shorten, Peter L; Gould, Gregory C; Markert, Ronald J; Barnett, Michael D; Laughlin, Richard T

2014-11-01

Surgical treatment of insertional Achilles tendinopathy often involves detachment and debridement of the Achilles tendon insertion. A recent study has shown that knotted suture bridge fixation of the Achilles to the calcaneus is biomechanically superior to single-row fixation, but there is an absence of literature on the use of different suture bridge constructs to repair the Achilles tendon. There will be no significant difference in the load to failure, mode of failure, tendon strain, tendon stiffness, repair site gapping, or footprint size when comparing knotted suture bridge repair to knotless suture bridge repair of the Achilles tendon after detachment for insertional Achilles tendinopathy. Controlled laboratory study. A single specimen from each pair of 10 cadaveric Achilles tendons was randomized to 1 of 2 Achilles insertion repair groups: knotted (n = 10) or knotless (n = 10) suture bridge repair. Repaired footprint size was measured, and then cyclic testing from 10 to 100 N for 2000 cycles was performed. This was followed by measurement of tendon strain, repair site displacement, load to failure, and tendon stiffness. The knotted suture bridge repair had a significantly higher load to failure compared with the knotless suture bridge (mean ± SD, 317.8 ± 93.6 N vs 196.1 ± 12.1 N, respectively; P = .001). All constructs failed at the tendon-suture interface. Tendon strain after cyclic testing was significantly greater in the knotless (1.20 ± 1.05) compared with the knotted (0.39 ± 0.4) suture repair groups (P = .011). There was no significant difference in footprint size between the knotted (230.3 ± 63.3 mm(2)) and knotless (248.5 ± 48.8 mm(2)) groups (P = .40). There was also no significant difference in stiffness (knotted = 76.4 ± 8.0 N/mm; knotless = 69.6 ± 10.9 N/mm; P = .17) and repair site displacement after cyclic testing (knotted = 2.8 ± 1.2 mm; knotless = 3.6 ± 1.1 mm; P = .17). During suture bridge repair of the Achilles tendon after detachment, knots at the proximal suture anchors significantly improve the biomechanical strength of the repair. This study demonstrated that the knotless suture bridge repair had a significantly lower load to failure than the knotted suture bridge. Surgeons should be aware of these biomechanical differences, as they influence the postoperative rehabilitation protocol and may lead to higher surgical complication rates. © 2014 The Author(s).

Comparison of carpal tunnel injection techniques: a cadaver study.

PubMed

Ozturk, Kahraman; Esenyel, Cem Zeki; Sonmez, Mesut; Esenyel, Meltem; Kahraman, Sinan; Senel, Berna

2008-01-01

The purpose of the study was to evaluate the accuracy of injections into the carpal tunnel using three different portals in cadavers, and to define safe guidelines. In this study, 150 wrists of 75 cadavers (54 male, 21 female) were included. To compare three injection sites, 50 wrists of 25 cadavers were used for each technique; we used 23 gauge needles, and acrylic dye. The first injection technique: the needle was inserted 1cm proximal to the wrist crease and directed distally by roughly 45 in an ulnar direction through the flexor carpi radialis tendon. The second injection technique: the needle was inserted into the carpal tunnel from a point just ulnar to the palmaris longus tendon and 1cm proximal to the wrist crease. The third injection technique: the needle was inserted just distal to the distal skin crease of the wrist in line with the fourth ray. The first injection technique gave the highest accuracy rate, and this was also the safest injection site. Median nerve injuries caused by injection was seen mostly with the second technique. Although a steroid injection may provide symptomatic relief in patients with carpal tunnel syndrome, the median nerve and other structures in the carpal tunnel are at risk of injury. Because of that, the injection should be given using the correct technique by physicians skilled in carpal tunnel surgery.

A large format in operando wound cell for analysing the structural dynamics of lithium insertion materials

NASA Astrophysics Data System (ADS)

Brant, William R.; Roberts, Matthew; Gustafsson, Torbjörn; Biendicho, Jordi Jacas; Hull, Stephen; Ehrenberg, Helmut; Edström, Kristina; Schmid, Siegbert

2016-12-01

This paper presents a large wound cell for in operando neutron diffraction (ND) from which high quality diffraction patterns are collected every 15 min while maintaining conventional electrochemical performance. Under in operando data collection conditions the oxygen atomic displacement parameters (ADPs) and cell parameters were extracted for Li0.18Sr0.66Ti0.5Nb0.5O3. Analysis of diffraction data collected under in situ conditions revealed that the lithium is located on the (0.5 0.5 0) site, corresponding to the 3c Wyckoff position in the cubic perovskite unit cell, after the cell is discharged to 1 V. When the cell is discharged under potentiostatic conditions the quantity of lithium on this site increases, indicating a potential position where lithium becomes pinned in the thermodynamically stable phase. During this potentiostatic step the oxygen ADPs reduce significantly. On discharge, however, the oxygen ADPs were observed to increase gradually as more lithium is inserted into the structure. Finally, the rate of unit cell expansion changed by ∼44% once the lithium content approached ∼0.17 Li per formula unit. A link between lithium content and degree of mobility, disorder of the oxygen positions and changing rate of unit cell expansion at various stages during lithium insertion and extraction is thus presented.

Fecal Fistula Communicating with a Femur Shaft Fracture Secondary to a Malpositioned Suprapubic Catheter: A Case Report

PubMed Central

Guled, Uday; Goni, Vijay G.; Honnurappa, Arjun R.H.; John, Rakesh; Vardhana, Harsha; Sharma, Gaurav; Pattabhiraman, Kirubakaran S.

2015-01-01

Patient: Male, 18 Final Diagnosis: Fecal fistula communicating with fracture shaft femur secondary to malpositioned SPC Symptoms: — Medication: — Clinical Procedure: Advertisement and rail fixator application Specialty: Orthopedics and Trauamtology Objective: Diagnostic/therapeutic accidents Background: Suprapubic catheter (SPC) insertion is a common urological procedure. Though considered a simple and safe procedure, complications are bound to occur if proper precautions are not taken during the procedure. The reported complications include gross hematuria, post-obstruction diuresis, insertion site skin-related complications, and intra-abdominal visceral injuries. Iatrogenic bowel injuries have been reported to occur as a complication in around 2.5% of cases. Case Report: We report a very rare case of a bowel injury due to improper insertion of a SPC leading to fecal matter tracking along the muscle planes to reach the fracture site of the femur shaft and formation of an external fecal fistula along the lateral aspect of thigh, which according to us is the first reported case in the literature. Conclusions: This case report shows the devastating complication of a technically simple procedure done in an improper manner and successful management of a rare case of femur fracture with communicating fecal fistula. The purpose of this case report is to highlight the importance of taking proper precautions before the procedure. PMID:26439133

Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903.

PubMed Central

Grindley, N D; Joyce, C M

1980-01-01

The kanamycin resistance transposon Tn903 consists of a unique region of about 1000 base pairs bounded by a pair of 1050-base-pair inverted repeat sequences. Each repeat contains two Pvu II endonuclease cleavage sites separated by 520 base pairs. We have constructed derivatives of Tn903 in which this 520-base-pair fragment is deleted from one or both repeats. Those derivatives that lack both 520-base-pair fragments cannot transpose, whereas those that lack just one remain transposition proficient. One such transposable derivative, Tn903 delta I, has been selected for further study. We have determined the sequence of the intact inverted repeat. The 18 base pairs at each end are identical and inverted relative to one another, a structure characteristic of insertion sequences. Additional experiments indicate that a single inverted repeat from Tn903 can, in fact, transpose; we propose that this element be called IS903. To correlate the DNA sequence with genetic activities, we have created mutations by inserting a 10-base-pair DNA fragment at several sites within the intact repeat of Tn903 delta 1, and we have examined the effect of such insertions on transposability. The results suggest that IS903 encodes a 307-amino-acid polypeptide (a "transposase") that is absolutely required for transposition of IS903 or Tn903. Images PMID:6261245

Isolation of CYP3A5P cDNA from human liver: a reflection of a novel cytochrome P-450 pseudogene.

PubMed

Schuetz, J D; Guzelian, P S

1995-03-14

We have isolated, from a human liver cDNA library, a 1627 bp CYP3A5 cDNA variant (CYP3A5P) that contains several large insertions, deletions, and in-frame termination codons. By comparison with the genomic structure of other CYP3A genes, the major insertions in CYP3A5P cDNA demarcate the inferred sites of several CYP3A5 exons. The segments inserted in CYP3A5P have no homology with splice donor acceptor sites. It is unlikely that CYP3A5P cDNA represents an artifact of the cloning procedures since Southern blot analysis of human genomic DNA disclosed that CYP3A5P cDNA hybridized with a DNA fragment distinct from fragments that hybridized with either CYP3A5, CYP3A3 or CYP3A4. Moreover, analysis of adult human liver RNA on Northern blots hybridized with a CYP3A5P cDNA fragment revealed the presence of an mRNA with the predicted size of CYP3A5P. We conclude that CYP3A5P cDNA was derived from a separate gene, CYP3A5P, most likely a pseudogene evolved from CYP3A5.

Identification of a novel selD homolog from Eukaryotes, Bacteria, and Archaea: Is there an autoregulatory mechanism in selenocysteine metabolism?

PubMed Central

Guimarães, M. Jorge; Peterson, David; Vicari, Alain; Cocks, Benjamin G.; Copeland, Neal G.; Gilbert, Debra J.; Jenkins, Nancy A.; Ferrick, David A.; Kastelein, Robert A.; Bazan, J. Fernando; Zlotnik, Albert

1996-01-01

Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme’s putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life. PMID:8986768

Tumor necrosis factor alpha induces gamma-glutamyltransferase expression via nuclear factor-kappaB in cooperation with Sp1.

PubMed

Reuter, Simone; Schnekenburger, Michael; Cristofanon, Silvia; Buck, Isabelle; Teiten, Marie-Hélène; Daubeuf, Sandrine; Eifes, Serge; Dicato, Mario; Aggarwal, Bharat B; Visvikis, Athanase; Diederich, Marc

2009-02-01

Gamma-glutamyltransferase (GGT) cleaves the gamma-glutamyl moiety of glutathione (GSH), an endogenous antioxidant, and is involved in mercapturic acid metabolism and in cancer drug resistance when overexpressed. Moreover, GGT converts leukotriene (LT) C4 into LTD4 implicated in various inflammatory pathologies. So far the effect of inflammatory stimuli on regulation of GGT expression and activity remained to be addressed. We found that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) induced GGT promoter transactivation, mRNA and protein synthesis, as well as enzymatic activity. Remicade, a clinically used anti-TNFalpha antibody, small interfering RNA (siRNA) against p50 and p65 nuclear factor-kappaB (NF-kappaB) isoforms, curcumin, a well characterized natural NF-kappaB inhibitor, as well as a dominant negative inhibitor of kappaB alpha (IkappaBalpha), prevented GGT activation at various levels, illustrating the involvement of this signaling pathway in TNFalpha-induced stimulation. Over-expression of receptor of TNFalpha-1 (TNFR1), TNFR-associated factor-2 (TRAF2), TNFR-1 associated death domain (TRADD), dominant negative (DN) IkappaBalpha or NF-kappaB p65 further confirmed GGT promoter activation via NF-kappaB. Linker insertion mutagenesis of 536 bp of the proximal GGT promoter revealed NF-kappaB and Sp1 binding sites at -110 and -78 relative to the transcription start site, responsible for basal GGT transcription. Mutation of the NF-kappaB site located at -110 additionally inhibited TNFalpha-induced promoter induction. Chromatin immunoprecipitation (ChIP) assays confirmed mutagenesis results and further demonstrated that TNFalpha treatment induced in vivo binding of both NF-kappaB and Sp1, explaining increased GGT expression, and led to RNA polymerase II recruitment under inflammatory conditions.

Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ.

PubMed

Yockey, Oliver P; Jha, Vikash; Ghodke, Pratibha P; Xu, Tianzuo; Xu, Wenyan; Ling, Hong; Pradeepkumar, P I; Zhao, Linlin

2017-11-20

DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N 2 -2'-deoxyguanosine (N 2 -dG) and N 6 -2'-deoxyadenosine (N 6 -dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N 2 -dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.

Automated location detection of injection site for preclinical stereotactic neurosurgery procedure

NASA Astrophysics Data System (ADS)

Abbaszadeh, Shiva; Wu, Hemmings C. H.

2017-03-01

Currently, during stereotactic neurosurgery procedures, the manual task of locating the proper area for needle insertion or implantation of electrode/cannula/optic fiber can be time consuming. The requirement of the task is to quickly and accurately find the location for insertion. In this study we investigate an automated method to locate the entry point of region of interest. This method leverages a digital image capture system, pattern recognition, and motorized stages. Template matching of known anatomical identifiable regions is used to find regions of interest (e.g. Bregma) in rodents. For our initial study, we tackle the problem of automatically detecting the entry point.

PEG tube insertion -- discharge

MedlinePlus

... be treated with medicine. Caring for the PEG-tube Site Drainage from around the PEG tube is common for the first 1 or 2 ... cotton swab or gauze. Try to remove any drainage or crusting on the skin and tube. Be gentle. If you used soap, gently clean ...

75 FR 62807 - Northern Border Pipeline Company; Notice of Availability of the Environmental Assessment for the...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-13

... or group of customers. a pig \\2\\ launcher assembly located adjacent to the Northern Border Kasbeer side valve site; and \\2\\ A ``pig'' is a tool that is inserted into and moves through the pipeline, and...

BAC Modification through Serial or Simultaneous Use of CRE/Lox Technology

PubMed Central

Parrish, Mark; Unruh, Jay; Krumlauf, Robb

2011-01-01

Bacterial Artificial Chromosomes (BACs) are vital tools in mouse genomic analyses because of their ability to propagate large inserts. The size of these constructs, however, prevents the use of conventional molecular biology techniques for modification and manipulation. Techniques such as recombineering and Cre/Lox methodologies have thus become heavily relied upon for such purposes. In this work, we investigate the applicability of Lox variant sites for serial and/or simultaneous manipulations of BACs. We show that Lox spacer mutants are very specific, and inverted repeat variants reduce Lox reaction rates through reducing the affinity of Cre for the site, while retaining some functionality. Employing these methods, we produced serial modifications encompassing four independent changes which generated a mouse HoxB BAC with fluorescent reporter proteins inserted into four adjacent Hox genes. We also generated specific, simultaneous deletions using combinations of spacer variants and inverted repeat variants. These techniques will facilitate BAC manipulations and open a new repertoire of methods for BAC and genome manipulation. PMID:21197414