Frequency Of Excision Research Articles

A Retrospective Review of the Frequency of Incomplete Excision and Upstaging of Early-Stage Melanomas at a Single Academic Institution.

A Retrospective Review of the Frequency of Incomplete Excision and Upstaging of Early-Stage Melanomas at a Single Academic Institution.

Differential diagnoses of benign eyelid tumors in children and adolescents

The clinical diagnosis as well as the treatment approach of periocular tumors in childhood and adolescence can be challenging. Knowledge of the most important differential diagnoses and their clinicopathological correlation is helpful for the treatment approach. The clinical and histological characteristics of various eyelid tumors in childhood and adolescence are presented taking the excision frequencies into consideration. The frequencies and clinicopathologic correlation of the most important eyelid tumors (n = 485) are presented based on the data of the ophthalmopathology laboratory of the University Eye Hospital Bonn from 1998-2023. The most frequent tumor in childhood and adolescence is chalazion (57.3%), followed by dermoid cysts (16.7%) and molluscum contagiosum (9.6%). Other lesions of childhood and adolescence include pilomatrixoma (2.1%), hemangioma and other vascular malformations (4.7%) and rare differential diagnoses, such as subcutaneous calcifying nodules and xanthogranuloma. Guidance on the approach in different age groups is presented in the form of adecision tree. Tumors in children and adolescents are mostly benign, yet there are important indications for excision. A histological examination of any excised tissue in childhood and adolescence is obligatory because unexpected findings are not uncommon and the spectrum of lesions also differs from that in adulthood. Knowledge of the histological picture can be very helpful in the preoperative clinical classification and for planning further procedures.

Transposase expression, element abundance, element size, and DNA repair determine the mobility and heritability of PIF/Pong/Harbinger transposable elements.

Introduction: Class II DNA transposable elements account for significant portions of eukaryotic genomes and contribute to genome evolution through their mobilization. To escape inactivating mutations and persist in the host genome over evolutionary time, these elements must be mobilized enough to result in additional copies. These elements utilize a "cut and paste" transposition mechanism that does not intrinsically include replication. However, elements such as the rice derived mPing element have been observed to increase in copy number over time. Methods: We used yeast transposition assays to test several parameters that could affect the excision and insertion of mPing and its related elements. This included development of novel strategies for measuring element insertion and sequencing insertion sites. Results: Increased transposase protein expression increased the mobilization frequency of a small (430bp) element, while overexpression inhibition was observed for a larger (7,126bp) element. Smaller element size increased both the frequency of excision and insertion of these elements. The effect of yeast ploidy on element excision, insertion, and copy number provided evidence that homology dependent repair allows for replicative transposition. These elements were found to preferentially insert into yeast rDNA repeat sequences. Discussion: Identifying the parameters that influence transposition of these elements will facilitate their use for gene discovery and genome editing. These insights in to the behavior of these elements also provide important clues into how class II transposable elements have shaped eukaryotic genomes.

Active in vivo translocation of the Methanosarcina mazei Gö1 Casposon.

Casposons are transposable elements containing the CRISPR associated gene Cas1solo. Identified in many archaeal genomes, casposons are discussed as the origin of CRISPR-Cas systems due to their proposed Cas1solo-dependent translocation. However, apart from bioinformatic approaches and the demonstration of Cas1solo integrase and endonuclease activity in vitro, casposon transposition has not yet been shown in vivo. Here, we report on active casposon translocations in Methanosarcina mazei Gö1 using two independent experimental approaches. First, mini-casposons, consisting of a R6Kγ origin and two antibiotic resistance cassettes, flanked by target site duplications (TSDs) and terminal inverted repeats (TIRs), were generated, and shown to actively translocate from a suicide plasmid and integrate into the chromosomal MetMaz-C1 TSD IS1a. Second, casposon excision activity was confirmed in a long-term evolution experiment using a Cas1solo overexpression strain in comparison to an empty vector control under four different treatments (native, high temperature, high salt, mitomycin C) to study stress-induced translocation. Analysis of genomic DNA using a nested qPCR approach provided clear evidence of casposon activity in single cells and revealed significantly different casposon excision frequencies between treatments and strains. Our results, providing the first experimental evidence for in vivo casposon activity are summarized in a modified hypothetical translocation model.

Influence of class 2 integron integrase concentration on gene cassette insertion and excision in vivo.

Integron can capture and express antimicrobial resistance gene cassettes and plays important roles in horizontal gene transfer. The establishment of a complete in vitro reaction system will help to reveal integron integrase mediated site-specific recombination process and regulation mechanism. As an enzymatic reaction, the concentration of integrase is assumed to have a great influence on the reaction rate. To determine the influence of different concentrations of integrase on the reaction rate and to find the best range of enzyme concentration were essential to optimizing the in vitro reaction system. In this study, plasmids with gradient transcription levels of class 2 integron integrase gene intI2 under different promoters were constructed. Among plasmids pI2W16, pINTI2N, pI2W, and pI2NW, intI2 transcription levels ranged from about 0.61-fold to 49.65-fold of that in pINTI2N. And the frequencies of gene cassette sat2 integration and excision catalyzed by IntI2 were positively correlated with the transcription levels of intI2 within this range. Western blotting results indicated high expression of IntI2 partly existed in the form of an inclusion body. When compared with Pc of class 1 integron, the spacer sequence of PintI2 can increase the strength of PcW but decrease the strength of PcS. In conclusion, the frequencies of gene cassette integration and excision were positively correlated with the concentration of IntI2. intI2 driving by PcW with PintI2 spacer sequence can obtain the optimum IntI2 concentration required to achieve the maximum recombination efficiency in vivo in this study.

Cre-mediated autoexcision of selectable marker genes in soybean, cotton, canola and maize transgenic plants.

Efficient selectable marker gene autoexcision in transgenic plants of soybean, cotton, canola, and maize is achieved by effective Cre recombinase expression. Selectable marker genes are often required for efficient generation of transgenic plants in plant transformation but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from using the soybean RSP1 promoter for cre expression. The cre gene expressed by soybean RSP1 promoter with an Arabidopsis AtpE intron delivered the best autoexcision frequency (69%) in cotton transformation. The cre gene expressed by the embryo-specific eUSP88 promoter from Vicia faba conferred the best marker excision frequency (32%) in canola transformation. Finally, the cre gene expressed by the rice CDC45-1 promoter resulted in 44% autoexcision in maize transformation. The Cre/loxP recombinase system enables the generation of selectable marker-free transgenic plants for commercial product development in four agriculturally important crops and provides further improvement opportunities for more specific and better marker excision efficiency.

Surgical Outcome of Pure Endonasal Transsphenoidal Approach for Pituitary Tumors

Objective: To determine the frequency of complete tumor excision by endoscopic skull base (transsphenoidal) approach in pituitary adenoma patients presenting to a neurosurgical department.
 Materials & Methods: This descriptive case series was conducted at the Department of Neurosurgery Unit II, Lahore General Hospital Lahore from May 2019 to November 2019. The study included60 patients with pituitary adenoma from the outpatient department who qualified for the inclusion criteria. All patients went through the endoscopic binarial transsphenoidal approach for pituitary adenectomy. All patients were treated according to the department protocols and were followed for 6 weeks to determine the outcome i.e. no residual tumor left as determined by Magnetic Resonance Imaging.
 Results: The mean age of pituitary adenoma patients studied was 50.02 ± 8.2 years. 43 patients (71.7%) were male.56 patients (93.3%) showed complete resection on MRI using the endoscopic binarial transsphenoidal approach. The outcome was not affected by patients' gender and age. Tumor size significantly affected the outcome i.e. complete resection.
 Conclusion: It is concluded that the endoscopic skull base (transsphenoidal) approach for excision of pituitary adenomas gives desirable results as depicted by 93.3% success rate in terms of complete resection of the tumor. We did not observe a significant effect of patients' gender and age on the clinical outcome in the studied sample of the population.

Variation in human herpesvirus 6B telomeric integration, excision, and transmission between tissues and individuals.

Human herpesviruses 6A and 6B (HHV-6A/6B) are ubiquitous pathogens that persist lifelong in latent form and can cause severe conditions upon reactivation. They are spread by community-acquired infection of free virus (acqHHV6A/6B) and by germline transmission of inherited chromosomally integrated HHV-6A/6B (iciHHV-6A/6B) in telomeres. We exploited a hypervariable region of the HHV-6B genome to investigate the relationship between acquired and inherited virus and revealed predominantly maternal transmission of acqHHV-6B in families. Remarkably, we demonstrate that some copies of acqHHV-6B in saliva from healthy adults gained a telomere, indicative of integration and latency, and that the frequency of viral genome excision from telomeres in iciHHV-6B carriers is surprisingly high and varies between tissues. In addition, newly formed short telomeres generated by partial viral genome release are frequently lengthened, particularly in telomerase-expressing pluripotent cells. Consequently, iciHHV-6B carriers are mosaic for different iciHHV-6B structures, including circular extra-chromosomal forms that have the potential to reactivate. Finally, we show transmission of an HHV-6B strain from an iciHHV-6B mother to her non-iciHHV-6B son. Altogether, we demonstrate that iciHHV-6B can readily transition between telomere-integrated and free virus forms.

Incomplete excision of basal cell carcinoma and its associated risk factors

Background: Basal cell carcinoma (BCC) is the most common type of skin tumor. Surgical removal has remained the gold standard of treatment for BCC, but incomplete removal is an important clinical challenge. The goal of our study was to evaluate the incomplete excision of BCC and the factors affecting it. Methods: In this retrospective study, 361 histological samples of BCC excisions referred to the Pathology Department of Imam Reza Hospital, Mashhad, Iran, from 2004 to 2018 were reviewed. Data including age, gender, tumor site, size, histopathologic subtype, surgical margin involvement, and the surgeon’s specialty were recorded. Results: The incomplete removal rate was 20%. Ten percent of the lesions had deep margin involvement, 5.8% had lateral margin involvement, and both margins were involved in 4.2% of cases. Incomplete excision had a significant relationship with the following variables: nodular, mixed, and sclerosing types; the 60-70 years age group; lesions larger than 2 cm, location in nose and canthi; and excision by an otorhinolaryngologist, ophthalmologist, or dermatologist. Conclusion: In this study, the frequency of incomplete excision was relatively high compared with other studies. Therefore, it is suggested to perform a diagnostic biopsy before the complete excision of BCC. Excision with a wider margin or using Mohs surgery is recommended for midface lesions, older individuals, nodular, mixed, or sclerosing lesions, and for tumors larger than 2 cm.

Comparison of Class 2 Integron Integrase Activities.

Integrons play important roles in the dissemination of antimicrobial resistant genes among bacteria. Class 2 integrons usually has an internal stop codon, TAA, in integrase genes (intI2), leading to a truncated integrase, IntI2*. However, a few class 2 integrons with a natural full-length integrase have been reported. In this study, the sequences of natural full-length intI2 were extracted from INTEGRALL database and analyzed. A total of 236 sequences of intI2 were retrieved from INTEGRALL database, only seven of which were natural full-length intI2 genes and could be divided into five types according to their coding amino acid sequence. Quantitative real-time PCR was used to detect gene cassette sat2 integration and excision efficiency catalyzed by different natural full-length IntI2s. The results showed that all five IntI2s could catalyze attI2 × attCsat2 integration and attCdfrA1/sat2 × attCsat2/aadA1 excision in Escherichia coli. Integration and excision frequency catalyzed by IntI2A176 was highest and was about twofold as high as those catalyzed by IntI2S175_A176. The secondary structure of the IntI2 was predicted by online software. Polymorphisms of these five IntI2s were limited within residues 172, 174, 175, 176 and 256, and these residues were all far away from the predicted DNA binding regions or catalyzed sites. Influence of amino acid sequence polymorphisms of these natural full-length IntI2s on their catalyzed activities is limited.

Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants.

Gene targeting (GT) enables precise genome modification—e.g., the introduction of base substitutions—using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously, we demonstrated marker excision via a piggyBac transposon from GT-modified loci in rice. However, piggyBac-mediated marker excision has the limitation that it recognizes only the sequence TTAA. Recently, we proposed a novel and universal precise genome editing system consisting of GT with subsequent single-strand annealing (SSA)-mediated marker excision, which has, in principle, no limitation of target sequences. In this study, we introduced base substitutions into the microRNA miR172 target site of the OsCly1 gene—an ortholog of the barley Cleistogamy1 gene involved in cleistogamous flowering. To ensure efficient SSA, the GT vector harbors 1.2-kb overlapped sequences at both ends of a selection marker. The frequency of positive–negative selection-mediated GT using the vector with overlapped sequences was comparable with that achieved using vectors for piggyBac-mediated marker excision without overlapped sequences, with the frequency of SSA-mediated marker excision calculated as ~40% in the T0 generation. This frequency is thought to be adequate to produce marker-free cells, although it is lower than that achieved with piggyBac-mediated marker excision, which approaches 100%. To date, introduction of precise substitutions in discontinuous multiple bases of a targeted gene using base editors and the prime editing system based on CRISPR/Cas9 has been quite difficult. Here, using GT and our SSA-mediated marker excision system, we succeeded in the precise base substitution not only of single bases but also of artificial discontinuous multiple bases in the miR172 target site of the OsCly1 gene. Precise base substitution of miRNA target sites in target genes using this precise genome editing system will be a powerful tool in the production of valuable crops with improved traits.

Marker-Free Transplastomic Plants by Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences.

Excision of marker genes using DNA direct repeats makes use of the efficient native homologous recombination pathway present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and green algae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes (plastomes). Release of selection allows the accumulation of marker-free plastomes generated by marker excision, which is a spontaneous and unidirectional process. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastomes during growth, development and flowering of T0 plants allows for the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The procedure enables precise plastome engineering involving insertion of transgenes, point mutations and deletion of genes without the inclusion of any extraneous DNA. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.

Risk of Postoperative Stenosis after Segmental Resection versus Disk Excision for Deep Endometriosis Infiltrating the Rectosigmoid: A Retrospective Study

To assess the prevalence, risk factors, and management of bowel stenosis after surgery for deep infiltrating endometriosis of the rectosigmoid using either disk excision (DE) or segmental resection (SR). Retrospective study using data from consecutive cases recorded in the North-West Inter Regional Female Cohort for Patients with Endometriosis database. University tertiary referral center. Four hundred thirty-one consecutive patients managed for rectosigmoid endometriosis were enrolled in our study. Laparoscopic SR or DE. One hundred sixty-five patients underwent DE, and 266 patients underwent SR. Large nodules ≥3 cm in diameter were more frequent in the SR group (73.3% vs 66.1%), whereas nodules infiltrating the low rectum were 3 times more frequent in the DE group (35.9% vs 11.3%). The frequency of vaginal excision (67.9% vs 62%) and stoma (46.7% vs 44.4%) were comparable between the DE and SR groups. Twenty-three patients presented with postoperative colorectal stenosis after SR (8.6%) versus none after DE (p <.001). Treatment of colorectal stenosis involved dilatation in 20 (87%) cases and SR in 4 (17.4%) cases. For 1 patient, dilatation resulted in rectosigmoid injury requiring SR, followed by rectovaginal fistula. The logistic regression model identified a diverting stoma as the sole risk factor independently related to the risk of postoperative stenosis after SR. Bowel stenosis after surgery for deep infiltrating endometriosis occurred in patients who underwent SR, most of them with a diverting stoma, whereas no cases of stenosis were reported in patients who underwent DE, with or without stoma.

Genomic Stability of Composite SCCmec ACME and COMER-Like Genetic Elements in Staphylococcus epidermidis Correlates With Rate of Excision.

The epidemiological success of methicillin-resistant Staphylococcus aureus USA300 has been associated with the presence of two mobile elements, the arginine catabolic mobile element (ACME) and the copper and mercury resistance (COMER) element. These two mobile elements are associated with resistance to copper, which has been related to host fitness and survival within macrophages. Several studies found that ACME is more prevalent, and exhibits greater diversity, in Staphylococcus epidermidis while COMER has not been identified in S. epidermidis or any other staphylococcal species. We aimed in this study to evaluate the presence and diversity of ACME and COMER-like elements in our S. epidermidis clinical isolates. The genomes of 58 S. epidermidis clinical isolates, collected between 2009 and 2018 in a Scottish hospital, were sequenced. A core-genome phylogenetic tree and genome based MLST typing showed that more than half of the isolates belong to the clinically predominant sequence type2 (ST2) and these isolates have been found to split into two lineages within the phylogenetic tree. Analysis showed the presence of SCCmec in the majority of isolates. Comparative analysis identified a cluster of ACME-positive isolates with most of them belonging to ST48. ACME showed high variation even between isolates of the same ACME type and ST. COMER-like elements have been identified in one of the two major hospital adapted drug resistant ST2 lineages; and showed high stability. This difference in stability at the genomic level correlates well with the up to one hundred times higher excision frequency found for the SCCmec elements in ACME-containing isolates compared to COMER-like element containing isolates. ACME/COMER-like element positive isolates did not show a significant phenotype of decreased copper susceptibility, while resistance to mercury was over-represented in COMER-like element positive isolates. To the best of our knowledge, this is the first molecular characterization of COMER-like elements in S. epidermidis isolates. The presence of the COMER-like elements is the most prominent accessory genome feature of these successful lineages suggesting that this chromosomal island contributes to the success and wide clinical distribution of ST2 S. epidermidis.

Pathogenicity island excision during an infection by Salmonella enterica serovar Enteritidis is required for crossing the intestinal epithelial barrier in mice to cause systemic infection.

Pathogenicity island excision is a phenomenon that occurs in several Salmonella enterica serovars and other members of the family Enterobacteriaceae. ROD21 is an excisable pathogenicity island found in the chromosome of S. Enteritidis, S. Dublin and S. Typhi among others, which contain several genes encoding virulence-associated proteins. Excision of ROD21 may play a role in the ability of S. Enteritidis to cause a systemic infection in mice. Our previous studies have shown that Salmonella strains unable to excise ROD21 display a reduced ability to colonize the liver and spleen. In this work, we determined the kinetics of ROD21 excision in vivo in C57BL/6 mice and its effect on virulence. We quantified bacterial burden and excision frequency in different portions of the digestive tract and internal organs throughout the infection. We observed that the frequency of ROD21 excision was significantly increased in the bacterial population colonizing mesenteric lymph nodes at early stages of the infective cycle, before 48 hours post-infection. In contrast, excision frequency remained very low in the liver and spleen at these stages. Interestingly, excision increased drastically after 48 h post infection, when intestinal re-infection and mortality begun. Moreover, we observed that the inability to excise ROD21 had a negative effect on S. Enteritidis capacity to translocate from the intestine to deeper organs, which correlates with an abnormal transcription of invA in the S. Enteritidis strain unable to excise ROD21. These results suggest that excision of ROD21 is a genetic mechanism required by S. Enteritidis to produce a successful invasion of the intestinal epithelium, a step required to generate systemic infection in mice.

Nuclear export signal (NES) of transposases affects the transposition activity of mariner-like elements Ppmar1 and Ppmar2 of moso bamboo

Ppmar1 and Ppmar2 are two active mariner-like elements (MLEs) cloned from moso bamboo (Phyllostachys edulis (Carrière) J. Houz) genome possessing transposases that harbour nuclear export signal (NES) domain, but not any nuclear localization signal (NLS) domain. To understand the functions of NES in transposon activity, we have conducted two experiments, fluorescence and excision frequency assays in the yeast system. For this, by site-directed mutagenesis, three NES mutants were developed from each of the MLE. In the fluorescence assay, the mutants, NES-1, 2 and 3 along with the wild types (NES-0) were fused with fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) were co-transformed into yeast system. To differentiate protein localisation under the NES influence, ECFP alone was fused to wild and mutant NES domains either on N- or C-terminal and not to EYFP. Fluorescence assay revealed that blue fluorescence of ECFP was more intense than the red fluorescence of the EYFP in the yeast cell matrix. Further, ECFP had a wider localisation in the cellular matrix, but EYFP was largely located in the nucleus. The NES-1 domain was related to the comparatively high spread of ECFP, while NES-2 and NES-3 indicated a low spread, implying that NES activity on nuclear export increased when the NES is made leucine-rich, while the signalling activity was reduced when the leucine content was lowered in the NES domain. In the transposon excision assay, the mutant and wild type NES of both the Ppmar elements were integrated into an Ade2 vector, and within the Ade2 gene. Co-transformation of the vector together with non-autonomous Ppmar transposons and NES-lacking transposases was used to assess the differential excision frequencies of the mutants NES domains. In both the MLEs, NES-1 had the highest excision suppression, which was less than half of the excision frequency of the wild type. NES-2 and NES-3 elements showed, up to three times increase in transposon excision than the wild types. The results suggested that NES is an important regulator of nuclear export of transposase in Ppmar elements and the mutation of the NES domains can either increase or decrease the export signalling. We speculate that in moso bamboo, NESs regulates the transposition activity of MLEs to maintain the genome integrity.

Inter-surgeon Variability in Cystic Artery Lymph Node Excision during Laparoscopic Cholecystectomy.

IntroductionExpert opinion recommends that surgeons perform a laparoscopic cholecystectomy (LC) in a standardized manner by dissecting the hepatobiliary triangle lateral to the cystic artery lymph node (LN) to minimize the rate of a major bile duct injury.MethodsTo determine whether surgeons performed a laparoscopic cholecystectomy in a standardized manner, the study assessed the variability in the frequency of an LN excision. All LCs that were performed at a single hospital were identified from a prospective dataset. The presence of an LN was retrospectively determined from the histology report.ResultsTwenty-seven surgeons were recorded to have performed 2332 laparoscopic cholecystectomies. Out of the total number of patients, 76.8% were female. The median patient age was 42.4 years. About 60.8% of the LCs were elective, while 39.2% of them were acute. Nineteen pathologists reported that in 99% of the specimens – the LN status of 1831 (78.5%) gallbladders was reported and analyzed. Overall, the LN yield per surgeon varied from 0% to 50% (mean 18.7%).Conclusion The high inter-surgeon variability in the rate of LN excision during laparoscopic cholecystectomy shows that surgeons dissect the hepatobiliary triangle differently. The LN yield may also represent a surrogate marker of surgical technique (which is easy to measure).

Precise excision of a selectable marker gene in transgenic Coccomyxa strains by the piggyBac transposase

The piggyBac transposon isolated from the cabbage looper moth Trichoplusia ni, is integrated into the host genome, and then excised from it without leaving a footprint. The piggyBac transposon system has been used as a genomic engineering tool in a variety of organisms. In this study, we used two improved versions of the piggyBac transposase (PBase) to create marker-free transgenic strains of the unicellular green alga Coccomyxa sp. strain KJ as follows: Uracil-auxotrophic (Ura−) mutants of strain KJ defective in the gene for uridine monophosphate synthase (KJUMPS) were isolated on agar plates containing 5-fluoroorotic acid. Subsequently, cDNA of KJUMPS (cKJUMPS) was cloned between the promoter and terminator of the elongation factor 1 alpha gene to construct a cKJUMPS expression cassette. A DNA fragment carrying the cKJUMPS expression cassette flanked with piggyBac transposon terminal repeats was then constructed (TR_cKJUMPS) and introduced into an Ura− mutant, and Ura+ transformants were isolated. One of the Ura+ transformants was named strain TR2-7. Hyperactive PBase (hyPBase) is a mutant PBase with increased excision and integration frequencies. Herein, we synthesized a coding sequence for hyPBase (KJhyPBase), which has optimized codons for expression in strain KJ, and its expression cassette was introduced into strain TR2-7. Fourteen transformants stably carried the KJhyPBase expression cassette, and TR_cKJUMPS was excised from seven of these. We also introduced an expression cassette of KJhyPBase_Ex, which encodes the excision-competent/integration-defective R372A/K375A/D450N mutant of KJhyPBase (KJhyPBase_Ex), into strain TR2-7, and found that the excision frequency of TR_cKJUMPS in KJhyPBase_Ex transformants was significantly higher than that in KJhyPBase transformants. In further experiments, we purified His-tagged KJhyPBase_Ex, and transfected it into strain TR2-7 using electroporation. Under these conditions, TR_cKJUMPS was precisely excised at a frequency of 8.8×10−8cell−1. The present data extend applications of the present piggyBac transposase-catalyzed excision system in green algae.

Direct andInvertedRepeat stimulatedexcision (DIRex): Simple, single-step, and scar-free mutagenesis of bacterial genes.

The need for generating precisely designed mutations is common in genetics, biochemistry, and molecular biology. Here, I describe a new λ Red recombineering method (Direct and Inverted Repeat stimulated excision; DIRex) for fast and easy generation of single point mutations, small insertions or replacements as well as deletions of any size, in bacterial genes. The method does not leave any resistance marker or scar sequence and requires only one transformation to generate a semi-stable intermediate insertion mutant. Spontaneous excision of the intermediate efficiently and accurately generates the final mutant. In addition, the intermediate is transferable between strains by generalized transductions, enabling transfer of the mutation into multiple strains without repeating the recombineering step. Existing methods that can be used to accomplish similar results are either (i) more complicated to design, (ii) more limited in what mutation types can be made, or (iii) require expression of extrinsic factors in addition to λ Red. I demonstrate the utility of the method by generating several deletions, small insertions/replacements, and single nucleotide exchanges in Escherichia coli and Salmonella enterica. Furthermore, the design parameters that influence the excision frequency and the success rate of generating desired point mutations have been examined to determine design guidelines for optimal efficiency.

Is cystic artery lymph node excision during laparoscopic cholecystectomy a marker of technique?

In order to minimize bile duct injury, experts suggest that dissection during laparoscopic cholecystectomy (LC) should be performed lateral to the lymph node (LN). This study aims to determine whether the frequency of excision of the LN is related to patient factors, disease severity or surgical difficulty. All LCs performed or supervised by one surgeon were identified from a prospective database. The presence of LN was retrospectively determined by reviewing the gallbladder histology report. The LN was identified in 10.4% of 1332 cholecystectomies. The American Society of Anesthesiologists class 3 was associated with a lower rate of LN excision compared with class 1 (odds ratio: 0.36; P = 0.049) as was the presence of a senior surgical trainee (odds ratio: 0.18; P < 0.001). Rate of LN excision was independent of patient demographic and clinical characteristics, including indication for cholecystectomy, conversion to open, gallbladder perforation, cholangiography, bile duct exploration and overall surgical difficulty. The frequency with which the LN is excised during LC by the one surgeon is independent of the majority of clinical and surgical factors and may represent a surrogate marker of surgical technique. Whether this is related to the rate of bile duct injury remains to be determined.