Excision Site Research Articles

Reactive oxygen species regulate auxin levels to mediate adventitious root induction in Arabidopsis hypocotyl cuttings.

Adventitious root (AR) formation from leafy stem cuttings is critical for breeding of many forest and horticultural species. In addition to the plant hormone auxin, wound-induced signaling caused by the cutting excision is also essential for AR initiation. Here we found that reactive oxygen species (ROS) are rapidly generated at the excision site as a wound-induced signal and propagated throughout the hypocotyl cutting after excision of the Arabidopsis (Arabidopsis thaliana) primary root. ROS propagation was not observed in the presence of an NADPH oxidase inhibitor (diphenylene iodonium chloride) or in a knockout mutant of the NADPH oxidase gene respiratory burst oxidase homolog protein D (RBOHD). Respiratory burst oxidase homolog protein D was specifically upregulated in hypocotyl cuttings at 0.5 h post excision (hpe). Together, these data suggest that RBOHD mediates ROS propagation in hypocotyl cuttings. We also found that auxin levels increased significantly in the shoot apex at 5 hpe and at the base of the cutting at 6 hpe; these effects were blocked by treatment with ROS scavengers. Consistent with this, transcript levels of auxin biosynthesis and polar-transport genes generally increased between 1 to 6 hpe. Collectively, our results suggest that wound-induced ROS participate in AR induction through regulation of auxin biosynthesis and transport.

Rab7 regulates primary cilia disassembly through cilia excision.

The primary cilium is a sensory organelle that protrudes from the cell surface. Primary cilia undergo dynamic transitions between assembly and disassembly to exert their function in cell signaling. In this study, we identify the small GTPase Rab7 as a novel regulator of cilia disassembly. Depletion of Rab7 potently induced spontaneous ciliogenesis in proliferating cells and promoted cilia elongation during quiescence. Moreover, Rab7 performs an essential role in cilia disassembly; knockdown of Rab7 blocked serum-induced ciliary resorption, and active Rab7 was required for this process. Further, we demonstrate that Rab7 depletion significantly suppresses cilia tip excision, referred to as cilia ectocytosis, which has been identified as required for cilia disassembly. Mechanically, the failure of F-actin polymerization at the site of excision of cilia tips caused suppression of cilia ectocytosis on Rab7 depletion. Overall, our results suggest a novel function for Rab7 in regulating cilia ectocytosis and cilia disassembly via control of intraciliary F-actin polymerization.

Conjunctival Benign Fibrous Histiocytoma in a 57-year-old man

Purpose. To report a case of benign fibrous histiocytoma of the conjunctiva involving the cornea, an uncommon ocular surface tumor. Methods. A 57-year-old patient came in our service complaining of a progressively enlarging conjunctival mass temporally to the limbus and invading the adjacent cornea of the left eye. Results. The approach consisted in surgical excision followed by cryotherapy on the edges and on the base of the excision site and amniotic membrane patch reconstruction of the ocular surface defect. Pathologic examination and immunohistochemistry were performed in order to establish the diagnosis. No recurrences appeared in 8 months of follow up. Conclusions. Fibrous histiocytoma might be easily misdiagnosed as it is exceedingly rare. Complete resection with careful inspection of edges is advised. Cryotherapy at the base and borders of the resection site is recommended as both benign and malignant tumors can show recurrence. Amniotic membrane should always be regarded as an efficient option in reconstruction of broad surface defects after tumor resection. Abbreviations: FH = fibrous histiocytoma, CIN = corneal intraepithelial neoplasia, SSCA = squamous cell carcinoma, AM = amniotic membrane, MMC = topical mitomycin-C, 5-FU = 5-fluorouracil, BCVA = best corrected visual acuity.

Local excision followed by early radical surgery in rectal cancer: long-term outcome

BackgroundIn rectal cancers, radical surgery should follow local excisions, in cases of unexpected, unfavorable tumor characteristics. The oncological results of this completion surgery are inconsistent.This retrospective cohort study assessed the clinical and long-term oncological outcomes of patients that underwent completion surgery to clarify whether a local excision compromised the results of radical surgery.MethodsForty-six patients were included, and the reasons for completion surgery, intraoperative complications, residual tumors, local recurrences (LRs), distant metastases, and cancer-specific survival (CSS) were assessed. The results were compared to 583 patients that underwent primary surgery without adjuvant therapy, treated with a curative intention during the same time period.ResultsThe median follow-up was 14.6 years. The reasons for undergoing completion surgery were positive resection margins (24%), high-risk cancer (30%), or both (46%). Intraoperative perforations occurred in 10/46 (22%) cases. Residual tumor in the rectal wall or lymph node involvement occurred in 12/46 (26%) cases. The risk of intraoperative perforation and residual tumor increased with the pT category. Intraoperative perforations did not increase postoperative complications, but they increased the risk of LRs in cases of intramural residual tumors (p = 0.003). LRs occurred in 2.6% of pT1/2 and 29% of pT3 tumors. Both the 5- and 10-year CSS rates were 88.8% (95% CI 80.0–98.6). Moreover, the LRs of patients with pT1/2 cancers were lower in patients with completion surgery than in patients with primary surgery.ConclusionsRectal wall perforations at the local excision site and residual cancer were the main risks for poor oncological outcomes associated with completion surgery. Local excisions followed by early radical surgery did not appear to compromise outcomes compared to patients with primary surgery for pT1/2 rectal cancer. Improvements in clinical staging should allow more appropriate selection of patients that are eligible for a local excision of rectal cancer.

Emulating evolutionary processes to morph aureothin-type modular polyketide synthases and associated oxygenases

Polyketides produced by modular type I polyketide synthases (PKSs) play eminent roles in the development of medicines. Yet, the production of structural analogs by genetic engineering poses a major challenge. We report an evolution-guided morphing of modular PKSs inspired by recombination processes that lead to structural diversity in nature. By deletion and insertion of PKS modules we interconvert the assembly lines for related antibiotic and antifungal agents, aureothin (aur) and neoaureothin (nor) (aka spectinabilin), in both directions. Mutational and functional analyses of the polyketide-tailoring cytochrome P450 monooxygenases, and PKS phylogenies give contradictory clues on potential evolutionary scenarios (generalist-to-specialist enzyme evolution vs. most parsimonious ancestor). The KS-AT linker proves to be well suited as fusion site for both excision and insertion of modules, which supports a model for alternative module boundaries in some PKS systems. This study teaches important lessons on the evolution of PKSs, which may guide future engineering approaches.

Leukemia inhibitory factor promotes the regeneration of rat uterine horns with full-thickness injury.

Severe uterine injuries may lead to infertility or pregnancy complications. There is a lack of effective methods to restore the structure and function of seriously injured uteri. Leukemia inhibitory factor (LIF), which plays a crucial role in blastocyst implantation, promotes the process of regeneration after injury in several different tissues. In this study, we explored the effect of LIF on the regeneration of rat uterine horns following full-thickness injury. One hundred and twenty four female Sprague-Dawley rats were assigned to three groups, including a sham-operated group (n = 34 uterine horns), a PBS/collagen group (n = 90 uterine horns), and a LIF/collagen group (n = 124 uterine horns). The regenerated uterine horns were collected at 1, 2, 4, 8, or 12 weeks after the surgery. The results showed that LIF/collagen scaffolds increased the number of endometrial cells and neovascularization 2 weeks after uterine full-thickness defect in excision sites (p < 0.001 vs PBS/collagen). Eight weeks after the surgery, the number of endometrial glands was dramatically higher in the LIF/collagen scaffolds group (35.2 ± 4.1/field) than in the PBS/collagen scaffolds (15.1 ± 1.4/field). The percentage of a-smooth muscle actin (a-SMA)-positive areas in the LIF/collagen scaffolds (88.8% ± 9.8%) was also significantly higher than that in the PBS/collagen group (52.9% ± 3.7%). Moreover, LIF improved the pregnancy rate and fetus number. We also found that LIF inhibited the infiltration of inflammatory cells and down-regulated the pro-inflammatory cytokine IL-12 expression while up-regulating the anti-inflammatory cytokine IL-10 expression in the injured part of the uterine horns. Our results indicate that LIF promotes regeneration of the uterus after injury, and this is at least partially due to its immunomodulatory properties. In addition, it is worth to explore further the possibility for LIF/collagen to be an alternative therapeutic approach for uterine damage in the clinic in near future.

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

SummaryWe describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.

The efficacy of polyglycolic acid felt to prevent the pancreatic fistula after distal pancreatectomy ∼A multicenter prospective cohort study∼

Introduction: The pancreas fistula (PF) after distal pancreatectomy (DP) could be a fatal complication, and it is necessary to prevent. Various methods have been devised to prevent PF so far, but the optimal method has not yet been known. It has been proven by animal experiments that fibrin glue formulation interferes with tissue replacement of polyglycolic acid (PGA) felt which is usually used in combination with fibrin glue formulation. Therefore, we designed a multicenter prospective cohort study of remnant closure by stapling plus PGA felt patch after distal pancreatectomy. Method: 30 patients, scheduled for DP in our group, were prospectively enrolled, and received remnant closure by stapling plus PGA felt patch after DP. Four patients were unresected at laparotomy findings, and therefore 26 patients were undergone DP and analyzed the results. Result: Primary diseases were followed; 17 cases were pancreatic adenocarcinoma, 3 pancreatic neuroendocrine neoplasm, 2 mucinous cystic neoplasm, 2 solid pseudopapillary neoplasm, and 2 others. The mean diameter of main pancreatic duct at the excision site was 2.3 mm (1.0 to 10.0 mm), and the mean thickness of pancreatic parenchyma was 12.3 mm (7.3 to 24.5 mm). PF was found in 9 cases (34.6%) as biochemical leak, in 9 cases (33.9%) as grade B, and no case as grade C of ISGPF (2016). The mean postoperative drainage duration was 6 days (3 to 35 days), and the mean postoperative hospital stay was 16 days (9 to 40 days). Conclusion: The reduction of a fibrin glue formulation from PGA felt and standard stapled remnant closure did not increase the rate or severity of pancreatic fistula in patients undergoing distal pancreatectomy.

A New Method for Partial Breast Reconstruction: 3-Year New Zealand Experience.

In New Zealand, oncoplastic surgery is common, but partial breast reconstruction presents challenges for radiation therapy targeting. Tissue rearrangement creates ambiguity when targeting the tumor bed, with resultant overestimation of treatment volumes. Thus, adoption of advanced methods of radiation therapy have been hindered. This pilot study describes use of a novel three-dimensional implant that provides a scaffolding for tissue ingrowth during partial breast reconstruction and delineates the tumor bed more precisely to assist radiation planning and mammographic surveillance. After informed consent, 15 women were implanted with the three-dimensional bioabsorbable implant. The device was sutured to the tumor bed during lumpectomy, and tissue flaps were mobilized and attached to the implant. Visualization of the marker and radiation treatment volumes were recorded and compared. The implant provided volume replacement and helped to maintain breast contour. Cosmetic outcomes were excellent; no device- or radiation-related complications occurred. One patient had a postoperative hematoma that resolved after percutaneous drainage; there were no postoperative infections. Three-year follow-up shows no tumor recurrences and no untoward effects. When compared to conventional radiation targeting, use of the implant showed that a greater than 50 percent reduction in treatment volume was possible in some cases. Three-year mammograms show no significant artifact, normal tissue ingrowth, and minimal fibrosis. This study describes a method of oncoplastic breast reconstruction using an implantable device that marks the site of tumor excision and provides for volume replacement with tissue ingrowth. Patients tolerated it well, and radiation therapy planning, positioning, and treatment were facilitated.

Papillary Fibroelastoma: A Unique Case of Distant Recurrence

Primary cardiac tumors are rare, with an incidence of 0.001%to 0.28% in autopsy series.(1) The third most common primary tumor, although likely under diagnosed, is the cardiac papillary fibroelastoma (CPF).(2) These tumors are benign, do not exhibit local infiltration on histopathologic examination, and carry a low likelihood of recurrence.(3) After excision, recurrence is rare, with a total of4 reported cases of recurrence. Tamin and colleagues2re-ported 3 cases, occurring at 1, 5, and 6 years after initial surgery, that they described as at or near the previous surgical excision site. Kammerer and associates (3) described a case of mitral valve recurrence after 3 years but hypothesized that incomplete resection in attempt to preserve the valve was the likely underlying mechanism. We report a unique case of recurrent CPF after previous resection,occurring at multiple alternate sites throughout the left ventricle (LV).

Resistance to anti-PD-1-based immunotherapy in basal cell carcinoma: a case report and review of the literature

BackgroundImmunotherapy with immune checkpoint inhibitors has radically changed the management of a broad spectrum of tumors. In contrast, only very limited information is available about the efficacy of these therapies in non-melanoma skin cancers, especially in basal cell carcinoma. The latter malignancy is often associated with both an impairment of the host immune response and a high mutation burden, suggesting that immune checkpoint inhibitor-based immunotherapy may be effective in the treatment of this tumor.Case presentationA 78-year-old woman was diagnosed with a metastatic non-small-cell-lung-cancer. Following the lack of response to two lines of systemic chemotherapy, she was treated with the anti-PD-1 monoclonal antibody nivolumab, obtaining a prolonged stable disease. Under nivolumab treatment, the patient developed a basal cell carcinoma of the nose. The latter was surgically resected. Immunohistochemical staining of tumor tissue showed a PD-L1 expression < 1% and lack of human leukocyte antigen class I subunit (i.e. heavy and light chain) expression on tumor cells. In addition, a limited number of T cells (CD3+) was present in the tumor microenvironment, with a higher number of regulatory T cells (Foxp3+) and macrophages (Cd11b+) as compared to a low infiltration of activated cytotoxic T cells (CD8+/ Granzyme B+). Two months following the surgical removal of the tumor, while still on nivolumab treatment, the patient relapsed with a basal cell carcinoma in the same anatomic site of the previous surgical excision. The tumor displayed the same pathological characteristics.ConclusionPreclinical lines of evidence suggest a potential role of immune checkpoint inhibitors for basal cell carcinoma treatment. However, limited clinical data is available. In the patient we have described administration of the immune checkpoint inhibitor nivolumab for the treatment of a responsive non-small cell carcinoma was associated with the development and relapse of a basal cell carcinoma tumor. This association is likely to reflect the resistance of basal cell carcinoma cells to anti-PD-1 based immunotherapy because of a “cold” tumor microenvironment characterized by lack of human leukocyte antigen class I expression, low PD-L1 expression and high number of immune regulatory cells.

Morphogenic Regulator-Mediated Transformation of Maize Inbred B73.

Maize B73 is a reference genome and has long been a major resource for genetics and molecular biology research. We have developed an efficient B73 transformation protocol by enabling somatic embryogenesis through differential co-expression of maize morphogenic regulators BBM and WUS2. We describe a successful protocol that utilizes Agrobacterium tumefaciens strain AGL1 harboring binary vector PHP78891 that comprises a BBM and WUS2 expression cassette as well as a green fluorescent protein (GFP) reporter cassette. The PHP78891 vector also contains, within the T-DNA region, a CRE/lox recombination system flanking the CRE/BBM/WUS2 co-expression cassette driven by the desiccation inducible RAB17 promoter that allows removal of the BBM/WUS2 cassette. Introduction and co-expression of BBM and WUS2 induced direct somatic embryogenesis (SE) in non-regenerable maize B73 from immature embryo explants. Removal of the CRE/BBM/WUS2 cassette is essential to allow regeneration to fertile plants. The GFP expression cassette outside the lox excision sites is retained in the transgenic plant genome, allowing subsequent phenotypic analysis of calli and regenerated transgenic events. This transformation system enables a selectable marker-free transformation process by taking advantage of BBM/WUS2-induced SE as a developmental selection system. © 2018 by John Wiley & Sons, Inc.

Correlation Between Surgical and Histologic Margins in Melanoma Wide Excision Specimens.

Wide surgical excision is the standard treatment for localized primary cutaneous melanomas, with a narrow histologic margin associated with an increased risk of local recurrence. The correlation between surgical and histologic margins is poorly documented in the literature. An audit was performed to (1) document the shrinkage of formalin-fixed specimens, and (2) use a precisely measured surgical margin in vivo to predict the histologic margin. For patients presenting for wide excision of melanomas and other malignant skin tumors, measured surgical margin, in vivo and ex vivo specimen width, and histologic margins after formalin fixation were recorded. The effects of clinicopathologic characteristics, including age, sex, body mass index (BMI), tumor type, anatomic site, and presence of visible tumor in predicting specimen shrinkage and histologic margin were assessed. In total, 252 specimens were evaluated. When compared with measured width in vivo, the formalin-fixed specimens showed a mean shrinkage of 14% (R2 = 0.98), regardless of patient age, sex, BMI, or site of the lesion. The measured surgical margin was not a strong predictor of the histologic margin, with a high degree of variability (R2 = 0.55) not explained by patient factors, tumor subtype, or presence of visible tumor at the time of excision (p > 0.05). A consistent 14% shrinkage rate of wide excision specimens was found across all patients and excision sites, and we propose a clinically useful 15% correction factor that will account for fixation and shrinkage of cutaneous excision specimens. Excision margins measured by the surgeon were a poor predictor of the histologic margins.

Prevention of Earlobe Keloid Recurrence After Excision: Assessment of the Value of Presurgical Injection of Triamcinolone

The ear is commonly involved in keloid formation, being often pierced. This study estimates the value of pre-excision triamcinolone injection of earlobe keloid, by comparing recurrence rates in patients who only received postexcision triamcinolone injection to those who had it administered before and after excision.The prospective intervention study involved 18 keloid lesions in as many consenting patients who presented with pedunculated earlobe keloid from January 2005 to January 2007.Triamcinolone doses were administered 10 mg/cm of lesion or sutured excision site. Each patient was followed-up for 10 years. Aggregate length of keloid lesions at presentation was 24.4 cm in Group A and 26.5 cm in Group B. Aggregate length of postexcision sutured wound in Group A was 16.1 cm, and 14.8 cm in Group B. Group A patients received a total of 1610 mg of triamcinolone, while a total of 4660 mg of same drug was administered to Group B patients, among whom 3180 mg was delivered presurgically. Total number of clinic visits during the course of treatment in Group A was 10, while that of Group B was 22. No patient with recurrent keloid was recorded in both groups.The authors concluded that there is no advantage to presurgical injection of triamcinolone when excision is considered as part of treatment protocol of a keloid lesion. This study helps to eliminate an ostensibly needless part of a commonly used treatment protocol.

Combined transanal excision (TAE) and transanal minimally invasive surgery (TAMIS) for a full thickness excision of a giant tubulovillous adenoma

Transanal minimally invasive surgery (TAMIS) is a rapidly developing minimally invasive method for high quality excision of rectal polyps and early rectal neoplasia. Treatment via TAMIS offers more advantages over other surgical modalities including endoscopic submucosal dissection (ESD), endoscopic mucosal resection (EMR), or a conventional transanal excision (TAE). We present a case of a successful excision of a giant 10 cm × 8 cm tubulovillous adenoma (TVA) with high grade dysplasia (HGD) located in the mid and low rectum using the TAMIS technique after an initial debulking. A 69-year-old male was found to have a large mid and low rectal polyp with raised components, located 1–1.5 cm from the dentate line, encompassing the lateral and posterior aspects of the rectum. Biopsy revealed a TVA with HGD. After colorectal evaluation and endoscopic rectal ultrasound (ERUS), no submucosal invasion was found, and the patient was offered a TAE via TAMIS. During the procedure, snare polypectomy was initially used to decrown the raised components of the polyp, so that the distal boundaries of the lesion could be identified and the TAMIS port inserted. Full thickness excision of the polyp with primary closure of the remaining rectal defect was successfully performed thereafter using a combined TAMIS and transanal technique. Follow up flexible sigmoidoscopy demonstrated no recurrence at the excision site.

Management of Large Cirsoid Aneurysms of the Scalp Using Tissue Expanders, Intravascular Occlusion, and En Bloc Excision

Background: Arteriovenous malformations of the scalpconsist of abnormally connecting arterial feeding vessels anddraining veins that are devoid of a normal capillary bed withinthe subcutaneous fatty layer of the scalp. The name cirsoidin cirsoid aneurysm is derived from the Greek word kirsosmeaning varix or varicose vein. The en bloc excision of scalptissues affected by aneurysm is preferable to selective ligationof the feeding and draining vessels. Because the managementof cirsoid aneurysm is an elective procedure, it is best to usetissue expanders to create sufficient scalp flaps to reconstructthe site of the excised lesion in the first stage. Preoperativeembolization greatly reduces blood loss during resection.Aim of the Work: To present the successful managementof cirsoid aneurysms of the scalp using tissue expanders,endovascular occlusion, and en bloc excision.Material and Methods: Five patients who had presentedcirsoid aneurysms of the scalp (two temporoparietal, twofrontal, and one occipital) were managed successfully usingthree stages of intervention. The first stage was the applicationof one or two tissue expanders, in which expanders wereapplied under the normal (non-affected) scalp in the subgalealplane; expansion was then performed weekly for 3-4 months.The second stage involved endovascular occlusion throughendovascular neuroradiology. The third stage was performedthe day after occlusion and included en bloc excision, thedelivery of tissue expanders, and reconstruction of the site ofexcision using scalp flaps. The postoperative period wasuneventful. Six months to three years of following-up showedno recurrence.Conclusion:We conclude that the three-stage managementof large cirsoid aneurysms of the scalp (application of tissueexpanders, endovascular occlusion, then en bloc excision andreconstruction) provides excellent results.

DNA identification of human remains in Disaster Victim Identification (DVI): An efficient sampling method for muscle, bone, bone marrow and teeth

In disaster victim identification (DVI), DNA profiling is considered to be one of the most reliable and efficient means to identify bodies or separated body parts. This requires a post mortem DNA sample, and an ante mortem DNA sample of the presumed victim or their biological relative(s). Usually the collection of an adequate ante mortem sample is technically simple, but the acquisition of a good quality post mortem sample under unfavourable DVI circumstances is complicated due to the variable degree of preservation of the human remains and the high risk of DNA (cross) contamination. This paper provides the community with an efficient method to collect post-mortem DNA samples from muscle, bone, bone marrow and teeth, with a minimal risk of contamination. Our method has been applied in a recent, challenging DVI operation (i.e. the identification of the 298 victims of the MH17 airplane crash in 2014). 98,2% of the collected PM samples provided the DVI team with highly informative DNA genotyping results without the risk of contamination and consequent mistyping the victim’s DNA. Moreover, the method is easy, cheap and quick. This paper provides the DVI community with a step-wise instructions with recommendations for the type of tissue to be sampled and the site of excision (preferably the upper leg). Although initially designed for DVI purposes, the method is also suited for the identification of individual victims.

Reconstruction of cheek mucosal defect with a buccal fat pad flap in a squamous cell carcinoma patient: a case report and literature review

BackgroundSquamous cell carcinoma (SCC) is the most commonly occurring malignant tumor in the oral cavity. In South Korea, it occurs most frequently in the mandible, tongue, maxilla, buccal mucosa, other areas of the oral cavity, and lips. Radial forearm free flap (RFFF) is the most widely used reconstruction method for the buccal mucosal defect. The scar of the forearm donor, however, is highly visible and unsightly, and a secondary surgical site is needed when such technique is applied. For these reasons, buccal fat pad (BFP) flap has been commonly used for closing post-surgical excision sites since the recent decades because of its reliability, ease of harvest, and low complication rate.Case presentationIn the case reported herein, BFP flap was used to reconstruct a cheek mucosal defect after excision. The defect was completely covered by the BFP flap, without any complications.ConclusionDiscussed herein is the usefulness of BFP flap for the repair of the cheek mucosal defect. Also, further studies are needed to determine the possibility of using BFP flap when the defect is deep, and the maximum volume that can be harvested considering the changes in volume with age.

Transcriptome-wide Interrogation of the Functional Intronome by Spliceosome Profiling

Full understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although RNA sequencing (RNA-seq) provides much of this information, the low abundance of many spliced transcripts (often due to their rapid cytoplasmic decay) limits the ability of RNA-seq alone to reveal the full repertoire of spliced species. Here, we present "spliceosome profiling," a strategy based on deep sequencing of RNAs co-purifying with late-stage spliceosomes. Spliceosome profiling allows for unambiguous mapping of intron ends to single-nucleotide resolution and branchpoint identification at unprecedented depths. Our data reveal hundreds of new introns in S.pombe and numerous others that were previously misannotated. By providing a means to directly interrogatesites of spliceosome assembly and catalysis genome-wide, spliceosome profiling promises to transform our understanding of RNA processing in the nucleus, much as ribosome profiling has transformed our understanding mRNA translation in the cytoplasm.

The Effect of Hobo Transposon Excision on DNA repair in Drosophila melanogaster

As the repository of genetic information, it is critical to organisms that DNA damage is repaired quickly and with high fidelity. When DNA damage is improperly repaired, it may cause diseases such as cancer and severe combined immunodeficiency. DNA can be mutated by numerous agents both external and internal. One internal source of DNA damage is from the transposition of transposable elements, including the P and hobo elements. Transposable elements “jump” out from one section of the DNA and reinsert into another, leaving double strand DNA breaks at the excision site. Hobo excisions are preferentially repaired by nonhomologous end‐joining, NHEJ. DNA repair and transposable elements themselves can be studied in Drosophila melanogaster. This study looked at the hobo on P, or HOP, element. The P element is located inside of the Hobo element; HOP8 is nonautonomous and needs hobo transposase to be supplied in trans. Curly winged, glazed eye female flies that have an inducible source of hobo transposase were crossed with HOP8 male flies and heat shocked to trigger transposition, leaving a double strand DNA break requiring repair behind. The resulting mosaic eyed male flies were then crossed with wild type female flies because mosaic eyes mark meiotic recombination in the P element. Female flies with white eyes indicating that hobo transposition occurred, were collected and stored in a freezer. Experiments are underway to determine the original location and new insertion sites of hobo using inverse PCR to further understand DNA repair.Support or Funding InformationAlbion College Foundation for Undergraduate Research, Scholarship, and Creative Activity (FURSCA)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.