Exploration and analysis of R-loop mapping data with RLBase.

R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA. In 2012, Ginno et al. introduced the first R-loop mapping method. Since that time, dozens of R-loop mapping studies have been conducted, yielding hundreds of publicly available datasets. Current R-loop databases provide only limited access to these data. Moreover, no web tools for analyzing user-supplied R-loop datasets have yet been described. In our recent work, we reprocessed 810 R-loop mapping samples, building the largest R-loop data resource to date. We also defined R-loop consensus regions and developed a framework for R-loop data analysis. Now, we introduce RLBase, a user-friendly database that provides the capability to (i) explore hundreds of public R-loop mapping datasets, (ii) explore R-loop consensus regions, (iii) analyze user-supplied data and (iv) download standardized and reprocessed datasets. RLBase is directly accessible via the following URL: https://gccri.bishop-lab.uthscsa.edu/shiny/rlbase/.

Generating Molecular Fragmentation Graphs with Autoregressive Neural Networks.

The accurate prediction of tandem mass spectra from molecular structures has the potential to unlock new metabolomic discoveries by augmenting the community's libraries of experimental reference standards. Cheminformatic spectrum prediction strategies use a "bond-breaking" framework to iteratively simulate mass spectrum fragmentations, but these methods are (a) slow due to the need to exhaustively and combinatorially break molecules and (b) inaccurate as they often rely upon heuristics to predict the intensity of each resulting fragment; neural network alternatives mitigate computational cost but are black-box and not inherently more accurate. We introduce a physically grounded neural approach that learns to predict each breakage event and score the most relevant subset of molecular fragments quickly and accurately. We evaluate our model by predicting spectra from both public and private standard libraries, demonstrating that our hybrid approach offers state-of-the-art prediction accuracy, improved metabolite identification from a database of candidates, and higher interpretability when compared to previous breakage methods and black-box neural networks. The grounding of our approach in physical fragmentation events shows especially great promise for elucidating natural product molecules with more complex scaffolds.

Morbidity and Outcomes of Primary Tumor Management in Patients with Widely Metastatic Well-Differentiated Small Bowel Neuroendocrine Tumors.

BACKGROUND: The benefit of primary tumor resection in distant metastatic small bowel neuroendocrine tumors (SBNETs) is controversial, with treatment-based morbidity not well-defined. We aimed to determine the impact of primary tumor resection on development of disease-specific complications in patients with metastatic well-differentiated SBNETs. PATIENTS AND METHODS: A retrospective analysis was performed of patients diagnosed with metastatic well-differentiated jejunal/ileal SBNETs at a single tertiary care cancer center from 1980 to 2016. Outcomes were compared on the basis of treatment selected at diagnosis between patients who underwent initial medical treatment or primary tumor resection. RESULTS: Among 180 patients, 71 underwent medical management and 109 primary tumor resection. Median follow-up was 116 months. Median event-free survival did not differ between treatment approaches (log-rank p = 0.2). In patients medically managed first, 16/71 (23%) required surgery due to obstruction, perforation, or bleeding. These same complications led to resection at presentation in 31/109 (28%) surgically treated patients. Development of an obstruction from the primary tumor was not associated with disease progression/recurrence (HR 1.14, 95% CI 0.75-1.75) with all patients recovering postoperatively. Ongoing tumor progression requiring secondary laparotomy was associated with worse mortality (HR 7.51, 95% CI 3.3-16.9; p < 0.001) and occurred in 20/109 (18%) primary tumor resection and 7/16 (44%) initially medically treated patients. CONCLUSIONS: Rates of event-free survival among patients with metastatic SBNETs do not differ on the basis of primary tumor management. The development of an obstruction from the primary tumor was not associated with worse outcomes with all patients salvaged. Regardless of initial treatment selected, patients with metastatic SBNET should be closely followed for early signs of primary tumor complications.

Genomic virulence features of Beauveria bassiana as a biocontrol agent for the mountain pine beetle population.

BACKGROUND: The mountain pine beetle, Dendroctonus ponderosae, is an irruptive bark beetle that causes extensive mortality to many pine species within the forests of western North America. Driven by climate change and wildfire suppression, a recent mountain pine beetle (MPB) outbreak has spread across more than 18 million hectares, including areas to the east of the Rocky Mountains that comprise populations and species of pines not previously affected. Despite its impacts, there are few tactics available to control MPB populations. Beauveria bassiana is an entomopathogenic fungus used as a biological agent in agriculture and forestry and has potential as a management tactic for the mountain pine beetle population. This work investigates the phenotypic and genomic variation between B. bassiana strains to identify optimal strains against a specific insect. RESULTS: Using comparative genome and transcriptome analyses of eight B. bassiana isolates, we have identified the genetic basis of virulence, which includes oosporein production. Genes unique to the more virulent strains included functions in biosynthesis of mycotoxins, membrane transporters, and transcription factors. Significant differential expression of genes related to virulence, transmembrane transport, and stress response was identified between the different strains, as well as up to nine-fold upregulation of genes involved in the biosynthesis of oosporein. Differential correlation analysis revealed transcription factors that may be involved in regulating oosporein production. CONCLUSION: This study provides a foundation for the selection and/or engineering of the most effective strain of B. bassiana for the biological control of mountain pine beetle and other insect pests populations.

LongStitch: high-quality genome assembly correction and scaffolding using long reads.

BACKGROUND: Generating high-quality de novo genome assemblies is foundational to the genomics study of model and non-model organisms. In recent years, long-read sequencing has greatly benefited genome assembly and scaffolding, a process by which assembled sequences are ordered and oriented through the use of long-range information. Long reads are better able to span repetitive genomic regions compared to short reads, and thus have tremendous utility for resolving problematic regions and helping generate more complete draft assemblies. Here, we present LongStitch, a scalable pipeline that corrects and scaffolds draft genome assemblies exclusively using long reads. RESULTS: LongStitch incorporates multiple tools developed by our group and runs in up to three stages, which includes initial assembly correction (Tigmint-long), followed by two incremental scaffolding stages (ntLink and ARKS-long). Tigmint-long and ARKS-long are misassembly correction and scaffolding utilities, respectively, previously developed for linked reads, that we adapted for long reads. Here, we describe the LongStitch pipeline and introduce our new long-read scaffolder, ntLink, which utilizes lightweight minimizer mappings to join contigs. LongStitch was tested on short and long-read assemblies of Caenorhabditis elegans, Oryza sativa, and three different human individuals using corresponding nanopore long-read data, and improves the contiguity of each assembly from 1.2-fold up to 304.6-fold (as measured by NGA50 length). Furthermore, LongStitch generates more contiguous and correct assemblies compared to state-of-the-art long-read scaffolder LRScaf in most tests, and consistently improves upon human assemblies in under five hours using less than 23 GB of RAM. CONCLUSIONS: Due to its effectiveness and efficiency in improving draft assemblies using long reads, we expect LongStitch to benefit a wide variety of de novo genome assembly projects. The LongStitch pipeline is freely available at https://github.com/bcgsc/longstitch .

Narrow excision margins are appropriate for Merkel cell carcinoma when combined with adjuvant radiation: Analysis of 188 cases of localized disease and proposed management algorithm.

BACKGROUND: Merkel cell carcinoma (MCC) management typically includes surgery with or without adjuvant radiation therapy (aRT). Major challenges include determining surgical margin size and whether aRT is indicated. OBJECTIVE: To assess the association of aRT, surgical margin size, and MCC local recurrence. METHODS: Analysis of 188 MCC cases presenting without clinical nodal involvement. RESULTS: aRT-treated patients tended to have higher-risk tumors (larger diameter, positive microscopic margins, immunosuppression) yet had fewer local recurrences (LRs) than patients treated with surgery only (1% vs 15%; P = .001). For patients who underwent surgery alone, 7 of 35 (20%) treated with narrow margins (defined as ≤1.0 cm) developed LR, whereas 0 of 13 patients treated with surgical margins greater than 1.0 cm developed LR (P = .049). For aRT-treated patients, local control was excellent regardless of surgical margin size; only 1% experienced recurrence in each group (1 of 70 with narrow margins ≤1 cm and 1 of 70 with margins >1 cm; P = .56). LIMITATIONS: This was a retrospective study. CONCLUSIONS: Among patients treated with aRT, local control was superb even if significant risk factors were present and margins were narrow. We propose an algorithm for managing primary MCC that integrates risk factors and optimizes local control while minimizing morbidity.

Comparison of Trendelenburg Angles in Vaginal, Laparoscopic, and Robotic Uterovaginal Apical Prolapse Repairs.

STUDY OBJECTIVE: To compare the Trendelenburg angle used in laparoscopic uterovaginal apical prolapse repairs with the angles used in vaginal and robotic uterovaginal apical prolapse repairs. DESIGN: Prospective, multicenter cohort study from May 2015 to December 2016. SETTING: Two academic teaching hospitals. PATIENTS: Sixty patients who underwent vaginal high uterosacral ligament suspension, laparoscopic sacrocolpopexy, or robotic sacrocolpopexy performed by 6 surgeons board-certified in female pelvic medicine and reconstructive surgery. INTERVENTIONS: Measurement of Trendelenburg angle and time spent in Trendelenburg during surgery. MEASUREMENTS AND MAIN RESULTS: Twenty patients were enrolled in each procedure group. The median maximum angle of Trendelenburg was significantly greater in the laparoscopic group (22° [20-25]) than in the vaginal group (15° [6-19]; p <.001) and the robotic group (19° [16-21]; p = .02). The participants in the laparoscopic group spent significantly more time overall in Trendelenburg (176 minutes [143-221]) than those in the robotic group (150 minutes [127-161]; p = .01) and those in the vaginal group (120 minutes [86-128]; p <.001). The participants in the laparoscopic and robotic groups spent similar amounts of time in maximum Trendelenburg (116 minutes [52-164] and 117 minutes [61-134], respectively; p = .56), whereas the participants in the vaginal group spent significantly less time in maximum Trendelenburg (10 minutes [7-38]) than those in the laparoscopic group (p <.001). The total median operative time was highest for the laparoscopic approach (211 minutes [173-270]), followed by the robotic approach (181 minutes [165-201]) and the vaginal approach (162 minutes [128-186]; p = .008). CONCLUSION: The median maximum angle of Trendelenburg was highest in laparoscopic sacrocolpopexy-followed by robotic sacrocolpopexy-and lowest in vaginal high uterosacral ligament suspension. Patients who underwent robotic sacrocolpopexy spent less time in Trendelenburg than those who underwent the laparoscopic approach. Prolonged, steep Trendelenburg is often not required for any of the 3 surgical procedures, but a vaginal approach should be considered for those at high risk of complications from Trendelenburg position.

Review of Common Alternative Herbal "Remedies" for Skin Cancer.

BACKGROUND: Alternative herbal remedies for skin cancer are commonly found on the Internet. Many websites contain inaccurate or false information regarding side effects and efficacy. OBJECTIVE: To review the evidence behind several commonly advertised herbal remedies that claim to cure skin cancer: black salve, eggplant, frankincense, cannabis, black raspberry, milk thistle, St. John's wort, and turmeric. METHODS: A PubMed search was performed using the common and scientific names of frequently advertised herbal remedies along with the terms "nonmelanoma skin cancer," or "basal cell carcinoma" or "squamous cell carcinoma," or "melanoma." RESULTS: Some preclinical studies have shown positive evidence that these substances can induce apoptosis in skin cancer, but clinical studies proving efficacy are either insufficient, nonexistent, or show negative evidence. Botanicals that were excluded are those that do not have published studies of their efficacy as skin cancer treatments. CONCLUSION: Online advertising may tempt patients to use botanical agents while citing efficacy found in preclinical studies. However, many agents lack strong clinical evidence of efficacy. Dermatologists must be aware of common herbal alternatives for skin cancer treatment to maintain effective patient communication and education.

Fire Safety in Mohs Micrographic Surgery.

BACKGROUND: Surgical fire is a rare event, but one with potentially devastating patient outcomes. OBJECTIVE: This study was conducted to investigate the incidence, risk factors, and outcome of surgical fires experienced by members of the American College of Mohs Surgeons (ACMS). METHODS: An internet survey was developed and sent to ACMS members. Data collected included total years of experience, total number of cases, typical management of supplemental oxygen, and surgical fires experienced. RESULTS: Eighty participants contributed data on 886,200 cases of MMS. Nine surgeons (11%) reported at least 1 surgical fire, yielding an estimated incidence of 1 fire per 88,620 cases (0.001%). The most common site of involvement was the scalp (67%). Common ignition sources included monopolar electrosurgical devices (78%) and battery-powered thermal cautery (22%). Fuel sources included towels or drapes, gauze, isopropyl alcohol, aluminum chloride, hairspray, and diethyl ether. Supplemental oxygen was not involved in any of the cases. Five patients suffered singed hair while 4 patients did not suffer any injuries. None suffered any permanent functional or aesthetic deformities. CONCLUSION: The overall risk of surgical fire in MMS is minimal. However, safety measures and greater awareness of fire risks are necessary to prevent patient harm.

Novel Regulatory Mechanisms for the SoxC Transcriptional Network Required for Visual Pathway Development.

What pathways specify retinal ganglion cell (RGC) fate in the developing retina? Here we report on mechanisms by which a molecular pathway involving Sox4/Sox11 is required for RGC differentiation and for optic nerve formation in mice in vivo, and is sufficient to differentiate human induced pluripotent stem cells into electrophysiologically active RGCs. These data place Sox4 downstream of RE1 silencing transcription factor in regulating RGC fate, and further describe a newly identified, Sox4-regulated site for post-translational modification with small ubiquitin-related modifier (SUMOylation) in Sox11, which suppresses Sox11's nuclear localization and its ability to promote RGC differentiation, providing a mechanism for the SoxC familial compensation observed here and elsewhere in the nervous system. These data define novel regulatory mechanisms for this SoxC molecular network, and suggest pro-RGC molecular approaches for cell replacement-based therapies for glaucoma and other optic neuropathies.SIGNIFICANCE STATEMENT Glaucoma is the most common cause of blindness worldwide and, along with other optic neuropathies, is characterized by loss of retinal ganglion cells (RGCs). Unfortunately, vision and RGC loss are irreversible, and lead to bilateral blindness in ∼14% of all diagnosed patients. Differentiated and transplanted RGC-like cells derived from stem cells have the potential to replace neurons that have already been lost and thereby to restore visual function. These data uncover new mechanisms of retinal progenitor cell (RPC)-to-RGC and human stem cell-to-RGC fate specification, and take a significant step toward understanding neuronal and retinal development and ultimately cell-transplant therapy.

Mohs Mapping Fidelity: Optimizing Orientation, Accuracy, and Tissue Identification in Mohs Surgery.

BACKGROUND: Mohs micrographic surgery (MMS) is a highly effective process that requires consistent accuracy in resection, mapping, and histologic interpretation. Although the general sequence in MMS is similar, there are numerous variations among Mohs surgeons as to how this process is performed. OBJECTIVE: This article aims to review the process of MMS, with the intent to identify and mitigate the potential errors at each step. Existing variations will be discussed and protocols offered to minimize error and optimize accuracy. METHODS: A Pubmed search was performed for publications on methods of tissue mapping, orienting, and processing in MMS. RESULTS: Our literature review highlights various techniques for tissue orientation, transfer, flattening, inking, mapping, and processing of later stages and multiple specimens. We discuss our system, which reduces error during tissue transfer, tissue identification in vivo and ex vivo, and tissue flattening. Furthermore, we discuss adaptations to increase the accuracy during reexcisions of subsequent Mohs layers. CONCLUSION: Variations in MMS reflects the diverse training and creativity among Mohs surgeons. Unless potential errors are addressed, however, false negatives will occur and undermine the superior cure rate of MMS.

Autoimmune dermatologic toxicities from immune checkpoint blockade with anti-PD-1 antibody therapy: a report on bullous skin eruptions.

Monoclonal antibodies against the immune checkpoint programmed cell death receptor 1 (PD-1) improve the hosts' antitumor immune response and have showed tremendous promise in the treatment of advanced solid tumors and hematologic malignancies. Reports of serious autoimmune dermatologic toxicities from immune checkpoint blockade therapy, however, are emerging. We report our experience with five patients who presented with pruritic vesicles and blisters on the skin while treated with anti-PD-1 antibody immunotherapy with either nivolumab or pembrolizumab. Four of the patients' skin biopsies revealed subepidermal bullae with immunohistochemical study for type IV collagen labeling the floor of the blister cavity and direct immunofluorescence studies (in three of the four patients tested) decorated linear IgG and C3 immune deposits on the blister roof, diagnostic of bullous pemphigoid. One patient developed bullous erythema multiforme. All patients had partial or complete resolution of skin lesions following treatment with systemic corticosteroid and cessation of checkpoint blockade. Recognition and treatment of rare immune-related bullous dermatologic toxicities will become increasingly important as more patients are treated with effective and newer immune checkpoint blockade therapy.

Voiding trial outcome following pelvic floor repair without incontinence procedures.

INTRODUCTION AND HYPOTHESIS: Our aim was to identify predictors of postoperative voiding trial failure among patients who had a pelvic floor repair without a concurrent incontinence procedure in order to identify low-risk patients in whom postoperative voiding trials may be modified. METHODS: We conducted a retrospective cohort study of women who underwent pelvic floor repair without concurrent incontinence procedures at two institutions from 1 November 2011 through 13 October 2013 after abstracting demographic and clinical data from medical records. The primary outcome was postoperative retrograde voiding trial failure. We used modified Poisson regression to calculate the risk ratio (RR) and 95 % confidence interval (CI). RESULTS: Of the 371 women who met eligibility criteria, 294 (79.2 %) had complete data on the variables of interest. Forty nine (16.7 %) failed the trial, and those women were less likely to be white (p = 0.04), more likely to have had an anterior colporrhaphy (p = 0.001), and more likely to have had a preoperative postvoid residual (PVR) ≥150 ml (p = 0.001). After adjusting for race, women were more likely to fail their voiding trial if they had a preoperative PVR of ≥150 ml (RR: 1.9; 95 % CI: 1.1-3.2); institution also was associated with voiding trial failure (RR: 3.0; 95 % CI: 1.6-5.4). CONCLUSIONS: Among our cohort, postoperative voiding trial failure was associated with a PVR of ≥150 ml and institution at which the surgery was performed.

A review of melasma treatment focusing on laser and light devices.

Melasma is a pigmentary disorder of unclear etiology with numerous treatment options and high recurrence rates. Laser and light therapies may be utilized cautiously as second- or third-line options for recalcitrant melasma, but low-energy settings are preferred due to the risk of postinflammatory hyperpigmentation and melasma stimulation. Commonly used lasers include the low-fluence 1064-nm Q-switched neodymium-doped yttrium aluminum garnet laser, nonablative fractionated lasers, and intense pulsed light. Strict sun protection, concomitant use of bleaching agents, and maintenance treatments are necessary. A variety of other treatments that may also help to improve results are now being more widely adopted, including oral tranexamic acid, pulsed dye laser, antioxidants, and laser-assisted drug delivery.

Combined melanocytic and sweat gland neoplasm: cell subsets harbor an identical HRAS mutation in phacomatosis pigmentokeratotica.

Phacomatosis pigmentokeratotica (PPK) is characterized by the co-existence of epidermal nevi and large segmental speckled lentiginous nevi of the papulosa type. PPK, previously explained as 'twin spot' mosaicism due to the postzygotic crossing-over of two homozygous recessive mutations, has recently been shown to derive from one postzygotic activating RAS mutation. Epidermal nevi, including those in PPK, are known to give rise to neoplasms such as trichoblastoma and basal cell carcinoma. Within speckled lentiginous nevi, Spitz nevi and melanoma have been well documented. We report a case of PPK with a combined melanocytic and adnexal neoplasm presenting where the nevi conjoined. Using next-generation sequencing techniques, we were able to identify the same HRAS G13R mutation within both components of the tumor, and to show the absence of additional mutated modifier genes in a panel of 300 cancer-related genes. Given the genetic findings in this rare tumor-type, we suggest that this case may be used as a model for understanding the development of biphenotypic neoplasia or intratumoral heterogeneity in some cases.

Multicenter case series of indolent small/medium-sized CD8+ lymphoid proliferations with predilection for the ear and face.

We report 7 cases of a CD8 lymphoid proliferation of the ear and face with a cytotoxic T-cell phenotype, but an indolent clinical course. All patients presented with stable or slowly growing asymptomatic lesions on the ear, nose, or lower eyelid. Histopathology showed a dense diffuse dermal infiltrate of small- to medium-sized atypical lymphocytes without destructive features. The lymphocytes were positive for CD3, CD8, ß-F1, and TIA-1 and negative for CD4, CD30, CD56, granzyme B, and PD-1. Of note, the proliferation index was low in available cases. All patients remained in complete remission at median follow-up of 14 months regardless of treatment modality. Staging was negative for extracutaneous disease in all patients. The clinically indolent behavior and histopathologic phenotype together with a low proliferation index (10%-15%) emphasize the importance of accurate diagnosis and appropriate clinical management to avoid overtreatment and complications of therapy.

Hepatic cytoreduction for lung and renal neuroendocrine tumor metastases.

BACKGROUND: Patients with hepatic metastases from lung and renal neuroendocrine tumors are rare. Outcome data on treatment of hepatic metastases for these types of tumors are lacking. We report the outcomes of hepatic cytoreduction operations for these tumors. METHODS: Records of patients undergoing hepatic cytoreduction operations of at least 70 â€‹% of the hepatic tumors for well differentiated lung and renal neuroendocrine tumors were reviewed. Data collected included primary tumor type, number and size of metastases resected, tumor grade, percentage of hepatic cytoreduction, presence of extra-hepatic disease, and status at last follow up. RESULTS: Twenty-one patients were identified. Ninety percent had extrahepatic metastases. Median-time to liver progression was 66 months. The five-year survival rate was 65 â€‹%. Liver failure was the predominant cause of death. No prognostic factors for survival could be identified among the variables collected. CONCLUSION: Hepatic cytoreduction operations for lung and renal neuroendocrine tumors do not yield as good of survival rates as observed with small bowel and pancreatic neuroendocrine tumors, but are considerably better than those obtained with complete resection of colorectal metastases.

A case-control study of clinicopathologic features, prognosis, and therapeutic responses in patients with granulomatous mycosis fungoides.

BACKGROUND: Granulomatous mycosis fungoides (GMF) is an uncommon variant of mycosis fungoides (MF). OBJECTIVE: We sought to analyze the relative frequency, clinicopathologic characteristics, prognosis, and therapeutic responses of GMF. METHODS: We conducted a retrospective case-control study of patients with GMF and age- and stage-matched patients with classic MF between 1981 and 2012. RESULTS: A total of 27 patients with GMF were identified, representing 6.3% of all patients with MF at our center. Skin manifestations were similar to classic MF having an atypical lichenoid CD4(+) CD8(-) lymphocytic infiltrate with interstitial histiocytes and/or perivascular granulomas with giant cells. Fewer patients with GMF achieved a partial response or complete response with topical (57% vs 83%; P = .002) or ultraviolet light (62% vs 90%; P = .006) therapy. The 5- and 10-year progression-free survival rates were significantly lower in patients with GMF (59% and 33%) compared with patients with classic MF (84% and 56%; P = .02), but overall survival was similar between groups (86% and 72% vs 85% and 85%; P = .54). LIMITATIONS: The retrospective methodology may underestimate the frequency of GMF. The median follow-up time may be too short to detect possible differences in overall survival. CONCLUSION: More frequent disease progression and poorer response to skin-directed therapies were observed in patients with GMF. Our findings may be helpful in selecting the most appropriate treatment for these patients.