Excess of guide RNA reduces knockin efficiency and drastically increases on-target large deletions.

CRISPR-Cas9 cleavage efficacy and accuracy are the main challenges gene editing faces, and they are particularly affected by the optimal formation of the ribonucleoprotein (RNP) complex. We used nano differential scanning fluorimetry, a label and immobilization-free assay, to demonstrate that an equimolar ratio of Cas9 and guide RNA (gRNA) is optimal for RNP complex formation. We almost achieved 50% of green fluorescent protein (GFP) to blue fluorescent protein (BFP) conversion using a biallelic homozygous GFP human induced pluripotent stem cell line, when 0.4 µM of Cas9, equimolar Cas9/gRNA ratio and 2 µM of single-stranded oligonucleotide, were used and showed that increasing Cas9/gRNA ratio did not further improve KI efficiency. Additionally, excess gRNA decreased point mutation KI efficiency in rat embryos and drastically increased the occurrence of on-target large deletions. These findings highlight the importance of CRISPR/Cas9 stoichiometric optimization to ensure efficient and accurate KI generation, which will be applicable to other in vitro as well as in vivo models.

Directions for 3D User Interface Research from Consumer VR Games.

With the continuing development of affordable immersive virtual reality (VR) systems, there is now a growing market for consumer content. The current form of consumer systems is not dissimilar to the lab-based VR systems of the past 30 years: the primary input mechanism is a head-tracked display and one or two tracked hands with buttons and joysticks on hand-held controllers. Over those 30 years, a very diverse academic literature has emerged that covers design and ergonomics of 3D user interfaces (3DUIs). However, the growing consumer market has engaged a very broad range of creatives that have built a very diverse set of designs. Sometimes these designs adopt findings from the academic literature, but other times they experiment with completely novel or counter-intuitive mechanisms. In this paper and its online adjunct, we report on novel 3DUI design patterns that are interesting from both design and research perspectives: they are highly novel, potentially broadly re-usable and/or suggest interesting avenues for evaluation. The supplemental material, which is a living document, is a crowd-sourced repository of interesting patterns. This paper is a curated snapshot of those patterns that were considered to be the most fruitful for further elaboration.

Data for training and testing radiation detection algorithms in an urban environment.

The detection, identification, and localization of illicit nuclear materials in urban environments is of utmost importance for national security. Most often, the process of performing these operations consists of a team of trained individuals equipped with radiation detection devices that have built-in algorithms to alert the user to the presence nuclear material and, if possible, to identify the type of nuclear material present. To encourage the development of new detection, radioisotope identification, and source localization algorithms, a dataset consisting of realistic Monte Carlo-simulated radiation detection data from a 2 in. × 4 in. × 16 in. NaI(Tl) scintillation detector moving through a simulated urban environment based on Knoxville, Tennessee, was developed and made public in the form of a Topcoder competition. The methodology used to create this dataset has been verified using experimental data collected at the Fort Indiantown Gap National Guard facility. Realistic signals from special nuclear material and industrial and medical sources are included in the data for developing and testing algorithms in a dynamic real-world background.

Aggressive fibromatosis of the oropharynx: a multidisciplinary approach to a benign disease.

We present the case of a 23-year-old woman with aggressive fibromatosis of the oropharynx that was initially treated elsewhere as a peritonsillar abscess. We discuss the characteristics of this rare tumor and review the literature, stressing the importance of postoperative follow-up for peritonsillar abscesses to avoid missing other important diagnoses, such as the one described here.

The Atkinson Morley's Hospital joint neurosurgical-maxillofacial procedures: cranioplasty case series 1985-2003.

AIMS: To present a retrospective case series resulting from the co-operation in cranioplasty procedures between neurosurgeons and maxillofacial surgeons of the Atkinson Morley's and Royal Marsden Hospitals of London, UK for the period 1985-2003. MATERIALS AND METHODS: The cranioplasty case series is part of an integrated analysis of the complete Atkinson Morley's Hospital craniofacial procedure database. Cases included both cosmetic and functional procedures. The latter followed a variety of conditions such as tumour recurrence, craniectomy-associated neurological symptoms, wound infection and infection of previous prosthesis or bone flap. RESULTS: Fourty-eight procedures (27 functional, 21 cosmetic) were performed in 43 patients (mean age: 44.99 years, SD: 18.1 years). Our case notes analysis reviewed symptoms on presentation and duration, previous neurosurgical procedures and previous histopathology, nature and length of operative procedure, imaging studies, post-operative complications and management, and patient follow-up and survival data. CONCLUSION: Analysis of the international literature highlights the paucity and poor quality of evidence on the subject of cranioplasty. The authors hope this work adds to the body of knowledge, despite its retrospective nature.

Recent technological developments: in situ histopathological interrogation of surgical tissues and resection margins.

OBJECTIVES: The tumour margin is an important surgical concept significantly affecting patient morbidity and mortality. We aimed in this prospective study to apply the microendoscope on tissue margins from patients undergoing surgery for oral cancer in vivo and ex vivo and compare it to the gold standard "paraffin wax", inter-observer agreement was measured; also to present the surgical pathologist with a practical guide to the every day use of the microendoscope both in the clinical and surgical fields. MATERIALS AND METHODS: Forty patients undergoing resection of oral squamous cell carcinoma were recruited. The surgical margin was first marked by the operator followed by microendoscopic assessment. Biopsies were taken from areas suggestive of close or positive margins after microendoscopic examination. These histological samples were later scrutinized formally and the resection margins revisited accordingly when necessary. RESULTS: Using the microendoscope we report our experience in the determination of surgical margins at operation and later comparison with frozen section and paraffin section margins "gold standard". We were able to obtain a sensitivity of 95% and a specificity of 90%. Inter-observer Kappa scores comparing the microendoscope with formal histological analysis of normal and abnormal mucosa were 0.85. CONCLUSION: The advantage of this technique is that a large area of mucosa can be sampled and any histomorphological changes can be visualized in real time allowing the operator to make important informed decisions with regards the intra-operative resection margin at the time of the surgery.

The management of carotid artery rupture.

Carotid artery rupture is fortunately an uncommon complication of head and neck cancer treatment. Eleven episodes of carotid artery rupture following irradiation and major head and neck resection were identified over a 6-year period. We review our experience and discuss the predisposing factors that can cause this complication, important aspects of management and outcome. During this 6-year period, 11 episodes of carotid artery rupture were treated in our unit. All patients had received prior irradiation (more than 60 Gy) and undergone a major surgical resection or resections. The average age was 59 years; all patients had a salivary fistula, local infection and a manifest 'herald bleed' just before their major carotid artery rupture. These patients were resuscitated, taken to theatre and the neck explored, with control of the vessel and debridement of necrotic tissue. Soft tissue coverage was in the form of a flap. Many of the factors predisposing to carotid artery rupture can be ameliorated or treated early in order to avoid this complication. Early and aggressive nutritional support together with correction of haematological abnormalities promote wound healing and prevent tissue breakdown. The detection and treatment of infection also reduces fistula formation and wound compromise. We present our protocol for the early, aggressive management of these patients with carotid artery rupture.

Le Fort I osteotomy and low-dose rate Ir192 brachytherapy for treatment of recurrent nasopharyngeal tumours.

BACKGROUND: Treatment of recurrent nasopharyngeal carcinoma is a difficult clinical problem. External beam re-irradiation is associated with a long-term cure in a proportion of cases but this may be associated with severe radiation injury. METHODS: Eighteen patients with post-nasal space tumours were treated between 1986 and 2001 with surgical excision and nasopharyngeal brachytherapy via a Le Fort I osteotomy approach. Low-dose rate (LDR) and high-dose rate (HDR) brachytherapy was used. Data was prospectively collected. Local control and overall survival were measured. Acute and late complications were assessed using the RTOG system. RESULTS: The overall survival was 67% at 2 years and 33.5% at 5 years. The corresponding local control rates were 42 and 31.5%, respectively. The T stage at relapse was a significant prognostic factor for local control (P=0.004) but not overall survival. Acute toxicity was mild. RTOG grade >/=3 late complications occurred in 40% of patients treated with the HDR, and 0% treated with LDR. CONCLUSIONS: The results of the Le Fort osteotomy, tumour debulking and post-operative brachytherapy gives local control rates similar to those achieved with wide-field re-irradiation. Complication rates are acceptable and are lower than that reported with other methods of radiation therapy. The surgical technique was well tolerated. HDR brachytherapy with this technique had a high complication rate. This approach is a viable option in the treatment of recurrent nasopharyngeal tumours.