CasTuner: a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression

Certain cellular processes are dose-dependent, requiring a specific quantity of gene products or a defined stoichiometry between them. This is exemplified by haploinsufficiency or by the need for dosage compensation for X-linked genes between the sexes in many species. Understanding dosage-sensitive processes requires the ability to perturb endogenous gene products in a quantitative manner. Here we present CasTuner, a CRISPR-based toolkit that allows analog tuning of endogenous gene expression. In the CasTuner system, activity of Cas-derived repressors is controlled through a FKBP12F36V degron domain and can thereby be quantitatively tuned by titrating the small molecule degrader dTAG-13. The toolkit can be applied at the transcriptional level, using the histone deacetylase hHDAC4 fused to dCas9, or at the post-transcriptional level, using the RNA-targeting CasRx. To optimise efficiency, inducibility and homogeneity of repression we target a fluorescently tagged endogenous gene, Esrrb, in mouse embryonic stem cells. Through flow cytometry, we show that CasTuner allows analog tuning of the target gene in a homogeneous manner across cells, as opposed to the widely used KRAB repressor domain, which exhibits a digital mode of action. We quantify repression and derepression dynamics for CasTuner and use it to measure dose-response curves between the pluripotency factor NANOG and several of its target genes, providing evidence for target-specific dose dependencies. CasTuner thus provides an easy-to-implement tool to perturb gene expression in an inducible, tunable and reversible manner and will be useful to study dose-responsive processes within their physiological contex

Laparoscopic approach to Meckel's diverticulum

Aim.To retrospective review the laparoscopic management of Meckel Diverticulum (MD) in two Italian Pediatric Surgery Centers. METHODS: Between January 2002 and December 2012, 19 trans-umbilical laparoscopic-assisted (TULA) procedures were performed for suspected MD. The children were hospitalized for gastrointestinal bleeding and/or recurrent abdominal pain. Median age at diagnosis was 5.4 years (range 6 mo-15 years). The study included 15 boys and 4 girls. All patients underwent clinical examination, routine laboratory tests, abdominal ultrasound and technetium-99m pertechnetate scan, and patients with bleeding underwent gastrointestinal endoscopy. The abdominal exploration was performed with a 10 mm operative laparoscope. Pneumoperitoneum was established based on the body weight. Systematic overview of the peritoneal cavity allowed the ileum to be grasped with an atraumatic instrument. The complete exploration and surgical treatment of MD were performed extracorporeally, after intestinal exteriorization through the umbilicus. All patients' demographics, main clinical features, diagnostic investigations, operative time, histopathology reports, conversion rate, hospital stay and complications were registered and analyzed. RESULTS: MD was identified in 17 patients, while 1 had an ileal duplication and 1 a jejunal hemangioma. Fifteen patients had painless intestinal bleeding, while 4 had recurrent abdominal pain and exhibited cyst like structures in an ultrasound study. Eleven patients had a positive technetium-99m pertechnetate scan. In the patients with bleeding, gastrointestinal endoscopy did not name the source of hemorrhage. All patients were subjected to a TULA surgical procedure. An intestinal resection/anastomosis was performed in 14 patients, while 4 had a wedge resection of the diverticulum and 1 underwent stapling diverticulectomy. All surgical procedures were performed without conversion to open laparotomy. Mean operative time was 75 min (range 40-115 min). No major surgical complications were recorded. The median hospital stay was 5-7 d (range 4-13 d). All patients are asymptomatic at a median follow up of 4, 5 years (range 10 mo-10 years). CONCLUSION: Trans-umbilical laparoscopic-assisted Meckel's diverticulectomy is safe and effective in the treatment of MD, with excellent results. KEYWORDS: Gastrointestinal bleeding; Ileal duplication; Jejunal hemangioma; Laparoscopy; Meckel’s diverticulum; Minimal invasive surgery; One trocar surger

Development of large radii half-wave plates for CMB satellite missions

The successful European Space Agency (ESA) Planck mission has mapped the Cosmic Microwave Background (CMB) temperature anisotropy with unprecedented accuracy. However, Planck was not designed to detect the polarised components of the CMB with comparable precision. The BICEP2 collaboration has recently reported the first detection of the B-mode polarisation. ESA is funding the development of critical enabling technologies associated with B-mode polarisation detection, one of these being large diameter half-wave plates. We compare different polarisation modulators and discuss their respective trade-offs in terms of manufacturing, RF performance and thermo-mechanical properties. We then select the most appropriate solution for future satellite missions, optimized for the detection of B-modes.Comment: 16 page

Persistent nonbilious vomiting in a child: Possible duodenal webbing

An association between malrotation and congenital duodenal webbing is rare. We present our experience with four patients at two centers, and a review of published reports. There are currently 94 reported cases of duodenal pathology associated with malrotation. However, only 15 of the 94 cases (15.9%) include patients with malrotation and a duodenal web. We suggest that nonbilious vomiting in a child must prompt the surgeon to consider duodenal pathology even in the presence of malrotation

Gastric transposition as a valid surgical option for esophageal replacement in pediatric patients: Experience from three Italian medical centers

Background: Esophageal replacement in children is an option that is confined to very few situations including long-gap esophageal atresia and esophageal strictures unresponsive to other therapies (peptic or caustic ingestion). The purpose of our work was to describe the experience of gastric transposition in three Italian centers. Methods: This is a retrospective study. The data were extrapolated from a prospective database. We included all patients who had undergone gastric transposition in the last 15 years. Results: In the 15-year period, eight infants and children (3 males and 5 females) underwent gastric transposition for esophageal replacement. Six patients had long-gap esophageal atresia, and two had caustic esophageal stenosis. There were no deaths in the series. Three patients had an early postoperative complication: Two had a self-limited salivary fistula at three weeks, and one (a patient with jejunostomy) had a jejunal perforation treated surgically. One late complication, anastomotic stricture, was recorded that required two endoscopic dilatations. The median follow-up was 60 months (range: 18-144 months). At final clinical follow-up, six patients had no eating problems, and two patients had some difficulties with eating (jejunostomy in situ), but they underwent logopedic therapy with improved outcomes. All patients had an increase in body weight and height postoperatively. Conclusion: Our small study reports the clinical experience of three Italian centers in which gastric transposition was performed with excellent results, both in terms of surgical technique (simplicity, reproducibility, complication rate) and clinical follow-up (good oral feeding of young patients, normal social life and regular growth curves)

SOLE Project – Demonstration of a Multistatic and Multiband Coherent Radar Network

The aim of the NATO-SPS SOLE project is demonstrating the feasibility and the high performance of a radar network thanks to photonics. Indeed, the coherence offered by photonics makes the proposed distributed radar system capable of an efficient implementation of MIMO processing and ISAR imaging, enhancing the performance in terms of resolution and precision. The advantage of a fully coherent, multistatic radar system here is experimentally proven by a 5-time cross-range resolution enhancement thanks to MIMO processing, and in an efficient focusing in ISAR imaging

Planck pre-launch status: HFI beam expectations from the optical optimisation of the focal plane

Planck is a European Space Agency (ESA) satellite, launched in May 2009, which will map the cosmic microwave background anisotropies in intensity and polarisation with unprecedented detail and sensitivity. It will also provide full-sky maps of astrophysical foregrounds. An accurate knowledge of the telescope beam patterns is an essential element for a correct analysis of the acquired astrophysical data. We present a detailed description of the optical design of the High Frequency Instrument (HFI) together with some of the optical performances measured during the calibration campaigns. We report on the evolution of the knowledge of the pre-launch HFI beam patterns when coupled to ideal telescope elements, and on their significance for the HFI data analysis procedure