Iterative deep learning-design of human enhancers exploits condensed sequence grammar to achieve cell type-specificity.

An important and largely unsolved problem in synthetic biology is how to target gene expression to specific cell types. Here, we apply iterative deep learning to design synthetic enhancers with strong differential activity between two human cell lines. We initially train models on published datasets of enhancer activity and chromatin accessibility and use them to guide the design of synthetic enhancers that maximize predicted specificity. We experimentally validate these sequences, use the measurements to re-optimize the predictor, and design a second generation of enhancers with improved specificity. Our design methods embed relevant transcription factor binding site (TFBS) motifs with higher frequencies than comparable endogenous enhancers while using a more selective motif vocabulary, and we show that enhancer activity is correlated with transcription factor expression at the single cell level. Finally, we characterize causal features of top enhancers via perturbation experiments and show enhancers as short as 50bp can maintain specificity.

Ultrasound-guided percutaneous insertion of Broviac lines in infants less than 5kg: Prospective study of 100 consecutive procedures.

AIM: Ultrasound-guided (USG) percutaneous insertion of Broviac lines (cuffed tunnelled silastic central venous catheters, TCVC) has increasingly been adopted throughout the UK. However, vascular access remains a challenge in small babies and in some units is still performed by open cutdown. Our vascular access team, established in 2004, consists of consultant surgeons, anaesthetists and interventional radiologists, who provide all permanent vascular access by the USG technique. We reviewed the outcome in our last 100 patients less than 5 kg. METHOD: A prospective database of TCVC insertions in patients <5 kg weight recorded age, gestation, weight, diagnosis, type of catheter and complications within 28 days of insertion. A standardised technique of USG insertion is used by all operators. RESULTS: One-hundred patients <5 kg had TCVC inserted between 1/1/2018 and 31/3/2020. Median age 46(range0-316)days, gestation 36.5(23-42)weeks, weight 3(0.66 to 5)kg. INDICATION: parenteral nutrition(75), long term antibiotics(14), cardiac medication(6), chemotherapy(3), other(2). All were tunnelled silicone lines of single 2.7fr(51) and 4.2fr(46) or double lumen 7fr(3). Uncomplicated insertion in 94/100 cases. In 6 patients difficulties were encountered with cannulating the vein. In 4 cases an experienced colleague was called and managed to cannulate the vein; in 1 case a new successful attempt was made on the opposite internal jugular vein, and in 1 the femoral vein was used. No patient required an open cutdown. There were no cases of line sepsis requiring removal but 1 replacement was required for blockage within 28days. CONCLUSION: The USG approach in infants<5 kg is safe and can be used exclusively for venous access even in the most tiny babies. It is, however, a technically challenging procedure therefore we would recommend establishing a consultant delivered vascular access team to provide this service. Open venous cutdown in a tertiary children's hospital is no longer necessary for the insertion of TCVC and should be abandoned altogether. LEVELS OF EVIDENCE: Level I Prognosis Study.

Central venous catheters in the left-sided superior vena cava: clinical implications.

AIM: Left-sided superior vena cava (LSVC) is a congenital venous anomaly with an incidence of about 0.3%, and which is sometimes discovered during vascular intervention [Le Cat. Histoire de l'acadroyale des sciences .Paris 1738:62, I Steinberg, W Dubilier, D Lucas. Persistence of left superior vena cava. Dis Chest 1953;24:479-88]. There is little clear guidance on what to do in this event. METHODS: Children with LSVC were identified from our prospectively collected database of percutaneous central venous catheter (CVC) insertions between 2004 and 2011. If a LSCV was suspected, usually a venogram was performed. All available documentation was reviewed to identify complications. RESULTS: Eleven children with LSVC had 12 CVCs during the study period. Mean age at operation was 5.8 years (range 27 days to 15 years). Cardiovascular anomalies were already known in three cases. After CVC insertion, the line tip lay in the LSVC in eight cases and in the RA in 4. In 11 cases there was no immediate problem, but in 1 case there were postoperative bradyarrhythmias, which caused the CVC to be removed on day 1. This case was later found to have abdominal sepsis. The mean duration the CVCs were retained was 331days (range 1 day to 4 years). Other reasons for CVC removal were infection (n=1), damaged line (n=2), no longer required (n=2), and death unrelated to CVC (n=2). CONCLUSIONS: Our experience suggests that a LSVC (i) is often first discovered during CVC insertion, (ii) can be safely used for parenteral nutrition or chemotherapy, and (iii) the best practice would be to leave the CVC tip high in the LSVC or in the RA via another route.

Combined liver-intestine grafts compared with isolated intestinal transplantation in children: a single-center experience.

BACKGROUND: Intestinal transplantation is known to be associated with a high risk of early complications and mortality. METHODS: We analyzed prospective data of 51 primary small bowel transplantations from December 1999 to August 2009 and identified perioperative factors that impact on early mortality (≤6 months after transplantation) after isolated intestinal (IITx; n=12) and combined liver-intestinal transplantation (CLITx group; n=39). RESULTS: Ten patients died during the first 6 months after transplantation, all of them in CLITx group (n=10/51, 19%). Multivariate analyses demonstrated intraoperative red blood cell transfusion greater than 70 mL/kg (P=0.019, odds ratio [OR]=13.79) and base excess 30-min after reperfusion less than -16 (P=0.001, OR=14.05), thrombocytopenia (<50,000 per dL) between day 1 and day 15 after transplantation (P=0.047, OR=5.22), and occurrence of vascular complications (P=0.003, OR=8.96) during the posttransplantation period as predictors of early mortality in CLITx group. CONCLUSION: Risk of mortality at 6 months after intestinal transplantation increased when the liver is included as combined graft. Strategies to reduce mortality such as refining selection for transplantation and early referral before the development of liver failure should be a priority.

A dedicated consultant-led vascular access team significantly reduces out-of-hours and emergency permanent central venous access insertions.

PURPOSE: The insertion of a permanent central venous access device (PCVAD i.e. Broviac or Hickman lines and vascuports) is often considered a low priority and performed as an emergency. In 2004, a vascular access team (VAT), responsible for all PCVAD insertions, was established in our institution to address this. METHODS: Data were collected retrospectively on all PCVAD insertions in 2 periods (January 2002-December 2003 and January 2005-December 2006). This included procedure, list type, surgeon grade, and operative time. RESULTS: During 2002 to 2003 and 2005 to 2006, 465 and 569 PCVADs were inserted, respectively (22% increase). After introduction of the VAT, the percentage of lines inserted during emergency lists fell from 24% (n = 112) to 13% (n = 72), and out-of-hours fell from 6% (n = 29) to 3% (n = 17; P < .05), respectively. Median time taken for PCVAD insertion in 2005 to 2006 was significantly less if using an elective list compared with insertions performed on an emergency list (elective, 67 [56-82] minutes vs emergency, 85 (65-110) minutes; P < .05). DISCUSSION: Introduction of a VAT has led to a significant decrease in emergency and out-of-hours PCVAD insertions, despite an increase in overall activity for the period. The median time taken for elective insertions is significantly less than emergency insertions, which has increased the efficiency of vascular access provision at our institution.

Ultrasound-guided percutaneous insertion of Hickman lines in children. Prospective study of 500 consecutive procedures.

AIM: The ultrasound-guided percutaneous technique of Hickman line insertion has not been widely adopted in pediatric surgical practice. We wished to review our own experience of using this technique for insertion into the internal jugular vein. METHODS: Our vascular access team consists of a consultant surgeon and 2 consultant anesthetists. All procedures were prospectively recorded on a database and were either performed or directly supervised by our team. RESULTS: Five hundred consecutive Hickman lines were inserted between June 2004 and October 2006. Patients' ages ranged from 14 days to 19 years (median, 44 months). Patients weighed between 600 g to more than 100 kg. Lines inserted were all tunneled silicone Hickman lines with a Dacron cuff (size 2.7F-10F, with 1-3 lumens), of which 60% were 7F double-lumen lines. Successful cannulation occurred in 99.8%. Perioperative complications (within 30 days) occurred in 12 patients (2.4%) and were all treated conservatively with no need for either blood transfusion or chest drain. Catheter-related sepsis rate was 3.16 per 1000 line days. DISCUSSION: 1. The technique of ultrasound-guided percutaneous insertion of Hickman line to the internal jugular vein is safe and is applicable to all children regardless of size, age, or diagnosis. 2. Pediatric surgeons and anesthetists can learn this technique without specific training in interventional radiology. 3. A learning curve does exist, and we recommend concentrating pediatric vascular access procedures to a specialist team.

MRI-guided ultrasonic heating allows spatial control of exogenous luciferase in canine prostate.

The need for efficient and controlled delivery is one of the major obstacles to clinical use of gene therapy. In this study, we investigated the use of magnetic resonance imaging-monitored ultrasound (US) to induce expression of luciferase after local injection of the construct Ad-HSP-Luc, an adenoviral vector containing a transgene encoding firefly luciferase under the control of the human hsp70B promoter. The hsp promoter allows induction of the associated transgene only in areas that are subsequently heated after infection. US imaging was used to guide the injection of purified virus into both lobes of the prostates of three beagles. At 48 h after injection, the left lobe of the prostate was heated using a 1.5-MHz US transducer driven by a multichannel radiofrequency system and employing an magnetic resonance imaging guidance system. High levels of luciferase expression were observed only in areas exposed to ultrasonic heating. This study demonstrates the feasibility of using ultrasonic heating to control transgene expression spatially using a minimally-invasive approach.

Spatial and temporal control of transgene expression through ultrasound-mediated induction of the heat shock protein 70B promoter in vivo.

Adenoviral vectors have been constructed that express the transgenes luciferase (Adeno-HSP-Luc) or Fas ligand (Adeno-HSP-FasL) under the control of the heat shock protein 70B (hsp70B) promoter. Cultures infected with Adeno-HSP-Luc transiently expressed high levels of luciferase after heat shock. When cultures infected with Adeno-HSP-FasL were maintained at 37 degrees C, no transgene expression was observed, but when cultures were exposed to heat stress, transgene expression resulted in apoptotic cell death. In vivo, transgene expression was induced by ultrasound-mediated heating of adenovirus-infected tissue. In mice or rats injected with the Adeno-HSP-Luc construct, high levels of localized expression of luciferase activity were observed in regions subjected to ultrasound-mediated irradiation. Adeno-HSP-FasL was administered systemically to mice via the tail vein to evaluate safety. Animals receiving Adeno-HSP-FasL in the absence of ultrasound treatment did not display liver toxicity, whereas animals receiving ultrasound treatment to induce the expression of Fas ligand from the hsp70B promoter had significant increases in serum levels of liver enzymes. These data demonstrate that combining the inducible hsp70B promoter with ultrasound induction allows safe local expression of cytotoxic genes with possible therapeutic utility.