Establishment of a Transgenic Zebrafish Line for Superficial Skin Ablation and Functional Validation of Apoptosis Modulators In Vivo

BACKGROUND: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)(cy17) (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+) signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+) fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling. CONCLUSION/SIGNIFICANCE: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo

Reducing implant infection in orthopaedics (RIIiO): results of a pilot study comparing the influence of forced air and resistive fabric warming technologies on post-operative infections following orthopaedic implant surgery

BACKGROUND Active warming during surgery prevents perioperative hypothermia but the effectiveness and post-operative infection rates may differ between warming technologies. We report results of a pilot study in patients over the age of 65 undergoing hemiarthroplasty following fractured neck of femur. AIM To establish the recruitment and data management strategies needed for a full trial comparing post-operative infection rates associated with forced air versus resistive fabric warming. METHODS Participants were randomised 1:1 in permuted blocks to forced air or resistive fabric warming. Hypothermia was defined as a temperature of <36ºC at the end of surgery. Primary outcomes were the number of participants recruited and the number with definitive deep surgical site infections. FINDINGS 515 participants were randomised at 6 sites over a period of 18 months. Follow-up was completed for 70.1%. Thirty-seven participants were hypothermic (7.5% in the FAW group; 9.7 % in the RFW group). The mean temperatures before anaesthesia and at the end of surgery were similar. For the primary clinical outcome, there were 4 deep surgical site infections in the forced air warming group and 3 in the resistive fabric warming group. All participants who developed a post-operative infection had antibiotic prophylaxis, a cemented prosthesis and were operated under laminar airflow; none were hypothermic. There were no serious adverse events related to warming. CONCLUSION Surgical site infections were identified in both groups. Progression from the pilot to the full trial is possible but will need to take account of the high attrition rate. TRIAL REGISTRATION ISRCTN 74612906 (http://www.isrctn.com/ISRCTN74612906)

Reducing Implant Infection in Orthopaedics (RIIiO): a pilot study for a randomised controlled trial comparing the influence of forced air versus resistive fabric warming technologies on postoperative infection rates following orthopaedic implant surgery in adults

Background Approximately 70,000 to 75,000 proximal femoral fracture repairs take place in the UK each year. Hemiarthroplasty is the preferred treatment for adults aged over 60 years. Postoperative infection affects up to 3% of patients and is the single most common reason for early return to theatre. Ultraclean ventilation was introduced to help mitigate the risk of infection, but it may also contribute to inadvertent perioperative hypothermia, which itself is a risk for postoperative infection. To counter this, active intraoperative warming is used for all procedures that take 30 min or more. Forced air warming (FAW) and resistive fabric warming (RFW) are the two principal techniques used for this purpose; they are equally effective in prevention of inadvertent perioperative hypothermia, but it is not known which is associated with the lowest infection rates. Deep surgical site infection doubles operative costs, triples investigation costs and quadruples ward costs. The Reducing Implant Infection in Orthopaedics (RIIiO) study seeks to compare infection rates with FAW versus RFW after hemiarthroplasty for hip fracture. A cost-neutral intervention capable of reducing postoperative infection rates would likely lead to a change in practice, yield significant savings for the health economy, reduce overall exposure to antibiotics and improve outcomes following hip fracture in the elderly. The findings may be transferable to other orthopaedic implant procedures and to non-orthopaedic surgical specialties. Methods RIIiO is a parallel group, open label study randomising hip fracture patients over 60 years of age who are undergoing hemiarthroplasty to RFW or FAW. Participants are followed up for 3 months. Definitive deep surgical site infection within 90 days of surgery, the primary endpoint, is determined by a blinded endpoint committee. Discussion Hemiarthroplasty carries a risk of deep surgical site infection of approximately 3%. In order to provide 90% power to demonstrate an absolute risk reduction of 1%, using a 5% significance level, a full trial would need to recruit approximately 8630 participants. A pilot study is being conducted in the first instance to demonstrate that recruitment and data management strategies are appropriate and robust before embarking on a large multi-centre trial

The rhabdoviruses: Biodiversity, phylogenetics, and evolution

Rhabdoviruses (family Rhabdoviridae) include a diversity of important pathogens of animals and plants. They share morphology and genome organization. The understanding of rhabdovirus phylogeny, ecology and evolution has progressed greatly during the last 30 years, due to enhanced surveillance and improved methodologies of molecular characterization. Along with six established genera, several phylogenetic groups at different levels were described within the Rhabdoviridae. However, comparative relationships between viral phylogeny and taxonomy remains incomplete. with multiple representatives awaiting further genetic characterization. The same is true for rhabdovirus evolution. To date, rather simplistic molecular clock models only partially describe the evolutionary dynamics of postulated viral lineages. Ongoing progress in viral evolutionary and ecological investigations will provide the platform for future studies of this diverse family. Published by Elsevier B.V

Evaluation of the Operator Protection Factors Offered by Positive Pressure Air Suits against Airborne Microbiological Challenge

Laboratories throughout the world that perform work with Risk Group 4 Pathogens generally adopt one of two approaches within BSL-4 environments: either the use of positive pressure air-fed suits or using Class III microbiological safety cabinets and isolators for animal work. Within the UK at present, all laboratories working with Risk Group 4 agents adopt the use of Class III microbiological safety cabinet lines and isolators. Operator protection factors for the use of microbiological safety cabinets and isolators are available however; there is limited published data on the operator protection factors afforded by the use of positive pressure suits. This study evaluated the operator protection factors provided by positive pressure air suits against a realistic airborne microbiological challenge. The suits were tested, both intact and with their integrity compromised, on an animated mannequin within a stainless steel exposure chamber. The suits gave operator protection in all tests with an intact suit and with a cut in the leg. When compromised by a cut in the glove, a very small ingress of the challenge was seen as far as the wrist. This is likely to be due to the low airflow in the gloves of the suit. In all cases no microbiological penetration of the respiratory tract was observed. These data provide evidence on which to base safety protocols for use of positive pressure suits within high containment laboratories