Mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti.

Multiple viruses, including pathogenic viruses, bacteriophages, and even plant viruses, cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the nonstructural polyproteins (P123 and P1234) into individual proteins (nsP1-nsP4), forming the viral replication complex. Taking a synthetic biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titers, and transmission potential in mosquitoes.

The Acquisition and Retention of Lumpy Skin Disease Virus by Blood-Feeding Insects Is Influenced by the Source of Virus, the Insect Body Part, and the Time since Feeding.

Lumpy skin disease virus (LSDV) is a poxvirus that causes severe systemic disease in cattle and is spread by mechanical arthropod-borne transmission. This study quantified the acquisition and retention of LSDV by four species of Diptera (Stomoxys calcitrans, Aedes aegypti, Culex quinquefasciatus, and Culicoides nubeculosus) from cutaneous lesions, normal skin, and blood from a clinically affected animal. The acquisition and retention of LSDV by Ae. aegypti from an artificial membrane feeding system was also examined. Mathematical models of the data were generated to identify the parameters which influence insect acquisition and retention of LSDV. For all four insect species, the probability of acquiring LSDV was substantially greater when feeding on a lesion compared with feeding on normal skin or blood from a clinically affected animal. After feeding on a skin lesion LSDV was retained on the proboscis for a similar length of time (around 9 days) for all four species and for a shorter time in the rest of the body, ranging from 2.2 to 6.4 days. Acquisition and retention of LSDV by Ae. aegypti after feeding on an artificial membrane feeding system that contained a high titer of LSDV was comparable to feeding on a skin lesion on a clinically affected animal, supporting the use of this laboratory model as a replacement for some animal studies. This work reveals that the cutaneous lesions of LSD provide the high-titer source required for acquisition of the virus by insects, thereby enabling the mechanical vector-borne transmission. IMPORTANCE Lumpy skin disease virus (LSDV) is a high consequence pathogen of cattle that is rapidly expanding its geographical boundaries into new regions such as Europe and Asia. This expansion is promoted by the mechanical transmission of the virus via hematogenous arthropods. This study quantifies the acquisition and retention of LSDV by four species of blood-feeding insects and reveals that the cutaneous lesions of LSD provide the high titer virus source necessary for virus acquisition by the insects. An artificial membrane feeding system containing a high titer of LSDV was shown to be comparable to a skin lesion on a clinically affected animal when used as a virus source. This promotes the use of these laboratory-based systems as replacements for some animal studies. Overall, this work advances our understanding of the mechanical vector-borne transmission of LSDV and provides evidence to support the design of more effective disease control programmes.

Protecting Sleep Hygiene in the PICU: A Quality Improvement Project.

OBJECTIVES: To increase the number of nights without sleep interruptions for routine tasks in recovering PICU patients. DESIGN: Prospective quality improvement project. SETTING: Single-center, free-standing, tertiary children's hospital. PATIENTS: Patients admitted to the PICU for greater than 72 hours and eligible for early mobilization. INTERVENTIONS: A multidisciplinary sleep hygiene team was created to improve sleep hygiene in critically ill patients eligible for early mobilization. This team rewrote local nursing policies to avoid routine tasks between 11 pm and 5 am . The team provided periodic control chart updates to staff detailing progress made protecting sleep. Discussions of sleep hygiene were added to the daily goal sheet and a sleep hygiene order set was created. Finally, the PICU quality dashboard was modified to show whether a sleep hygiene order set was initiated in eligible patients. MEASUREMENTS AND MAIN RESULTS: Routine tasks were defined as daily chest radiographs, baths, routine tracheostomy care, central line dressing changes, twice daily medications, weights, and Foley care. After a year of data collection, avoidance of routine pupillary examinations was added to the sleep protection criteria. Baseline data was collected for 2 months prior to the creation of the sleep hygiene team. Screening of eligible patients occurred 1 week each month. The data were analyzed utilizing control charts. Baseline data demonstrated 32% of PICU patients without sleep interruptions. The centerline increased to 58% after the initial interventions but dropped to 33% after inclusion of pupillary checks. Following the introduction of the daily goal sheet, sleep hygiene order set, and tracking on the quality board, 49% of patients went without interruptions. CONCLUSIONS: The initiation of a sleep hygiene team along with retiming routine tasks, daily discussions on rounds with the daily goal sheet, introduction of a sleep hygiene order set, and transparent tracking improved the percentage of patients with protected sleep.

IV Versus Subcutaneous Enoxaparin in Critically Ill Infants and Children: Comparison of Dosing, Anticoagulation Quality, Efficacy, and Safety Outcomes.

OBJECTIVE: Subcutaneous enoxaparin is the mainstay anticoagulant in critically ill pediatric patients although it poses several challenges in this patient population. Enoxaparin infused IV over 30 minutes represents an attractive alternative, but there is limited experience with this route of administration in children. In this study, we assess dosing, anticoagulation quality, safety, and clinical efficacy of IV enoxaparin compared to subcutaneous enoxaparin in critically ill infants and children. DESIGN: Retrospective single-center study comparing dosing, anticoagulation quality, safety, and clinical efficacy of two different routes of enoxaparin administration (IV vs subcutaneous) in critically ill infants and children. Key outcome measures included dose needed to achieve target antifactor Xa levels, time required to achieve target antifactor Xa levels, proportion of patients achieving target anticoagulation levels on initial dosing, number of dose adjustments, duration spent in the target antifactor Xa range, anticoagulation-related bleeding complications, anticoagulation failure, and radiologic response to anticoagulation. SETTING: Tertiary care pediatric hospital. PATIENTS: All children admitted to the cardiac ICU, PICU, or neonatal ICU who were prescribed enoxaparin between January 2014 and March 2016 were studied. INTERVENTIONS: One hundred ten patients were identified who had received IV or subcutaneous enoxaparin and had at least one postadministration peak antifactor Xa level documented. MEASUREMENTS AND MAIN RESULTS: Of the 139 courses of enoxaparin administered, 96 were therapeutic dose courses (40 IV and 56 subcutaneous) and 43 were prophylactic dose courses (20 IV and 23 subcutaneous). Dosing, anticoagulation quality measurements, safety, and clinical efficacy were not significantly different between the two groups. CONCLUSIONS: Our study suggests that anticoagulation with IV enoxaparin infused over 30 minutes is a safe and an equally effective alternative to subcutaneous enoxaparin in critically ill infants and children.

AePUb promoter length modulates gene expression in Aedes aegypti.

Molecular tools for modulating transgene expression in Aedes aegypti are few. Here we demonstrate that adjustments to the AePUb promoter length can alter expression levels of two reporter proteins in Ae. aegypti cell culture and in mosquitoes. This provides a simple means for increasing or decreasing expression of a gene of interest and easy translation from cells to whole insects.

Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti.

Introduction: Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects' biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos. Results: Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard. Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti. These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control.

Modes of action of alpha-hederin and thymoquinone, active constituents of Nigella sativa, against HEp-2 cancer cells.

Our previous studies on active constituents of Nigella sativa have indicated that cell death induced by thymoquinone and alpha-hederin was dose- and time-dependent, in a range of four cancer cell lines. Both compounds elicited necrosis and apoptosis with a higher incidence of the latter induced by thymoquinone. As HEp-2 human laryngeal carcinoma cells were the most susceptible, we sought to better understand the mechanisms involved by using buthionine sulfoximine (BSO), a selective inhibitor of glutathione (GSH) synthesis, to determine the importance of GSH in the apoptosis elicited, using cisplatin as internal standard. BSO significantly enhanced alpha-hederin- and cisplatin- mediated toxicity as assessed by the MIT assay, without changes in apoptosis or necrosis levels. Although the MTI assay did not indicate BSO potentiation of thymoquinone, apoptosis levels were significantly enhanced following this combination, without changes in necrosis. Thymoquinone and cisplatin significantly decreased GSH levels in a dose-dependent manner, with BSO pre-treatment synergistically depleting GSH levels in only thymoquinone- treated cells. As the caspase 3 inhibitor, Z-DEVD-fmk significantly decreased thymoquinone- and cisplatin-induced apoptosis, GSH depletion and caspase 3-activation mediate thymoquinone-induced apoptosis, in this cell line.

Effects of alpha-hederin and thymoquinone, constituents of Nigella sativa, on human cancer cell lines.

The separate effects of alpha-hederin and thymoquinone, the two principal bioactive constituents of Nigella sativa, on four human cancer cell lines [A549 (lung carcinoma), HEp-2 (larynx epidermoid carcinoma), HT-29 (colon adenocarcinoma) and MIA PaCa-2 (pancreas carcinoma)] were investigated. Alpha-hederin was also examined as a pro-drug. Each assessment quantified both cytotoxic and apoptotic/necrotic effects. Alpha-hederin and thymoquinone separately induced a dose- and time-dependent effect on the cell lines tested. HEp-2 cells were the most sensitive, exhibiting apoptosis with a higher incidence following thymoquinone treatment. Pre-treatment of cells with alpha-hederin, followed by thymoquinone or cisplatin, did not enhance the cytotoxicity or apoptosis induced by either drug. So, the membrane-perforating properties associated with saponins, here represented by alpha-hederin, enhance neither cytotoxicity nor apoptosis of these cancer cells.