Modelling cost-effectiveness of replacement strategies for ambulance services in the Ministry of Health Malaysia.

BACKGROUND: Emergency Medical Service (EMS) is a very crucial aspect of the healthcare system in providing urgent management and transportation of patients during emergencies. The sustainability of the services is however greatly impacted by the quality and age of ambulances. While this has led to numerous replacement policy recommendations, the implementations are often limited due to a lack of evidence and financial constraints. This study thus aims to develop a cost-effectiveness model and testing the model by evaluating the cost-effectiveness of 10-year and 15-year compulsory ambulance replacement strategies in public healthcare for the Malaysian Ministry of Health (MOH). METHODS: A Markov model was developed to estimate the cost and outcomes ambulance replacement strategies over a period of 20 years. The model was tested using two alternative strategies of 10-year and 15-year. Model inputs were derived from published literature and local study. Model development and economic analysis were accomplished using Microsoft Excel 2016. The outcomes generated were costs per year, the number of missed trips and the number of lives saved, in addition to the Incremental Cost-Effectiveness Ratio (ICER). One-Way Deterministic Sensitivity Analysis (DSA) and Probabilistic Sensitivity Analysis (PSA) were conducted to identify the key drivers and to assess the robustness of the model. RESULTS: Findings showed that the most expensive strategy, which is the implementation of 10 years replacement strategy was more cost-effective than 15 years ambulance replacement strategy, with an ICER of MYR 11,276.61 per life saved. While an additional MYR 13.0 million would be incurred by switching from a 15- to 10-year replacement strategy, this would result in 1,157 deaths averted or additional live saved per year. Sensitivity analysis showed that the utilization of ambulances and the mortality rate of cases unattended by ambulances were the key drivers for the cost-effectiveness of the replacement strategies. CONCLUSIONS: The cost-effectiveness model developed suggests that an ambulance replacement strategy of every 10 years should be considered by the MOH in planning sustainable EMS. While this model may have its own limitation and may require some modifications to suit the local context, it can be used as a guide for future economic evaluations of ambulance replacement strategies and further exploration of alternative solutions.

Pseudomonas aeruginosa Infections Are Associated with Infection Recurrence in Arteriovenous Grafts Treated with Revision.

Background and Objectives: This study was conducted to investigate whether Pseudomonas aeruginosa (PA) infections of arteriovenous grafts (AVGs) recur more frequently than other bacterial infections following treatment with revision. Materials and Methods: Operative procedures, including total excision, subtotal excision, and revision, were performed on 60 patients to treat 65 AVG infections. Final outcomes were classified as no infection recurrence, infection recurrence, and death without prior recurrence. In the competing risk setting, the cumulative incidence was estimated using the cumulative incidence function and Gray's test, and the associations between outcomes and different variables were estimated using a subdistribution hazard (SDH) model. Results: Comparing AVG infections with and without recurrence, PA infection was not associated with a higher risk of infection recurrence (p = 0.13); however, the first operative procedure type was associated with infection recurrence (p = 0.04). AVGs with PA infection were associated with a higher total number of surgical interventions (p < 0.05) than AVGs without PA infection. Regarding the cumulative incidences of outcomes, for AVGs treated with subtotal excision or revision, the cumulative incidence of recurrent infection was 3.3-fold higher for those with PA infection than without one year after the first surgery. However, when AVGs were treated with revision alone, the cumulative incidence was 4.1-fold. After excluding AVGs treated with total excision, the SDH model was applied, obtaining a hazard ratio for infection recurrence of 16.05 (p = 0.02) for AVGs with PA infection compared with AVGs without PA infection. No other variables were significantly associated with infection recurrence. Conclusions: For subtotal resection and revision, AVGs infected with PA had a higher recurrence rate than those infected with other species. However, revision surgery may aggravate the recurrence rate.

Stage one endometrioid endometrial adenocarcinoma: is there a role of traditional hospital follow-up in the detection of cancer recurrence in women after treatment?

OBJECTIVE: To investigate the rate of asymptomatic recurrence of stage 1 endometrioid endometrial cancer and assess the role of routine hospital follow-up after treatment. METHODS: We performed a retrospective case-note review study of women who were diagnosed with stage 1 endometrioid endometrial adenocarcinoma at Queen's Hospital, Romford, between January 2008 and December 2016. RESULTS: We included 299 patients with a median follow-up period of 44.4 months. All the patients underwent total hysterectomy and bilateral salpingo-oophorectomy. Adjuvant radiotherapy was offered to the patients subsequent to discussions in the multidisciplinary team meeting in accordance with the risk stratification criteria. There was no significant correlation between the risk factors and disease recurrence. In total, 11 patients presented with recurrent disease with original staging: 1a, n=6/199; and 1b, n=5/100. Four patients presented with vaginal bleeding due to vault recurrence and one patient with abdominal pain due to pelvic mass. Locoregional recurrence was an incidental finding in two other patients. Four patients presented with symptomatic distant metastases to the lung (n=2), liver (n=1), and bone (n=1). No asymptomatic recurrences were identified on routine follow-ups, despite several hospital appointments and clinical examinations. The recurrence rate for patients with stage 1a and 1b, grade 1, and grade 2 disease was 3.53%, and that for patients with stage 1a, grade 1, and grade 2 disease was 2.7%. CONCLUSION: Routine clinical examinations have a low yield in finding recurrence in asymptomatic women and should be questioned for their value, considering the limited resources of the National Health Service (NHS). Larger studies are required to support a stratified follow-up, which will include telephone and patient-initiated follow-up.

A Novel Tool for a Challenging Disease: Stasis Leg Ulcers Assessed Using QFlow in Triggered Angiography Noncontrast Enhanced Magnetic Resonance Imaging.

Imaging characteristics of stasis leg ulcers (SLUs) are not easily demonstrated through existing diagnostic tools. Early diagnosis and treatment are crucial. This pilot study was conducted to assess the quantitative flow (QFlow) in triggered angiography noncontrast enhanced (TRANCE) magnetic resonance imaging (MRI) to identify the hemodynamics of victims with stasis leg ulcers (SLUs). This study included 33 patients with SLUs and 14 healthy controls (HC). The 33 patients with SLUs were divided into a reflux (15 patients) and a nonreflux group (18 patients). QFlow was done in the reflux, the nonreflux, and the HC. The stroke volume (SV), forward flow volume (FFV), absolute flow volume (AFV), mean flow (MF), and mean velocity (MV) were higher in the reflux than in the HC group in most segments, namely the external iliac vein (EIV), popliteal vein (PV), and great saphenous vein (GSV) (SV, p = 0.008; FFV, p = 0.008; absolute stroke volume (ASV), p = 0.008; MF, p = 0.002; MV, p = 0.009). No differences in the QFlow patterns were found in the GSV segment between the nonreflux group and the HC. Excellent performance in discriminating SLU with superficial venous reflux was reported for SV in the EIV and the PV (area under the curve (AUC) = 0.851 and 0.872), FFV in the EIV and PV (AUC = 0.854 and 0.869), ASV in the EIV and PV (AUC = 0.848 and 0.881), and MF in the EIV and PV (AUC = 0.866 and 0.868). The cutoff levels of SV/FFV/ASV/MF in the EIV/FV/PV/GSV for discriminating the SLU with superficial venous reflux were identified (p < 0.005). In conclusion, SLUs present different QFlow patterns by different etiology. The QFlow parameters of all vessel segments were higher in the morbid limbs of the reflux group than HC. The GSV segment of the nonreflux group displayed a pattern like the HC.

Non-clinical similarity of biosimilar ABP 798 with rituximab reference product.

ABP 798 is a biosimilar to Rituxan® (rituximab reference product [RP]). Non-clinical assessments relevant to the primary and secondary mechanisms of action (MOA) contribute to the totality of the evidence (TOE) in supporting biosimilarity and are critical in providing scientific evidence for extrapolation of indications. Similarity of ABP 798 with rituximab RP was investigated across a range of biological activities which have potential impact on pharmacokinetics and clinical efficacy with non-clinical assessments relevant to MOA such as CD20 internalization, trogocytosis, binding to primary human natural killer (NK) cells as well as the ability to induce antibody-dependent cellular phagocytosis (ADCP) in peripheral blood mononuclear cells. Additionally, in vitro synergy of ABP 798 or RP with chemotherapeutic agents, in vivo xenograft studies in mice, and toxicological assessments in cynomolgus monkeys (including B cell depletion and toxicokinetics) were also conducted. Results from these non-clinical assessments contribute to the TOE supporting the biosimilarity between ABP 798 and rituximab RP across a range of primary and secondary MOAs and support justification for extrapolation to all indications of use for ABP 798 for which the RP is approved.

The Ferric Citrate Uptake System Encoded in a Novel bla CTX-M-3- and bla TEM-1-Harboring Conjugative Plasmid Contributes to the Virulence of Escherichia coli.

Escherichia coli is one major cause of bacterial infections and can horizontally acquire antimicrobial resistance and virulence genes through conjugation. Because conjugative plasmids can rapidly spread among bacteria of different species, the plasmids carrying both antimicrobial resistance and virulence genes may pose a significant threat to public health. Therefore, the identification and characterization of these plasmids may facilitate a better understanding of E. coli pathogenesis and the development of new strategies against E. coli infections. Because iron uptake ability is a potential virulence trait of bacteria, we screened for E. coli conjugative plasmids able to confer both iron uptake ability and ampicillin resistance. The plasmid pEC41, which was derived from the bacteremia clinical isolate EC41, was identified. EC41, which carried the fimH27 allele, belonged to sequence type (ST) 405 and phylogroup D. According to the sequencing analyses, pEC41 was 86 kb in size, and its backbone structure was almost identical to that of another highly conjugative plasmid, pCTX-M3, in which the extended-spectrum ß-lactamase gene bla CTX-M-3 was originally identified. pEC41 carried bla CTX-M-3 and bla TEM-1. The ferric citrate uptake (fec) system was identified in pEC41 and was responsible for conferring iron uptake ability. The fec system contributes to the pathogenesis of EC41 in systemic infections but not in urinary tract infections (UTIs). However, this system promoted competitive fitness of a cystitis-associated clinical isolate to colonize urinary tracts. Additionally, the distribution of the fec system was related to E. coli isolates associated with human bacteremia and UTIs. In summary, the present study identified a novel conjugative plasmid, pEC41, which conferred both antimicrobial resistance and an extra iron uptake ability to E. coli. The iron uptake ability was encoded in the fec system and contributed to E. coli pathogenesis. This study is the first to show that the fec system is a virulence factor in E. coli.

Aortic dissection assessment by 4D phase-contrast MRI with hemodynamic parameters: the impact of stent type.

BACKGROUND: To explore the diagnostic performance of 4-dimensional phase-contrast magnetic resonance imaging (4D PC-MRI) in evaluating aortic dissection in different clinical scenarios. METHODS: The study group comprised 32 patients with a known aortic dissection who each underwent computed tomography angiography (CTA), and then 4D PC-MRI with a 1.5-T MR scanner. The 4D PC-MRI images were compared with the CTA images to evaluate the aortic size, branch identification, and iliac and femoral arterial access. RESULTS: The patients were divided into three groups: (I) patients diagnosed with Type B aortic dissection but did not undergo intervention (n=8); (II) patients with residual aortic dissection after open repair of Type A dissection (n=7); (III) patients who underwent endovascular aortic repair with or without open surgery (n=17). Without radiation or contrast media injection, 4D PC-MRI provided similar aortic images for patients in Group 1 and most of those in Group 2. In Group 3, stainless steel stents affected image quality in three patients. High-quality 4D PC-MRI images were obtained for the remaining 14 patients in Group 3, who had non-stainless steel stents, and provided major aortic information comparable to that provided by CTA with contrast media. The hemodynamic parameters of true and false lumens were evaluated between three patients with Type B aortic dissections and three patients who underwent thoracic endovascular aortic repair for their aortic dissection. The stroke volume was higher in the true lumen of the patients with stent-grafts than in the patients with Type B aortic dissection without intervention. The regurgitant fraction, an indicator of nonlaminar flow, was higher in the false lumens than in the true lumens. All 32 patients in this study tolerated 4D PC-MRI without adverse events. CONCLUSIONS: 4D PC-MRI is radiation- and contrast media-free option for imaging aortic dissection. It not only provided images comparable in quality to those obtained with CTA but also provided information on hemodynamic parameters, including endoleak detection after thoracic endovascular aortic repair. 4D PC-MRI was safe and accurate in evaluating chronic Type B aortic dissection and residual aortic dissection after surgery for acute Type A aortic dissection. Therefore, it could be a potential tool in treating pathology in aortic dissection, especially for patients with malperfusion syndrome of visceral vessels and in young patients with renal function impairment. However, certain endograft materials, especially stainless steel, may prevent the further application of 4D PC-MRI and should be avoided.

Dispersal history of Miniopterus fuliginosus bats and their associated viruses in east Asia.

In this study, we examined the role of the eastern bent-winged bat (Miniopterus fuliginosus) in the dispersion of bat adenovirus and bat alphacoronavirus in east Asia, considering their gene flows and divergence times (based on deep-sequencing data), using bat fecal guano samples. Bats in China moved to Jeju Island and/or Taiwan in the last 20,000 years via the Korean Peninsula and/or Japan. The phylogenies of host mitochondrial D-loop DNA was not significantly congruent with those of bat adenovirus (m2XY = 0.07, p = 0.08), and bat alphacoronavirus (m2XY = 0.48, p = 0.20). We estimate that the first divergence time of bats carrying bat adenovirus in five caves studied (designated as K1, K2, JJ, N2, and F3) occurred approximately 3.17 million years ago. In contrast, the first divergence time of bat adenovirus among bats in the 5 caves was estimated to be approximately 224.32 years ago. The first divergence time of bats in caves CH, JJ, WY, N2, F1, F2, and F3 harboring bat alphacoronavirus was estimated to be 1.59 million years ago. The first divergence time of bat alphacoronavirus among the 7 caves was estimated to be approximately 2,596.92 years ago. The origin of bat adenovirus remains unclear, whereas our findings suggest that bat alphacoronavirus originated in Japan. Surprisingly, bat adenovirus and bat alphacoronavirus appeared to diverge substantially over the last 100 years, even though our gene-flow data indicate that the eastern bent-winged bat serves as an important natural reservoir of both viruses.

Comparison of Microbiological Characteristics and Genetic Diversity between Burkholderia cepacia Complex Isolates from Vascular Access and Other Clinical Infections.

Burkholderia cepacia complex (BCC) is a group of closely related bacteria with widespread environmental distribution. BCC bacteria are opportunistic pathogens that cause nosocomial infections in patients, especially cystic fibrosis (CF). Multilocus sequence typing (MLST) is used nowadays to differentiate species within the BCC complex. This study collected 41 BCC isolates from vascular access infections (VAIs) and other clinical infections between 2014 and 2020. We preliminarily identified bacterial isolates using standard biochemical procedures and further conducted recA gene sequencing and MLST for species identification. We determined genetic diversity indices using bioinformatics software. We studied 14 isolates retrieved from patients with VAIs and observed that Burkholderia cepacia was the predominant bacterial species, and B. contaminans followed by B. cenocepacia were mainly retrieved from patients with other infections. According to MLST data, we identified that all B. contaminans isolates belonged to ST102, while a wide variety of sequence types (STs) were found in B. cenocepacia isolates. In summary, the high diversity and easy transmission of BCC increase BCC infections, which provides insights into their potential clinical effects in non-CF infections.

Novel Diagnostic Options without Contrast Media or Radiation: Triggered Angiography Non-Contrast-Enhanced Sequence Magnetic Resonance Imaging in Treating Different Leg Venous Diseases.

OBJECTIVES: Venous diseases in the lower extremities long lacked an objective diagnostic tool prior to the advent of the triggered angiography non-contrast-enhanced (TRANCE) technique. METHODS: An observational study with retrospective data analysis. MATERIALS: Between April 2017 and June 2019, 66 patients were evaluated for venous diseases through TRANCE-magnetic resonance imaging (MRI) and were grouped according to whether they had occlusive venous (OV) disease, a static venous ulcer (SU), or symptomatic varicose veins (VV). The clinical appliance of TRANCE-MRI was analysed by groups. RESULTS: In total, 63 patients completed the study. TRANCE-MRI could identify venous thrombosis, including that of the abdominal and pelvic vessels, and it enabled the timely treatment of underlying diseases in patients with OV disease. TRANCE-MRI was statistically compared with the duplex scan, the gold standard to exclude deep vein thrombosis (DVT) in the legs, with regard to their abilities to detect venous thrombosis by using Cohen's kappa coefficient at a compatible value of 0.711. It could provide the occlusion degree of the peripheral artery for treating an SU. Finally, TRANCE-MRI can be used to outline all collateral veins and occult thrombi before treating symptomatic or recurrent VV to ensure a perfect surgical plan and to avoid complications. CONCLUSIONS: TRANCE-MRI is an innovative tool in the treatment of versatile venous pathology in the lower extremities and is widely used for vascular diseases in our institution.

Caregiver Stress, Coping Strategies, and Support Needs of Mothers Caring for their Children Who Are Undergoing Active Cancer Treatments.

The diagnosis of childhood cancer not only affects the life of the child but also impacts the lives of the caregivers as well. This study aims to explore the caregiving stress, coping strategies, and support needs of mothers caring for children/adolescents with cancer during the active treatment phase. Individual semi-structured interviews were conducted, and two authors independently and thematically analyzed data. Caregiving mothers went through a process of emotional changes and a change in lifestyles when their children were diagnosed with cancer and undergoing treatments. It is important to ensure that caregiving mothers of children/adolescents with cancer are well supported by family, friends, and healthcare professionals. Healthcare professionals can develop informational booklets on cancer treatment protocols and work together with mothers. Parent support groups and plans for psychoeducational and spiritual care programs for mothers as forms of informational and emotional support may also be established.

Salmonella-Infected Aortic Aneurysm: Investigating Pathogenesis Using Salmonella Serotypes.

Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes - Typhimurium, Enteritidis, and Choleraesuis - in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1ß, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy - significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1ß secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1ß secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes ­ Typhimurium, Enteritidis, and Choleraesuis ­ in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1ß, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy ­ significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1ß secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1ß secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.

Drug Repurposing: The Anthelmintics Niclosamide and Nitazoxanide Are Potent TMEM16A Antagonists That Fully Bronchodilate Airways.

There is an unmet need in severe asthma where approximately 40% of patients exhibit poor ß-agonist responsiveness, suffer daily symptoms and show frequent exacerbations. Antagonists of the Ca2+-activated Cl- channel, TMEM16A, offers a new mechanism to bronchodilate airways and block the multiple contractiles operating in severe disease. To identify TMEM16A antagonists we screened a library of ∼580,000 compounds. The anthelmintics niclosamide, nitazoxanide, and related compounds were identified as potent TMEM16A antagonists that blocked airway smooth muscle depolarization and contraction. To evaluate whether TMEM16A antagonists resist use- and inflammatory-desensitization pathways limiting ß-agonist action, we tested their efficacy under harsh conditions using maximally contracted airways or airways pretreated with a cytokine cocktail. Stunningly, TMEM16A antagonists fully bronchodilated airways, while the ß-agonist isoproterenol showed only partial effects. Thus, antagonists of TMEM16A and repositioning of niclosamide and nitazoxanide represent an important additional treatment for patients with severe asthma and COPD that is poorly controlled with existing therapies. It is of note that drug repurposing has also attracted wide interest in niclosamide and nitazoxanide as a new treatment for cancer and infectious disease. For the first time we identify TMEM16A as a molecular target for these drugs and thus provide fresh insights into their mechanism for the treatment of these disorders in addition to respiratory disease.

Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers.

Plant genetic engineering is an important tool used in current efforts in crop improvement, pharmaceutical product biosynthesis and sustainable agriculture. However, conventional genetic engineering techniques target the nuclear genome, prompting concerns about the proliferation of foreign genes to weedy relatives. Chloroplast transformation does not have this limitation, since the plastid genome is maternally inherited in most plants, motivating the need for organelle-specific and selective nanocarriers. Here, we rationally designed chitosan-complexed single-walled carbon nanotubes, utilizing the lipid exchange envelope penetration mechanism. The single-walled carbon nanotubes selectively deliver plasmid DNA to chloroplasts of different plant species without external biolistic or chemical aid. We demonstrate chloroplast-targeted transgene delivery and transient expression in mature Eruca sativa, Nasturtium officinale, Nicotiana tabacum and Spinacia oleracea plants and in isolated Arabidopsis thaliana mesophyll protoplasts. This nanoparticle-mediated chloroplast transgene delivery tool provides practical advantages over current delivery techniques as a potential transformation method for mature plants to benefit plant bioengineering and biological studies.

A Nanobionic Light-Emitting Plant.

The engineering of living plants for visible light emission and sustainable illumination is compelling because plants possess independent energy generation and storage mechanisms and autonomous self-repair. Herein, we demonstrate a plant nanobionic approach that enables exceptional luminosity and lifetime utilizing four chemically interacting nanoparticles, including firefly luciferase conjugated silica (SNP-Luc), d-luciferin releasing poly(lactic-co-glycolic acid) (PLGA-LH2), coenzyme A functionalized chitosan (CS-CoA) and semiconductor nanocrystal phosphors for longer wavelength modulation. An in vitro kinetic model incorporating the release rates of the nanoparticles is developed to maximize the chemiluminescent lifetimes to exceed 21.5 h. In watercress (Nasturtium officinale) and other species, the nanoparticles circumvent limitations such as luciferin toxicity above 400 µM and colocalization of enzymatic reactions near high adenosine triphosphate (ATP) production. Pressurized bath infusion of nanoparticles (PBIN) is introduced to deliver a mixture of nanoparticles to the entire living plant, well described using a nanofluidic mathematical model. We rationally design nanoparticle size and charge to control localization within distinct tissues compartments with 10 nm nanoparticles localizing within the leaf mesophyll and stomata guard cells, and those larger than 100 nm segregated in the leaf mesophyll. The results are mature watercress plants that emit greater than 1.44 × 1012 photons/sec or 50% of 1 µW commercial luminescent diodes and modulate "off" and "on" states by chemical addition of dehydroluciferin and coenzyme A, respectively. We show that CdSe nanocrystals can shift the chemiluminescent emission to 760 nm enabling near-infrared (nIR) signaling. These results advance the viability of nanobionic plants as self-powered photonics, direct and indirect light sources.

Nitroaromatic detection and infrared communication from wild-type plants using plant nanobionics.

Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors-single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal-embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm-1 leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.

Goatpoxvirus ATPase activity is increased by dsDNA and decreased by zinc ion.

Viral-encoded ATPase can act as a part of molecular motor in genome packaging of DNA viruses, such as vaccinia virus and adenovirus, by ATP hydrolysis and interaction with DNA. Poxviral ATPase (also called A32) is involved in genomic double-stranded DNA (dsDNA) encapsidation, and inhibition of the expression of A32 causes formation of immature virions lacking viral DNA. However, the role of A32 in goatpoxvirus genome packaging and its dsDNA binding property are not known. In this study, purified recombinant goatpoxvirus A32 protein (rA32) was examined for its dsDNA binding property as well as the effect of dsDNA on ATP hydrolysis. We found that rA32 could bind dsDNA, and its ATPase activity was significant increased with dsDNA binding. Effects of magnesium and calcium ions on ATP hydrolysis were investigated also. The ATPase activity was dramatically enhanced by dsDNA in the presence of Mg(2+); in contrast, ATPase function was not altered by Ca(2+). Furthermore, the enzyme activity of rA32 was completely blocked by Zn(2+). Regarding DNA-protein interaction, the rA32-ATP-Mg(2+) showed lower dsDNA binding affinity than that of rA32-ATP-Ca(2+). The DNA-protein binding was stronger in the presence of zinc ion. Our results implied that A32 may play a role in viral genome encapsidation and DNA condensation.

Lipid Exchange Envelope Penetration (LEEP) of Nanoparticles for Plant Engineering: A Universal Localization Mechanism.

Nanoparticles offer clear advantages for both passive and active penetration into biologically important membranes. However, the uptake and localization mechanism of nanoparticles within living plants, plant cells, and organelles has yet to be elucidated.1 Here, we examine the subcellular uptake and kinetic trapping of a wide range of nanoparticles for the first time, using the plant chloroplast as a model system, but validated in vivo in living plants. Confocal visible and near-infrared fluorescent microscopy and single particle tracking of gold-cysteine-AF405 (GNP-Cys-AF405), streptavidin-quantum dot (SA-QD), dextran and poly(acrylic acid) nanoceria, and various polymer-wrapped single-walled carbon nanotubes (SWCNTs), including lipid-PEG-SWCNT, chitosan-SWCNT and 30-base (dAdT) sequence of ssDNA (AT)15 wrapped SWCNTs (hereafter referred to as ss(AT)15-SWCNT), are used to demonstrate that particle size and the magnitude, but not the sign, of the zeta potential are key in determining whether a particle is spontaneously and kinetically trapped within the organelle, despite the negative zeta potential of the envelope. We develop a mathematical model of this lipid exchange envelope and penetration (LEEP) mechanism, which agrees well with observations of this size and zeta potential dependence. The theory predicts a critical particle size below which the mechanism fails at all zeta potentials, explaining why nanoparticles are critical for this process. LEEP constitutes a powerful particulate transport and localization mechanism for nanoparticles within the plant system.

Dual ATPase and GTPase activity of the replication-associated protein (Rep) of beak and feather disease virus.

BACKGROUND: Psittacine beak and feather disease affects parrots resulting in an immunosuppressive disease that is often characterized by an abnormal shape and growth of the animal's beak, feathers, and claws. Beak and feather disease virus (BFDV) is a single-stranded circular DNA virus and is classified as a member of the Circoviridae family. Two major open reading frames (ORFs) are known to encode the replication-associated (Rep) protein and the capsid-associated (Cap) protein. METHODS: The Rep and Cap genes of BFDV were fused with tags and then expressed and purified, respectively. Both the ATPase and GTPase activities of the recombinant Rep protein are measured. The substrate and ion preference, the optimal conditions, the effects of ATPase and GTPase inhibitors and the presence of Cap protein on the ATPase and GTPase activity of the Rep protein are examined. Finally, the effects of the Walker A motif, the Walker B motif, and a novel GYDG motif of the Rep protein on the ATPase and GTPase activities are studied by various mutants. RESULTS: The recombinant Rep protein could display ATPase activity and GTPase activity. The Rep protein was able to hydrolyze both deoxyribonucleotides and ribonucleotides. Among nucleoside triphosphates and deoxynucleoside triphosphates, the substrate preference orders were found to be ATP>GTP>CTP≥UTP and dATP>dGTP>dTTP>dCTP, respectively. Both the ATPase and GTPase activity of the BFDV Rep protein required magnesium ions and the presence of calcium ions significantly inhibited the ATPase and GTPase activity of the Rep protein. The optimal temperatures for ATPase activity and GTPase activity were both 56 °C, while their optimal pH values were both pH 7.5. Both the ATPase activity and GTPase activity of the BFDV Rep protein were significantly down-regulated by polynucleotides and the dsDNA of 36 bp (located in origin of replication) of BFDV genome. The ATPase activity of the BFDV Rep protein was found to be more sensitive to sodium azide than sodium orthovanadate and N-ethylmaleimide. Linoleic acid more strongly inhibited the GTPase activity of the Rep protein than dynasore. This suggested the Rep protein of BFDV should be classified as an F-type ATPase and polyunsaturated fatty acid-sensitive GTPase. In the presence of 10 ng of the Cap protein, the ATPase activity and GTPase activity of the BFDV Rep protein were significantly increased. Furthermore, the BFDV Rep protein contains the Walker A motif, the Walker B motif and a novel GYDG motif. Both the ATPase activity and the GTPase activity of various deletion and site-directed mutants of the Rep protein affecting these motifs were significantly reduced, suggesting all the three motifs contribute to the ATPase and GTPase activities; specifically. In addition, the ATPase activity and GTPase activity of the deletion mutants of the Rep protein were reversed in the presence of the Cap protein. This is the first example of dual ATPase and GTPase activity being reported in the Rep protein of BFDV.

Suppression of influenza virus infection by the orf virus isolated in Taiwan.

Orf virus (ORFV), a member of parapoxvirus, is an enveloped virus with genome of double-stranded DNA. ORFV causes contagious pustular dermatitis or contagious ecthyma in sheep and goats worldwide. In general, detection of viral DNA and observing ORFV virion in tissues of afflicted animals are two methods commonly used for diagnosis of orf infection; however, isolation of the ORFV in cell culture using virus-containing tissue as inoculum is known to be difficult. In this work, the ORFV (Hoping strain) isolated in central Taiwan was successfully grown in cell culture. We further examined the biochemical characteristic of our isolate, including viral genotyping, viral mRNA and protein expression. By electron microscopy, one unique form of viral particle from ORFV infected cellular lysate was demonstrated in the negative-stained field. Moreover, immunomodulating and anti-influenza virus properties of this ORFV were investigated. ORFV stimulated human monocytes (THP-1) secreting proinflammatory cytokines IL-8 and TNF-α. And, pre-treatment of ORFV-infected cell medium prevents A549 cells from subsequent type A influenza virus (IAV) infection. Similarly, mice infected with ORFV via both intramuscular and subcutaneous routes at two days prior to IAV infection significantly decreased the replication of IAV. In summary, the results of a current study indicated our Hoping strain harbors the immune modulator property; with such a bio-adjuvanticity, we further proved that pre-exposure of ORFV protects animals from subsequent IAV infection.