Combating Acute Myeloid Leukemia via Sphingosine Kinase 1 Inhibitor-Nanomedicine Combination Therapy with Cytarabine or Venetoclax.

MP-A08 is a novel sphingosine kinase 1 (SPHK1) inhibitor with activity against acute myeloid leukemia (AML). A rationally designed liposome-based encapsulation and delivery system has been shown to overcome the physicochemical challenges of MP-A08 and enable its effective delivery for improved efficacy and survival of mice engrafted with human AML in preclinical models. To establish therapies that overcome AML's heterogeneous nature, here we explored the combination of MP-A08-loaded liposomes with both the standard chemotherapy, cytarabine, and the targeted therapy, venetoclax, against human AML cell lines. Cytarabine (over the dose range of 0.1-0.5 µM) in combination with MP-A08 liposomes showed significant synergistic effects (as confirmed by the Chou-Talalay Combination Index) against the chemosensitised human AML cell lines MV4-11 and OCI-AML3. Venetoclax (over the dose range of 0.5-250 nM) in combination with MP-A08 liposomes showed significant synergistic effects against the chemosensitised human AML cell lines, particularly in venetoclax-resistant human AML cells. This strong synergistic effect is due to multiple mechanisms of action, i.e., inhibiting MCL-1 through SPHK1 inhibition, leading to ceramide accumulation, activation of protein kinase R, ATF4 upregulation, and NOXA activation, ultimately resulting in MCL-1 degradation. These combination therapies warrant further consideration and investigation in the search for a more comprehensive treatment strategy for AML.

Targeting Acute Myeloid Leukemia Using Sphingosine Kinase 1 Inhibitor-Loaded Liposomes.

Acute myeloid leukemia (AML) kills 75% of patients and represents a major clinical challenge with a need to improve on current treatment approaches. Targeting sphingosine kinase 1 with a novel ATP-competitive-inhibitor, MP-A08, induces cell death in AML. However, limitations in MP-A08's "drug-like properties" (solubility, biodistribution, and potency) hinder its pathway to the clinic. This study demonstrates a liposome-based delivery system of MP-A08 that exhibits enhanced MP-A08 potency against AML cells. MP-A08-liposomes increased MP-A08 efficacy against patient AML cells (>140-fold) and significantly prolonged overall survival of mice with human AML disease (P = 0.03). The significant antileukemic property of MP-A08-liposomes could be attributed to its enhanced specificity, bioaccessibility, and delivery to the bone marrow, as demonstrated in the pharmacokinetic and biodistribution studies. Our findings indicate that MP-A08-liposomes have potential as a novel treatment for AML.

Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

Vasculogenic properties of adventitial Sca-1+CD45+ progenitor cells in mice: a potential source of vasa vasorum in atherosclerosis.

The cellular origins of vasa vasorum are ill-defined and may involve circulating or local progenitor cells. We previously discovered that murine aortic adventitia contains Sca-1+CD45+ progenitors that produce macrophages. Here we investigated whether they are also vasculogenic. In aortas of C57BL/6 mice, Sca-1+CD45+ cells were localised to adventitia and lacked surface expression of endothelial markers (<1% for CD31, CD144, TIE-2). In contrast, they did show expression of CD31, CD144, TIE-2 and VEGFR2 in atherosclerotic ApoE-/- aortas. Although Sca-1+CD45+ cells from C57BL/6 aorta did not express CD31, they formed CD31+ colonies in endothelial differentiation media and produced interconnecting vascular-like cords in Matrigel that contained both endothelial cells and a small population of macrophages, which were located at branch points. Transfer of aortic Sca-1+CD45+ cells generated endothelial cells and neovessels de novo in a hindlimb model of ischaemia and resulted in a 50% increase in perfusion compared to cell-free control. Similarly, their injection into the carotid adventitia of ApoE-/- mice produced donor-derived adventitial and peri-adventitial microvessels after atherogenic diet, suggestive of newly formed vasa vasorum. These findings show that beyond its content of macrophage progenitors, adventitial Sca-1+CD45+ cells are also vasculogenic and may be a source of vasa vasorum during atherogenesis.

Loss of EfnB1 in the osteogenic lineage compromises their capacity to support hematopoietic stem/progenitor cell maintenance.

The bone marrow stromal microenvironment contributes to the maintenance and function of hematopoietic stem/progenitor cells (HSPCs). The Eph receptor tyrosine kinase family members have been implicated in bone homeostasis and stromal support of HSPCs. The present study examined the influence of EfnB1-expressing osteogenic lineage on HSPC function. Mice with conditional deletion of EfnB1 in the osteogenic lineage (EfnB1OB-/-), driven by the Osterix promoter, exhibited a reduced prevalence of osteogenic progenitors and osteoblasts, correlating to lower numbers of HSPCs compared with Osx:Cre mice. Long-term culture-initiating cell (LTC-IC) assays confirmed that the loss of EfnB1 within bone cells hindered HSPC function, with a significant reduction in colony formation in EfnB1OB-/- mice compared with Osx:Cre mice. Human studies confirmed that activation of EPHB2 on CD34+ HSPCs via EFNB1-Fc stimulation enhanced myeloid/erythroid colony formation, whereas functional blocking of either EPHB1 or EPHB2 inhibited the maintenance of LTC-ICs. Moreover, EFNB1 reverse signaling in human and mouse stromal cells was found to be required for the activation of the HSPC-promoting factor CXCL12. Collectively, the results of this study confirm that EfnB1 contributes to the stromal support of HSPC function and maintenance and may be an important factor in regulating the HSPC niche.

The osteoprogenitor-specific loss of ephrinB1 results in an osteoporotic phenotype affecting the balance between bone formation and resorption.

The present study investigated the effects of conditional deletion of ephrinB1 in osteoprogenitor cells driven by the Osterix (Osx) promoter, on skeletal integrity in a murine model of ovariectomy-induced (OVX) osteoporosis. Histomorphometric and µCT analyses revealed that loss of ephrinB1 in sham Osx:cre-ephrinB1fl/fl mice caused a reduction in trabecular bone comparable to OVX Osx:Cre mice, which was associated with a significant reduction in bone formation rates and decrease in osteoblast numbers. Interestingly, these observations were not exacerbated in OVX Osx:cre-ephrinB1fl/fl mice. Furthermore, sham Osx:cre-ephrinB1fl/fl mice displayed significantly higher osteoclast numbers and circulating degraded collagen type 1 compared to OVX Osx:Cre mice. Confirmation studies found that cultured monocytes expressing EphB2 formed fewer TRAP+ multinucleated osteoclasts and exhibited lower resorption activity in the presence of soluble ephrinB1-Fc compared to IgG control. This inhibition of osteoclast formation and function induced by ephrinB1-Fc was reversed in the presence of an EphB2 chemical inhibitor. Collectively, these observations suggest that ephrinB1, expressed by osteoprogenitors, influences bone loss during the development of osteoporosis, by regulating both osteoblast and osteoclast formation and function, leading to a loss of skeletal integrity.

EphA5 and EphA7 forward signaling enhances human hematopoietic stem and progenitor cell maintenance, migration, and adhesion via Rac1 activation.

The proliferation, differentiation, adhesion, and migration of hematopoietic stem and progenitor cells (HSPCs) are dependent upon bone marrow stromal cells (BMSCs). In this study, we found that human primitive HSPCs (CD34+CD38-), but not lineage-committed hematopoietic cell populations, express the tyrosine kinase receptors EphA5 and EphA7. Moreover, we found that the ephrinA5 ligand, the high-affinity binding partner of EphA5 and EphA7, is highly expressed by primary human BMSCs. Previous studies have reported that interactions between EphA and ephrinA play important roles in hematopoietic cell trafficking; however, their role in BMSC support of hematopoiesis had not been described previously. Herein, we show that stimulating EphA5 and/or EphA7 forward signaling in HSPCs using soluble ephrinA5-Fc molecules promoted human HSPC-derived colony formation significantly and was associated with increased expression of granulocyte macrophage colony-stimulating factor receptor on HSPCs. Studies using functional blocking peptides to EphA5/7 found that disruption of EphA5/ephrinA5 and/or EphA7/ephrinA5 interactions inhibited HSPC function in BMSC-dependent long-term culture-initiating cell assays. Furthermore, the adhesion and migration of HSPCs was increased significantly in the presence of ephrinA5-Fc molecules compared with human immunoglobulin G-treated controls. Conversely, blocking EphA5 activation led to a reduction of HSPC adhesion, whereas inhibiting EphA5 and/or EphA7 activation hindered HSPC migration. Analysis of HSPC cultured in the presence of ephrinA5-Fc showed that EphA forward signaling stimulated Rac1 gene and protein expression and the Rac1 target molecule WAVE1. Moreover, a significant reduction of ephrinA5-mediated HSPC adhesion and migration was observed in the presence of Rac1 inhibitor. These findings suggest that interactions between EphA and ephrinA5 are important in maintaining the HSPC niche mediated in part by activation of Rac1 signaling.

Loss of ephrinB1 in osteogenic progenitor cells impedes endochondral ossification and compromises bone strength integrity during skeletal development.

The EphB receptor tyrosine kinase family and their ephrinB ligands have been implicated as mediators of skeletal development and bone homeostasis in humans, where mutations in ephrinB1 contribute to frontonasal dysplasia and coronal craniosynostosis. In mouse models, ephrinB1 has been shown to be a critical factor mediating osteoblast function. The present study examined the functional importance of ephrinB1 during endochondral ossification using the Cre recombination system with targeted deletion of ephrinB1 (EfnB1fl/fl) in osteogenic progenitor cells, under the control of the osterix (Osx:Cre) promoter. The Osx:EfnB1-/- mice displayed aberrant bone growth during embryonic and postnatal skeletal development up to 4weeks of age, when compared to the Osx:Cre controls. Furthermore, compared to the Osx:Cre control mice, the Osx:EfnB1-/- mice exhibited significantly weaker and less rigid bones, with a reduction in trabecular/ cortical bone formation, reduced trabecular architecture and a reduction in the size of the growth plates at the distal end of the femora from newborn through to 4weeks of age. The aberrant bone formation correlated with increased numbers of tartrate resistant acid phosphatase positive osteoclasts and decreased numbers of bone lining osteoblasts in 4week old Osx:EfnB1-/- mice, compared to Osx:Cre control mice. Taken together, these observations demonstrate the importance of ephrinB1 signalling between cells of the skeleton required for endochondral ossification.

In situ monitoring, separation, and characterization of gold nanorod transformation during seed-mediated synthesis.

The control of gold nanorod (GNR) solution-based syntheses has been hindered in part by the inability to examine and control the conversion of precursor seed populations to anisotropic materials, which have resulted in low yields of desired products and limited their commercial viability. The advantages offered by tandem separation and characterization methods utilizing asymmetric-flow field flow fractionation (A4F) are principally achieved as a result of their non-disruptive nature (minimizing artefacts), fast throughput, and in-situ analysis. With hyphenated A4F methods, resolved populations of seeds and secondary products, up to long aspect ratio rods, have been achieved and exemplify progress towards elucidating mechanistic aspects of formation and thus rational design. While there have been previously reported studies on A4F separation of GNRs, to our knowledge, this is the first published investigation of in situ GNR growth, separation, and characterization based on A4F, where its utilization in this capacity goes beyond traditional separation analysis. By using hydroquinone as the reducing agent, the conversion of the initial seed population to a distribution of products, including the GNRs, could be monitored in real time using A4F hyphenated with a diode array detector. Transmission electron microscopy confirms that the number of peaks observed during fractionation corresponds with size and shape dispersity. This proof-of-principle study introduces A4F as a technique that establishes a foundation for future mechanistic studies on the growth of GNRs from gold seeds, including conversion of the seed population to initial products, a topic highly relevant to advancing progress in nanomanufacturing.

The role of Eph/ephrin molecules in stromal­hematopoietic interactions.

Bone marrow mesenchymal stromal/stem cells(BMSC) are fundamental regulatory elements of the hematopoietic stem cell niche; however, the molecular signals that mediate BMSC support of hematopoiesis are poorly understood. Recent studies indicate that BMSC and hematopoietic stem/progenitors cells differentially express the Eph cell surface tyrosine kinase receptors, and their ephrinligands. Eph/ephrin interactions are thought to mediate cross-talk between BMSC and different hematopoietic cell populations to influence cell development, migration and function. This review summarizes Eph/ephrin interactions in the regulation of BMSC communication with hematopoietic stem/progenitor cells and discusses Eph/ephrintargeted therapeutic strategies that are currently being pursued or various hematotological malignancies.

Unexpected Changes in Functionality and Surface Coverage for Au Nanoparticle PEI Conjugates: Implications for Stability and Efficacy in Biological Systems.

Cationic polyethylenimine conjugated gold nanoparticles (AuNP-PEI) are a widely studied vector for drug delivery and an effective probe for interrogating NP-cell interactions. However, an inconsistent body of literature currently exists regarding the reproducibility of physicochemical properties, colloidal stability, and efficacy for these species. To address this gap, we systematically examined the preparation, stability, and formation mechanism of PEI conjugates produced from citrate-capped AuNPs. We considered the dependence on relative molar mass, Mr, backbone conformation, and material source. The conjugation mechanism of Au-PEI was probed using attenuated total reflectance FTIR and X-ray photoelectron spectroscopy, revealing distinct fates for citrate when interacting with different PEI species. The differences in residual citrate, PEI properties, and sample preparation resulted in distinct products with differentiated stability. Overall, branched PEI (25 kDa) conjugates exhibited the greatest colloidal stability in all media tested. By contrast, linear PEI (25 kDa) induced agglomeration. Colloidal stability of the products was also observed to correlate with displaced citrate, which supports a glaring knowledge gap that has emerged regarding the role of this commonly used carboxylate species as a "place holder" for conjugation with ligands of broad functionalities. We observed an unexpected and previously unreported conversion of amine functional groups to quaternary ammonium species for 10 kDa branched conjugates. Results suggest that the AuNP surface catalyzes this conversion. The product is known to manifest distinct processes and uptake in biological systems compared to amines and may lead to unintentional toxicological consequences or decreased efficacy as delivery vectors. Overall, comprehensive physicochemical characterization (tandem spectroscopy methods combined with physical measurements) of the conjugation process provides a methodology for elucidating the contributing factors of colloidal stability and chemical functionality that likely influence the previously reported variations in conjugate properties and biological response models.

EphB4 Expressing Stromal Cells Exhibit an Enhanced Capacity for Hematopoietic Stem Cell Maintenance.

The tyrosine kinase receptor, EphB4, mediates cross-talk between stromal and hematopoietic populations during bone remodeling, fracture repair and arthritis, through its interactions with the ligand, ephrin-B2. This study demonstrated that transgenic EphB4 mice (EphB4 Tg), over-expressing EphB4 under the control of collagen type-1 promoter, exhibited higher frequencies of osteogenic cells and hematopoietic stem/progenitor cells (HSC), correlating with a higher frequency of long-term culture-initiating cells (LTC-IC), compared with wild type (WT) mice. EphB4 Tg stromal feeder layers displayed a greater capacity to support LTC-IC in vitro, where blocking EphB4/ephrin-B2 interactions decreased LTC-IC output. Similarly, short hairpin RNA-mediated EphB4 knockdown in human bone marrow stromal cells reduced their ability to support high ephrin-B2 expressing CD34(+) HSC in LTC-IC cultures. Notably, irradiated EphB4 Tg mouse recipients displayed enhanced bone marrow reconstitution capacity and enhanced homing efficiency of transplanted donor hematopoietic stem/progenitor cells relative to WT controls. Studies examining the expression of hematopoietic supportive factors produced by stromal cells indicated that CXCL12, Angiopoietin-1, IL-6, FLT-3 ligand, and osteopontin expression were more highly expressed in EphB4 Tg stromal cells compared with WT controls. These findings indicate that EphB4 facilitates stromal-mediated support of hematopoiesis, and constitute a novel component of the HSC niche.

Conductive polymer nanotube patch for fast and controlled ex vivo transdermal drug delivery.

AIM: To uptake and release hydrophilic model drugs and insulin in a novel conductive polymer (CP) nanotube transdermal patch. MATERIALS & METHODS: The externally controlled transdermal delivery of model drugs and insulin were tested ex vivo and results were compared with CP films. The unique intrinsic properties of CPs provide electrostatic interaction between the model drugs and polymer backbone. RESULTS & DISCUSSION: When a pulsed potential was applied, the drug delivery release profile mimics that of injection delivery. With a constant potential applied, the release rate constants of the patch system were up to three-times faster than the control (0 V) and released approximately 80% more drug molecules over 24 h. CONCLUSION: The CP nanotube transdermal patch represents a new and promising drug method, specifically for hydrophilic molecules, which have been a large obstacle for conventional transdermal drug delivery systems.

PEGylated gold nanorod separation based on aspect ratio: characterization by asymmetric-flow field flow fractionation with UV-Vis detection.

The development of highly efficient asymmetric-flow field flow fractionation (A4F) methodology for biocompatible PEGylated gold nanorods (GNR) without the need for surfactants in the mobile phase is presented. We report on the potential of A4F for rapid separation by evaluating the efficiency of functionalized surface coverage in terms of fractionation, retention time (t R ) shifts, and population analysis. By optimizing the fractionation conditions, we observed that the mechanism of separation for PEGylated GNRs by A4F is the same as that for CTAB stabilized GNRs (i.e., according to their AR) which confirms that the elution mechanism is not dependent on the surface charge of the analytes and/or the membrane. In addition, we demonstrated that A4F can distinguish different surface coverage populations of PEGylated GNRs. The data established that a change in Mw of the functional group and/or surface orientation can be detected and fractionated by A4F. The findings in this study provide the foundation for a complete separation and physicochemical analysis of GNRs and their surface coatings, which can provide accurate and reproducible characterization critical to advancing biomedical research.

EphB and Ephrin-B interactions mediate human mesenchymal stem cell suppression of activated T-cells.

Mesenchymal stromal/stem cells (MSC) express the contact-dependent erythropoietin-producing hepatocellular (Eph) receptor tyrosine kinase family and their cognate ephrin ligands, which are known to regulate thymocyte maturation and selection, T-cell transendothelial migration, activation, co-stimulation, and proliferation. However, the contribution of Eph/ephrin molecules in mediating human MSC suppression of activated T-cells remains to be determined. In the present study, we showed that EphB2 and ephrin-B2 are expressed by ex vivo expanded MSC, while the corresponding ligands, ephrin-B1 and EphB4, respectively, are highly expressed by T-cells. Initial studies demonstrated that EphB2-Fc and ephrin-B2-Fc molecules suppressed T-cell proliferation in allogeneic mixed lymphocyte reaction (MLR) assays compared with human IgG-treated controls. While the addition of a third-party MSC population demonstrated dramatic suppression of T-cell proliferation responses in the MLR, blocking the function of EphB2 or EphB4 receptors using inhibitor binding peptides significantly increased T-cell proliferation. Consistent with these observations, shRNA EphB2 or ephrin-B2 knockdown expression in MSC reduced their ability to inhibit T-cell proliferation. Importantly, the expression of immunosuppressive factors, indoleamine 2, 3-dioxygenase, transforming growth factor-ß1, and inducible nitric oxide synthase expressed by MSC, was up-regulated after stimulation with EphB4 and ephrin-B1 in the presence of interferon (IFN)-γ, compared with untreated controls. Conversely, key factors involved in T-cell activation and proliferation, such as interleukin (IL)-2, IFN-γ, tumor necrosis factor-α, and IL-17, were down-regulated by T-cells treated with EphB2 or ephrin-B2 compared with untreated controls. Studies utilizing signaling inhibitors revealed that inhibition of T-cell proliferation is partly mediated through EphB2-induced ephrin-B1 reverse signaling or ephrin-B2-mediated EphB4 forward signaling by activating Src, PI3Kinase, Abl, and JNK kinase pathways, activated by tyrosine phosphorylation. Taken together, these observations suggest that EphB/ephrin-B interactions play an important role in mediating human MSC inhibition of activated T cells.

Electrochemical synthesis and one step modification of PMProDot nanotubes and their enhanced electrochemical properties.

Poly (3,4-(2-methylene)propylenedioxythiophene) (PMProDot) nanotubes were synthesized within the pores of polycarbonate and were further modified with styrene and vinylcarbazole by a one step electrochemical method through the methylene functional group. The enhanced electrochemical and electrochromic properties of composite nanotubes were investigated using FTIR, UV/Vis absorbance spectroscopy, and AFM.

Nanodetoxification: emerging role of nanomaterials in drug intoxication treatment.

Treatment for intoxication involves the neutralization or clearance of a toxic compound, but the current methods of treatment are limited in their ability to safely and effectively detoxify the patient. Emerging research has focused on using nanoparticles as parenteral detoxifying agents to circulate through the body and capture toxins. The variable compositions of these nanoparticles control the mechanism in which they capture and remove specific compounds. As discussed in this article, the recent methods for utilizing nanoparticles for detoxification show great potential for intoxication treatment. However, several challenges must be overcome before a universal nanoparticle detoxification method is available for clinical use.

Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells.

Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation.

Potential impact of peripheral intravenous catheter placement on resource use in the pediatric emergency department.

OBJECTIVES: In an era of pediatric emergency department (PED) overcrowding and diminishing health care resources, routine peripheral intravenous (PIV) catheter placement in the pediatric population requires evaluation because it might directly impact PED efficiency. This study aims to determine the utility of routine PIV catheter placement during phlebotomy. METHODS: Electronic medical and billing records from 2 tertiary care PEDs during 1 year in patients 21 years or younger were analyzed. Data on the presence of PIV catheter placement in the PED, subsequent PIV catheter usage, chief complaint, and demographics were tabulated and analyzed. RESULTS: During the study period, there were 131,003 PED visits analyzed and 26,776 PIV catheters placed. Of those placed, 12,475 (47%) were not used. The median age of the patients who received a PIV catheter that was not subsequently used was 36 months. The frequency of unused PIV catheters correlates with lower initial triage acuity. The highest rate of unused PIV catheter was in those 1 to 6 months old (63%), followed by that in groups younger than 1 month (57%), older than 6 to 24 months (52%), and older than 24 months (41%). CONCLUSIONS: Nearly half of the PIV catheters placed in the PED were unused. Unused PIV catheters represent an inefficient use of limited resources that could be redistributed to improve ED efficiency, flow, and resource use.

Multiplex-ready PCR: a new method for multiplexed SSR and SNP genotyping.

BACKGROUND: Microsatellite (SSR) and single nucleotide polymorphism (SNP) markers are widely used in plant breeding and genomic research. Thus, methods to improve the speed and efficiency of SSR and SNP genotyping are highly desirable. Here we describe a new method for multiplex PCR that facilitates fluorescence-based SSR genotyping and the multiplexed preparation of DNA templates for SNP assays. RESULTS: We show that multiplex-ready PCR can achieve a high (92%) success rate for the amplification of published sequences under standardised reaction conditions, with a PCR specificity comparable to that of conventional PCR methods. We also demonstrate that multiplex-ready PCR supports an improved level of multiplexing in plant genomes of varying size and ploidy, without the need to carefully optimize assay conditions. Several advantages of multiplex-ready PCR for SSR and SNP genotyping are demonstrated and discussed. These include the uniform amplification of target sequences within multiplexed reactions and between independent assays, and the ability to label amplicons during PCR with specialised moieties such fluorescent dyes and biotin. CONCLUSION: Multiplex-ready PCR provides several technological advantages that can facilitate fluorescence-based SSR genotyping and the multiplexed preparation of DNA templates for SNP assays. These advantages can be captured at several points in the genotyping process, and offer considerable cost and labour savings. Multiplex-ready PCR is broadly applicable to plant genomics and marker assisted breeding, and should be transferable to any animal or plant species.