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Fedratinib hydrochloride is a selective Janus kinase 2 (JAK2) inhibitor approved by the U.S. Food and Drug Administration (FDA) in August 2019 for intermediate- 2 or high-risk primary or secondary myelofibrosis. The approval of this novel oral agent was based on the phase II and III JAKARTA-2 and JAKARTA trials, which both showed significant reduction in splenomegaly and myelofibrosis symptom burden. The most common adverse effects associated with fedratinib include anemia, gastrointestinal symptoms and elevation in liver transaminases. Early clinical trial data was concerning for an increased incidence of Wernicke's encephalopathy (WE), which led the FDA to place a clinical hold on further drug development. However, upon further investigation it was determined that there was no clear evidence that fedratinib causes WE, and the clinical hold was lifted in 2017. This inclusive review provides insight into the pharmacology, safety and efficacy, and future direction of fedratinib use in myeloproliferative neoplasms.

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Intracellular delivery of mRNA, DNA, and other large macromolecules into cells plays an essential role in an array of biological research and clinical therapies. However, current methods yield a wide variation in the amount of material delivered, as well as limitations on the cell types and cargoes possible. Here, we demonstrate quantitatively controlled delivery into a range of primary cells and cell lines with a tight dosage distribution using a nanostraw-electroporation system (NES). In NES, cells are cultured onto track-etched membranes with protruding nanostraws that connect to the fluidic environment beneath the membrane. The tight cell-nanostraw interface focuses applied electric fields to the cell membrane, enabling low-voltage and nondamaging local poration of the cell membrane. Concurrently, the field electrophoretically injects biomolecular cargoes through the nanostraws and into the cell at the same location. We show that the amount of material delivered is precisely controlled by the applied voltage, delivery duration, and reagent concentration. NES is highly effective even for primary cell types or different cell densities, is largely cargo agnostic, and can simultaneously deliver specific ratios of different molecules. Using a simple cell culture well format, the NES delivers into >100,000 cells within 20 s with >95% cell viability, enabling facile, dosage-controlled intracellular delivery for a wide variety of biological applications.

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BACKGROUND: Tobacco (Nicotiana tabacum) is an important plant model system that has played a key role in the early development of molecular plant biology. The tobacco genome is large and its characterisation challenging because it is an allotetraploid, likely arising from hybridisation between diploid N. sylvestris and N. tomentosiformis ancestors. A draft assembly was recently published for N. tabacum, but because of the aforementioned genome complexities it was of limited utility due to a high level of fragmentation. RESULTS: Here we report an improved tobacco genome assembly, which, aided by the application of optical mapping, achieves an N50 size of 2.17 Mb and enables anchoring of 64% of the genome to pseudomolecules; a significant increase from the previous value of 19%. We use this assembly to identify two homeologous genes that explain the differentiation of the burley tobacco market class, with potential for greater understanding of Nitrogen Utilization Efficiency and Nitrogen Use Efficiency in plants; an important trait for future sustainability of agricultural production. CONCLUSIONS: Development of an improved genome assembly for N. tabacum enables what we believe to be the first successful map-based gene discovery for the species, and demonstrates the value of an improved assembly for future research in this model and commercially-important species.

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ABCG2 (ATP binding cassette subfamily G, member 2) mediates resistance to a variety of cytotoxic agents. Although human ABCG2 is well characterized, the function of canine ABCG2 has not been studied previously. Feline ABCG2 has an amino acid substitution in the adenosine triphosphate-binding domain that decreases its transport capacity relative to human ABCG2. Our goal was to compare canine ABCG2-mediated chemotherapeutic drug resistance to feline ABCG2-mediated chemotherapeutic drug resistance. HEK-293 cells stably transfected with plasmid containing canine ABCG2, feline ABCG2 or no ABCG2 were exposed to carboplatin, doxorubicin, mitoxantrone, toceranib or vincristine, and cell survival was subsequently determined. Canine ABCG2 conferred a greater degree of chemotherapy resistance than feline ABCG2 for mitoxantrone. Neither canine nor feline ABCG2 conferred resistance to doxorubicin, vincristine or toceranib. Canine, but not feline, ABCG2 conferred resistance to carboplatin, a drug that is not reported to be a substrate for ABCG2 in other species.

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AIM: The outcome of surgery for colorectal cancer in each unit in the UK is collated by the National Bowel Cancer Audit Project (NBOCAP). In 2008-2009 our unit had a raw 30-day postoperative mortality close to the national average, but when it was nationally adjusted it appeared to be an outlier. The purpose of this study was to identify reasons for this disparity. METHOD: All records were obtained for patients undergoing surgery for colorectal cancer over the 2 years. Data submitted to NBOCAP to determine adjusted rates were compared with actual data. RESULTS: There were major discordances between submitted and actual data for American Society of Anesthesiology grades and timing of surgery. This explained why the unit appeared to be an outlier. CONCLUSION: There is increasing emphasis on outcome of health service delivery, which has important implications. Submission of correct data is essential if objective comparison is to be made on which to base decisions on service delivery among units and within health regions.

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Black shank, caused by Phytophthora nicotianae, is one of the most important diseases affecting tobacco (Nicotiana tabacum) production worldwide. Many current tobacco cultivars possess immunity to race 0 of this pathogen conferred by introgressed dominant genetic factors. Novel alleles conditioning resistance to alternative races are desired. The objective of this research was to evaluate variability for black shank resistance within a collection of N. rustica germplasm using both soilborne disease nurseries and controlled race-specific (race 0 and race 1) inoculations. Nearly all of the 86 accessions studied exhibited very high resistance to race 0, and many displayed levels of race 1 resistance greater than that exhibited by the resistant flue-cured tobacco check, 'K 346'. Materials found to be highly resistant to race 0 and race 1 in growth-chamber experiments also had the best survivability in field disease nurseries. N. rustica accessions TR 6, TR 12, TR 16, TR 21, TR 20, TR 48, TR 54, TR 57, and TR 69 could be sources of novel alleles with large effects on black shank resistance, and could have value for burley and flue-cured tobacco breeding.

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BACKGROUND: Immediate breast reconstruction (IBR) using Free flaps is becoming increasingly popular. However, these are complex surgical procedures with more complications and longer recovery time, which can potentially delay adjuvant treatment. Our aim is to investigate the impact of free flap IBR on the timing of adjuvant treatment. METHODS: Details of all breast cancer patients undergoing mastectomy with (study group) and without (control group) free flap IBR, followed by adjuvant treatment between 2002 and 2007 were obtained. The time lapse between surgery and adjuvant therapy was calculated and the causes of delay were recorded. The results were compared between the two groups and with local and international guidelines. RESULTS: Twenty-seven and 139 patients were included in the study and control group, respectively. The mean time period between surgery and commencement of adjuvant treatment for the study group was 55 days compared with 40 days for the controls. Furthermore, significantly less IBR patients received their adjuvant treatment within 6, 8 or 10 weeks after surgery in comparison to the control group. Groups appeared similar however at 12-week point. The reason for the delays was reconstruction-related surgical complications. CONCLUSION: There is a significant delay in the commencement of adjuvant treatment after mastectomy and free flap IBR in comparison to mastectomy alone patients due to reconstruction related surgical complications. The effects of this delay on survival have not been fully investigated yet and may be significant for at least some of the patients.

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BACKGROUND: There has been a dramatic increase in the incidence of immediate breast reconstruction after mastectomy for breast cancer over the past few decades and autologous tissue flaps are being used with increasing frequency. Concern has been expressed that these complex procedures may lead to a delay in the delivery of adjuvant therapy, which in turn may adversely affect recurrence and survival rates. Several publications have looked into the effect of immediate reconstruction on the timing of delivery of adjuvant therapy, but all types of immediate breast reconstruction (IBR) tend to be examined as a homogenous group. AIM: The aim of this review was to search current literature and look specifically at the effect of autologous tissue reconstructions on adjuvant therapy, and identify possible causes of delay. DISCUSSION: From the data analysed, it appears that there may be a delay in the delivery of adjuvant therapy associated with autologous tissue IBR, especially transverse rectus abdominis muscle (TRAM) flaps. The studies available, however, examine small numbers of patients, which makes proving statistical significance difficult. Moreover, there also appears to be no consensus on what constitutes a delay in the delivery of adjuvant therapy. From 3% to 72% of autologous tissue IBR patients seem to receive their adjuvant treatment with a delay, according to the guidelines of the respective centres. Moreover, the period of time until chemotherapy appears generally increased from 13% to 36% compared with mastectomy alone patients. The most common reasons for delay are wound and flap complications. CONCLUSION: At the moment, despite the increasing popularity of autologous IBR, these procedures have not proved their oncological safety. Further studies looking at the effect of IBR, especially TRAM flap reconstruction, are needed.

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The recent development of microsatellite markers for tobacco, Nicotiana tabacum L., may be valuable for genetic studies within the genus Nicotiana. The first objective was to evaluate transferability of 100 N. tabacum microsatellite primer combinations to 5 diploid species closely related to tobacco. The number of primer combinations that amplified scorable bands in these species ranged from 42 to 56. Additional objectives were to assess levels of genetic diversity amongst available accessions of diploid relatives closely related to tobacco (species of sections Sylvestres and Tomentosae), and to evaluate the efficacy of microsatellite markers for establishing species relationships in comparison with existing phylogenetic reconstructions. A subset of 46 primer combinations was therefore used to genotype 3 synthetic tobaccos and an expanded collection of 51 Nicotiana accessions representing 15 species. The average genetic similarity for 7 diverse accessions of tobacco was greater than the average similarity for N. otophora accessions, but lower than the average genetic similarities for N. sylvestris, N. tomentosa, N. kawakamii, and N. tomentosiformis accessions. A microsatellite-based phylogenetic tree was largely congruent with taxonomic representations based on morphological, cytological, and molecular observations. Results will be useful for selection of parents for creation of diploid mapping populations and for germplasm introgression activities.

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Stem rot and target spot of tobacco, caused by Rhizoctonia solani and its teleomorph Thanatephorus cucumeris, respectively, can cause serious problems in production of tobacco (Nicotiana tabacum) seedlings. Previous screens for genetic resistance in tobacco have been limited. The objective of this study was to evaluate 97 genotypes composing several classes of tobacco and related Nicotiana spp. for seedling resistance to stem rot and target spot. Significant differences in disease incidence initially were observed among the genotypes for both stem rot and target spot; however, resistance to target spot was not observed when disease pressure was high. Partial resistance to stem rot was observed in several genotypes in repeated tests. These accessions may be useful as a source of resistance to R. solani in future breeding efforts.

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Resistance to tobacco mosaic virus (TMV) is controlled by the single dominant gene N in Nicotiana glutinosa L. This gene has been transferred to cultivated tobacco (N. tabacum L.) by interspecific hybridization and backcrossing, but has historically been associated with reduced yields and/or quality in flue-cured tobacco breeding materials. Past researchers have suggested the role of pleiotropy and/or linkage drag effects in this unfavorable relationship. Introduction of the cloned N gene into a TMV-susceptible tobacco genotype (cultivar 'K326') via plant transformation permitted investigation of the relative importance of these possibilities. On average, yield and cash return ($ ha(-1)) of 14 transgenic NN lines of K326 were significantly higher relative to an isoline of K326 carrying N introduced via interspecific hybridization and backcrossing. The negative effects of tissue culture-induced genetic variation confounded comparisons with the TMV-susceptible cultivar, K326, however. Backcrossing the original transgenic lines to non-tissue cultured K326 removed many of these unfavorable effects, and significantly improved their performance for yield and cash return. Comparisons of the 14 corresponding transgenic NN backcross-derived lines with K326 indicated that linkage drag is the main factor contributing to reduced yields in TMV-resistant flue-cured tobacco germplasm. On average, these transgenic lines outyielded the conventionally-developed TMV-resistant K326 isoline by 427 kg ha(-1) (P < 0.05) and generated $1,365 ha(-1) more (P < 0.05). Although transgenic tobacco cultivars are currently not commercially acceptable, breeding strategies designed to reduce the amount of N. glutinosa chromatin linked to N may increase the likelihood of developing high-yielding TMV-resistant flue-cured tobacco cultivars.

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Germplasm from closely related diploid relatives of tobacco (Nicotiana tabacum L.) could be of value for continued genetic modification of this species and for mapping quantitative trait loci (QTLs). We examined near isogenic tobacco lines and hybrids differing for an introgressed genomic region from N. tomentosa Ruiz and Pavon designated as Many Leaves that exhibits a large influence on leaf number and correlated traits. Within a 'Red Russian' genetic background, the region acted in an additive to partially dominant fashion to delay flowering time, and increase leaf number, plant height, and green leaf yield. Evidence of epistasis was observed as the region affected these traits to varying degrees in diverse near isogenic hybrids. Fifteen amplified fragment length polymorphism (AFLP) markers of N. tomentosa origin were mapped within a single linkage group of 34.5 cM using a population of 207 BC(1)F(1) individuals segregating for Many Leaves. Composite interval mapping produced 2-LOD confidence intervals for likely QTL positions influencing leaf number (3.1 cM region), plant height (2.9 cM region), and days to flowering (3.3 cM region). These intervals were overlapping. Results demonstrate that genomic regions with large genetic effects can be transferred to tobacco from closely related diploid relatives, and that sufficient recombination within these regions may permit mapping of genes controlling quantitative traits. Materials and results described here may be useful in future research to gain insight on the genetic control of the transition from vegetative to reproductive development in Nicotiana.

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A disomic chromosome addition line of tobacco, Nicotiana tabacum L., was established previously that possesses a single chromosome pair from N. africana [Merxm. and Buttler]. This addition chromosome carries a gene that confers increased resistance to severe strains of potato virus Y (PVY). Methods to increase the probability of gene transfer from alien chromosomes to tobacco (2n=48) are desired. In the research described here, the PVY resistance gene was transferred to a tobacco chromosome from the N. africana addition chromosome in seven independent cases. One introgression event was obtained using conventional backcrossing of the disomic addition line to N. tabacum cv. Petite Havana, while the remaining six events were obtained using a scheme that involved exposure of explants of the addition line to tissue culture. Twenty-six derived 2n=48 individuals heterozygous for PVY resistance were found to exhibit 24 bivalents or 23 bivalents + 2 univalents at metaphase I. Ovular transmission rates for the PVY resistance factor ranged from 25% to 52%, while pollen transmission rates were much lower, ranging from 0 to 39%. Fifty-one random amplified polymorphic DNA (RAPD) markers specific for the intact addition chromosome were identified and used to characterize derived 2n=48/PVY-resistant genotypes. Variability was observed among these plants with respect to the total number of N. africana RAPD markers that were present, which is an indication that crossing over was occurring within each of the seven introgressed chromosome segments. A limited molecular marker-assisted backcrossing experiment allowed for selection of a 2n=48/PVY-resistant individual that possessed only 6 of the 51 original N. africana RAPD markers. In vitro culture is potentially a valuable system for increasing the rate of alien gene transfer in tobacco, and the successful transfer of PVY resistance from N. africana may allow for an increased level and range of resistance to this virus in tobacco.

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Recently, we reported that L-cysteine attached to polymeric biomaterials, without prior nitrosation, enhances the hemocompatibility of biomaterials via exploiting endogenous nitric oxide (NO). As part of the polymer optimization process to further enhance platelet inhibition, a kinetic model is being developed to predict the release rate of NO. A key model parameter is the immobilized concentration of L-cysteine. This article demonstrates how several chemiluminescence-based assays, previously utilized for measuring thiols in solution, were successfully adapted to quantify immobilized L-cysteine. The assays showed that the immobilized L-cysteine on the modified PET sample is within the range of 4.1 to 6.5 nmol/cm(2). An advantage of using the more successful chemiluminescence-based assay is that it can accurately measure molar concentrations of any thiol-containing compound with a detection limit in the pmol range. The major disadvantage is that L-cysteine must first be broken off of the polymer and released into solution prior to measurement, therefore leaving the sample unable to be reused. Other thiol-measuring techniques, such as fluorescence microscopy and X-ray photoelectron spectroscopy (XPS), were used to provide qualitative and semiquantitative analysis to substantiate the polymer development.

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[Ca(2+)](i) (intracellular Ca(2+) concentration) oscillations play a central role in the activation of T-lymphocytes by antigen. Oscillations in T-cells are absolutely dependent on Ca(2+) influx through store-operated CRAC channels (Ca(2+)-release-activated Ca(2+) channels), and evidence suggests that they arise from delayed interactions between these channels and Ca(2+) stores. Their potential functions have been explored by creating controlled [Ca(2+)](i) oscillations with pulses of Ca(2+) entry or pulses of Ins(1,4,5)P(3). Oscillations enhance both the efficiency and specificity of signalling through the Ca(2+)-dependent transcription factors nuclear factor of activated T-cells (NFAT), Oct/Oap and nuclear factor kappa B (NF kappa B) in ways that are consistent with each factor's Ca(2+) dependence and kinetics of activation and deactivation. These studies show how [Ca(2+)](i) oscillations may enhance signalling to the nucleus, and suggest a possible cellular mechanism for extracting information encoded in oscillation frequency.

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Exotic maize ( Zea mays L.) germplasm may allow for increased flexibility and greater long-term progress from selection if it can be incorporated at high rates into U.S. breeding programs. Crosses were made between a temperate line, NC262A, and each of eight different lines consisting of 100% temperate-adapted tropical germplasm. Pedigree selection was used to generate a set of 148 F(5)S(2) lines that were evaluated in testcrosses with FR992/FR1064 in nine North Carolina environments. Several entries had grain yield, grain moisture content and standability that were comparable to three commercial checks. The best testcrosses outyielded the cross NC262A x FR992/FR1064 by 9.5 to 10.9%, suggesting that a significant amount of tropical germplasm was retained in these lines and that this germplasm combined well with the Stiff Stalk tester. Previous researchers had suggested that tropical alleles could be rapidly lost during inbreeding in populations derived from tropical x temperate bi-parental crosses, leading to the development of lines that possess significantly less than 50% tropical germplasm. F(5)S(5) sub-lines corresponding to the 14 best testcrosses were genotyped at 47 to 49 polymorphic simple sequence repeat (SSR) loci across all ten chromosomes to estimate the amount of tropical germplasm that was retained. The estimated genetic contribution from the tropical parent ranged from 32 to 70%, with the average being 49%. Only two of the 14 lines deviated significantly from a 50%-tropical/50%-temperate ratio, suggesting limited overall selection against germplasm from the tropical parents. These experiments collectively demonstrated that tropical maize germplasm can be incorporated at high rates into a temperate line via pedigree breeding methods in order to derive new inbred lines with acceptable agronomic performance.

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The inferred crystallographic class of circumstellar silicon carbide based on astronomical infrared spectra is controversial. We have directly determined the polytype distribution of circumstellar SiC from transmission electron microscopy of presolar silicon carbide from the Murchison carbonaceous meteorite. Only two polytypes (of a possible several hundred) were observed: cubic 3C and hexagonal 2H silicon carbide and their intergrowths. We conclude that this structural simplicity is a direct consequence of the low pressures in circumstellar outflows and the corresponding low silicon carbide condensation temperatures.

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1. The effects of the IP(3)-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined. 2. 2-APB elicited both stimulatory and inhibitory effects on Ca(2+) influx through CRAC channels. At concentrations of 1-5 microM, 2-APB enhanced Ca(2+) entry in intact cells and increased I(CRAC) amplitude by up to fivefold. At levels > or = 10 microM, 2-APB caused a transient enhancement of I(CRAC) followed by inhibition. 3. 2-APB altered the kinetics of fast Ca(2+)-dependent inactivation of I(CRAC). At concentrations of 1-5 microM, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (> or = 10 microM) reduced or completely blocked inactivation. 4. 2-APB inhibited Ca(2+) efflux from mitochondria. 5. 2-APB inhibited I(CRAC) more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect potentiation or inhibition. Thus, 2-APB may have an extracellular site of action. 6. Neither I(CRAC) activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 microg ml(-1) heparin. 7. I(CRAC) is present in wild-type as well as mutant DT40 B cells lacking all three IP(3) receptor isoforms. 2-APB also potentiates and inhibits I(CRAC) in both cell types, indicating that 2-APB exerts its effects independently of IP(3) receptors. 8. Our results show that CRAC channel activation does not require physical interaction with IP(3) receptors as proposed in the conformational coupling model. Potentiation of I(CRAC) by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.

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Vascular thrombosis is regulated via the release of several constituents from the vascular endothelium, including nucleoside triphosphate diphosphohydrolases (NTPDases or ectonucleotidases), nitric oxide (NO), and eicosanoids. Currently, it is unknown how these constituents interact in the inhibition of platelet aggregation and adhesion. To investigate the combined effects of NO and NTPDase on platelet deposition sequestration, an in vitro study was performed to compare inhibition of platelet deposition to a biomaterial by NO in the absence or presence of soluble NTPDase. Results of the platelet inhibition studies with NO and NTPDase conclusively show that the inhibitory effects of NTPDase and NO are additive. The platelet inhibitory potency in the presence of NO was enhanced by NTPDase in a dose-dependent manner, for a given NO exposure. This augmentation is independent of aspirin; the ability of NTPDase or NO alone to inhibit platelet deposition is also independent of aspirin. Clearly, NO and NTPDase independently contribute to platelet inhibition via different mechanisms. The inaction of NO on the activity of NTPDase confirmed that NO or reaction products in the presence of O(2) do not interact with NTPDase directly.

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Elevation of intracellular free Ca(2+) is one of the key triggering signals for T-cell activation by antigen. A remarkable variety of Ca(2+) signals in T cells, ranging from infrequent spikes to sustained oscillations and plateaus, derives from the interactions of multiple Ca(2+) sources and sinks in the cell. Following engagement of the T cell receptor, intracellular channels (IP3 and ryanodine receptors) release Ca(2+) from intracellular stores, and by depleting the stores trigger prolonged Ca(2+) influx through store-operated Ca(2+) (CRAC) channels in the plasma membrane. The amplitude and dynamics of the Ca(2+) signal are shaped by several mechanisms, including K(+) channels and membrane potential, slow modulation of the plasma membrane Ca(2+)-ATPase, and mitochondria that buffer Ca(2+) and prevent the inactivation of CRAC channels. Ca(2+) signals have a number of downstream targets occurring on multiple time scales. At short times, Ca(2+) signals help to stabilize contacts between T cells and antigen-presenting cells through changes in motility and cytoskeletal reorganization. Over periods of minutes to hours, the amplitude, duration, and kinetic signature of Ca(2+) signals increase the efficiency and specificity of gene activation events. The complexity of Ca(2+) signals contains a wealth of information that may help to instruct lymphocytes to choose between alternate fates in response to antigenic stimulation.

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