Retrospective review of SGLT2 inhibitor exposures reported to 13 poison centers.

BACKGROUND: SGLT2 inhibitors are a new class of oral antidiabetics prescribed in the United States since 2013. They act by inhibiting reabsorption of glucose in the proximal convoluted tubule of the kidney, allowing excess glucose to be excreted. Little has been reported regarding effects of non-therapeutic exposure to this class of medication. METHODS: Retrospective records from 13 poison centers were examined for human exposures to SGLT2 inhibitors between 1st January 2013 and 31st December 2016. Exclusion criteria included multi-substance exposures and exposures without any follow-up call. Data examined included patient age, chronicity of exposure, clinical effects, management site, treatments administered, duration of follow-up, and outcome. RESULTS: Eighty-eight cases met inclusion criteria. Patient age ranged from 1 to 75 years; 49 were evaluated in a health care facility with 18 admissions. No symptoms developed in 80 (91%) patients, 6 (7%) developed minor symptoms, and 2 (2%) developed moderate symptoms. Hypoglycemia was not observed. Mean time to final follow-up was 9.3 h, ranging from 1 to 42 h; median was 6 h. Of the two patients who developed moderate symptoms, one was a 65 year old male who developed metabolic acidosis and hypokalemia while taking canagliflozin therapeutically; the other a 43-year-old female who developed tachycardia and mild hypertension following the intentional ingestion of 6000 mg of canagliflozin. DISCUSSIONS: The number of patients evaluated in a health care facility is most likely reflective of a cautious approach to dealing with a new class of drug. Exposures were generally well-tolerated and managed with minimal intervention. CONCLUSIONS: In this retrospective series, acute ingestions of SGLT2 inhibitors were well-tolerated with no hypoglycemia and only minor effects. For young children with unintentional ingestions, a reasonable approach to home management would include at least one follow-up for signs and symptoms of possible toxicity including mental status changes, polyuria, or tachypnea.

Comparative ultrastructure of fruit plastids in three genetically diverse genotypes of apple (Malus × domestica Borkh.) during development.

KEY MESSAGE: Comparative ultrastructural developmental time-course analysis has identified discrete stages at which the fruit plastids undergo structural and consequently functional transitions to facilitate subsequent development-guided understanding of the complex plastid biology. Plastids are the defining organelle for a plant cell and are critical for myriad metabolic functions. The role of leaf plastid, chloroplast, is extensively documented; however, fruit plastids-chromoplasts-are poorly understood, especially in the context of the diverse metabolic processes operating in these diverse plant organs. Recently, in a comparative study of the predicted plastid-targeted proteomes across seven plant species, we reported that each plant species is predicted to harbor a unique set of plastid-targeted proteins. However, the temporal and developmental context of these processes remains unknown. In this study, an ultrastructural analysis approach was used to characterize fruit plastids in the epidermal and collenchymal cell layers at 11 developmental timepoints in three genotypes of apple (Malus × domestica Borkh.): chlorophyll-predominant 'Granny Smith', carotenoid-predominant 'Golden Delicious', and anthocyanin-predominant 'Top Red Delicious'. Plastids transitioned from a proplastid-like plastid to a chromoplast-like plastid in epidermis cells, while in the collenchyma cells, they transitioned from a chloroplast-like plastid to a chloro-chromo-amyloplast plastid. Plastids in the collenchyma cells of the three genotypes demonstrated a diverse array of structures and features. This study enabled the identification of discrete developmental stages during which specific functions are most likely being performed by the plastids as indicated by accumulation of plastoglobuli, starch granules, and other sub-organeller structures. Information regarding the metabolically active developmental stages is expected to facilitate biologically relevant omics studies to unravel the complex biochemistry of plastids in perennial non-model systems.

The expanding footprint of CRISPR/Cas9 in the plant sciences.

CRISPR/Cas9 has evolved and transformed the field of biology at an unprecedented pace. From the initial purpose of introducing a site specific mutation within a genome of choice, this technology has morphed into enabling a wide array of molecular applications, including site-specific transgene insertion and multiplexing for the simultaneous induction of multiple cleavage events. Efficiency, specificity, and flexibility are key attributes that have solidified CRISPR/Cas9 as the genome-editing tool of choice by scientists from all areas of biology. Within the field of plant biology, several CRISPR/Cas9 technologies, developed in other biological systems, have been successfully implemented to probe plant gene function and to modify specific crop traits. It is anticipated that this trend will persist and lead to the development of new applications and modifications of the CRISPR technology, adding to an ever-expanding collection of genome-editing tools. We envision that these tools will bestow plant researchers with new utilities to alter genome complexity, engineer site-specific integration events, control gene expression, generate transgene-free edited crops, and prevent or cure plant viral disease. The successful implementation of such utilities will represent a new frontier in plant biotechnology.

An Observational Study of the Factor Xa Inhibitors Rivaroxaban and Apixaban as Reported to Eight Poison Centers.

STUDY OBJECTIVE: Rivaroxaban and apixaban are part of a new group of oral anticoagulants targeting factor Xa and approved by the Food and Drug Administration in 2011 and 2012. These oral anticoagulants are administered at fixed daily doses, without the need for laboratory-guided adjustments. There are limited data available on supratherapeutic doses or overdose of the oral Xa inhibitors. This study characterizes the clinical effect in patients exposed to rivaroxaban and apixaban. METHODS: A retrospective study collected data from 8 regional poison centers covering 9 states. Cases were initially identified by a search of the poison centers' databases for case mentions involving a human exposure to Xarelto, rivaroxaban, Eliquis, or apixaban. Inclusion criteria included single-substance exposure. Exclusion criteria were animal exposure, polysubstance exposure, or information call. Data for the study were collected by individual chart review, including case narratives, and compiled into a single data set. RESULTS: There were 223 patients: 124 (56%) were female patients, mean age was 60 years, and 20 were children younger than 12 years (9%). One hundred ninety-eight patients ingested rivaroxaban (89%) and 25 ingested apixaban (11%). Dose was reported in 182 rivaroxaban patients, with a mean dose of 64.5 mg (range 15 to 1,200 mg), and in 21 apixaban patients, with a mean dose of 9.6 mg (range 2.5 to 20 mg). For rivaroxaban, prothrombin time was measured in 49 patients (25%) and elevated in 7; partial thromboplastin time, measured in 49 (25%) and elevated in 5; and international normalized ratio, measured in 61 (31%) and elevated in 13. For apixaban, prothrombin time was measured in 6 patients (24%) and elevated in none; partial thromboplastin time, measure in 6 (24%) and elevated in none; and international normalized ratio, measured in 5 patients (20%) and elevated in none. Bleeding was reported in 15 patients (7%): 11 rivaroxaban and 4 apixaban. The site of bleeding was gastrointestinal (8), oral (2), nose (1), bruising (1), urine (1), and subdural (1). The subdural bleeding occurred after fall and head injury. All cases with bleeding involved long-term ingestions. Coagulation test results were normal in most patients with bleeding: prothrombin time 5 of 6 (83%), partial thromboplastin time 5 of 6 (83%), and international normalized ratio 5 of 9 (55%). Blood products were used in 7 rivaroxaban patients (1 suicide) and 3 apixaban patients. No bleeding or altered coagulation test results occurred in children, which all involved a one-time ingestion. All 12 suicide attempts involved rivaroxaban: altered coagulation test results occurred for 5 patients (42%), no bleeding occurred in any suicide attempt patient, 1 patient was treated with fresh frozen plasma (international normalized ratio 12.47), and dose by patient history did not predict risk of altered coagulation or bleeding. Two rivaroxaban patients experienced elevation of hepatic transaminase levels greater than 1,000 U/L. CONCLUSION: Bleeding after Xa inhibitor ingestion as a single agent is uncommon. Prothrombin time, partial thromboplastin time, or international normalized ratio may be elevated in a minority of cases but appears unreliable to measure risk of bleeding. Massive acute ingestion in suicide attempt may result in significant anticoagulation. Single exploratory ingestion by children was not associated with toxicity.

CRISPR/Cas9-mediated genome editing and gene replacement in plants: Transitioning from lab to field.

The CRISPR/Cas9 genome engineering system has ignited and swept through the scientific community like wildfire. Owing largely to its efficiency, specificity, and flexibility, the CRISPR/Cas9 system has quickly become the preferred genome-editing tool of plant scientists. In plants, much of the early CRISPR/Cas9 work has been limited to proof of concept and functional studies in model systems. These studies, along with those in other fields of biology, have led to the development of several utilities of CRISPR/Cas9 beyond single gene editing. Such utilities include multiplexing for inducing multiple cleavage events, controlling gene expression, and site specific transgene insertion. With much of the conceptual CRISPR/Cas9 work nearly complete, plant researchers are beginning to apply this gene editing technology for crop trait improvement. Before rational strategies can be designed to implement this technology to engineer a wide array of crops there is a need to expand the availability of crop-specific vectors, genome resources, and transformation protocols. We anticipate that these challenges will be met along with the continued evolution of the CRISPR/Cas9 system particularly in the areas of manipulation of large genomic regions, transgene-free genetic modification, development of breeding resources, discovery of gene function, and improvements upon CRISPR/Cas9 components. The CRISPR/Cas9 editing system appears poised to transform crop trait improvement.

Comparative analysis of predicted plastid-targeted proteomes of sequenced higher plant genomes.

Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome (Malus × domestica) to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis (Arabidopsis thaliana) plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding plastid biology, preferentially based on model systems is deficient. New plant genomes are expected to enable the identification of potentially new plastid-targeted proteins that will aid in studying novel roles of plastids.

Evaluation of dabigatran exposures reported to poison control centers.

BACKGROUND: Dabigatran is a novel oral anticoagulant for which a well-defined range of toxicity and proven antidote has not been established. OBJECTIVE: The primary objective of this study was to characterize dabigatran exposures reported to poison centers by dose ingested, clinical effects, treatments used, and managment sites to gain a better understanding of patient outcomes. METHODS: A retrospective database review was conducted for dabigatran exposures reported to the National Poison Data System for the American Association of Poison Control Centers (AAPCC) over the period October 2010 to December 2012. RESULTS: There were 802 human dabigatran exposures involving adults predominantly (91% of cases). Exposure chronicity was acute in 43%, acute-on-chronic in 46%, and chronic in 11%, with the most common reason for an exposure call being an unintentional therapeutic error (70.6%). The most common management sites were on-site in 72% of cases and within a health care facility for 26%. Bleeding events and coagulopathies were the most commonly observed clinical effects. Treatments administered included activated charcoal, blood and coagulation products, hemodialysis, and supportive measures. Confirmed outcomes included death in 13 patients (1.6%), major effects in 23 (2.9%), and moderate effects in 50 (6.2%). More severe outcomes were significantly associated with adverse drug reactions, patients ≥65 years of age, those treated with blood and coagulation products and/or dialysis, and renal dysfunction (P < .05). Children experienced few moderate effects and no major effects or deaths. CONCLUSIONS: Severe outcomes from dabigatran exposures were not common, occurring in approximately 5% of cases.

Application of Cydia pomonella expressed sequence tags: Identification and expression of three general odorant binding proteins in codling moth.

The codling moth, Cydia pomonella, is one of the most important pests of pome fruits in the world, yet the molecular genetics and the physiology of this insect remain poorly understood. A combined assembly of 8 341 expressed sequence tags was generated from Roche 454 GS-FLX sequencing of eight tissue-specific cDNA libraries. Putative chemosensory proteins (12) and odorant binding proteins (OBPs) (18) were annotated, which included three putative general OBP (GOBP), one more than typically reported for other Lepidoptera. To further characterize CpomGOBPs, we cloned cDNA copies of their transcripts and determined their expression patterns in various tissues. Cloning and sequencing of the 698 nt transcript for CpomGOBP1 resulted in the prediction of a 163 amino acid coding region, and subsequent RT-PCR indicated that the transcripts were mainly expressed in antennae and mouthparts. The 1 289 nt (160 amino acid) CpomGOBP2 and the novel 702 nt (169 amino acid) CpomGOBP3 transcripts are mainly expressed in antennae, mouthparts, and female abdomen tips. These results indicate that next generation sequencing is useful for the identification of novel transcripts of interest, and that codling moth expresses a transcript encoding for a new member of the GOBP subfamily.

Comparative genomics analysis in Prunoideae to identify biologically relevant polymorphisms.

Prunus is an economically important genus with a wide range of physiological and biological variability. Using the peach genome as a reference, sequencing reads from four almond accessions and one sweet cherry cultivar were used for comparative analysis of these three Prunus species. Reference mapping enabled the identification of many biological relevant polymorphisms within the individuals. Examining the depth of the polymorphisms and the overall scaffold coverage, we identified many potentially interesting regions including hundreds of small scaffolds with no coverage from any individual. Non-sense mutations account for about 70 000 of the 13 million identified single nucleotide polymorphisms (SNPs). Blast2GO analyses on these non-sense SNPs revealed several interesting results. First, non-sense SNPs were not evenly distributed across all gene ontology terms. Specifically, in comparison with peach, sweet cherry is found to have non-sense SNPs in two 1-aminocyclopropane-1-carboxylate synthase (ACS) genes and two 1-aminocyclopropane-1-carboxylate oxidase (ACO) genes. These polymorphisms may be at the root of the nonclimacteric ripening of sweet cherry. A set of candidate genes associated with bitterness in almond were identified by comparing sweet and bitter almond sequences. To the best of our knowledge, this is the first report in plants of non-sense SNP abundance in a genus being linked to specific GO terms.

Multiple lead appendoliths following ingestion of lead shot: time course and removal by laporoscopic appendectomy.

Systemic lead poisoning may occur from ingested lead foreign bodies. Rarely, these may cause a retained appendolith. The risks of lead toxicity from these appendoliths, the time course of retention in the appendix, the rapidity of inhibition of heme synthesis, and the best approach to removal remain controversial. Fourteen days post-ingestion of a "handful" of lead shot, a 15-year-old male was admitted for elevated lead level; an x-ray showed aggregation of many pellets in the teen's appendix. A laparoscopic appendectomy was performed; examination of the removed appendix revealed greater than 50 retained pellets. An abdominal film obtained post-procedure showed removal of all but three of the pellets. Results from admission blood work found a blood lead level of 41 mcg/dL and free erythrocyte porphyrin (FEP) level of 114 µmol/mol heme (reference normal <70). After a short recovery the patient was discharged on succimer chelation therapy and on follow up, his lead level was <5 mcg/dL.

Rapid gene-based SNP and haplotype marker development in non-model eukaryotes using 3'UTR sequencing.

BACKGROUND: Sweet cherry (Prunus avium L.), a non-model crop with narrow genetic diversity, is an important member of sub-family Amygdoloideae within Rosaceae. Compared to other important members like peach and apple, sweet cherry lacks in genetic and genomic information, impeding understanding of important biological processes and development of efficient breeding approaches. Availability of single nucleotide polymorphism (SNP)-based molecular markers can greatly benefit breeding efforts in such non-model species. RNA-seq approaches employing second generation sequencing platforms offer a unique avenue to rapidly identify gene-based SNPs. Additionally, haplotype markers can be rapidly generated from transcript-based SNPs since they have been found to be extremely utile in identification of genetic variants related to health, disease and response to environment as highlighted by the human HapMap project. RESULTS: RNA-seq was performed on two sweet cherry cultivars, Bing and Rainier using a 3' untranslated region (UTR) sequencing method yielding 43,396 assembled contigs. In order to test our approach of rapid identification of SNPs without any reference genome information, over 25% (10,100) of the contigs were screened for the SNPs. A total of 207 contigs from this set were identified to contain high quality SNPs. A set of 223 primer pairs were designed to amplify SNP containing regions from these contigs and high resolution melting (HRM) analysis was performed with eight important parental sweet cherry cultivars. Six of the parent cultivars were distantly related to Bing and Rainier, the cultivars used for initial SNP discovery. Further, HRM analysis was also performed on 13 seedlings derived from a cross between two of the parents. Our analysis resulted in the identification of 84 (38.7%) primer sets that demonstrated variation among the tested germplasm. Reassembly of the raw 3'UTR sequences using upgraded transcriptome assembly software yielded 34,620 contigs containing 2243 putative SNPs in 887 contigs after stringent filtering. Contigs with multiple SNPs were visually parsed to identify 685 putative haplotypes at 335 loci in 301 contigs. CONCLUSIONS: This approach, which leverages the advantages of RNA-seq approaches, enabled rapid generation of gene-linked SNP and haplotype markers. The general approach presented in this study can be easily applied to other non-model eukaryotes irrespective of the ploidy level to identify gene-linked polymorphisms that are expected to facilitate efficient Gene Assisted Breeding (GAB), genotyping and population genetics studies. The identified SNP haplotypes reveal some of the allelic differences in the two sweet cherry cultivars analyzed. The identification of these SNP and haplotype markers is expected to significantly improve the genomic resources for sweet cherry and facilitate efficient GAB in this non-model crop.

The genome of the domesticated apple (Malus × domestica Borkh.).

We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.

A beta-barrel outer membrane protein facilitates cellular uptake of polychlorophenols in Cupriavidus necator.

The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a beta-barrel outer membrane protein, similar to the Escherichia coli outer membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an outer membrane protein that facilitates the passing of polychlorophenols across the outer membrane of C. necator. Similarly, other COG4313 proteins are possibly outer membrane transporters of hydrophobic aromatic compounds.

Multiple seizures after bupropion overdose in a small child.

Unintentional ingestion of bupropion in young children has generally resulted in limited toxicity. We report a case of pediatric bupropion ingestion resulting in multiple seizures. The patient experienced hallucinations, agitation, vomiting, tachycardia and seizures after ingestion of 1050 (48 mg/kg) of extended-release bupropion. The potential for severe toxicity in the setting of pediatric overdose should be recognized.

Salicylate toxicity associated with administration of Percy medicine in an infant.

Percy Medicine is a nonprescription gastrointestinal suspension containing bismuth subsalicylate as the active ingredient (1050 mg/10-ml dose). A 3-month-old infant with colic developed salicylate toxicity requiring hospitalization in the pediatric intensive care unit (PICU) as a result of continued administration of this medicine. Bismuth subsalicylate has an aspirin equivalency conversion factor of 0.479 (approximately half the strength of aspirin). For 3.5 weeks the infant's parents administered the medicine, which provided the equivalent of aspirin 57-84 mg/kg/day with no reported problems. However, on the day of admission the baby presented with central nervous system depression and respiratory distress. Assessment at a local emergency facility revealed metabolic acidosis; his serum salicylate concentration was 747 mg/L. After acute management, the patient was transferred to our hospital, where he was treated with whole bowel irrigation and alkalinization therapy. Subsequently, the baby required 4 days of management in the PICU and 2 additional days of observation in a general nursing unit before he was discharged home without incident. The parents had chosen Percy Medicine based on the picture of a baby on the front of the package and because of its placement on the shelf next to a drug their family physician had recommended previously. Salicylate-containing products are not routinely recommended for children aged 1 year or younger. The general public may assume that over-the-counter products are safe because they do not require a prescription. Health care professionals must be responsible for educating the public regarding risks associated with over-the-counter products and the need to read and follow label directions.