Design details for overdose education and take-home naloxone kits: Codesign with family medicine, emergency department, addictions medicine and community.

INTRODUCTION: Overdose education and naloxone distribution (OEND) programmes equip and train people who are likely to witness an opioid overdose to respond with effective first aid interventions. Despite OEND expansion across North America, overdose rates are increasing, raising questions about how to improve OEND programmes. We conducted an iterative series of codesign stakeholder workshops to develop a prototype for take-home naloxone (THN)-kit (i.e., two doses of intranasal naloxone and training on how to administer it). METHODS: We recruited people who use opioids, frontline healthcare providers and public health representatives to participate in codesign workshops covering questions related to THN-kit prototypes, training on how to use it, and implementation, including refinement of design artefacts using personas and journey maps. Completed over 9 months, the workshops were audio-recorded and transcribed with visible results of the workshops (i.e., sticky notes, sketches) archived. We used thematic analyses of these materials to identify design requirements for THN-kits and training. RESULTS: We facilitated 13 codesign workshops to identify and address gaps in existing opioid overdose education training and THN-kits and emphasize timely response and stigma in future THN-kit design. Using an iterative process, we created 15 prototypes, 3 candidate prototypes and a final prototype THN-kit from the synthesis of the codesign workshops. CONCLUSION: The final prototype is available for a variety of implementation and evaluation processes. The THN-kit offers an integrated solution combining ultra-brief training animation and physical packaging of nasal naloxone to be distributed in family practice clinics, emergency departments, addiction medicine clinics and community settings. PATIENT OR PUBLIC CONTRIBUTION: The codesign process was deliberately structured to involve community members (the public), with multiple opportunities for public contribution. In addition, patient/public participation was a principle for the management and structuring of the research team.

Genomic regions associated with physiological, biochemical and yield-related responses under water deficit in diploid potato at the tuber initiation stage revealed by GWAS.

Water deficit, which is increasing with climate change, is a serious threat to agricultural sustainability worldwide. Dissection of the genetic architecture of water deficit responses is highly desirable for developing water-deficit tolerant potato cultivars and enhancing the resilience of existing cultivars. This study examined genetic variation in response to water deficit in a panel of diploid potato and identified the QTL governing this trait via a genome-wide association study (GWAS). A panel of 104 diploid potato accessions were evaluated under both well-watered and water deficit treatments at tuber initiation stage. Drought stress index (DTI) was calculated to assess tolerance of the diploid potato genotypes to water deficit. The GWAS was conducted using a matrix of 47K single nucleotide polymorphisms (SNP), recently available for this population. We are reporting 38 QTL, seven for well-watered conditions, twenty-two for water deficit conditions and nine for DTI which explain between 12.6% and 44.1% of the phenotypic variance. A set of 6 QTL were found to be associated with more than one variable. Marker WDP-9.21 was found associated with tuber fresh weigh under WD and gene annotation analysis revealed co-localization with the Glucan/water dikinase (GWD) gene. Of the nine QTL detected from DTI on chromosomes 2,3,5,8,10 and 12, three candidate genes with a feasible role in water deficit response were identified. The findings of this study can be used in marker-assisted selection (MAS) for water- deficit tolerance breeding in potato.

Natronolimnobius aegyptiacus sp. nov., an extremely halophilic alkalithermophilic archaeon isolated from the athalassohaline Wadi An Natrun, Egypt.

An obligately aerobic extremely halophilic alkalithermophilic archaeon, strain JW/NM-HA 15T, was isolated from the sediments of Wadi An Natrun in Egypt. Phylogenetic analysis based on 16S rRNA and rpoB' gene sequences indicated that it belongs to the family Natrialbaceae of the order Natrialbales. The closest relatives were Natronolimnobius baerhuensis IHC-005T and Natronolimnobius innermongolicus N-1311T (95.3 and 94.5 % 16S rRNA gene sequence similarity, respectively). Genome relatedness between strain JW/NM-HA 15T and its neighbours was evaluated using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity with the values of 75.7-85.0, 18.1-20.0, and 70.2-71.0%, respectively. Cells were obligately aerobic, rod-shaped, non-motile, Gram-stain-negative and chemo-organotrophic. The strain grew in the presence of 2.57 M to saturating Na+ (optimum 3.25-4.60 M Na+), at pH55 °C 7.5-10.5 (optimum pH55 °C 9.0-9.5), and at 30-56 °C (optimum 52 °C). The major polar lipids consisted of phosphatidylglycerol, methylated phosphatidylglycerolphosphate and two phospholipids. The complete genome size of strain JW/NM-HA 15T is approximately 3.93 Mb, with a DNA G+C content of 64.1 mol%. On the basis of phylogenetic features, genomic relatedness, phenotypic and chemotaxonomic data, strain JW/NM-HA 15T was thus considered to represent a novel species within the genus Natronolimnobius, for which the name Natronolimnobius aegyptiacus sp. nov. is proposed. The type strain is JW/NM-HA 15T (=ATCC BAA-2088T =DSM 23470T).

Novel SNP markers in InvGE and SssI genes are associated with natural variation of sugar contents and frying color in Solanum tuberosum Group Phureja.

BACKGROUND: Potato frying color is an agronomic trait influenced by the sugar content of tubers. The candidate gene approach was employed to elucidate the molecular basis of this trait in Solanum tuberosum Group Phureja, which is mainly diploid and represents an important genetic resource for potato breeding. The objective of this research was to identify novel genetic variants related with frying quality in loci with key functions in carbohydrate metabolism, with the purpose of discovering genetic variability useful in breeding programs. Therefore, an association analysis was implemented with 109 SNP markers identified in ten candidate genes. RESULTS: The analyses revealed four associations in the locus InvGE coding for an apoplastic invertase and one association in the locus SssI coding for a soluble starch synthase. The SNPs SssI-C 45711901 T and InvGE-C 2475454 T were associated with sucrose content and frying color, respectively, and were not found previously in tetraploid genotypes. The rare haplotype InvGE-A 2475187 C 2475295 A 2475344 was associated with higher fructose contents. Our study allowed a more detailed analysis of the sequence variation of exon 3 from InvGE, which was not possible in previous studies because of the high frequency of insertion-deletion polymorphisms in tetraploid potatoes. CONCLUSION: The association mapping strategy using a candidate gene approach in Group Phureja allowed the identification of novel SNP markers in InvGE and SssI associated with frying color and the tuber sugar content measured by High Performance Liquid Chromatography (HPLC). These novel associations might be useful in potato breeding programs for improving quality traits and to increase crop genetic variability. The results suggest that some genes involved in the natural variation of tuber sugar content and frying color are conserved in both Phureja and tetraploid germplasm. Nevertheless, the associated variants in both types of germplasm were present in different regions of these genes. This study contributes to the understanding of the genetic architecture of tuber sugar contents and frying color at harvest in Group Phureja.

StWRKY8 transcription factor regulates benzylisoquinoline alkaloid pathway in potato conferring resistance to late blight.

The resistance to late blight is either qualitative or quantitative in nature. Quantitative resistance is durable, but challenging due to polygenic inheritance. In the present study, the diploid potato genotypes resistant and susceptible to late blight, were profiled for metabolites. Tissue specific metabolite analysis of benzylisoquinoline alkaloids (BIAs) in response to pathogen infection revealed increased accumulation of morphinone, codeine-6-glucuronide and morphine-3-glucuronides. These BIAs are antimicrobial compounds and possibly involved in cell wall reinforcement, especially through cross-linking cell wall pectins. Quantitative reverse transcription-PCR studies revealed higher expressions of TyDC, NCS, COR-2 and StWRKY8 transcription factor genes, in resistant genotypes than in susceptible genotype, following pathogen inoculation. A luciferase transient expression assay confirmed the binding of the StWRKY8 TF to promoters of downstream genes, elucidating a direct regulatory role on BIAs biosynthetic genes. Sequence analysis of StWRKY8 in potato genotypes revealed polymorphism in the WRKY DNA binding domain in the susceptible genotype, which is important for the regulatory function of this gene. A complementation assay of StWRKY8 in Arabidopsis wrky33 mutant background was associated with decreased fungal biomass. In conclusion, StWRKY8 regulates the biosynthesis of BIAs that are both antimicrobial and reinforce cell walls to contain the pathogen to initial infection.

Natural variation of sucrose, glucose and fructose contents in Colombian genotypes of Solanum tuberosum Group Phureja at harvest.

BACKGROUND: Potato frying quality is a complex trait influenced by sugar content in tubers. Good frying quality requires low content of reducing sugars to avoid the formation of dark pigments. Solanum tuberosum Group Phureja is a valuable genetic resource for breeding and for genetic studies. The sugar content after harvest was analyzed in a germplasm collection of Group Phureja to contribute to the understanding of the natural variation of this trait. RESULTS: Sucrose, glucose and fructose genotypic mean values ranged from 6.39 to 29.48 g kg(-1) tuber dry weight (DW), from 0.46 to 28.04 g kg(-1) tuber DW and from 0.29 to 27.23 g kg(-1) tuber DW, respectively. Glucose/fructose and sucrose/reducing sugars ratios ranged from 1.01 to 6.67 mol mol(-1) and from 0.15 to 7.78 mol mol(-1) , respectively. Five clusters of genotypes were recognized, three of them with few genotypes and extreme phenotypic values. CONCLUSION: Sugar content showed a wide variation, representing the available variability useful for potato breeding. The results provide a quantitative approach to analyze the frying quality trait and are consistent with frying color. The analyzed germplasm presents extreme phenotypes, which will contribute to the understanding of the genetic basis of this trait. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cold and Hot Extremozymes: Industrial Relevance and Current Trends.

The development of enzymes for industrial applications relies heavily on the use of microorganisms. The intrinsic properties of microbial enzymes, e.g., consistency, reproducibility, and high yields along with many others, have pushed their introduction into a wide range of products and industrial processes. Extremophilic microorganisms represent an underutilized and innovative source of novel enzymes. These microorganisms have developed unique mechanisms and molecular means to cope with extreme temperatures, acidic and basic pH, high salinity, high radiation, low water activity, and high metal concentrations among other environmental conditions. Extremophile-derived enzymes, or extremozymes, are able to catalyze chemical reactions under harsh conditions, like those found in industrial processes, which were previously not thought to be conducive for enzymatic activity. Due to their optimal activity and stability under extreme conditions, extremozymes offer new catalytic alternatives for current industrial applications. These extremozymes also represent the cornerstone for the development of environmentally friendly, efficient, and sustainable industrial technologies. Many advances in industrial biocatalysis have been achieved in recent years; however, the potential of biocatalysis through the use of extremozymes is far from being fully realized. In this article, the adaptations and significance of psychrophilic, thermophilic, and hyperthermophilic enzymes, and their applications in selected industrial markets will be reviewed. Also, the current challenges in the development and mass production of extremozymes as well as future prospects and trends for their biotechnological application will be discussed.

Tn-seq explorer: a tool for analysis of high-throughput sequencing data of transposon mutant libraries.

Tn-seq is a high throughput technique for analysis of transposon mutant libraries. Tn-seq Explorer was developed as a convenient and easy-to-use package of tools for exploration of the Tn-seq data. In a typical application, the user will have obtained a collection of sequence reads adjacent to transposon insertions in a reference genome. The reads are first aligned to the reference genome using one of the tools available for this task. Tn-seq Explorer reads the alignment and the gene annotation, and provides the user with a set of tools to investigate the data and identify possibly essential or advantageous genes as those that contain significantly low counts of transposon insertions. Emphasis is placed on providing flexibility in selecting parameters and methodology most appropriate for each particular dataset. Tn-seq Explorer is written in Java as a menu-driven, stand-alone application. It was tested on Windows, Mac OS, and Linux operating systems. The source code is distributed under the terms of GNU General Public License. The program and the source code are available for download at http://www.cmbl.uga.edu/downloads/programs/Tn_seq_Explorer/ and https://github.com/sina-cb/Tn-seqExplorer.

Metabolomics deciphers quantitative resistance mechanisms in diploid potato clones against late blight.

Resistance to late blight in potato is either qualitative or quantitative in nature. The quantitative resistance is durable, but the molecular and biochemical mechanisms underlying quantitative resistance are poorly understood, and are not efficiently utilised in potato breeding. A non-targeted metabolomics, using high resolution hybrid mass spectrometry, was applied to decipher the mechanisms of resistance in the advanced breeding diploid potato genotypes (Solanum tuberosum L. Group Phureja), with valuable sources of genetic diversity. The metabolomics profiles of resistant genotypes (AC04 and AC09) were compared with a susceptible commercial genotype (Criolla Colombia), following Phytophthora infestans or mock-inoculation, to identify the resistance related (RR) metabolites. Metabolites belonging to phenylpropanoids, flavonoid and alkaloid chemical groups were highly induced in resistant genotypes relative to susceptible. Concurrently, the biosynthetic genes, tyrosine decarboxylase (TyDC) and tyramine hydroxycinnamoyl transferase (THT), involved in the biosynthesis of hydroxycinnamic acid amides (HCAAs), and chalcone synthase (CHS) and flavonol synthase (FLS), involved in flavonoid biosynthesis, were also upregulated, as confirmed by quantitative real-time PCR. Probable genes coding for these enzymes were sequenced and nonsynonymous single-nucleotide polymorphisms (nsSNPs) were identified. The resistance to late blight observed in this study was mainly associated with cell wall thickening due to deposition of HCAAs, flavonoids and alkaloids.

Diversity of the DNA replication system in the Archaea domain.

The precise and timely duplication of the genome is essential for cellular life. It is achieved by DNA replication, a complex process that is conserved among the three domains of life. Even though the cellular structure of archaea closely resembles that of bacteria, the information processing machinery of archaea is evolutionarily more closely related to the eukaryotic system, especially for the proteins involved in the DNA replication process. While the general DNA replication mechanism is conserved among the different domains of life, modifications in functionality and in some of the specialized replication proteins are observed. Indeed, Archaea possess specific features unique to this domain. Moreover, even though the general pattern of the replicative system is the same in all archaea, a great deal of variation exists between specific groups.

Genetic confirmation of the role of sulfopyruvate decarboxylase in coenzyme M biosynthesis in Methanococcus maripaludis.

Coenzyme M is an essential coenzyme for methanogenesis. The proposed biosynthetic pathway consists of five steps, of which the fourth step is catalyzed by sulfopyruvate decarboxylase (ComDE). Disruption of the gene comE by transposon mutagenesis resulted in a partial coenzyme M auxotroph, which grew poorly in the absence of coenzyme M and retained less than 3% of the wild type level of coenzyme M biosynthesis. Upon coenzyme M addition, normal growth of the mutant was restored. Moreover, complementation of the mutation with the wild type comE gene in trans restored full growth in the absence of coenzyme M. These results confirm that ComE plays an important role in coenzyme M biosynthesis. The inability to yield a complete CoM auxotroph suggests that either the transposon insertion failed to completely inactivate the gene or M. maripaludis possesses a promiscuous activity that partially complemented the mutation.

Genome-scale analysis of gene function in the hydrogenotrophic methanogenic archaeon Methanococcus maripaludis.

A comprehensive whole-genome analysis of gene function by transposon mutagenesis and deep sequencing methodology has been implemented successfully in a representative of the Archaea domain. Libraries of transposon mutants were generated for the hydrogenotrophic, methanogenic archaeon Methanococcus maripaludis S2 using a derivative of the Tn5 transposon. About 89,000 unique insertions were mapped to the genome, which allowed for the classification of 526 genes or about 30% of the genome as possibly essential or strongly advantageous for growth in rich medium. Many of these genes were homologous to eukaryotic genes that encode fundamental processes in replication, transcription, and translation, providing direct evidence for their importance in Archaea. Some genes classified as possibly essential were unique to the archaeal or methanococcal lineages, such as that encoding DNA polymerase PolD. In contrast, the archaeal homolog to the gene encoding DNA polymerase B was not essential for growth, a conclusion confirmed by construction of an independent deletion mutation. Thus PolD, and not PolB, likely plays a fundamental role in DNA replication in methanococci. Similarly, 121 hypothetical ORFs were classified as possibly essential and likely play fundamental roles in methanococcal information processing or metabolism that are not established outside this group of prokaryotes.

The BBX subfamily IV: additional cogs and sprockets to fine-tune light-dependent development.

Plants depend on light during all phases of its life cycle, and have evolved a complex signaling network to constantly monitor its surroundings. Photomorphogenesis, a process during which the plant reprograms itself in order to dwell life in presence of light is one of the most studied phenomena in plants. Recent mutant analyses using model plant Arabidopsis thaliana and protein interaction assays have unraveled a new set of players, an 8-member subfamily of B-box proteins, known as BBX subfamily IV. For the members of this subfamily, positive (BBX21, BBX22) as well as negative (BBX24) functions have been described for its members, showing a strong association to two major players of the photomorphogenic cascade, HY5 and COP1. The roles of these new BBX regulators are not restricted to photomorphogenesis, but also have functions in other facets of light-dependent development. Therefore this newly identified set of regulators has opened up new insights into the understanding of the fine-tuning of this complex process.

Genetic systems for hydrogenotrophic methanogens.

Methanogens are obligate anaerobic Archaea that produce energy from the biosynthesis of methane. These lithotrophic microorganisms are widely distributed in oxygen-free environments and participate actively in the carbon cycle. Indeed, methanogenesis plays a major role in the last step of the anoxic degradation of organic substances, transforming acetate, CO(2), and H(2) to methane. The vast majority of the known methanogens are classified as hydrogenotrophic because they use principally H(2) as the electron donor to drive the reduction of CO(2). Unlike many other cultured Archaea, many methanogens thrive in neutral pH, low salinity, and temperate environments. This has been a great advantage in cultivating these organisms in laboratory conditions and in the development of genetic tools. Moreover, the hydrogenotroph Methanococcus maripaludis is currently a model organism among Archaea, not only for its utility in genetic but also for biochemical and physiological studies. Over time, a broad spectrum of genetic tools and techniques has been developed for methanococci, such as site-directed mutagenesis, selectable markers, transformation methods, and reporter genes. These tools have contributed greatly to the overall understanding of this group of microorganisms and the processes that govern its life style. In this chapter, we describe in detail the available genetic tools for the hydrogenotrophic methanogens.

Putative storage root specific promoters from cassava and yam: cloning and evaluation in transgenic carrots as a model system.

A prerequisite for biotechnological improvements of storage roots is the availability of tissue-specific promoters enabling high expression of transgenes. In this work, we cloned two genomic fragments, pMe1 and pDJ3S, controlling the expression of a gene with unknown function from cassava (Manihot esculenta) and of the storage protein dioscorin 3 small subunit gene from yam (Dioscorea japonica), respectively. Using beta-glucuronidase as a reporter, the activities of pMe1 and pDJ3S were evaluated in independent transgenic carrot lines and compared to the constitutive CaMV35S and the previously described cassava p15 promoters. Activities of pMe1 and pDJ3S in storage roots were assessed using quantitative GUS assays that showed pDJ3S as the most active one. To determine organ specificities, uidA transcript levels in leaves, stems and roots were measured by real-time RT-PCR analyses showing highest storage root specificity for pDJ3S. Root cross sections revealed that pMe1 was highly active in secondary xylem. In contrast, pDJ3S was active in all root tissues except for the central xylem. The expression patterns caused by the cassava p15 promoter in carrot storage roots were consistent with its previously described activities for the original storage organ. Our data demonstrate that the pDJ3S and, to a lesser extent, the pMe1 regulatory sequences represent feasible candidates to drive high and preferential expression of genes in carrot storage roots.

Does light taste salty?

As research advances acquisition of new data reveals novel aspects on already investigated issues. This is the case for SALT TOLERANCE (STO), an Arabidopsis protein that confers tolerance to high salt concentrations when ectopically expressed in yeast cells. For the last years, STO was considered to participate mainly in the response and tolerance of Arabidopsis to high salinity, as it does in yeast. However, recent investigations using gain- and loss-of-function mutants revealed a major role for STO as negative regulator of photomorphogenesis. Interestingly, and contrary to other negative regulators of light dependent inhibition of hypocotyl elongation, STO protein instability is controlled by COP1 activity in etiolated seedlings. Thus, light stabilizes STO protein levels during de-etiolation. Whether STO participates in other signaling cascades different from light signaling, as it has been shown in yeast and proposed in plants or not, is still an open question.