Phaseolus vulgaris STP13.1 is an H+-coupled monosaccharide transporter, present in source leaves and seed coats, with higher substrate affinity at depolarized potentials.

Sugar transport proteins (STPs) are high-affinity H+-coupled hexose symporters. Recently, the contribution of STP13 to bacterial and fungal pathogen resistance across multiple plant species has garnered significant interest. Quantitative PCR analysis of source leaves, developing embryos, and seed coats of Phaseolus vulgaris L. (common bean) revealed that PvSTP13.1 was expressed in source leaves and seed coats throughout seed development. In contrast, PvSTP13.1 transcripts were detected at exceedingly low levels in developing embryos. To characterize the transport mechanism, PvSTP13.1 was expressed in Xenopus laevis oocytes, and inward-directed currents were analyzed using two-electrode voltage clamping. PvSTP13.1 was shown to function as an H+-coupled monosaccharide symporter exhibiting a unique high affinity for hexoses and aldopentoses at depolarized membrane potentials. Specifically, of the 31 assessed substrates, which included aldohexoses, deoxyhexoses, fructose, 3-O-methyl-D-glucose, aldopentoses, polyols, glycosides, disaccharides, trisaccharides, and glucuronic acid, PvSTP13.1 displayed the highest affinity (K 0.5) for glucose (43 µM), mannose (92 µM), galactose (145 µM), fructose (224 µM), xylose (1.0 mM), and fucose (3.7 mM) at pH 5.6 at a depolarized membrane potential of -40 mV. The results presented here suggest PvSTP13.1 contributes to retrieval of hexoses from the apoplasmic space in source leaves and coats of developing seeds.

Miniature Inverted-Repeat Transposable Elements: Small DNA Transposons That Have Contributed to Plant MICRORNA Gene Evolution.

Angiosperms form the largest phylum within the Plantae kingdom and show remarkable genetic variation due to the considerable difference in the nuclear genome size of each species. Transposable elements (TEs), mobile DNA sequences that can amplify and change their chromosome position, account for much of the difference in nuclear genome size between individual angiosperm species. Considering the dramatic consequences of TE movement, including the complete loss of gene function, it is unsurprising that the angiosperms have developed elegant molecular strategies to control TE amplification and movement. Specifically, the RNA-directed DNA methylation (RdDM) pathway, directed by the repeat-associated small-interfering RNA (rasiRNA) class of small regulatory RNA, forms the primary line of defense to control TE activity in the angiosperms. However, the miniature inverted-repeat transposable element (MITE) species of TE has at times avoided the repressive effects imposed by the rasiRNA-directed RdDM pathway. MITE proliferation in angiosperm nuclear genomes is due to their preference to transpose within gene-rich regions, a pattern of transposition that has enabled MITEs to gain further transcriptional activity. The sequence-based properties of a MITE results in the synthesis of a noncoding RNA (ncRNA), which, after transcription, folds to form a structure that closely resembles those of the precursor transcripts of the microRNA (miRNA) class of small regulatory RNA. This shared folding structure results in a MITE-derived miRNA being processed from the MITE-transcribed ncRNA, and post-maturation, the MITE-derived miRNA can be used by the core protein machinery of the miRNA pathway to regulate the expression of protein-coding genes that harbor homologous MITE insertions. Here, we outline the considerable contribution that the MITE species of TE have made to expanding the miRNA repertoire of the angiosperms.

Deletion of Sod1 in Motor Neurons Exacerbates Age-Related Changes in Axons and Neuromuscular Junctions in Mice.

Whole-body knock-out of Cu,Zn superoxide dismutase (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with neuromuscular junction (NMJ) breakdown similar to sarcopenia. In order to determine whether altered redox in motor neurons underlies this phenotype, an inducible neuron-specific deletion of Sod1 (i-mnSod1KO) was compared with wild-type (WT) mice of different ages (adult, mid-age, and old) and whole-body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Tamoxifen-induced deletion of neuronal Sod1 from two months of age. No specific effect of a lack of neuronal Sod1 was seen on markers of nerve oxidation (electron paramagnetic resonance of an in vivo spin probe, protein carbonyl, or protein 3-nitrotyrosine contents). i-mnSod1KO mice showed increased denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice displayed a simpler structure than that seen in adult or old WT mice. Thus, previous work showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, and we report that this deletion leads to a specific nerve phenotype including reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity. Other changes in nerve and NMJ structure seen in the old i-mnSod1KO mice reflect aging of the mice.

Genetic Variants Associated with Long-Terminal Repeats Can Diagnostically Classify Cannabis Varieties.

Cannabis sativa (Cannabis) has recently been legalized in multiple countries globally for either its recreational or medicinal use. This, in turn, has led to a marked increase in the number of Cannabis varieties available for use in either market. However, little information currently exists on the genetic distinction between adopted varieties. Such fundamental knowledge is of considerable value and underpins the accelerated development of both a nascent pharmaceutical industry and the commercial recreational market. Therefore, in this study, we sought to assess genetic diversity across 10 Cannabis varieties by undertaking a reduced representation shotgun sequencing approach on 83 individual plants to identify variations which could be used to resolve the genetic structure of the assessed population. Such an approach also allowed for the identification of the genetic features putatively associated with the production of secondary metabolites in Cannabis. Initial analysis identified 3608 variants across the assessed population with phylogenetic analysis of this data subsequently enabling the confident grouping of each variety into distinct subpopulations. Within our dataset, the most diagnostically informative single nucleotide polymorphisms (SNPs) were determined to be associated with the long-terminal repeat (LTRs) class of retroelements, with 172 such SNPs used to fully resolve the genetic structure of the assessed population. These 172 SNPs could be used to design a targeted resequencing panel, which we propose could be used to rapidly screen different Cannabis plants to determine genetic relationships, as well as to provide a more robust, scientific classification of Cannabis varieties as the field moves into the pharmaceutical sphere.

Skeletal muscle transcriptomics identifies common pathways in nerve crush injury and ageing.

Motor unit remodelling involving repeated denervation and re-innervation occurs throughout life. The efficiency of this process declines with age contributing to neuromuscular deficits. This study investigated differentially expressed genes (DEG) in muscle following peroneal nerve crush to model motor unit remodelling in C57BL/6 J mice. Muscle RNA was isolated at 3 days post-crush, RNA libraries were generated using poly-A selection, sequenced and analysed using gene ontology and pathway tools. Three hundred thirty-four DEG were found in quiescent muscle from (26mnth) old compared with (4-6mnth) adult mice and these same DEG were present in muscle from adult mice following nerve crush. Peroneal crush induced 7133 DEG in muscles of adult and 699 DEG in muscles from old mice, although only one DEG (ZCCHC17) was found when directly comparing nerve-crushed muscles from old and adult mice. This analysis revealed key differences in muscle responses which may underlie the diminished ability of old mice to repair following nerve injury.

Molecular Manipulation of the miR396 and miR399 Expression Modules Alters the Response of Arabidopsis thaliana to Phosphate Stress.

In plant cells, the molecular and metabolic processes of nucleic acid synthesis, phospholipid production, coenzyme activation and the generation of the vast amount of chemical energy required to drive these processes relies on an adequate supply of the essential macronutrient, phosphorous (P). The requirement of an appropriate level of P in plant cells is evidenced by the intricately linked molecular mechanisms of P sensing, signaling and transport. One such mechanism is the posttranscriptional regulation of the P response pathway by the highly conserved plant microRNA (miRNA), miR399. In addition to miR399, numerous other plant miRNAs are also required to respond to environmental stress, including miR396. Here, we exposed Arabidopsis thaliana (Arabidopsis) transformant lines which harbor molecular modifications to the miR396 and miR399 expression modules to phosphate (PO4) starvation. We show that molecular alteration of either miR396 or miR399 abundance afforded the Arabidopsis transformant lines different degrees of tolerance to PO4 starvation. Furthermore, RT-qPCR assessment of PO4-starved miR396 and miR399 transformants revealed that the tolerance displayed by these plant lines to this form of abiotic stress most likely stemmed from the altered expression of the target genes of these two miRNAs. Therefore, this study forms an early step towards the future development of molecularly modified plant lines which possess a degree of tolerance to growth in a PO4 deficient environment.

Cannabis sativa: Interdisciplinary Strategies and Avenues for Medical and Commercial Progression Outside of CBD and THC.

Cannabis sativa (Cannabis) is one of the world's most well-known, yet maligned plant species. However, significant recent research is starting to unveil the potential of Cannabis to produce secondary compounds that may offer a suite of medical benefits, elevating this unique plant species from its illicit narcotic status into a genuine biopharmaceutical. This review summarises the lengthy history of Cannabis and details the molecular pathways that underpin the production of key secondary metabolites that may confer medical efficacy. We also provide an up-to-date summary of the molecular targets and potential of the relatively unknown minor compounds offered by the Cannabis plant. Furthermore, we detail the recent advances in plant science, as well as synthetic biology, and the pharmacology surrounding Cannabis. Given the relative infancy of Cannabis research, we go on to highlight the parallels to previous research conducted in another medically relevant and versatile plant, Papaver somniferum (opium poppy), as an indicator of the possible future direction of Cannabis plant biology. Overall, this review highlights the future directions of cannabis research outside of the medical biology aspects of its well-characterised constituents and explores additional avenues for the potential improvement of the medical potential of the Cannabis plant.

MicroRNA-Mediated Responses to Cadmium Stress in Arabidopsis thaliana.

In recent decades, the presence of cadmium (Cd) in the environment has increased significantly due to anthropogenic activities. Cd is taken up from the soil by plant roots for its subsequent translocation to shoots. However, Cd is a non-essential heavy metal and is therefore toxic to plants when it over-accumulates. MicroRNA (miRNA)-directed gene expression regulation is central to the response of a plant to Cd stress. Here, we document the miRNA-directed response of wild-type Arabidopsis thaliana (Arabidopsis) plants and the drb1, drb2 and drb4 mutant lines to Cd stress. Phenotypic and physiological analyses revealed the drb1 mutant to display the highest degree of tolerance to the imposed stress while the drb2 mutant was the most sensitive. RT-qPCR-based molecular profiling of miRNA abundance and miRNA target gene expression revealed DRB1 to be the primary double-stranded RNA binding (DRB) protein required for the production of six of the seven Cd-responsive miRNAs analyzed. However, DRB2, and not DRB1, was determined to be required for miR396 production. RT-qPCR further inferred that transcript cleavage was the RNA silencing mechanism directed by each assessed miRNA to control miRNA target gene expression. Taken together, the results presented here reveal the complexity of the miRNA-directed molecular response of Arabidopsis to Cd stress.

Molecular Manipulation of the miR399/PHO2 Expression Module Alters the Salt Stress Response of Arabidopsis thaliana.

In Arabidopsis thaliana (Arabidopsis), the microRNA399 (miR399)/PHOSPHATE2 (PHO2) expression module is central to the response of Arabidopsis to phosphate (PO4) stress. In addition, miR399 has been demonstrated to also alter in abundance in response to salt stress. We therefore used a molecular modification approach to alter miR399 abundance to investigate the requirement of altered miR399 abundance in Arabidopsis in response to salt stress. The generated transformant lines, MIM399 and MIR399 plants, with reduced and elevated miR399 abundance respectively, displayed differences in their phenotypic and physiological response to those of wild-type Arabidopsis (Col-0) plants following exposure to a 7-day period of salt stress. However, at the molecular level, elevated miR399 abundance, and therefore, altered PHO2 target gene expression in salt-stressed Col-0, MIM399 and MIR399 plants, resulted in significant changes to the expression level of the two PO4 transporter genes, PHOSPHATE TRANSPORTER1;4 (PHT1;4) and PHT1;9. Elevated PHT1;4 and PHT1;9 PO4 transporter levels in salt stressed Arabidopsis would enhance PO4 translocation from the root to the shoot tissue which would supply additional levels of this precious cellular resource that could be utilized by the aerial tissues of salt stressed Arabidopsis to either maintain essential biological processes or to mount an adaptive response to salt stress.

HyPer2 imaging reveals temporal and heterogeneous hydrogen peroxide changes in denervated and aged skeletal muscle fibers in vivo.

To determine the role of denervation and motor unit turnover in the age-related increase in skeletal muscle oxidative stress, the hydrogen peroxide (H2O2) specific, genetically-encoded, fluorescent cyto-HyPer2 probe was expressed in mouse anterior tibialis (AT) muscle and compared with ex vivo measurements of mitochondrial oxidant generation. Crush of the peroneal nerve induced increased mitochondrial peroxide generation, measured in permeabilised AT fibers ex vivo and intra vital confocal microscopy of cyto-HyPer2 fluorescence showed increased cytosolic H2O2 in a sub-set (~24%) of individual fibers associated with onset of fiber atrophy. In comparison, mitochondrial peroxide generation was also increased in resting muscle from old (26 month) mice compared with adult (6-8 month) mice, but no age effect on fiber cytosolic H2O2 in vivo was seen. Thus ageing is associated with an increased ability of muscle fibers to maintain cytosolic redox homeostasis in the presence of denervation-induced increase in mitochondrial peroxide generation.

DRB1, DRB2 and DRB4 Are Required for Appropriate Regulation of the microRNA399/PHOSPHATE2 Expression Module in Arabidopsis thaliana.

Adequate phosphorous (P) is essential to plant cells to ensure normal plant growth and development. Therefore, plants employ elegant mechanisms to regulate P abundance across their developmentally distinct tissues. One such mechanism is PHOSPHATE2 (PHO2)-directed ubiquitin-mediated degradation of a cohort of phosphate (PO4) transporters. PHO2 is itself under tight regulation by the PO4 responsive microRNA (miRNA), miR399. The DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1, DRB2 and DRB4, have each been assigned a specific functional role in the Arabidopsis thaliana (Arabidopsis) miRNA pathway. Here, we assessed the requirement of DRB1, DRB2 and DRB4 to regulate the miR399/PHO2 expression module under PO4 starvations conditions. Via the phenotypic and molecular assessment of the knockout mutant plant lines, drb1, drb2 and drb4, we show here that; (1) DRB1 and DRB2 are required to maintain P homeostasis in Arabidopsis shoot and root tissues; (2) DRB1 is the primary DRB required for miR399 production; (3) DRB2 and DRB4 play secondary roles in regulating miR399 production, and; (4) miR399 appears to direct expression regulation of the PHO2 transcript via both an mRNA cleavage and translational repression mode of RNA silencing. Together, the hierarchical contribution of DRB1, DRB2 and DRB4 demonstrated here to be required for the appropriate regulation of the miR399/PHO2 expression module identifies the extreme importance of P homeostasis maintenance in Arabidopsis to ensure that numerous vital cellular processes are maintained across Arabidopsis tissues under a changing cellular environment.

Profiling the Abiotic Stress Responsive microRNA Landscape of Arabidopsis thaliana.

It is well established among interdisciplinary researchers that there is an urgent need to address the negative impacts that accompany climate change. One such negative impact is the increased prevalence of unfavorable environmental conditions that significantly contribute to reduced agricultural yield. Plant microRNAs (miRNAs) are key gene expression regulators that control development, defense against invading pathogens and adaptation to abiotic stress. Arabidopsis thaliana (Arabidopsis) can be readily molecularly manipulated, therefore offering an excellent experimental system to alter the profile of abiotic stress responsive miRNA/target gene expression modules to determine whether such modification enables Arabidopsis to express an altered abiotic stress response phenotype. Towards this goal, high throughput sequencing was used to profile the miRNA landscape of Arabidopsis whole seedlings exposed to heat, drought and salt stress, and identified 121, 123 and 118 miRNAs with a greater than 2-fold altered abundance, respectively. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was next employed to experimentally validate miRNA abundance fold changes, and to document reciprocal expression trends for the target genes of miRNAs determined abiotic stress responsive. RT-qPCR also demonstrated that each miRNA/target gene expression module determined to be abiotic stress responsive in Arabidopsis whole seedlings was reflective of altered miRNA/target gene abundance in Arabidopsis root and shoot tissues post salt stress exposure. Taken together, the data presented here offers an excellent starting platform to identify the miRNA/target gene expression modules for future molecular manipulation to generate plant lines that display an altered response phenotype to abiotic stress.

Incorporating X-ray summing into gamma-gamma signature quantification.

A method for quantifying coincidence signatures has been extended to incorporate the effects of X-ray summing, and tested using a high-efficiency γ-γ system. An X-ray library has been created, allowing all possible γ, X-ray and conversion electron cascades to be generated. The equations for calculating efficiency and cascade summing corrected coincidence signature probabilities have also been extended from a two γ, two detector 'special case' to an arbitrarily large system. The coincidence library generated is fully searchable by energy, nuclide, coincidence pair, γ multiplicity, cascade probability and the half-life of the cascade, allowing the user to quickly identify coincidence signatures of interest. The method and software described is inherently flexible, as it only requires evaluated nuclear data, an X-ray library, and accurate efficiency characterisations to quickly and easily calculate coincidence signature probabilities for a variety of systems. Additional uses for the software include the fast identification of γ coincidence signals with required multiplicities and branching ratios, identification of the optimal coincidence signatures to measure for a particular system, and the calculation of cascade summing corrections for single detector systems.

Perioperative management of diabetes in elective patients: a region-wide audit.

BACKGROUND: Ten percent of elective surgical patients have diabetes. These patients demonstrate excess perioperative morbidity and mortality. National guidance on the management of adults with diabetes undergoing surgery was published in 2011. We present a region-wide audit of adherence to this guidance across the North Western Deanery. METHODS: Local teams prospectively collected data according to a locally approved protocol. Pregnant, paediatric and non-elective patients were excluded from this audit. Patient characteristics, type of surgery and aspects of perioperative management were collated and centrally analysed against audit criteria based upon national recommendations. RESULTS: 247 patients with diabetes were identified. HbA1c was recorded in 71% of patients preoperatively; 9% of patients with an abnormal HbA1c were not known by, or referred to, the diabetes team. 17% of patients were admitted the evening preceding surgery. The mean fasting time was 12:20(4) h. Variable rate i.v. insulin infusions (VRIII) were not used when indicated in 11%. Only 8% of patients received the recommended substrate fluid, along with the VRIII (5% glucose in 0.45% saline). Intra-operative capillary blood glucose (CBG) was measured hourly in 56% of patients. Intra-operative CBG was within the acceptable range (4-12 mmol.L(-1)) in 85% of patients. 73% of patients had a CBG measurement performed in recovery. The WHO checklist was used in 95% of patients. CONCLUSIONS: National perioperative guidelines were not adhered to in a substantial proportion of patients with diabetes undergoing elective surgery. This study represents a template for future trainee networks.

A high-efficiency HPGe coincidence system for environmental analysis.

The Comprehensive Nuclear-Test-Ban Treaty (CTBT) is supported by a network of certified laboratories which must meet certain sensitivity requirements for CTBT relevant radionuclides. At the UK CTBT Radionuclide Laboratory (GBL15), a high-efficiency, dual-detector gamma spectroscopy system has been developed to improve the sensitivity of measurements for treaty compliance, greatly reducing the time required for each sample. Utilising list-mode acquisition, each sample can be counted once, and processed multiple times to further improve sensitivity. For the 8 key radionuclides considered, Minimum Detectable Activities (MDA's) were improved by up to 37% in standard mode (when compared to a typical CTBT detector system), with the acquisition time required to achieve the CTBT sensitivity requirements reduced from 6 days to only 3. When utilising the system in coincidence mode, the MDA for (60) Co in a high-activity source was improved by a factor of 34 when compared to a standard CTBT detector, and a factor of 17 when compared to the dual-detector system operating in standard mode. These MDA improvements will allow the accurate and timely quantification of radionuclides that decay via both singular and cascade γ emission, greatly enhancing the effectiveness of CTBT laboratories.

Nitric oxide availability is increased in contracting skeletal muscle from aged mice, but does not differentially decrease muscle superoxide.

Reactive oxygen and nitrogen species have been implicated in the loss of skeletal muscle mass and function that occurs during aging. Nitric oxide (NO) and superoxide are generated by skeletal muscle and where these are generated in proximity their chemical reaction to form peroxynitrite can compete with the superoxide dismutation to hydrogen peroxide. Changes in NO availability may therefore theoretically modify superoxide and peroxynitrite activities in tissues, but published data are contradictory regarding aging effects on muscle NO availability. We hypothesised that an age-related increase in NO generation might increase peroxynitrite generation in muscles from old mice, leading to an increased nitration of muscle proteins and decreased superoxide availability. This was examined using fluorescent probes and an isolated fiber preparation to examine NO content and superoxide in the cytosol and mitochondria of muscle fibers from adult and old mice both at rest and following contractile activity. We also examined the 3-nitrotyrosine (3-NT) and peroxiredoxin 5 (Prx5) content of muscles from mice as markers of peroxynitrite activity. Data indicate that a substantial age-related increase in NO levels occurred in muscle fibers during contractile activity and this was associated with an increase in muscle eNOS. Muscle proteins from old mice also showed an increased 3-NT content. Inhibition of NOS indicated that NO decreased superoxide bioavailability in muscle mitochondria, although this effect was not age related. Thus increased NO in muscles of old mice was associated with an increased 3-NT content that may potentially contribute to age-related degenerative changes in skeletal muscle.