Glutamate transporter dysfunction associated with nerve injury-induced pain in mice

Dysfunction at glutamatergic synapses has been proposed as a mechanism in the development of neuropathic pain. Here we sought to determine whether peripheral nerve injury-induced neuropathic pain results in functional changes to primary afferent synapses. Signs of neuropathic pain as well as an induction of glial fibrillary acidic protein in immunostained spinal cord sections 4 days after partial ligation of the sciatic nerve indicated the induction of neuropathic pain. We found that following nerve injury, no discernable change to kinetics of dl-α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) or N-methyl-d-aspartate receptor (NMDAR)-mediated evoked excitatory postsynaptic currents (eEPSCs) could be observed in dorsal horn (lamina I/II) neurons compared with those of naïve mice. However, we did find that nerve injury was accompanied by slowed decay of the early phase of eEPSCs in the presence of glutamate transporter inhibition by the competitive nontransportable inhibitor dl-threo-β-benzyloxyaspartic acid (TBOA). Concomitantly, expression patterns for the two major glutamate transporters in the spinal cord, excitatory amino acid transporters (EAAT) 1 and EAAT2, were found to be reduced at this time (4 days postinjury). We then sought to directly determine whether nerve injury results in glutamate spillover to NMDARs at dorsal horn synapses. By employing the use-dependent NMDAR blocker (±)MK-801 to block subsynaptic receptors, we found that although TBOA-induced spillover to extrasynaptic receptors trended to increased activation of these receptors after nerve injury, this was not significant compared with naïve mice. Together, these results suggest the development of neuropathic pain involves subtle changes to glutamate transporter expression and function that could contribute to neuropathic pain during excessive synaptic activity.NHMRC grant: 0569927 & 35144

Opioid-related (ORL1) receptors are enriched in a subpopulation of sensory neurons and prolonged activation produces no functional loss of surface N-type calcium channels.

The opioid-related receptor, ORL1, is activated by the neuropeptide nociceptin/orphanin FQ (N/OFQ) and inhibits high-voltage-activated (HVA) calcium channel currents (I(Ca)) via a G-protein-coupled mechanism. Endocytosis of ORL1 receptor during prolonged N/OFQ exposure was proposed to cause N-type voltage-gated calcium channel (VGCC) internalization via physical interaction between ORL1 and the N-type channel. However, there is no direct electrophysiological evidence for this mechanism in dorsal root ganglion (DRG) neurons or their central nerve terminals. The present study tested this using whole-cell patch-clamp recordings of HVA I(Ca) in rat DRG neurons and primary afferent excitatory synaptic currents (eEPSCs) in spinal cord slices. DRG neurons were classified on the basis of diameter, isolectin-B4 (IB4) binding and responses to capsaicin, N/OFQ and a μ-opioid agonist, DAMGO. IB4-negative neurons less than 20 μm diameter were selectively responsive to N/OFQ as well as DAMGO. In these neurons, ORL1 desensitization by a supramaximal concentration of N/OFQ was not followed by a decrease in HVA I(Ca) current density or proportion of whole-cell HVA I(Ca) contributed by N-type VGCC as determined using the N-type channel selective blocker, ω-conotoxin CVID. There was also no decrease in the proportion of N-type I(Ca) when neurons were incubated at 37°C with N/OFQ for 30 min prior to recording. In spinal cord slices, N/OFQ consistently inhibited eEPSCs onto dorsal horn neurons. As observed in DRG neurons, preincubation of slices in N/OFQ for 30 min produced no decrease in the proportion of eEPSCs inhibited by CVID. In conclusion, no internalization of the N-type VGCC occurs in either the soma or central nerve terminals of DRG neurons following prolonged exposure to high, desensitizing concentrations of N/OFQ.NHMRC Grant: 056992

Weed resistance to synthetic auxin herbicides

Herbicides classified as synthetic auxins have been most commonly used to control broadleaf weeds in a variety of crops and in non-cropland areas since the first synthetic auxin herbicide (SAH), 2,4-D, was introduced to the market in the mid-1940s. The incidence of weed species resistant to SAHs is relatively low considering their long-term global application with 29 broadleaf weed species confirmed resistant to date. An understanding of the context and mechanisms of SAH resistance evolution can inform management practices to sustain the longevity and utility of this important class of herbicides. A symposium was convened during the 2nd Global Herbicide Resistance Challenge (May 2017 in Denver, CO, USA) to provide an overview of the current state of knowledge of SAH resistance mechanisms including case studies of weed species resistant to SAHs and perspectives on mitigating resistance development in SAH-tolerant crops

Coccolithophores: Functional Biodiversity, Enzymes and Bioprospecting

Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an ‘in house’ enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase

Diagnostic Utility of Genome-wide DNA Methylation Testing in Genetically Unsolved Individuals with Suspected Hereditary Conditions.

Conventional genetic testing of individuals with neurodevelopmental presentations and congenital anomalies (ND/CAs), i.e., the analysis of sequence and copy number variants, leaves a substantial proportion of them unexplained. Some of these cases have been shown to result from DNA methylation defects at a single locus (epi-variants), while others can exhibit syndrome-specific DNA methylation changes across multiple loci (epi-signatures). Here, we investigate the clinical diagnostic utility of genome-wide DNA methylation analysis of peripheral blood in unresolved ND/CAs. We generate a computational model enabling concurrent detection of 14 syndromes using DNA methylation data with full accuracy. We demonstrate the ability of this model in resolving 67 individuals with uncertain clinical diagnoses, some of whom had variants of unknown clinical significance (VUS) in the related genes. We show that the provisional diagnoses can be ruled out in many of the case subjects, some of whom are shown by our model to have other diseases initially not considered. By applying this model to a cohort of 965 ND/CA-affected subjects without a previous diagnostic assumption and a separate assessment of rare epi-variants in this cohort, we identify 15 case subjects with syndromic Mendelian disorders, 12 case subjects with imprinting and trinucleotide repeat expansion disorders, as well as 106 case subjects with rare epi-variants, a portion of which involved genes clinically or functionally linked to the subjects\u27 phenotypes. This study demonstrates that genomic DNA methylation analysis can facilitate the molecular diagnosis of unresolved clinical cases and highlights the potential value of epigenomic testing in the routine clinical assessment of ND/CAs

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Oleaginous crops as integrated production platforms for food, feed, fuel and renewable industrial feedstock

The world faces considerable challenges including how to produce more biomass for food, feed, fuel and industrial feedstock without significantly impacting on our environment or increasing our consumption of limited resources such as water or petroleum-derived carbon. This has been described as sustainable intensification. Oleaginous crops have the potential to provide renewable resources for all these commodities, provided they can be engineered to meet end-use requirements, and that they can be produced on sufficient scale to meet current growing world population and industrial demand. Although traditional breeding methods have been used successfully to modify the fatty acid composition of oils, metabolic engineering provides a more rapid and direct method for manipulating plant lipid composition. Recent advances in our understanding of the biochemical mechanisms of seed oil biogenesis and the cloning of genes involved in fatty acid and oil metabolic pathways, have allowed the generation of oilseed crops that produce ‘designer oils’ tailored for specific applications and the conversion of high biomass crops into novel oleaginous crops. However, improvement of complex quantitative traits in oilseed crops remains more challenging as the underlying genetic determinants are still poorly understood. Technological advances in sequencing and computing have allowed the development of an association genetics method applicable to crops with complex genomes. Associative transcriptomics approaches and high throughput lipidomic profiling can be used to identify the genetic components controlling quantitative variation for lipid related traits in polyploid crops like oilseed rape and provide molecular tools for marker assisted breeding. In this review we are citing examples of traits with potential for bio-refining that can be harvested as co-products in seeds, but also in non-harvested biomass

High-voltage activated calcium current subtypes in mouse DRG neurons adapt in a subpopulation-specific manner after nerve injury

Changes in ion channel function and expression are characteristic of neuropathic pain. Voltage-gated calcium channels (VGCCs) are integral for neurotransmission and membrane excitability, but relatively little is known about changes in their expression after nerve injury. In this study, we investigate whether peripheral nerve ligation is followed by changes in the density and proportion of high-voltage-activated (HVA) VGCC current subtypes in dorsal root ganglion (DRG) neurons, the contribution of presynaptic N-type calcium channels in evoked excitatory postsynaptic currents (EPSCs) recorded from dorsal horn neurons in the spinal cord, and the changes in expression of mRNA encoding VGCC subunits in DRG neurons. Using C57BL/6 mice [8-to 11-wkold males (n = 91)] for partial sciatic nerve ligation or sham surgery, we performed whole cell patch-clamp recordings on isolated DRG neurons and dorsal horn neurons and measured the expression of all VGCC subunits with RT-PCR in DRG neurons. After nerve injury, the density of P/Q-type current was reduced overall in DRG neurons. There was an increase in the percentage of N-type and a decrease in that of P/Q-type current in medium-to large-diameter neurons. No changes were found in the contribution of presynaptic N-type calcium channels in evoked EPSCs recorded from dorsal horn neurons. The alpha 2 delta-1 subunit was upregulated by 1.7-fold and gamma-3, gamma-2, and beta-4 subunits were all downregulated 1.7-fold in injured neurons compared with sham-operated neurons. This comprehensive characterization of HVA VGCC subtypes in mouse DRG neurons after nerve injury revealed changes in N- and P/Q-type current proportions only in medium-to large-diameter neurons