Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

BACKGROUND: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. RESULTS: Expected relative increases in total fatty acids of 17-58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. CONCLUSIONS: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

A Feasibility Study Exploring Measures of Autonomic Function in Patients With Failed Back Surgery Syndrome Undergoing Spinal Cord Stimulation.

OBJECTIVES: Failed back surgery syndrome (FBSS) is associated with impaired autonomic tone, characterized by sympathetic prevalence and vagal withdrawal. Although spinal cord stimulation (SCS) alleviates pain in FBSS, there is limited research investigating how SCS affects measures of autonomic function. This was a prospective, open-label, feasibility study exploring measures of autonomic function in patients with FBSS receiving SCS therapy. MATERIALS AND METHODS: A total of 14 patients with FBSS were recruited for baseline measurements and underwent a trial of 10-kHz SCS. There were three failed trials, resulting in the remaining 11 participants receiving a fully implanted 10-kHz SCS system. One participant requested an explant, resulting in ten participants completing both baseline and follow-up (three to six months after SCS implant) measurements. Autonomic function was assessed using time- and frequency-domain heart rate variability (HRV), baroreceptor reflex sensitivity (BRS), and muscle sympathetic nerve activity (MSNA) using microneurography. Because this was a feasibility study, most of the analysis was descriptive. However, paired t-tests and Wilcoxon signed-rank tests tested for differences between baseline and follow-up. RESULTS: In the whole (N = 14) and final (N = 10) samples, there was between-participant variation in baseline and follow-up measures. This, combined with a small sample, likely contributed to finding no statistically significant differences in any of the measures between baseline and follow-up. However, plotting baseline and follow-up scores for individual participants revealed that those who showed increases in MSNA frequency, square root of the mean of the squared differences between adjacent RR intervals (RMSSD), percentage of the number of RR intervals >50 ms (pRR50), total power, and up BRS between baseline and follow-up had distinct clustering of baseline values compared with those who showed decreases in these measures. CONCLUSIONS: Findings from this feasibility study will aid with informing hypotheses for future research. A key aspect that should be considered in future research concerns exploring the role of baseline measures of autonomic function in influencing change in autonomic function with SCS therapy.

Comparison of Paresthesia Mapping With Anatomic Placement in Burst Spinal Cord Stimulation: Long-Term Results of the Prospective, Multicenter, Randomized, Double-Blind, Crossover CRISP Study.

OBJECTIVES: Spinal cord stimulation (SCS) is an effective therapy for chronic intractable pain. Conventional SCS involves electrode placement based on intraoperative paresthesia mapping; however, newer paradigms like burst may allow for anatomic placement of leads. Here, for the first time, we report the one-year safety and efficacy of burst SCS delivered using a lead placed with conventional, paresthesia mapping, or anatomic placement approach in subjects with chronic low back pain (CLBP). MATERIALS AND METHODS: Subjects with CLBP were implanted with two leads. The first lead was placed to cross the T8/T9 disc and active contacts for this lead were chosen through paresthesia mapping. The second lead was placed at the T9/T10 spinal anatomic landmark. Subjects initially underwent a four-week, double-blinded, crossover trial with a two-week testing period with burst SCS delivered through each lead in a random order. At the end of trial period, subjects expressed their preference for one of the two leads. Subsequently, subjects received burst SCS with the preferred lead and were followed up at 3, 6, and 12 months. Pain intensity (visual analog scale), quality-of-life (EuroQol-5D instrument), and disability (Oswestry Disability Index) were evaluated at baseline and follow-up. RESULTS: Forty-three subjects successfully completed the trial. Twenty-one preferred the paresthesia mapping lead and 21 preferred the anatomic placement lead. Anatomic placement lead was activated in one subject who had no preference. The pain scores (for back and leg) significantly improved from baseline for both lead placement groups at all follow-up time points, with no significant between-group differences. CONCLUSIONS: This study demonstrated that equivalent clinical benefits could be achieved with burst SCS using either paresthesia mapping or anatomic landmark-based approaches for lead placement. Nonparesthesia-based approaches, such as anatomic landmark-based lead placement investigated here, have the potential to simplify implantation of SCS and improve current surgical practice.

Men and Women Respond Equally Well to Spinal Cord and Dorsal Root Ganglion Stimulation.

OBJECTIVES: The influence of gender on outcomes in individuals undergoing treatment for chronic pain is unclear. This retrospective, single-site study explored the impact of gender on pain, quality of life (QoL), revisions, and explants in patients with failed back surgery syndrome or visceral pain, who received a fully implanted 10 kHz spinal cord stimulation (SCS), burst SCS, or dorsal root ganglion (DRG) stimulation system. MATERIALS AND METHODS: The following data were collected from paper and electronic records: gender, age, chronic pain diagnosis, system, baseline and follow-up scores (average pain [visual analog scale, VAS], worst pain [VAS], QoL [EQ-5D-3L]), revisions, and explants. Data were statistically analyzed by one-way ANCOVAs controlling for age, chi-square tests of independence, and logistic regression. RESULTS: The final sample comprised 387 patients (176 males and 211 females). Males were significantly older compared to females (mean difference: 2.33 years, p = 0.044). Controlling for age, baseline average pain was significantly lower in males than females (mean difference: -0.32, p = 0.049). Males and females responded equally well to 10 kHz SCS and burst SCS as well as DRG stimulation. A greater percentage of males (5%) than females (1%) had revisions due to lead fractures. Additionally, more females (13%) than males (6%) had an explant due to insufficient pain relief. Female gender and older age were associated with greater likelihood of having an explant compared to male gender and younger age. CONCLUSION: Gender may play an influential role in pain severity at baseline but have little effect at follow-up. To help identify which patients may undergo a revision or explant, gender and age could be important factors and should be further scrutinized. Even though men and women responded equally well to SCS and DRG stimulation, more men had a revision due to lead fractures, and more women were explanted due to insufficient pain relief.

Pain Relief and Safety Outcomes with Cervical 10 kHz Spinal Cord Stimulation: Systematic Literature Review and Meta-analysis.

BACKGROUND: Chronic pain in head, neck, shoulders and upper limbs is debilitating, and patients usually rely on pain medications or surgery to manage their symptoms. However, given the current opioid epidemic, non-pharmacological interventions that reduce pain, such as spinal cord stimulation (SCS), are needed. The purpose of this study was to review the evidence on paresthesia-free 10 kHz SCS therapy for neck and upper extremity pain. METHODS: Systematic literature search was performed for studies reporting outcomes for cervical 10 kHz SCS using date limits from May 2008 to November 2020. The study results were analyzed and described qualitatively. Additionally, when feasible, meta-analyses of the outcome data, with 95% confidence intervals (CIs), were conducted using both the fixed-effects (FE) and random-effects (RE) models. RESULTS: A total of 15 studies were eligible for inclusion. The proportion of patients who achieved ≥ 50% pain reduction was 83% (95% CI 77-89%) in both the FE and RE models. The proportion of patients who reduced/eliminated their opioid consumption was 39% (95% CI 31-46%) in the FE model and 39% (95% CI 31-48%) in the RE model. Pain or discomfort with the implant, lead migration, and infections were potential risks following cervical SCS. Explant rate was 0.1 (95% CI 0.0-0.2) events per 100 person-months, and no patients in the included studies experienced a neurological complication or paresthesia. CONCLUSION: Findings suggest 10 kHz SCS is a promising, safe, minimally invasive alternative for managing chronic upper limb and neck pain.

Changes in Leaf-Level Nitrogen Partitioning and Mesophyll Conductance Deliver Increased Photosynthesis for Lolium perenne Leaves Engineered to Accumulate Lipid Carbon Sinks.

Diacylglycerol acyl-transferase (DGAT) and cysteine oleosin (CO) expression confers a novel carbon sink (of encapsulated lipid droplets) in leaves of Lolium perenne and has been shown to increase photosynthesis and biomass. However, the physiological mechanism by which DGAT + CO increases photosynthesis remains unresolved. To evaluate the relationship between sink strength and photosynthesis, we examined fatty acids (FA), water-soluble carbohydrates (WSC), gas exchange parameters and leaf nitrogen for multiple DGAT + CO lines varying in transgene accumulation. To identify the physiological traits which deliver increased photosynthesis, we assessed two important determinants of photosynthetic efficiency, CO2 conductance from atmosphere to chloroplast, and nitrogen partitioning between different photosynthetic and non-photosynthetic pools. We found that DGAT + CO accumulation increased FA at the expense of WSC in leaves of L. perenne and for those lines with a significant reduction in WSC, we also observed an increase in photosynthesis and photosynthetic nitrogen use efficiency. DGAT + CO L. perenne displayed no change in rubisco content or Vcmax but did exhibit a significant increase in specific leaf area (SLA), stomatal and mesophyll conductance, and leaf nitrogen allocated to photosynthetic electron transport. Collectively, we showed that increased carbon demand via DGAT+CO lipid sink accumulation can induce leaf-level changes in L. perenne which deliver increased rates of photosynthesis and growth. Carbon sinks engineered within photosynthetic cells provide a promising new strategy for increasing photosynthesis and crop productivity.

One-Year Results of Prospective Research Study Using 10 kHz Spinal Cord Stimulation in Persistent Nonoperated Low Back Pain of Neuropathic Origin: Maiden Back Study.

PURPOSE: Spinal cord stimulation (SCS) is a recommended treatment for chronic neuropathic pain. Persistent nonoperative low back pain of neuropathic origin has profound negative impacts on patient's lives. This prospective, open label, research study aimed to explore the use of SCS in patients with associated features of central sensitisation such as allodynia and hyperalgesia. MATERIALS AND METHODS: Twenty-one patients with back pain and hyperalgesia or allodynia who had not had prior spinal surgery underwent a SCS trial followed by full implantation. SCS comprised administering electrical impulses epidurally at a frequency of 10 kHz and pulse width of 30 µsec. Patients attended follow-up visits after 6 and 12 months of SCS. Repeated measure ANOVAs/Friedman tests explored change after 6 and 12 months of 10 kHz SCS. Independent sample t-tests/Mann-Whitney U tests examined differences in response after 12 months of 10 kHz SCS. RESULTS: Back and leg pain, quality of life (QoL), pain-related disability, and morphine equivalence significantly improved compared with baseline following 6 and 12 months of 10 kHz SCS. There were no increases in the consumption of opioids, amitriptyline, gabapentin or pregabalin in any patient. After 12 months of treatment, 52% encountered ≥50% improvement in back pain, 44% achieved remission (0-3 cm back pain VAS), 40% reported ODI scores between 0 and 40 and 60% experienced a reduction of at least 10 ODI points. Patients reporting ≥10-point improvement in ODI had significantly longer pain history durations and experienced significantly greater improvements in back pain, leg pain and QoL than those reporting <10-point improvement in ODI. CONCLUSION: The 10 kHz SCS improved back and leg pain, QoL, pain-related disability and medication consumption in patients with nonoperative back pain of neuropathic origin. With further research incorporating a sham control arm, the efficacy of 10 kHz SCS in this patient cohort will become more established.

Comparison of Paresthesia Mapping to Anatomical Placement in Burst Spinal Cord Stimulation: Initial Trial Results of the Prospective, Multicenter, Randomized, Double-Blinded, Crossover, CRISP Study.

INTRODUCTION: In this prospective, multicenter, double-blinded, randomized, crossover study, we compared the therapeutic efficacy of burst SCS delivered using a lead implanted with the paresthesia mapping approach to a lead implanted with an anatomic placement approach. MATERIALS AND METHODS: Subjects with chronic low back pain were implanted with two leads, one using paresthesia-mapping approach (PM) and the second using anatomical placement procedure (AP). Stimulation contacts were chosen using the standard intraoperative paresthesia-testing procedure for the paresthesia-mapped lead or an activated bipole overlapping the T9-T10 junction for the anatomical lead. Amplitude for either lead was selected such that no sensory percepts were generated. Subjects were assessed at baseline and after a trial period during which they tested each lead for two weeks in random order. Eligible subjects had the option to receive permanent implants using their preferred AP or PM approach at end-of-trial. RESULTS: Of the 53 subjects who completed both trial periods, 43 (81.1%) experienced at least 50% back pain relief with at least one lead. Nearly half of these (20; 46.5%) were profound responders who experienced at least 80% back pain relief with either leads. Primary and secondary outcomes, at the end of trial, showed significant improvements for both AP and PM leads from baseline yet were not significantly different from each other. DISCUSSION: The trial results of this study suggest that similar clinical outcomes can be achieved in burst SCS when performing lead placement either using paresthesia mapping or anatomical placement with imaging references.

Storing carbon in leaf lipid sinks enhances perennial ryegrass carbon capture especially under high N and elevated CO2.

By modifying two genes involved in lipid biosynthesis and storage [cysteine oleosin (cys-OLE)/diacylglycerol O-acyltransferase (DGAT)], the accumulation of stable lipid droplets in perennial ryegrass (Lolium perenne) leaves was achieved. Growth, biomass allocation, leaf structure, gas exchange parameters, fatty acids, and water-soluble carbohydrates were quantified for a high-expressing cys-OLE/DGAT ryegrass transformant (HL) and a wild-type (WT) control grown under controlled conditions with 1-10 mM nitrogen (N) supply at ambient and elevated atmospheric CO2. A dramatic shift in leaf carbon (C) storage occurred in HL leaves, away from readily mobilizable carbohydrates and towards stable lipid droplets. HL exhibited an increased growth rate, mainly in non-photosynthetic organs, leading to a decreased leaf mass fraction. HL leaves, however, displayed an increased specific leaf area and photosynthetic rate per unit leaf area, delivering greater overall C capture and leaf growth at high N supply. HL also exhibited a greater photosynthesis response to elevated atmospheric CO2. We speculate that by behaving as uniquely stable microsinks for C, cys-OLE-encapsulated lipid droplets can reduce feedback inhibition of photosynthesis and drive greater C capture. Manipulation of many genes and gene combinations has been used to increase non-seed lipid content. However, the cys-OLE/DGAT technology remains the only reported case that increases plant biomass. We contrast cys-OLE/DGAT with other lipid accumulation strategies and discuss the implications of introducing lipid sinks into non-seed organs for plant energy homeostasis and growth.

Bolus intrathecal injection of ziconotide (Prialt®) to evaluate the option of continuous administration via an implanted intrathecal drug delivery (ITDD) system: a pilot study.

OBJECTIVES: This study evaluated efficacy and safety of bolus doses of ziconotide (Prialt®, Eisai Limited, Hertfordshire, UK) to assess the option of continuous administration of this drug via an implanted intrathecal drug delivery system. MATERIALS AND METHODS: Twenty adults with severe chronic pain who were under consideration for intrathecal (IT) therapy were enrolled in this open label, nonrandomized, pilot study. Informed consent was obtained. Demographics, medical/pain history, pain scores, and concomitant medications were recorded. A physical examination was performed. Creatine kinase was measured. Initial visual analog scale (VAS), blood pressure, heart rate, and respiratory rate were recorded. All patients received an initial bolus dose of 2.5 mcg ziconotide; the dose in the subsequent visits was modified according to response. Subsequent doses were 2.5 mcg, 1.2 mcg, or 3.75 mcg as per protocol. A good response (≥30% reduction in baseline pain VAS) with no side-effects on two occasions was considered a successful trial. Data were analyzed using a generalized estimating equations model, with pain VAS as the outcome and time (seven time points; preinjection and one to six hours postinjection) as the predictor. RESULTS: Generalized estimating equations analysis of summary measures showed a mean reduction of pain VAS of approximately 25% at the group level; of 11 responders, seven underwent pump implantation procedure, two withdrew because of adverse effects, one refused an implant, and one could not have an implant (lack of funding from the Primary Care Trust). CONCLUSIONS: Our data demonstrated that mean VAS was reduced by approximately 25% at the group level after IT ziconotide bolus. Treatment efficacy did not vary with sex, center, age, or pain etiology. Ziconotide bolus was generally well tolerated. Larger studies are needed to determine if bolus dosing with ziconotide is a good predictor of response to continuous IT ziconotide via an intrathecal drug delivery system.