Bacterial growth-mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming.

The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.

Is Personalized Dietary Therapy Effective for Individuals With Irritable Bowel Syndrome?

Introduction: Adverse reactions to foods and food additives have a critical role in clinical manifestations of irritable bowel syndrome (IBS). Personalized dietary modifications conducted under the supervision of a qualified health practitioner could considerably impact the clinical care and course of the condition. Objective: To investigate the clinical effectiveness of the Lifestyle Eating and Performance (LEAP) program based on the Leukocyte Activation Assay-MRT (LAA-MRT®) results in improving IBS symptoms and quality of life. Methods: The retrospective study included de-identified client records (n = 146) from private group practices seen by registered dietitians. The eligibility criteria were adults aged > 18 years old with an established diagnosis of IBS. Results: Participants were 46.7 ± 12.6 years old and had a BMI of 26.7 ± 6.1 kg/m2; the majority were female (87.0%) and followed-up by a registered dietitian for 10.1 ± 6.4 weeks. There was a significant reduction post-dietary intervention in overall Global Gastrointestinal Symptom Survey Scores (P < 0.001) and improvement in quality of life (P < 0.001). Conclusion: This study generates real-world evidence of an alternative treatment option for IBS using a personalized dietary approach. A more precise understanding of the effect of food intake reactions is vital for clinical improvements and enhancing health outcomes in IBS.

Proteomic Profiling of Interplay Between Agrobacterium tumefaciens and Nicotiana benthamiana for Improved Molecular Pharming Outcomes.

Transient expression of recombinant proteins in plants is being used as a platform for production of therapeutic proteins. Benefits of this system include a reduced cost of drug development, rapid delivery of new products to the market, and an ability to provide safe and efficacious medicines for diseases. Although plant-based production systems offer excellent potential for therapeutic protein production, barriers, such as plant host defense response, exist which negatively impact the yield of product. Here we provide a protocol using tandem mass tags and mass spectrometry-based proteomics to quickly and robustly quantify the change in abundance of host defense proteins produced during the production process. These proteins can then become candidates for genetic manipulation to create host plants with reduced plant defenses capable of producing higher therapeutic protein yields.

The emerging role of mass spectrometry-based proteomics in molecular pharming practices.

Molecular pharming relies on the integration of foreign genes into a plant system for production of the desired recombinant protein. The speed, scalability, and lack of contaminating human pathogens highlights plants as an enticing and feasible system to produce diverse protein-based products, including vaccines, antibodies, and enzymes. However, limitations of expression levels, host defense responses, and production irregularities underscore distinct areas for improvement within the molecular pharming pipeline. Within the past five years, mass spectrometry-based proteomics has begun to address these critical areas and show promise in advancing our understanding of the complex biological systems driving molecular pharming. Further, opportunities to leverage comprehensive proteome profiling have surfaced to meet good manufacturing practice regulations and move biopharmaceuticals derived from plants into mainstream production.

Wrist-mounted accelerometers provide objective evidence of disease and recovery in patients with frozen shoulder.

BACKGROUND: Commercially available wrist-mounted exercise monitors may offer objective data on disease and recovery. This study is the first to evaluate the potential of such devices in the assessment of frozen shoulder and the effects of treatment. METHODS: Twenty-one patients with isolated, unilateral frozen shoulder wore a wrist-mounted accelerometer (Fitbit Fire II, Fitbit Inc. 2007, California, USA) on each wrist for two separate seven-day periods, one week before and six months after treatment. The monitors produced an activity count for each 24-hour period, accounting for all movements of the upper limb. Three values were calculated for each time period: (1) the mean activity count for each limb, (2) the total activity count for both limbs, and (3) an activity count ratio calculated by dividing the activity of the frozen limb by the unaffected limb. Constant score, American Shoulder and Elbow Surgeons, visual analog scale-pain, and range of movement were recorded before and after treatment. RESULTS: Mean activity counts were significantly lower in the frozen shoulder limb than those in the unaffected limb over the initial seven-day period (6066 vs. 7516; P = .04). The activity count ratio significantly improved after treatment (0.83 vs. 096; p 0.01), whereas the mean total activity count remained similar before and after treatment (14915 vs. 12371; P = .18), demonstrating that activity transferred from the unaffected limb back to the previously frozen limb. Range of movement (P < .01), Constant (P < .01), American Shoulder and Elbow Surgeons (P < .01), and visual analog scale-pain (P < .01) scores all significantly improved after treatment, but there was no correlation with the data from the activity monitor. DISCUSSION: Wrist-mounted accelerometers are sufficiently sensitive to detect a difference in limb activity in patients affected by frozen shoulder. The movement deficit between the affected and unaffected limbs improved by 14% after treatment. These data could be used in conjunction with subjective scores to offer a clearer insight into patient disease burden and recovery.

Translational targeting of inflammation and fibrosis in frozen shoulder: Molecular dissection of the T cell/IL-17A axis.

Frozen shoulder is a common fibroproliferative disease characterized by the insidious onset of pain and restricted range of shoulder movement with a significant socioeconomic impact. The pathophysiological mechanisms responsible for chronic inflammation and matrix remodeling in this prevalent fibrotic disorder remain unclear; however, increasing evidence implicates dysregulated immunobiology. IL-17A is a key cytokine associated with inflammation and tissue remodeling in numerous musculoskeletal diseases, and thus, we sought to determine the role of IL-17A in the immunopathogenesis of frozen shoulder. We demonstrate an immune cell landscape that switches from a predominantly macrophage population in nondiseased tissue to a T cell-rich environment in disease. Furthermore, we observed a subpopulation of IL-17A-producing T cells capable of inducing profibrotic and inflammatory responses in diseased fibroblasts through enhanced expression of the signaling receptor IL-17RA, rendering diseased cells more sensitive to IL-17A. We further established that the effects of IL-17A on diseased fibroblasts was TRAF-6/NF-κB dependent and could be inhibited by treatment with an IKKß inhibitor or anti-IL-17A antibody. Accordingly, targeting of the IL-17A pathway may provide future therapeutic approaches to the management of this common, debilitating disease.

Enhanced Ability of Plant-Derived PGT121 Glycovariants To Eliminate HIV-1-Infected Cells.

The activity of broadly neutralizing antibodies (bNAbs) targeting HIV-1 depends on pleiotropic functions, including viral neutralization and the elimination of HIV-1-infected cells. Several in vivo studies have suggested that passive administration of bNAbs represents a valuable strategy for the prevention or treatment of HIV-1. In addition, different strategies are currently being tested to scale up the production of bNAbs to obtain the large quantities of antibodies required for clinical trials. Production of antibodies in plants permits low-cost and large-scale production of valuable therapeutics; furthermore, pertinent to this work, it also includes an advanced glycoengineering platform. In this study, we used Nicotiana benthamiana to produce different Fc-glycovariants of a potent bNAb, PGT121, with near-homogeneous profiles and evaluated their antiviral activities. Structural analyses identified a close similarity in overall structure and glycosylation patterns of Fc regions for these plant-derived Abs and mammalian cell-derived Abs. When tested for Fc-effector activities, afucosylated PGT121 showed significantly enhanced FcγRIIIa interaction and antibody dependent cellular cytotoxicity (ADCC) against primary HIV-1-infected cells, both in vitro and ex vivo. However, the overall galactosylation profiles of plant PGT121 did not affect ADCC activities against infected primary CD4+ T cells. Our results suggest that the abrogation of the Fc N-linked glycan fucosylation of PGT121 is a worthwhile strategy to boost its Fc-effector functionality. IMPORTANCE PGT121 is a highly potent bNAb and its antiviral activities for HIV-1 prevention and therapy are currently being evaluated in clinical trials. The importance of its Fc-effector functions in clearing HIV-1-infected cells is also under investigation. Our results highlight enhanced Fc-effector activities of afucosylated PGT121 MAbs that could be important in a therapeutic context to accelerate infected cell clearance and slow disease progression. Future studies to evaluate the potential of plant-produced afucosylated PGT121 in controlling HIV-1 replication in vivo are warranted.

Novel self-amplificatory loop between T cells and tenocytes as a driver of chronicity in tendon disease.

OBJECTIVES: Increasing evidence suggests that inflammatory mechanisms play a key role in chronic tendon disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence disease perpetuation. METHODS: T cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged tendon or interleukin-1ß was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes. RESULTS: Significant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte-T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio. CONCLUSIONS: Interaction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human tendon disorders.

Clinical and Anthropometric Improvements with a Tailored Dietary Approach in Pediatric Crohn's Disease.

Pediatric Crohn's disease (CD) is more common today than in prior decades. Therapeutic goals in children with CD are to reduce symptomatology, promote normal growth, and avoid nutritional deficiencies. Diet plays a crucial role in the treatment of CD. However, there is a lack of comprehensive guidelines and individualized dietary approaches. A 12-and-a-half-year-old boy presented with chronic diarrhea, intermittent fever, abdominal pain, and a history of eczema. At the first visit, his body mass index (BMI) was 14.4, and his BMI-for-age was in the 1st percentile, classifying him as underweight. He received the diagnosis of Crohn's ileocolitis and gastritis and was unresponsive to treatment for 10.5 months. The patient was placed on a tailored dietary approach based on the in vitro leukocyte activation assay-MRT (LAA-MRT®), known as the Lifestyle Eating and Performance (LEAP) program. The LEAP program is an elimination diet built on the selection of less-immune-reactive foods and chemicals identified by the LAA-MRT® results. After 39 days of following the LEAP program, his fecal calprotectin levels decreased from >2000 µg/g to 185.9 µg/g. A repeat esophagogastroduodenoscopy (EGD) and colonoscopy with 10 biopsies showed complete histologic remission 1 year after beginning diet therapy. At that point, the patient discontinued his pharmacologic therapy and maintained clinical remission of CD after more than 3 years of follow-up. In addition, his anthropometric measurements and laboratory biomarkers were normalized. This case presents evidence supporting the use of the LEAP program in clinical practice as an adjunctive, tailored dietary option to manage pediatric CD.

Comparison of Treatments for Frozen Shoulder: A Systematic Review and Meta-analysis.

Importance: There are a myriad of available treatment options for patients with frozen shoulder, which can be overwhelming to the treating health care professional. Objective: To assess and compare the effectiveness of available treatment options for frozen shoulder to guide musculoskeletal practitioners and inform guidelines. Data Sources: Medline, EMBASE, Scopus, and CINHAL were searched in February 2020. Study Selection: Studies with a randomized design of any type that compared treatment modalities for frozen shoulder with other modalities, placebo, or no treatment were included. Data Extraction and Synthesis: Data were independently extracted by 2 individuals. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline. Random-effects models were used. Main Outcomes and Measures: Pain and function were the primary outcomes, and external rotation range of movement (ER ROM) was the secondary outcome. Results of pairwise meta-analyses were presented as mean differences (MDs) for pain and ER ROM and standardized mean differences (SMDs) for function. Length of follow-up was divided into short-term (≤12 weeks), mid-term (>12 weeks to ≤12 months), and long-term (>12 months) follow-up. Results: From a total of 65 eligible studies with 4097 participants that were included in the systematic review, 34 studies with 2402 participants were included in pairwise meta-analyses and 39 studies with 2736 participants in network meta-analyses. Despite several statistically significant results in pairwise meta-analyses, only the administration of intra-articular (IA) corticosteroid was associated with statistical and clinical superiority compared with other interventions in the short-term for pain (vs no treatment or placebo: MD, -1.0 visual analog scale [VAS] point; 95% CI, -1.5 to -0.5 VAS points; P < .001; vs physiotherapy: MD, -1.1 VAS points; 95% CI, -1.7 to -0.5 VAS points; P < .001) and function (vs no treatment or placebo: SMD, 0.6; 95% CI, 0.3 to 0.9; P < .001; vs physiotherapy: SMD 0.5; 95% CI, 0.2 to 0.7; P < .001). Subgroup analyses and the network meta-analysis demonstrated that the addition of a home exercise program with simple exercises and stretches and physiotherapy (electrotherapy and/or mobilizations) to IA corticosteroid may be associated with added benefits in the mid-term (eg, pain for IA coritocosteriod with home exercise vs no treatment or placebo: MD, -1.4 VAS points; 95% CI, -1.8 to -1.1 VAS points; P < .001). Conclusions and Relevance: The findings of this study suggest that the early use of IA corticosteroid in patients with frozen shoulder of less than 1-year duration is associated with better outcomes. This treatment should be accompanied by a home exercise program to maximize the chance of recovery.

Treatment of periscapular tendinopathy with radiofrequency coblation: A case report.

Overuse injuries of the tendon - 'tendinopathy' - account for 30%-50% of all sporting injuries and a high proportion of orthopaedic referrals from primary care physicians. Tendinopathies often have a multifactorial aetiology and injury can be due to a combination of both acute and chronic trauma which contributes to loss of tissue integrity and eventual rupture. Our incomplete understanding of the mechanisms surrounding tendon pathophysiology continues to cause difficulties in treatments beyond loading regimes which can be unsuccessful in up to 30% of cases. We describe an uncommon case of tendinopathy affecting the periscapular muscle/tendon unit in a 35-year-old female with persistent pain around the inferior posterior pole of her right scapula. Magnetic resonance imaging findings confirmed oedema of the muscles around the inferior scapular margin in keeping with enthesopathy/tendinopathy and she was treated with radiofrequency coblation to the area. This case highlights radiofrequency ablation as a surgical option should non-operative treatments fail in the rare diagnosis of periscapular tendinopathy.

Fibroblast activation and inflammation in frozen shoulder.

INTRODUCTION: Frozen shoulder is a common, fibro-proliferative disease characterised by the insidious onset of pain and progressively restricted range of shoulder movement. Despite the prevalence of this disease, there is limited understanding of the molecular mechanisms underpinning the pathogenesis of this debilitating disease. Previous studies have identified increased myofibroblast differentiation and proliferation, immune cell influx and dysregulated cytokine production. We hypothesised that subpopulations within the fibroblast compartment may take on an activated phenotype, thus initiating the inflammatory processes observed in frozen shoulder. Therefore, we sought to evaluate the presence and possible pathogenic role of known stromal activation proteins in Frozen shoulder. METHODS: Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients undergoing shoulder stabilisation surgery. Fibroblast activation marker expression (CD248, CD146, VCAM and PDPN, FAP) was quantified using immunohistochemistry. Control and diseased fibroblasts were cultured for in vitro studies from capsule biopsies from instability and frozen shoulder surgeries, respectively. The inflammatory profile and effects of IL-1ß upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR. RESULTS: Immunohistochemistry demonstrated increased expression of fibroblast activation markers CD248, CD146, VCAM and PDPN in the frozen shoulder group compared with control (p < 0.05). Fibroblasts cultured from diseased capsule produced elevated levels of inflammatory protein (IL-6, IL-8 & CCL-20) in comparison to control fibroblasts. Exposing control fibroblasts to an inflammatory stimuli, (IL-1ß) significantly increased stromal activation marker transcript and protein expression (CD248, PDPN and VCAM). CONCLUSIONS: These results show that fibroblasts have an activated phenotype in frozen shoulder and this is associated with inflammatory cytokine dysregulation. Furthermore, it supports the hypothesis that activated fibroblasts may be involved in regulating the inflammatory and fibrotic processes involved in this disease.

The epidemiology of acromioclavicular joint excision.

BACKGROUND: With the development of arthroscopic procedures such as subacromial decompression (ASAD) and rotator cuff repair (RCR), it is hypothesized that there may have been a similar rise in the performance of acromioclavicular joint excision (ACJE). The purpose of this study was to investigate the epidemiology of ACJE to examine incidence, surgical technique, age, gender of patients and associated procedures in an urban population. METHODS: A prospectively collected surgical database was retrospectively examined to identify patients undergoing ACJE. Associated procedures such as ASAD or RCR were determined from these records. The demographic details (age and gender) were also recorded. RESULTS: A total of 411 ACJEs were performed over the study period (n = 216 males, n = 195 female). The overall incidence increased from 9.3 per 100,000 in 2009, to a peak of 19.6 per 1,00,000 in 2013. In 349 patients, ACJE was undertaken as part of an arthroscopic procedure, of which 332 were ASAD+ACJE alone. The prevalence of arthroscopic ACJE in ASADs was 23.7% (349/1400). ACJE was performed as an open procedure in 62 (15%) cases. Those undergoing open ACJE were younger than those undergoing an arthroscopic procedure (mean difference 6.2 years, 95% CI 3.2-9.2, p < 0.001). CONCLUSIONS: We demonstrate an increasing incidence of ACJE in the general population. The groups of patients most likely to undergo ACJE are women aged between 45 and 54 years old, men aged 55-64 years and the most socioeconomically deprived. The higher incidence of ACJE in the most deprived socioeconomic quintile may have public health implications. Level of Evidence: II; retrospective design: prognosis study.

S100A8 & S100A9: Alarmin mediated inflammation in tendinopathy.

Alarmins S100A8 and S100A9 are endogenous molecules released in response to environmental triggers and cellular damage. They are constitutively expressed in immune cells such as monocytes and neutrophils and their expression is upregulated under inflammatory conditions. The molecular mechanisms that regulate inflammatory pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. Based on our previous investigations highlighting tendinopathy as an alarmin mediated pathology we sought evidence of S100A8 & A9 expression in a human model of tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate release of inflammatory mediators and matrix synthesis in human tenocytes. Immunohistochemistry and quantitative RT-PCR showed S100A8 & A9 expression was significantly upregulated in tendinopathic tissue compared with control. Furthermore, treating primary human tenocytes with exogenous S100A8 & A9 significantly increased protein release of IL-6, IL-8, CCL2, CCL20 and CXCL10; however, no alterations in genes associated with matrix remodelling were observed at a transcript level. We propose S100A8 & A9 participate in early pathology by modulating the stromal microenvironment and influencing the inflammatory profile observed in tendinopathy. S100A8 and S100A9 may participate in a positive feedback mechanism involving enhanced leukocyte recruitment and release of pro-inflammatory cytokines from tenocytes that perpetuates the inflammatory response within the tendon in the early stages of disease.

Topical glyceryl trinitrate for the treatment of tendinopathies: a systematic review.

OBJECTIVE: To produce a best evidence synthesis of the clinical effects of topical glyceryl trinitrate (GTN) in the treatment of tendinopathies. DESIGN: A systematic review of published randomised controlled trials (RCTs) of the use of GTN in patients with tendinopathy. DATA SOURCES: MEDLINE, Embase, Scopus and CINAHL from database inception to January 2018. METHODS: We examined RCTs comparing the effects of topical GTN with either placebo or other treatments on tendinopathy. Overall quality of each eligible study was determined based on a combined assessment of internal validity, external validity and precision. The level of evidence for each assessed parameter was rated based on the system by van Tulder et al. RESULTS: A total of 10 eligible RCTs were identified including patients with tendinopathy of the rotator cuff (n=4), wrist extensors (n=3), Achilles (n=2) and patellar (n=1) tendons. For all tendinopathies, improvements in pain were significant when comparing GTN versus placebo in the short term (<8 weeks; poor evidence). Significant improvements in midterm outcomes for treatment with GTN versus placebo included the following: patient satisfaction (strong evidence); chances of being asymptomatic with activities of daily living (strong evidence); range of movement (moderate evidence); strength (moderate evidence); pain (at night and with activity; poor evidence) and local tenderness (poor evidence). Patients treated with topical GTN reported a higher incidence of headaches than those who received placebo (moderate evidence). CONCLUSIONS AND RELEVANCE: Treatment of tendinopathies with topical GTN for up to 6 months appears to be superior to placebo and may therefore be a useful adjunct to the treating healthcare professions.

Alarmins in Frozen Shoulder: A Molecular Association Between Inflammation and Pain.

BACKGROUND: The pathophysiological mechanisms behind proliferation of fibroblasts and deposition of dense collagen matrix in idiopathic frozen shoulder remain unclear. Alarmins (also known as danger signals) are endogenous molecules that are released into the extracellular milieu after infection or tissue injury and that signal cell and tissue damage. PURPOSE: To investigate whether the presence of alarmins is higher in patients with idiopathic frozen shoulder than in control subjects. STUDY DESIGN: Controlled laboratory study. METHODS: Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients with unstable shoulders (control). The samples were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry using antibodies against alarmin molecules including high-mobility group protein B1 (HMGB1), interleukin 33, S100A8, S100A9, and the peripheral nerve marker PGP9.5. Immunoreactivities were rated in a blinded fashion from "none" to "strong." Immunohistochemical distribution within the capsule was noted. Before surgery, patient-ranked pain frequency, severity, stiffness, and the range of passive shoulder motion were recorded and statistically analyzed. RESULTS: Compared with control patients, patients with frozen shoulder had greater frequency and severity of self-reported pain ( P = .02) and more restricted range of motion in all planes ( P < .05). H&E-stained capsular tissue from frozen shoulder showed fibroblastic hypercellularity and increased subsynovial vascularity. Immunoreactivity of alarmins was significantly stronger in frozen shoulder capsules compared with control capsules ( P < .05). Furthermore, the expression of the alarmin molecule HMGB1 significantly correlated ( r > 0.9, P < .05) with the severity of patient-reported pain. CONCLUSION: This study demonstrates a potential role for key molecular danger signals in frozen shoulder and suggests an association between the expression of danger molecules and the pain experienced by patients.

Glyphosate resistance in Ambrosia trifida: Part 1. Novel rapid cell death response to glyphosate.

BACKGROUND: Glyphosate-resistant (GR) Ambrosia trifida is now present in the midwestern United States and in southwestern Ontario, Canada. Two distinct GR phenotypes are known, including a rapid response (GR RR) phenotype, which exhibits cell death within hours after treatment, and a non-rapid response (GR NRR) phenotype. The mechanisms of resistance in both GR RR and GR NRR remain unknown. Here, we present a description of the RR phenotype and an investigation of target-site mechanisms on multiple A. trifida accessions. RESULTS: Glyphosate resistance was confirmed in several accessions, and whole-plant levels of resistance ranged from 2.3- to 7.5-fold compared with glyphosate-susceptible (GS) accessions. The two GR phenotypes displayed similar levels of resistance, despite having dramatically different phenotypic responses to glyphosate. Glyphosate resistance was not associated with mutations in EPSPS sequence, increased EPSPS copy number, EPSPS quantity, or EPSPS activity. CONCLUSION: These encompassing results suggest that resistance to glyphosate in these GR RR A. trifida accessions is not conferred by a target-site resistance mechanism. © 2017 Society of Chemical Industry.

Glyphosate resistance in Ambrosia trifida: Part 2. Rapid response physiology and non-target-site resistance.

BACKGROUND: The glyphosate-resistant rapid response (GR RR) resistance mechanism in Ambrosia trifida is not due to target-site resistance (TSR) mechanisms. This study explores the physiology of the rapid response and the possibility of reduced translocation and vacuolar sequestration as non-target-site resistance (NTSR) mechanisms. RESULTS: GR RR leaf discs accumulated hydrogen peroxide within minutes of glyphosate exposure, but only in mature leaf tissue. The rapid response required energy either as light or exogenous sucrose. The combination of phenylalanine and tyrosine inhibited the rapid response in a dose-dependent manner. Reduced glyphosate translocation was observed in GR RR, but only when associated with tissue death caused by the rapid response. Nuclear magnetic resonance studies indicated that glyphosate enters the cytoplasm and reaches chloroplasts, and it is not moved into the vacuole of GR RR, GR non-rapid response or glyphosate-susceptible A. trifida. CONCLUSION: The GR RR mechanism of resistance is not associated with vacuole sequestration of glyphosate, and the observed reduced translocation is likely a consequence of rapid tissue death. Rapid cell death was inhibited by exogenous application of aromatic amino acids phenylalanine and tyrosine. The mechanism by which these amino acids inhibit rapid cell death in the GR RR phenotype remains unknown, and it could involve glyphosate phytotoxicity or other agents generating reactive oxygen species. Implications of these findings are discussed. The GR RR mechanism is distinct from the currently described glyphosate TSR or NTSR mechanisms in other species. © 2017 Society of Chemical Industry.

Temporal lobe origin is common in patients who have undergone epilepsy surgery for hypermotor seizures.

RATIONALE: Hypermotor seizures are most often reported from the frontal lobe but may also have temporal, parietal, or insular origin. We noted a higher proportion of patients with temporal lobe epilepsy in our surgical cohort who had hypermotor seizures. We evaluated the anatomic localization and surgical outcome in patient with refractory hypermotor seizures who had epilepsy surgery in our center. METHODS: We identified twenty three patients with refractory hypermotor seizures from our epilepsy surgery database. We analyzed demographics, presurgical evaluation including semiology, MRI, PET scan, interictal/ictal scalp video-EEG, intracranial recording, and surgical outcomes. We evaluated preoperative variables as predictors of outcome. RESULTS: Most patients (65%) had normal brain MRI. Intracranial EEG was required in 20 patients (86.9%). Based on the presurgical evaluation, the resection was anterior temporal in fourteen patients, orbitofrontal in four patients, cingulate in four patients, and temporoparietal in one patient. The median duration of follow-up after surgery was 76.4months. Fourteen patients (60%) had been seizure free at the last follow up while 3 patients had rare disabling seizures. CONCLUSIONS: Hypermotor seizures often originated from the temporal lobe in this series of patients who had epilepsy surgery. This large proportion of temporal lobe epilepsy may be the result of a selection bias, due to easier localization and expected better outcome in temporal lobe epilepsy. With extensive presurgical evaluation, including intracranial EEG when needed, seizure freedom can be expected in the majority of patients.