Gut microbe-generated phenylacetylglutamine is an endogenous allosteric modulator of ß2-adrenergic receptors.

Allosteric modulation is a central mechanism for metabolic regulation but has yet to be described for a gut microbiota-host interaction. Phenylacetylglutamine (PAGln), a gut microbiota-derived metabolite, has previously been clinically associated with and mechanistically linked to cardiovascular disease (CVD) and heart failure (HF). Here, using cells expressing ß1- versus ß2-adrenergic receptors (ß1AR and ß2AR), PAGln is shown to act as a negative allosteric modulator (NAM) of ß2AR, but not ß1AR. In functional studies, PAGln is further shown to promote NAM effects in both isolated male mouse cardiomyocytes and failing human heart left ventricle muscle (contracting trabeculae). Finally, using in silico docking studies coupled with site-directed mutagenesis and functional analyses, we identified sites on ß2AR (residues E122 and V206) that when mutated still confer responsiveness to canonical ß2AR agonists but no longer show PAGln-elicited NAM activity. The present studies reveal the gut microbiota-obligate metabolite PAGln as an endogenous NAM of a host GPCR.

Planovalgus Foot Deformity in Patients Undergoing Total Hip Arthroplasty Is Associated With Increased Risk of Falls, Implant-Related Complications, and Revisions: A Case-Control Analysis.

BACKGROUND: Pes planus occurs due to the loss of the longitudinal arch of the foot, resulting in altered gait mechanics. This may lead to increased complications following total hip arthroplasty (THA). Thus, the aim of this study was to assess the effects that pes planus has on rates of falls, implant complications, fall-related injuries, and times to revision among THA patients. METHODS: A retrospective review of a private insurance claims database was conducted from 2010 to 2021. Patients who had a diagnosis of congenital or acquired pes planus and cases of THA were identified. Patients undergoing THA with a diagnosis of pes planus were matched to control patients 1:5 based on age, sex, and comorbidity profiles. Logistic regression was utilized to assess for differences in complication rates. RESULTS: A total of 3,622 pes planus patients were matched to 18,094 control patients. The pes planus group had significantly higher rates of falls than the control group (6.93 versus 2.97%, OR [odds ratio]: 2.43; CI [confidence interval]: 2.09 to 2.84; P < .001). Pes planus patients also had significantly greater odds of dislocation (OR: 1.89; CI: 1.58 to 2.27; P < .001), mechanical loosening (OR: 2.43; CI: 2.09 to 2.84; P = .019), and periprosthetic fracture (OR: 2.43; CI: 2.09 to 2.84; P < .001). The pes planus group had significantly greater rates of proximal humerus fractures (P = .008), but no difference was seen in distal radius fractures (P = .102). The time to revision was significantly shorter in the pes planus group (190 versus 554 days, P < .001). CONCLUSIONS: Pes planus in patients undergoing THA is associated with increased risk of complications and faster time to revision. These findings may allow orthopaedic surgeons to identify those patients at risk and allow for more educated patient counseling and operative planning.

Patient-Reported Outcomes for Spine Oncology: A Narrative Review.

Spine tumors, both primary and metastatic, impose significant morbidity and mortality on patients and physicians. Patient-reported outcomes are valuable tools to assess a patient's impression of their health status and enhance communication between physicians and patients. Various spine generic patient-reported outcome tools have traditionally been used but have not been validated in the spine tumor patient population. The Spine Oncology Study Group Outcome Questionnaire, which is disease-specific for the metastatic spine patient population, has been shown to have strong validity, even across multiple languages. Patient-Reported Outcomes Measurement Information System, which has recently been developed, employs computerized adaptive testing to assess multiple health domains. It has been shown to capture information in both generic and specific questionnaires and has the potential to be used as a universal tool in the spine oncology patient population. Further long-term studies, as well as, cross-cultural adaptations, are needed to validate Patient-Reported Outcomes Measurement Information System's applicability and effectiveness.

Accuracy of Intraarticular Injections: Blind vs. Image Guided Techniques-A Review of Literature.

Intra-articular injections are widely used for diagnostic and therapeutic purposes of joint pathologies throughout the body. These injections can be performed blind by utilizing anatomical landmarks or with the use of imaging modalities to directly visualize the joint space during injections. This review of the literature aims to comprehensively identify differences in the accuracy of intra-articular injections via palpation vs. image guidance in the most commonly injected joints in the upper and lower extremities. To our knowledge, there are no such comprehensive reviews available. A narrative literature review was performed using PubMed and Google Scholar databases to identify studies focusing on the accuracy of blind or image-guided intra-articular injections for each joint. A total of 75 articles was included in this review, with blind and image-guided strategies being discussed for the most commonly injected joints of the upper and lower extremities. Varying ranges of accuracy with blind and image-guided modalities were found throughout the literature, though an improvement in accuracy was seen in nearly all joints when using image guidance. Differences are pronounced, particularly in deep joints such as the hip or in the small joints such as those in the hand or foot. Image guidance is a useful adjunct for most intra-articular injections, if available. Though there is an increase in accuracy in nearly all joints, minor differences in accuracy seen in large, easily accessed joints, such as the knee, may not warrant image guidance.

Spinal fusion for postlaminectomy kyphosis following intramedullary spinal cord tumor resection: A 34-year follow-up.

Background: Resection of intramedullary spinal cord tumors (IMSCTs) in pediatric patients results in a high incidence of spinal deformity (i.e., kyphoscoliosis often requiring fusion). Here, a 6-year-old male underwent a spinal fusion to correct postlaminectomy thoracic kyphosis following resection of an IMSCT. Case Description: A 6-year-old male initially underwent multilevel thoracic laminectomies for resection of an IMSCT. Six months later, he presented with the onset of kyphoscoliosis. During adolescence he became increasingly paraparetic due to a thoracic kyphosis that had now progressed to 118°. He underwent a 360 decompression/fusion that included a T1-T9 laminectomy, a T5 to T11 anterior interbody arthrodesis/rib autograft, and posterolateral T2-T12 fusion/iliac crest autograft with Harrington rods placed from T5 to T12. Postoperative radiographs showed the thoracic kyphosis improved to 62°. However, 4.5 years later, X-rays showed the thoracic kyphosis newly progressed to 90° (i.e., from T3 to T12). Thirty-four years after this corrective surgery, he remained neurologically intact with only mild complaints of balance changes, and bladder/bowel urgency while radiographs confirmed continued stability. Further, the thoracic magnetic resonance imaging showed only chronic thoracic spine/cord changes. Conclusion: A 6-year-old child originally underwent a thoracic laminectomy for an IMSCT. As an adolescent, due to progressive postlaminectomy kyphosis, he underwent a successful secondary thoracic 360° decompression/ fusion. Notably, 34 years later, he did not require any surgical revision.

Exploratory Transcriptomic Profiling Reveals the Role of Gut Microbiota in Vascular Dementia.

Stroke is the second most common cause of cognitive impairment and dementia. Vascular dementia (VaD), a cognitive impairment following a stroke, is common and significantly impacts the quality of life. We recently demonstrated via gut microbe transplant studies that the gut microbe-dependent trimethylamine-N-oxide (TMAO) pathway impacts stroke severity, both infarct size and long-term cognitive outcomes. However, the molecular mechanisms that underly the role of the microbiome in VaD have not been explored in depth. To address this issue, we performed a comprehensive RNA-sequencing analysis to identify differentially expressed (DE) genes in the ischemic cerebral cortex of mouse brains at pre-stroke and post-stroke day 1 and day 3. A total of 4016, 3752 and 7861 DE genes were identified at pre-stroke and post-stroke day 1 and day 3, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated pathways of neurodegeneration in multiple diseases, chemokine signaling, calcium signaling, and IL-17 signaling as the key enriched pathways. Inflammatory response genes interleukin-1 beta (Il-1ß), chemokines (C-X-C motif chemokine ligand 10 (Cxcl10), chemokine ligand 2 (Ccl2)), and immune system genes (S100 calcium binding protein 8 (S100a8), lipocalin-2 (Lcn2)) were among the most significantly upregulated genes. Hypocretin neuropeptide precursor (Hcrt), a neuropeptide, and transcription factors such as neuronal PAS domain protein 4 (Npas4), GATA binding protein 3 (Gata3), and paired box 7 (Pax7) were among the most significantly downregulated genes. In conclusion, our results indicate that higher plasma TMAO levels induce differential mRNA expression profiles in the ischemic brain tissue in our pre-clinical stroke model, and the predicted pathways provide the molecular basis for regulating the TMAO-enhanced neuroinflammatory response in the brain.

The artificial sweetener erythritol and cardiovascular event risk.

Artificial sweeteners are widely used sugar substitutes, but little is known about their long-term effects on cardiometabolic disease risks. Here we examined the commonly used sugar substitute erythritol and atherothrombotic disease risk. In initial untargeted metabolomics studies in patients undergoing cardiac risk assessment (n = 1,157; discovery cohort, NCT00590200 ), circulating levels of multiple polyol sweeteners, especially erythritol, were associated with incident (3 year) risk for major adverse cardiovascular events (MACE; includes death or nonfatal myocardial infarction or stroke). Subsequent targeted metabolomics analyses in independent US (n = 2,149, NCT00590200 ) and European (n = 833, DRKS00020915 ) validation cohorts of stable patients undergoing elective cardiac evaluation confirmed this association (fourth versus first quartile adjusted hazard ratio (95% confidence interval), 1.80 (1.18-2.77) and 2.21 (1.20-4.07), respectively). At physiological levels, erythritol enhanced platelet reactivity in vitro and thrombosis formation in vivo. Finally, in a prospective pilot intervention study ( NCT04731363 ), erythritol ingestion in healthy volunteers (n = 8) induced marked and sustained (>2 d) increases in plasma erythritol levels well above thresholds associated with heightened platelet reactivity and thrombosis potential in in vitro and in vivo studies. Our findings reveal that erythritol is both associated with incident MACE risk and fosters enhanced thrombosis. Studies assessing the long-term safety of erythritol are warranted.

Gut Microbiota-Generated Phenylacetylglutamine and Heart Failure.

BACKGROUND: The gut microbiota-dependent metabolite phenylacetylgutamine (PAGln) is both associated with atherothrombotic heart disease in humans, and mechanistically linked to cardiovascular disease pathogenesis in animal models via modulation of adrenergic receptor signaling. METHODS: Here we examined both clinical and mechanistic relationships between PAGln and heart failure (HF). First, we examined associations among plasma levels of PAGln and HF, left ventricular ejection fraction, and N-terminal pro-B-type natriuretic peptide in 2 independent clinical cohorts of subjects undergoing coronary angiography in tertiary referral centers (an initial discovery US Cohort, n=3256; and a validation European Cohort, n=829). Then, the impact of PAGln on cardiovascular phenotypes relevant to HF in cultured cardiomyoblasts, and in vivo were also examined. RESULTS: Circulating PAGln levels were dose-dependently associated with HF presence and indices of severity (reduced ventricular ejection fraction, elevated N-terminal pro-B-type natriuretic peptide) independent of traditional risk factors and renal function in both cohorts. Beyond these clinical associations, mechanistic studies showed both PAGln and its murine counterpart, phenylacetylglycine, directly fostered HF-relevant phenotypes, including decreased cardiomyocyte sarcomere contraction, and B-type natriuretic peptide gene expression in both cultured cardiomyoblasts and murine atrial tissue. CONCLUSIONS: The present study reveals the gut microbial metabolite PAGln is clinically and mechanistically linked to HF presence and severity. Modulating the gut microbiome, in general, and PAGln production, in particular, may represent a potential therapeutic target for modulating HF. REGISTRATION: URL: https://clinicaltrials.gov/; Unique identifier: NCT00590200 and URL: https://drks.de/drks_web/; Unique identifier: DRKS00020915.

Abatement of microfibre pollution and detoxification of textile dye - Indigo by engineered plant enzymes.

Microfibres (diameter <5 mm) and textile dyes released from textile industries are ubiquitous, cause environmental pollution, and harm aquatic flora, fauna, animals and human life. Therefore, enzymatic abatement of microfibre pollution and textile dye detoxification is essential. Microbial enzymes for such application present major challenges of scale and affordability to clean up large scale pollution. Therefore, enzymes required for the biodegradation of microfibres and indigo dye were expressed in transplastomic tobacco plants through chloroplast genetic engineering. Integration of laccase and lignin peroxidase genes into the tobacco chloroplast genomes and homoplasmy was confirmed by Southern blots. Decolorization (up to 86%) of samples containing indigo dye (100 mg/L) was obtained using cp-laccase (0.5% plant enzyme powder). Significant (8-fold) reduction in commercial microbial cellulase cocktail was achieved in pretreated cotton fibre hydrolysis by supplementing cost effective cellulases (endoglucanases, ß-glucosidases) and accessory enzymes (swollenin, xylanase, lipase) and ligninases (laccase lignin peroxidase) expressed in chloroplasts. Microfibre hydrolysis using cocktail of Cp-cellulases and Cp-accessory enzymes along with minimal dose (0.25% and 0.5%) of commercial cellulase blend (Ctec2) showed 88%-89% of sugar release from pretreated cotton and microfibres. Cp-ligninases, Cp-cellulases and Cp-accessory enzymes were stable in freeze dried leaves up to 15 and 36 months respectively at room temperature, when protected from light. Use of plant powder for decolorization or hydrolysis eliminated the need for preservatives, purification or concentration or cold chain. Evidently, abatement of microfibre pollution and textile dye detoxification using Cp-enzymes is a novel and cost-effective approach to prevent their environmental pollution.

Diabetes Education and Support Tele-Visit Needs Differ in Duration, Content, and Satisfaction in Older Versus Younger Adults.

Background: Diabetes education and support are critical components of diabetes care. During the COVID-19 pandemic, when telemedicine took the place of in-person visits, remote Certified Diabetes Care and Education Specialist (CDCES) services were offered to address diabetes education and support. Specific needs for older adults, including the time required to provide education and support remotely, have not been previously reported. Methods: Adults with diabetes (primarily insulin-requiring) were referred to remote CDCESs. Utilization was individualized based on patient needs and preferences. Topics discussed, patient satisfaction, and time spent in each tele-visit were evaluated by diabetes type, age, sex, insurance type, glycosylated hemoglobin (HbA1c), pump, and continuous glucose monitor (CGM) usage. t-Tests, one-way analysis of variance, and Pearson correlations were employed as appropriate. Results: Adults (n = 982; mean age 48.4 years, 41.0% age ≥55 years) with type 1 diabetes (n = 846) and type 2 diabetes mellitus (n = 136, 86.0% insulin-treated), 50.8% female; 19.0% Medicaid, 29.1% Medicare, 48.9% private insurance; mean HbA1c 8.4% (standard deviation 1.9); and 46.6% pump and 64.5% CGM users had 2203 tele-visits with remote CDCESs over 5 months. Of those referred, 272 (21.7%) could not be reached or did not receive education/support. Older age (≥55 years), compared with 36-54 year olds and 18-35 year olds, respectively, was associated with more tele-visits (mean 2.6 vs. 2.2 and 1.8) and more time/tele-visits (mean 20.4 min vs. 16.5 min and 14.8 min; p < 0.001) as was coverage with Medicare (mean 2.8 visits) versus private insurance (mean 2.0 visits; p < 0.001) and lower participant satisfaction. The total mean time spent with remote CDCESs was 53.1, 37.4, and 26.2 min for participants aged ≥55, 36-54, and 18-35 years, respectively. During remote tele-visits, the most frequently discussed topics per participant were CGM and insulin pump use (73.4% and 49.7%). After adjustment for sex and diabetes type, older age was associated with lack of access to a computer, tablet, smartphone, or internet (p < 0.001), and need for more education related to CGM (p < 0.001), medications (p = 0.015), hypoglycemia (p = 0.044), and hyperglycemia (p = 0.048). Discussion: Most remote CDCES tele-visits were successfully completed. Older adults/those with Medicare required more time to fulfill educational needs. Although 85.7% of individual sessions lasted <30 min, which does not meet current Medicare requirements for reimbursement, multiple visits were common with a total time of >50 min for most older participants. This suggests that new reimbursement models are needed. Education/support needs of insulin-treated older adults should be a focus of future studies.

Novel allosteric ligands of the angiotensin receptor AT1R as autoantibody blockers.

While orthosteric ligands of the angiotensin II (AngII) type 1 receptor (AT1R) are available for clinical and research applications, allosteric ligands are not known for this important G protein-coupled receptor (GPCR). Allosteric ligands are useful tools to modulate receptor pharmacology and subtype selectivity. Here, we report AT1R allosteric ligands for a potential application to block autoimmune antibodies. The epitope of autoantibodies for AT1R is outside the orthosteric pocket in the extracellular loop 2. A molecular dynamics simulation study of AT1R structure reveals the presence of a druggable allosteric pocket encompassing the autoantibody epitope. Small molecule binders were then identified for this pocket using structure-based high-throughput virtual screening. The top 18 hits obtained inhibited the binding of antibody to AT1R and modulated agonist-induced calcium response of AT1R. Two compounds out of 18 studied in detail exerted a negative allosteric modulator effect on the functions of the natural agonist AngII. They blocked antibody-enhanced calcium response and reactive oxygen species production in vascular smooth muscle cells as well as AngII-induced constriction of blood vessels, demonstrating their efficacy in vivo. Our study thus demonstrates the feasibility of discovering inhibitors of the disease-causing autoantibodies for GPCRs. Specifically, for AT1R, we anticipate development of more potent allosteric drug candidates for intervention in autoimmune maladies such as preeclampsia, bilateral adrenal hyperplasia, and the rejection of organ transplants.

Phenylpropanoid Biosynthesis Gene Expression Precedes Lignin Accumulation During Shoot Development in Lowland and Upland Switchgrass Genotypes.

Efficient conversion of lignocellulosic biomass into biofuels is influenced by biomass composition and structure. Lignin and other cell wall phenylpropanoids, such as para-coumaric acid (pCA) and ferulic acid (FA), reduce cell wall sugar accessibility and hamper biochemical fuel production. Toward identifying the timing and key parameters of cell wall recalcitrance across different switchgrass genotypes, this study measured cell wall composition and lignin biosynthesis gene expression in three switchgrass genotypes, A4 and AP13, representing the lowland ecotype, and VS16, representing the upland ecotype, at three developmental stages [Vegetative 3 (V3), Elongation 4 (E4), and Reproductive 3 (R3)] and three segments (S1-S3) of the E4 stage under greenhouse conditions. A decrease in cell wall digestibility and an increase in phenylpropanoids occur across development. Compared with AP13 and A4, VS16 has significantly less lignin and greater cell wall digestibility at the V3 and E4 stages; however, differences among genotypes diminish by the R3 stage. Gini correlation analysis across all genotypes revealed that lignin and pCA, but also pectin monosaccharide components, show the greatest negative correlations with digestibility. Lignin and pCA accumulation is delayed compared with expression of phenylpropanoid biosynthesis genes, while FA accumulation coincides with expression of these genes. The different cell wall component accumulation profiles and gene expression correlations may have implications for system biology approaches to identify additional gene products with cell wall component synthesis and regulation functions.

The IgG3 subclass of ß1-adrenergic receptor autoantibodies is an endogenous biaser of ß1AR signaling.

Dysregulation of immune responses has been linked to the generation of immunoglobulin G (IgG) autoantibodies that target human ß1ARs and contribute to deleterious cardiac outcomes. Given the benefits of ß-blockers observed in patients harboring the IgG3 subclass of autoantibodies, we investigated the role of these autoantibodies in human ß1AR function. Serum and purified IgG3(+) autoantibodies from patients with onset of cardiomyopathy were tested using human embryonic kidney (HEK) 293 cells expressing human ß1ARs. Unexpectedly, pretreatment of cells with IgG3(+) serum or purified IgG3(+) autoantibodies impaired dobutamine-mediated adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) generation while enhancing biased ß-arrestin recruitment and Extracellular Regulated Kinase (ERK) activation. In contrast, the ß-blocker metoprolol increased AC activity and cAMP in the presence of IgG3(+) serum or IgG3(+) autoantibodies. Because IgG3(+) autoantibodies are specific to human ß1ARs, non-failing human hearts were used as an endogenous system to determine their ability to bias ß1AR signaling. Consistently, metoprolol increased AC activity, reflecting the ability of the IgG3(+) autoantibodies to bias ß-blocker toward G-protein coupling. Importantly, IgG3(+) autoantibodies are specific toward ß1AR as they did not alter ß2AR signaling. Thus, IgG3(+) autoantibody biases ß-blocker toward G-protein coupling while impairing agonist-mediated G-protein activation but promoting G-protein-independent ERK activation. This phenomenon may underlie the beneficial outcomes observed in patients harboring IgG3(+) ß1AR autoantibodies.

A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors.

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

Imaging Salt Uptake Dynamics in Plants Using PET.

Soil salinity is a global environmental challenge for crop production. Understanding the uptake and transport properties of salt in plants is crucial to evaluate their potential for growth in high salinity soils and as a basis for engineering varieties with increased salt tolerance. Positron emission tomography (PET), traditionally used in medical and animal imaging applications for assessing and quantifying the dynamic bio-distribution of molecular species, has the potential to provide useful measurements of salt transport dynamics in an intact plant. Here we report on the feasibility of studying the dynamic transport of 22Na in millet using PET. Twenty-four green foxtail (Setaria viridis L. Beauv.) plants, 12 of each of two different accessions, were incubated in a growth solution containing 22Na+ ions and imaged at 5 time points over a 2-week period using a high-resolution small animal PET scanner. The reconstructed PET images showed clear evidence of sodium transport throughout the whole plant over time. Quantitative region-of-interest analysis of the PET data confirmed a strong correlation between total 22Na activity in the plants and time. Our results showed consistent salt transport dynamics within plants of the same variety and important differences between the accessions. These differences were corroborated by independent measurement of Na+ content and expression of the NHX transcript, a gene implicated in sodium transport. Our results demonstrate that PET can be used to quantitatively evaluate the transport of sodium in plants over time and, potentially, to discern differing salt-tolerance properties between plant varieties. In this paper, we also address the practical radiation safety aspects of working with 22Na in the context of plant imaging and describe a robust pipeline for handling and incubating plants. We conclude that PET is a promising and practical candidate technology to complement more traditional salt analysis methods and provide insights into systems-level salt transport mechanisms in intact plants.

Rice Genome-Scale Network Integration Reveals Transcriptional Regulators of Grass Cell Wall Synthesis.

Grasses have evolved distinct cell wall composition and patterning relative to dicotyledonous plants. However, despite the importance of this plant family, transcriptional regulation of its cell wall biosynthesis is poorly understood. To identify grass cell wall-associated transcription factors, we constructed the Rice Combined mutual Ranked Network (RCRN). The RCRN covers >90% of annotated rice (Oryza sativa) genes, is high quality, and includes most grass-specific cell wall genes, such as mixed-linkage glucan synthases and hydroxycinnamoyl acyltransferases. Comparing the RCRN and an equivalent Arabidopsis network suggests that grass orthologs of most genetically verified eudicot cell wall regulators also control this process in grasses, but some transcription factors vary significantly in network connectivity between these divergent species. Reverse genetics, yeast-one-hybrid, and protoplast-based assays reveal that OsMYB61a activates a grass-specific acyltransferase promoter, which confirms network predictions and supports grass-specific cell wall synthesis genes being incorporated into conserved regulatory circuits. In addition, 10 of 15 tested transcription factors, including six novel Wall-Associated regulators (WAP1, WACH1, WAHL1, WADH1, OsMYB13a, and OsMYB13b), alter abundance of cell wall-related transcripts when transiently expressed. The results highlight the quality of the RCRN for examining rice biology, provide insight into the evolution of cell wall regulation, and identify network nodes and edges that are possible leads for improving cell wall composition.

Validation of leaf and microbial pectinases: commercial launching of a new platform technology.

Almost all current genetically modified plant commercial products are derived from seeds. The first protein product made in leaves for commercial use is reported here. Leaf pectinases are validated here with eight liquid commercial microbial enzyme products for textile or juice industry applications. Leaf pectinases are functional in broad pH/temperature ranges as crude leaf extracts, while most commercial enzyme products showed significant loss at alkaline pH or higher temperature, essential for various textile applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, leaf pectinase powder was stored up to 16 months at ambient temperature without loss of enzyme activity. Commercial pectinase products showed much higher enzyme protein PAGE than crude leaf extracts with comparable enzyme activity without protease inhibitors. Natural cotton fibre does not absorb water due to hydrophobic nature of waxes and pectins. After bioscouring with pectinase, measurement of contact-angle water droplet absorption by the FAMAS videos showed 33 or 63 (leaf pectinase), 61 or 64 (commercial pectinase) milliseconds, well below the 10-second industry requirements. First marker-free lettuce plants expressing pectinases were also created by removal of the antibiotic resistance aadA gene. Leaf pectinase powder efficiently clarified orange juice pulp similar to several microbial enzyme products. Commercial pilot scale biomass production of tobacco leaves expressing different pectinases showed that hydroponic growth at Fraunhofer yielded 10 times lower leaf biomass per plant than soil-grown plants in the greenhouse. Pectinase enzyme yield from the greenhouse plants was double that of Fraunhofer. Thus, this leaf-production platform offers a novel, low-cost approach for enzyme production by elimination of fermentation, purification, concentration, formulation and cold chain.

Validation of leaf enzymes in the detergent and textile industries: launching of a new platform technology.

Chemical catalysts are being replaced by biocatalysts in almost all industrial applications due to environmental concerns, thereby increasing their demand. Enzymes used in current industries are produced in microbial systems or plant seeds. We report here five newly launched leaf-enzyme products and their validation with 15 commercial microbial-enzyme products, for detergent or textile industries. Enzymes expressed in chloroplasts are functional at broad pH/temperature ranges as crude-leaf extracts, while most purified commercial enzymes showed significant loss at alkaline pH or higher temperature, required for broad range commercial applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, chloroplast enzymes as a leaf powder can be stored up to 16 months at ambient temperature without loss of enzyme activity. Chloroplast-derived enzymes are stable in crude-leaf extracts without addition of protease inhibitors. Leaf lipase/mannanase crude extracts removed chocolate or mustard oil stains effectively at both low and high temperatures. Moreover, leaf lipase or mannanase crude-extracts removed stain more efficiently at 70 °C than commercial microbial enzymes (<10% activity). Endoglucanase and exoglucanase in crude leaf extracts removed dye efficiently from denim surface and depilled knitted fabric by removal of horizontal fibre strands. Due to an increased demand for enzymes in the food industry, marker-free lettuce plants expressing lipase or cellobiohydrolase were created for the first time and site-specific transgene integration/homoplasmy was confirmed by Southern blots. Thus, leaf-production platform offers a novel low-cost approach by the elimination of fermentation, purification, concentration, formulation and cold-chain storage/transportation. This is the first report of commercially launched protein products made in leaves and validated with current commercial products.

Expression pattern of salt tolerance-related genes in Aegilops cylindrica.

Aegilops cylindrica, a salt-tolerant gene pool of wheat, is a useful plant model for understanding mechanism of salt tolerance. A salt-tolerant USL26 and a salt-sensitive K44 genotypes of A. cylindrica, originating from Uremia Salt Lake shores in Northwest Iran and a non-saline Kurdestan province in West Iran, respectively, were identified based on screening evaluation and used for this work. The objective of the current study was to investigate the expression patterns of four genes related to ion homeostasis in this species. Under treatment of 400 mM NaCl, USL26 showed significantly higher root and shoot dry matter levels and K+ concentrations, together with lower Na+ concentrations than K44 genotype. A. cylindrica HKT1;5 (AecHKT1;5), SOS1 (AecSOS1), NHX1 (AecNHX1) and VP1 (AecVP1) were partially sequenced to design each gene specific primer. Quantitative real-time PCR showed a differential expression pattern of these genes between the two genotypes and between the root and shoot tissues. Expressions of AecHKT1;5 and AecSOS1 was greater in the roots than in the shoots of USL26 while AecNHX1 and AecVP1 were equally expressed in both tissues of USL26 and K44. The higher transcripts of AecHKT1;5 in the roots versus the shoots could explain both the lower Na+ in the shoots and the much lower Na+ and higher K+ concentrations in the roots/shoots of USL26 compared to K44. Therefore, the involvement of AecHKT1;5 in shoot-to-root handover of Na+ in possible combination with the exclusion of excessive Na+ from the root in the salt-tolerant genotype are suggested.