A Light Touch: Solar Photocatalysis Detoxifies Oil Sands Process-Affected Waters Prior to Significant Treatment of Naphthenic Acids.

Environmental reclamation of Canada's oil sands tailings ponds is among the single largest water treatment challenges globally. The toxicity of oil sands process-affected water (OSPW) has been associated with its dissolved organics, a complex mixture of naphthenic acid fraction components (NAFCs). Here, we evaluated solar treatment with buoyant photocatalysts (BPCs) as a passive advanced oxidation process (P-AOP) for OSPW remediation. Photocatalysis fully degraded naphthenic acids (NAs) and acid extractable organics (AEO) in 3 different OSPW samples. However, classical NAs and AEO, traditionally considered among the principal toxicants in OSPW, were not correlated with OSPW toxicity herein. Instead, nontarget petroleomic analysis revealed that low-polarity organosulfur compounds, composing <10% of the total AEO, apparently accounted for the majority of waters' toxicity to fish, as described by a model of tissue partitioning. These findings have implications for OSPW release, for which a less extensive but more selective treatment may be required than previously expected.

Photocatalytic treatment of organoselenium in synthetic mine-impacted effluents.

Biological selenium reduction processes are commonly employed as the best available technology (BAT) for selenium removal; however, as a by-product they produce trace amounts of organoselenium compounds with orders of magnitude greater bioaccumulation potential and toxicity. Here, we assessed buoyant photocatalysts (BPCs) as a potential passive advanced oxidation process (P-AOP) for organoselenium treatment. Using a synthetic mine-impacted water solution, spiked with selenomethionine (96 µg/L) as a representative organoselenium compound, photocatalysis with BPCs fully eliminated selenomethionine to <0.01 µg/L with conversion to selenite and selenate. A theoretical reaction pathway was inferred, and a kinetics model developed to describe the treatment trends and intermediates. Given the known toxic responses of Lepomis macrochirus and Daphnia magna to organoselenium, it was estimated that photocatalysis could effectively eliminate organoselenium acute toxicity within a UV dose of 8 kJ/L (1-2 days solar equivalent exposure), by transformation of selenomethionine to less hazardous oxidized Se species. Solar photocatalysis may therefore be a promising passive treatment technology for selenium-impacted mine water management.

Controlling process instability for defect lean metal additive manufacturing.

The process instabilities intrinsic to the localized laser-powder bed interaction cause the formation of various defects in laser powder bed fusion (LPBF) additive manufacturing process. Particularly, the stochastic formation of large spatters leads to unpredictable defects in the as-printed parts. Here we report the elimination of large spatters through controlling laser-powder bed interaction instabilities by using nanoparticles. The elimination of large spatters results in 3D printing of defect lean sample with good consistency and enhanced properties. We reveal that two mechanisms work synergistically to eliminate all types of large spatters: (1) nanoparticle-enabled control of molten pool fluctuation eliminates the liquid breakup induced large spatters; (2) nanoparticle-enabled control of the liquid droplet coalescence eliminates liquid droplet colliding induced large spatters. The nanoparticle-enabled simultaneous stabilization of molten pool fluctuation and prevention of liquid droplet coalescence discovered here provide a potential way to achieve defect lean metal additive manufacturing.

Effects of Particle Size Distribution with Efficient Packing on Powder Flowability and Selective Laser Melting Process.

The powder bed-based additive manufacturing (AM) process contains uncertainties in the powder spreading process and powder bed quality, leading to problems in repeatability and quality of the additively manufactured parts. This work focuses on identifying the uncertainty induced by particle size distribution (PSD) on powder flowability and the laser melting process, using Ti6Al4V as a model material. The flowability test results show that the effect of PSDs on flowability is not linear, rather the PSDs near dense packing ratios cause significant reductions in flowability (indicated by the increase in the avalanche angle and break energy of the powders measured by a revolution powder analyzer). The effects of PSDs on the selective laser melting (SLM) process are identified by using in-situ high-speed X-ray imaging to observe the melt pool dynamics during the melting process. The results show that the powder beds made of powders with dense packing ratios exhibit larger build height during laser melting. The effects of PSD with efficient packing on powder flowability and selective laser melting process revealed in this work are important for understanding process uncertainties induced by feedstock powders and for designing mitigation approaches.

Uncertainties Induced by Processing Parameter Variation in Selective Laser Melting of Ti6Al4V Revealed by In-Situ X-ray Imaging.

Selective laser melting (SLM) additive manufacturing (AM) exhibits uncertainties, where variations in build quality are present despite utilizing the same optimized processing parameters. In this work, we identify the sources of uncertainty in SLM process by in-situ characterization of SLM dynamics induced by small variations in processing parameters. We show that variations in the laser beam size, laser power, laser scan speed, and powder layer thickness result in significant variations in the depression zone, melt pool, and spatter behavior. On average, a small deviation of only ~5% from the optimized/reference laser processing parameter resulted in a ~10% or greater change in the depression zone and melt pool geometries. For spatter dynamics, small variation (10 µm, 11%) of the laser beam size could lead to over 40% change in the overall volume of the spatter generated. The responses of the SLM dynamics to small variations of processing parameters revealed in this work are useful for understanding the process uncertainties in the SLM process.

Insulin signaling alters antioxidant capacity in the diabetic heart.

Diabetic cardiomyopathy is associated with an increase in oxidative stress. However, antioxidant therapy has shown a limited capacity to mitigate disease pathology. The molecular mechanisms responsible for the modulation of reactive oxygen species (ROS) production and clearance must be better defined. The objective of this study was to determine how insulin affects superoxide radical (O2•-) levels. O2•- production was evaluated in adult cardiomyocytes isolated from control and Akita (type 1 diabetic) mice by spin-trapping electron paramagnetic resonance spectroscopy. We found that the basal rates of O2•- production were comparable in control and Akita cardiomyocytes. However, culturing cardiomyocytes without insulin resulted in a significant increase in O2•- production only in the Akita group. In contrast, O2•- production was unaffected by high glucose and/or fatty acid supplementation. The increase in O2•- was due in part to a decrease in superoxide dismutase (SOD) activity. The PI3K inhibitor, LY294002, decreased Akita SOD activity when insulin was present, indicating that the modulation of antioxidant activity is through insulin signaling. The effect of insulin on mitochondrial O2•- production was evaluated in Akita mice that underwent a 1-week treatment of insulin. Mitochondria isolated from insulin-treated Akita mice produced less O2•- than vehicle-treated diabetic mice. Quantitative proteomics was performed on whole heart homogenates to determine how insulin affects antioxidant protein expression. Of 29 antioxidant enzymes quantified, thioredoxin 1 was the only one that was significantly enhanced by insulin treatment. In vitro analysis of thioredoxin 1 revealed a previously undescribed capacity of the enzyme to directly scavenge O2•-. These findings demonstrate that insulin has a role in mitigating cardiac oxidative stress in diabetes via regulation of endogenous antioxidant activity.

Evaluation of National Resident Matching Program and Electronic Residency Application Service Data in the Integrated Plastic Surgery Match.

INTRODUCTION: The National Resident Matching Program and Electronic Residency Application Service provide data for tracking trends among applicants in each specialty over the past 5 years. The purpose of this study was to examine this information and determine sex and race/ethnicity distribution over the past 5 years. METHODS: The National Resident Matching Program and Electronic Residency Application Service databases were surveyed for trends in the following categories: number of positions, number of applicants, percent of applicants per position, and number of applicants by sex and self-identified race/ethnicity. This information was analyzed graphically for visual representation of the changes in the field. RESULTS: While there has been a steady increase in number of positions offered, there has also been a significant decrease in number of applicants, resulting in an increase in number of positions offered per applicant. While female and Asian applicants have increased in number, rates of applications from other diverse groups have remained stagnant. CONCLUSIONS: The number of plastic surgery positions offered has increased, whereas the number of applicants has decreased, resulting in a reduction in the number of applicants per position. Lack of racial diversity remains a significant issue in the applicant pool.

Oxytocin and Addiction: Potential Glutamatergic Mechanisms.

Recently, oxytocin (OXT) has been investigated for its potential therapeutic role in addiction. OXT has been found to diminish various drug-seeking and drug-induced behaviors. Although its behavioral effects are well-established, there is not much consensus on how this neuropeptide exerts its effects. Previous research has given thought to how dopamine (DA) may be involved in oxytocinergic mechanisms, but there has not been as strong of a focus on the role that glutamate (Glu) has. The glutamatergic system is critical for the processing of rewards and the disruption of glutamatergic projections produces the behaviors seen in drug addicts. We introduce the idea that OXT has direct effects on Glu transmission within the reward processing pathway. Thus, OXT may reduce addictive behaviors by restoring abnormal drug-induced changes in the glutamatergic system and in its interactions with other neurotransmitters. This review offers insight into the mechanisms through which a potentially viable therapeutic target, OXT, could be used to reduce addiction-related behaviors.

Botulinum Toxin for the Treatment of Intractable Raynaud Phenomenon.

Raynaud phenomenon (RP) is a condition causing vasospasm in the fingers and toes of patients that can have a significant negative impact on quality of life. This can lead to pain, ulceration, and possible loss of digits. Several pharmacological options are available for treatment. However, RP can often be refractory to traditional modalities, leaving surgery or injections as the next available options. This article provides a review and update on the use of botulinum toxin as an effective therapy for the treatment of RP refractory to medical management.

Consensus, cooperative learning, and flocking for multiagent predator avoidance.

Multiagent coordination is highly desirable with many uses in a variety of tasks. In nature, the phenomenon of coordinated flocking is highly common with applications related to defending or escaping from predators. In this article, a hybrid multiagent system that integrates consensus, cooperative learning, and flocking control to determine the direction of attacking predators and learns to flock away from them in a coordinated manner is proposed. This system is entirely distributed requiring only communication between neighboring agents. The fusion of consensus and collaborative reinforcement learning allows agents to cooperatively learn in a variety of multiagent coordination tasks, but this article focuses on flocking away from attacking predators. The results of the flocking show that the agents are able to effectively flock to a target without collision with each other or obstacles. Multiple reinforcement learning methods are evaluated for the task with cooperative learning utilizing function approximation for state-space reduction performing the best. The results of the proposed consensus algorithm show that it provides quick and accurate transmission of information between agents in the flock. Simulations are conducted to show and validate the proposed hybrid system in both one and two predator environments, resulting in an efficient cooperative learning behavior. In the future, the system of using consensus to determine the state and reinforcement learning to learn the states can be applied to additional multiagent tasks.

Paraspinous Muscle Flaps for the Treatment of Complex Spinal Wounds.

MINI: This retrospective case series investigated paraspinous flaps for coverage of complex spinal wounds. 6.90% of patients developed postoperative wound infections and 0.00% of patients required instrumentation removal for infection. This suggests that these flaps may offer a long-term solution in wound management for patients with repeated spinal operations. STUDY DESIGN: Retrospective case series. OBJECTIVE: To investigate the efficacy and complication profile of the use of paraspinous muscle flaps for closure of complex spinal wounds. SUMMARY OF BACKGROUND DATA: Paraspinous muscle flap closure offers an innovative option in difficult-to-manage post-spinal surgery wounds. Current literature reports are mixed in terms of success and complication rates of these flap procedures, with most sources citing a wound complication rate of 20%. METHODS: This case series investigated the hospital course of 58 patients undergoing paraspinous flap closure after spinal surgery between the years 2014 and 2018. Information gathered includes: demographics, surgery indication, location, and length of incision on the spine, nutrition labs, previous spinal surgeries, preoperative wound class, operative times, length of hospital stay, and complication rates including reoperation, wound infection, and other postoperative complications. RESULTS: Of the 58 patients undergoing spinal muscle flap closure, 51 (87.93%) had undergone previous spinal surgery with an average of 2.12 previous surgeries in these patients. Mean albumin and prealbumin were 2.62 and 13.75, respectively. 4/58 (6.90%) developed a wound infection or experienced a continuation of their chronic osteomyelitis. Of the 57 patients that had spinal instrumentation, three (5.26%) had spinal implants removed at the time of surgery and two (3.51%) had it removed or replaced later for mechanical complications. No patients had instrumentation removed for chronic infections. One (1.72%) experienced reoperation for wound-related complications. These rates are lower than most complication rates in the current literature. CONCLUSION: The plastic and reconstructive paraspinous muscle flap has promising results as a closure option for complex spinal wounds following neurosurgical cases. Further investigation is called for to determine the applicability of these results to the general population. LEVEL OF EVIDENCE: 4.

Retrospective case series. To investigate the efficacy and complication profile of the use of paraspinous muscle flaps for closure of complex spinal wounds. Paraspinous muscle flap closure offers an innovative option in difficult-to-manage post-spinal surgery wounds. Current literature reports are mixed in terms of success and complication rates of these flap procedures, with most sources citing a wound complication rate of 20%. This case series investigated the hospital course of 58 patients undergoing paraspinous flap closure after spinal surgery between the years 2014 and 2018. Information gathered includes: demographics, surgery indication, location, and length of incision on the spine, nutrition labs, previous spinal surgeries, preoperative wound class, operative times, length of hospital stay, and complication rates including reoperation, wound infection, and other postoperative complications. Of the 58 patients undergoing spinal muscle flap closure, 51 (87.93%) had undergone previous spinal surgery with an average of 2.12 previous surgeries in these patients. Mean albumin and prealbumin were 2.62 and 13.75, respectively. 4/58 (6.90%) developed a wound infection or experienced a continuation of their chronic osteomyelitis. Of the 57 patients that had spinal instrumentation, three (5.26%) had spinal implants removed at the time of surgery and two (3.51%) had it removed or replaced later for mechanical complications. No patients had instrumentation removed for chronic infections. One (1.72%) experienced reoperation for wound-related complications. These rates are lower than most complication rates in the current literature. The plastic and reconstructive paraspinous muscle flap has promising results as a closure option for complex spinal wounds following neurosurgical cases. Further investigation is called for to determine the applicability of these results to the general population. Level of Evidence: 4.

Enhancing cardiac glycolysis causes an increase in PDK4 content in response to short-term high-fat diet.

The healthy heart has a dynamic capacity to respond and adapt to changes in nutrient availability. Metabolic inflexibility, such as occurs with diabetes, increases cardiac reliance on fatty acids to meet energetic demands, and this results in deleterious effects, including mitochondrial dysfunction, that contribute to pathophysiology. Enhancing glucose usage may mitigate metabolic inflexibility and be advantageous under such conditions. Here, we sought to identify how mitochondrial function and cardiac metabolism are affected in a transgenic mouse model of enhanced cardiac glycolysis (GlycoHi) basally and following a short-term (7-day) high-fat diet (HFD). GlycoHi mice constitutively express an active form of phosphofructokinase-2, resulting in elevated levels of the PFK-1 allosteric activator fructose 2,6-bisphosphate. We report that basally GlycoHi mitochondria exhibit augmented pyruvate-supported respiration relative to fatty acids. Nevertheless, both WT and GlycoHi mitochondria had a similar shift toward increased rates of fatty acid-supported respiration following HFD. Metabolic profiling by GC-MS revealed distinct features based on both genotype and diet, with a unique increase in branched-chain amino acids in the GlycoHi HFD group. Targeted quantitative proteomics analysis also supported both genotype- and diet-dependent changes in protein expression and uncovered an enhanced expression of pyruvate dehydrogenase kinase 4 (PDK4) in the GlycoHi HFD group. These results support a newly identified mechanism whereby the levels of fructose 2,6-bisphosphate promote mitochondrial PDK4 levels and identify a secondary adaptive response that prevents excessive mitochondrial pyruvate oxidation when glycolysis is sustained after a high-fat dietary challenge.

National Surgical Quality Improvement Program Analysis of 9110 Reduction Mammaplasty Patients: Identifying Risk Factors Associated With Complications in Patients Older Than 60 Years.

BACKGROUND: The purpose of this study was to identify preoperative risk factors in patients undergoing reduction mammoplasty as well as identify any increased complication risk in patients older than 60 years undergoing reduction mammoplasty. METHODS: The American College of Surgeons National Surgical Quality Improvement Program data from years 2013-2015 was reviewed. Patients were identified using Current Procedural Terminology code 19318 specific for reduction mammoplasty. Only patients undergoing bilateral procedures were included, and no reconstructive procedures were included. Patient demographics, comorbidities, and 30-day complications were analyzed. Comparative analysis was performed between patients younger than 60 years and patients 60 years and older, identifying risk factors associated with complications in the geriatric population. RESULTS: A total of 9110 patients undergoing reduction mammoplasty were identified. Of these 1442 (15.83%) were patients older than 60 years. Mean age of all patients was 42 years (range, 18-85 years). Eighty hundred fifty-nine patients were active smokers. Four hundred eighty-two patients were diabetic. Overall, 798 complications occurred with an incidence of 8.7%. Group 1 (<60 years) mean age was 39 years (range, 18-59). Group 2 (>60 years) mean age was 66 years (range, 60-85 years). The geriatric population showed a higher risk of cerebral vascular accidents (P < 0.00006), myocardial infarction (P < 0.02), and readmission (P < 0.03). Smoking was found to be a statistically significant risk factor for superficial surgical site infection, and deep space infection. Diabetes was found to be a statistically significant risk factor for readmission. CONCLUSIONS: Reduction mammoplasty is a common surgical procedure. It is not uncommon for patients older than 60 years to undergo elective reduction mammoplasty (15.83% incidence), resulting in a cumulative complication rate of 11.65% in the geriatric population compared with 8.89% in the group of patients younger than 60 years. Smoking and diabetes were found to be independent risk factors for complications, regardless of age.

Coenzyme A-mediated degradation of pyruvate dehydrogenase kinase 4 promotes cardiac metabolic flexibility after high-fat feeding in mice.

Cardiac energy is produced primarily by oxidation of fatty acids and glucose, with the relative contributions of each nutrient being sensitive to changes in substrate availability and energetic demand. A major contributor to cardiac metabolic flexibility is pyruvate dehydrogenase (PDH), which converts glucose-derived pyruvate to acetyl-CoA within the mitochondria. PDH is inhibited by phosphorylation dependent on the competing activities of pyruvate dehydrogenase kinases (PDK1-4) and phosphatases (PDP1-2). A single high-fat meal increases cardiac PDK4 content and subsequently inhibits PDH activity, reducing pyruvate utilization when abundant fatty acids are available. In this study, we demonstrate that diet-induced increases in PDK4 are reversible and characterize a novel pathway that regulates PDK4 degradation in response to the cardiac metabolic environment. We found that PDK4 degradation is promoted by CoA (CoASH), the levels of which declined in mice fed a high-fat diet and normalized following transition to a control diet. We conclude that CoASH functions as a metabolic sensor linking the rate of PDK4 degradation to fatty acid availability in the heart. However, prolonged high-fat feeding followed by return to a low-fat diet resulted in persistent in vitro sensitivity of PDH to fatty acid-induced inhibition despite reductions in PDK4 content. Moreover, increases in the levels of proteins responsible for ß-oxidation and rates of palmitate oxidation by isolated cardiac mitochondria following long-term consumption of high dietary fat persisted after transition to the control diet. We propose that these changes prime PDH for inhibition upon reintroduction of fatty acids.

Cardiac Insulin Signaling Regulates Glycolysis Through Phosphofructokinase 2 Content and Activity.

BACKGROUND: The healthy heart has a dynamic capacity to respond and adapt to changes in nutrient availability. Diabetes mellitus disrupts this metabolic flexibility and promotes cardiomyopathy through mechanisms that are not completely understood. Phosphofructokinase 2 (PFK-2) is a primary regulator of cardiac glycolysis and substrate selection, yet its regulation under normal and pathological conditions is unknown. This study was undertaken to determine how changes in insulin signaling affect PFK-2 content, activity, and cardiac metabolism. METHODS AND RESULTS: Streptozotocin-induced diabetes mellitus, high-fat diet feeding, and fasted mice were used to identify how decreased insulin signaling affects PFK-2 and cardiac metabolism. Primary adult cardiomyocytes were used to define the mechanisms that regulate PFK-2 degradation. Both type 1 diabetes mellitus and a high-fat diet induced a significant decrease in cardiac PFK-2 protein content without affecting its transcript levels. Overnight fasting also induced a decrease in PFK-2, suggesting it is rapidly degraded in the absence of insulin signaling. An unbiased metabolomic study demonstrated that decreased PFK-2 in fasted animals is accompanied by an increase in glycolytic intermediates upstream of phosphofructokianse-1, whereas those downstream are diminished. Mechanistic studies using cardiomyocytes showed that, in the absence of insulin signaling, PFK-2 is rapidly degraded via both proteasomal- and chaperone-mediated autophagy. CONCLUSIONS: The loss of PFK-2 content as a result of reduced insulin signaling impairs the capacity to dynamically regulate glycolysis and elevates the levels of early glycolytic intermediates. Although this may be beneficial in the fasted state to conserve systemic glucose, it represents a pathological impairment in diabetes mellitus.

Post-Synthetic Defucosylation of AGP by Aspergillus nidulans α-1,2-Fucosidase Expressed in Arabidopsis Apoplast Induces Compensatory Upregulation of α-1,2-Fucosyltransferases.

Cell walls are essential components of plant cells which perform a variety of important functions for the different cell types, tissues and organs of a plant. Besides mechanical function providing cell shape, cell walls participate in intercellular communication, defense during plant-microbe interactions, and plant growth. The plant cell wall consists predominantly of polysaccharides with the addition of structural glycoproteins, phenolic esters, minerals, lignin, and associated enzymes. Alterations in the cell wall composition created through either changes in biosynthesis of specific constituents or their post-synthetic modifications in the apoplast compromise cell wall integrity and frequently induce plant compensatory responses as a result of these alterations. Here we report that post-synthetic removal of fucose residues specifically from arabinogalactan proteins in the Arabidopsis plant cell wall induces differential expression of fucosyltransferases and leads to the root and hypocotyl elongation changes. These results demonstrate that the post-synthetic modification of cell wall components presents a valuable approach to investigate the potential signaling pathways induced during plant responses to such modifications that usually occur during plant development and stress responses.

A homology model of Xyloglucan Xylosyltransferase 2 reveals critical amino acids involved in substrate binding.

In dicotyledonous plants, xyloglucan (XyG) is the most abundant hemicellulose of the primary cell wall. The enzymes involved in XyG biosynthesis have been identified through reverse-genetics and activity was characterized by heterologous expression. Currently, there is no information on the atomic structures or amino acids involved in activity or substrate binding of any of the Golgi-localized XyG biosynthetic enzymes. A homology model of the xyloglucan xylosyltransferase 2 (XXT2) catalytic domain was built on the basis of the crystal structure of A64Rp. Molecular dynamics simulations revealed that the homology model retains the glycosyltransferase (GT)-A fold of the template structure used to build the homology model indicating that XXT2 likely has a GT-A fold. According to the XXT2 homology model, six amino acids (Phe204, Lys207, Asp228, Ser229, Asp230, His378) were selected and their contribution in catalytic activity was investigated. Site-directed mutagenesis studies show that Asp228, Asp230 and His378 are critical for XXT2 activity and are predicted to be involved in coordination of manganese ion. Lys207 was also found to be critical for protein activity and the homology model indicates a critical role in substrate binding. Additionally, Phe204 mutants have less of an impact on XXT2 activity with the largest effect when replaced with a polar residue. This is the first study that investigates the amino acids involved in substrate binding of the XyG-synthesizing xylosyltransferases and contributes to the understanding of the mechanisms of polysaccharide-synthesizing GTs and XyG biosynthesis.

Enzymatic Activity of Xyloglucan Xylosyltransferase 5.

Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a ß-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.

Protein-protein interactions among xyloglucan-synthesizing enzymes and formation of Golgi-localized multiprotein complexes.

Arabidopsis thaliana xyloglucan has an XXXG structure, with branches of xylosyl residues, ß-D-galacosyl-(1,2)-α-d-xylosyl motifs and fucosylated ß-D-galactosyl-(1,2)-α-D-xylosyl motifs. Most of the enzymes involved in xyloglucan biosynthesis in Arabidopsis have been identified, including the glucan synthase CSLC4 (cellulose synthase-like C4), three xylosyltransferases (XXT1, XXT2 and XXT5), two galactosyltransferases (MUR3 and XLT2) and the fucosyltransferase FUT1. The XXTs and CSLC4 form homo- and heterocomplexes and were proposed to co-localize in the same complex, but the organization of the other xyloglucan-synthesizing enzymes remains unclear. Here we investigate whether the glycosyltransferases MUR3, XLT2 and FUT1 interact with the XXT-CSLC4 complexes in the Arabidopsis Golgi. We used co-immunoprecipitation and bimolecular fluorescence complementation, with signal quantification by flow cytometry, to demonstrate that CSLC4 interacts with MUR3, XLT2 and FUT1. FUT1 forms homocomplexes and interacts with MUR3, XLT2, XXT2 and XXT5. XLT2 interacts with XXT2 and XXT5, but MUR3 does not. Co-immunoprecipitation assays showed that FUT1 forms a homocomplex through disulfide bonds, and formation of the heterocomplexes does not involve covalent interactions. In vitro pull-down assays indicated that interactions in the FUT1-MUR3 and FUT1-XXT2 complexes occur through the protein catalytic domains. We propose that enzymes involved in xyloglucan biosynthesis are functionally organized in multiprotein complexes localized in the Golgi.