Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism.

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

Metformin and small for gestational age babies: findings of a randomised placebo-controlled clinical trial of metformin in gestational diabetes (EMERGE).

AIMS/HYPOTHESIS: Gestational diabetes mellitus (GDM) is associated with adverse perinatal outcomes because of suboptimal glucose management and glucose control and excessive weight gain. Metformin can offset these factors but is associated with small for gestational age (SGA) infants. We sought to identify risk factors for SGA infants, including the effect of metformin exposure on SGA status. METHODS: In this prespecified secondary analysis of the EMERGE trial, which evaluated the effectiveness of metformin vs placebo in treating GDM and found reduced gestational weight gain and longer time to insulin initiation with metformin use, we included women with a live-born infant and known infant birthweight and gestational age at delivery. We compared the numbers of SGA infants in both groups and explored baseline predictive factors to help identify those at highest risk of delivering an SGA infant. RESULTS: Baseline maternal characteristics were similar between SGA and non-SGA pregnancies. On multivariable-adjusted regression, no baseline maternal variables were associated with SGA status. Mothers of SGA infants were more likely to develop pre-eclampsia or gestational hypertension (18.2% vs 2.0%, p=0.001; 22.7% vs 5.4%, p=0.005, respectively); after multivariable adjustment, pre-eclampsia was positively associated with SGA status). Among SGA pregnancies, important perinatal outcomes including preterm birth, Caesarean delivery and neonatal care unit admission did not differ between the metformin and placebo groups (20.0% vs 14.3%, p=1.00; 50.0% vs 28.6%, p=0.25; 13.3% vs 42.9%, p=0.27, respectively). CONCLUSIONS/INTERPRETATION: Pre-eclampsia was strongly associated with SGA infants. Metformin-exposed SGA infants did not display a more severe SGA phenotype than infants treated with placebo. TRIAL REGISTRATION: Clinical Trials.gov NCT02980276; EudraCT number: 2016-001644-19.

The involvement of allosteric effectors and post-translational modifications in the control of plant central carbon metabolism.

Plant metabolism is finely orchestrated to allow the occurrence of complementary and sometimes opposite metabolic pathways. In part this is achieved by the allosteric regulation of enzymes, which has been a cornerstone of plant research for many decades. The completion of the Arabidopsis genome and the development of the associated toolkits for Arabidopsis research moved the focus of many researchers to other fields. This is reflected by the increasing number of high-throughput proteomic studies, mainly focused on post-translational modifications. However, follow-up 'classical' biochemical studies to assess the functions and upstream signaling pathways responsible for such modifications have been scarce. In this work, we review the basic concepts of allosteric regulation of enzymes involved in plant carbon metabolism, comprising photosynthesis and photorespiration, starch and sucrose synthesis, glycolysis and gluconeogenesis, the oxidative pentose phosphate pathway and the tricarboxylic acid cycle. Additionally, we revisit the latest results on the allosteric control of the enzymes involved in these pathways. To conclude, we elaborate on the current methods for studying protein-metabolite interactions, which we consider will become crucial for discoveries in the future.

Study of two glycosyltransferases related to polysaccharide biosynthesis in Rhodococcus jostii RHA1.

The bacterial genus Rhodococcus comprises organisms performing oleaginous behaviors under certain growth conditions and ratios of carbon and nitrogen availability. Rhodococci are outstanding producers of biofuel precursors, where lipid and glycogen metabolisms are closely related. Thus, a better understanding of rhodococcal carbon partitioning requires identifying catalytic steps redirecting sugar moieties to storage molecules. Here, we analyzed two GT4 glycosyl-transferases from Rhodococcus jostii (RjoGlgAb and RjoGlgAc) annotated as α-glucan-α-1,4-glucosyl transferases, putatively involved in glycogen synthesis. Both enzymes were produced in Escherichia coli cells, purified to homogeneity, and kinetically characterized. RjoGlgAb and RjoGlgAc presented the "canonical" glycogen synthase activity and were actives as maltose-1P synthases, although to a different extent. Then, RjoGlgAc is a homologous enzyme to the mycobacterial GlgM, with similar kinetic behavior and glucosyl-donor preference. RjoGlgAc was two orders of magnitude more efficient to glucosylate glucose-1P than glycogen, also using glucosamine-1P as a catalytically efficient aglycon. Instead, RjoGlgAb exhibited both activities with similar kinetic efficiency and preference for short-branched α-1,4-glucans. Curiously, RjoGlgAb presented a super-oligomeric conformation (higher than 15 subunits), representing a novel enzyme with a unique structure-to-function relationship. Kinetic results presented herein constitute a hint to infer on polysaccharides biosynthesis in rhodococci from an enzymological point of view.

A cluster-randomised controlled feasibility trial evaluating the Cognitive Occupation-Based programme for people with Multiple Sclerosis (COB-MS).

INTRODUCTION: There is a high prevalence of cognitive difficulties in MS, but despite this, there are few programmes targeting cognition that focus on the ability to function well in everyday life. The Cognitive Occupation-Based programme for people with Multiple Sclerosis (COB-MS), an occupation-focused cognitive intervention, was developed to address this. It addresses both the functional difficulties and the wide-ranging symptoms that present in MS. OBJECTIVE: Here we report on the results of a cluster-randomised controlled feasibility trial (ISRCTN11462710; registered 4th September 2019) evaluating the COB-MS in terms of feasibility and initial efficacy as a cognitive intervention for people with MS. METHOD: The eight-session COB-MS intervention was delivered remotely by occupational therapists to participants with MS in the intervention group. Following the end of the trial the COB-MS was delivered to the wait-list control group. Data was collected from people with MS experiencing cognitive difficulties at baseline, post-intervention, 12-weeks, and 6-month follow-up. The primary outcome measure was the Goal Attainment Scaling at 12 weeks. Data was also collected in the domains of cognition, quality of life, and mood. RESULTS: One hundred and eighteen people with MS and cognitive difficulties were randomised to either usual care (n = 60) or COB-MS intervention (n = 58). Ninety-four participants were retained at 6-month follow-up. The COB-MS was found to be feasible, including trial procedures and protocol. Data indicates that the COB-MS is accepted by participants and had positive impacts on daily life. Those allocated to the COB-MS group had a significant improvement in the primary outcome compared to the control condition. Progression criteria set for the feasibility trial have been met therefore further testing of the COB-MS at a definitive trial is supported by the results. CONCLUSION: The results provide a strong basis for a pathway to a future definitive trial of COB-MS, with respect to both feasibility and preliminary, clinical efficacy. TRIAL REGISTRATION: ISRCTN11462710 Date of registration: 4th September 2019.

Efficacy and toxicity of primary re-irradiation for malignant spinal cord compression based on radiobiological modelling: a phase II clinical trial.

BACKGROUND: The efficacy and safety of primary re-irradiation for MSCC are not known. Our aim was to establish the efficacy and safety of biologically effective dose-based re-irradiation. METHODS: Patients presenting with MSCC at a previously irradiated spine segment, and not proceeding with surgical decompression, were eligible. A 3 Gray per fraction experimental schedule (minimum 18 Gy/6 fractions, maximum 30 Gy/10 fractions) was used, delivering a maximum cumulative spinal dose of 100 Gy2 if the interval since the last radiotherapy was within 6 months, or 130 Gy2 if longer. The primary outcome was a change in mobility from week 1 to week 5 post-treatment, as assessed by the Tomita score. The RTOG SOMA score was used to screen for spinal toxicity, and an MRI performed to assess for radiation-induced myelopathy (RIM). RESULTS: Twenty-two patients were enroled, of whom eleven were evaluable for the primary outcome. Nine of eleven (81.8%) had stable or improved Tomita scores at 5 weeks. One of eight (12.5%) evaluable for late toxicity developed RIM. CONCLUSIONS: Re-irradiation is an efficacious treatment for MSCC. There is a risk of RIM with a cumulative dose of 120 Gy2. CLINICAL TRIAL REGISTRATION: Cancer Trials Ireland (ICORG 07-11); NCT00974168.

Transforming growth factor ß1 upregulates 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-4 expression in A549 and MCF-10A cells.

Transforming growth factor ß1 (TGFß1) induces a cellular process known as epithelial-mesenchymal transition (EMT) associated with metabolic reprogramming, including enhanced glycolysis. Given the involvement of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFKFB) enzymes in glycolysis, we aimed to investigate whether TGFß1 regulates expressions of PFKFB genes and if PFKFBs are required for TGFß1-driven phenotypes. A549 and MCF-10A cell lines were used as TGFß1-driven EMT models. Messenger RNA expressions of PFKFB and EMT genes were determined by real-time quantitative polymerase chain reaction. A small interfering RNA approach was used to deplete PFKFB4 expression. A Matrigel invasion assay was conducted to assess the effect of PFKFB4 silencing on the TGFß1-enhanced invasion of A549 cells. F2,6BP levels were analyzed using an enzyme-coupled assay. Glucose and lactate concentrations were determined using colorimetric assays. TGFß1 robustly induced expression of the fourth isoform of PFKFBs, PFKFB4, in both cell lines. PFKFB4 depletion partially inhibits mesenchymal transdifferentiation caused by TGFß1 in A549 cells, as assessed by microscopy. Inductions of Snail in MCF-10A cells and Fibronectin in A549 cells and repressions of E-cadherin in both cell lines by TGFß1 are attenuated by PFKFB4 silencing. PFKFB4 silencing reduces F2,6BP and glycolytic activity, although TGFß1 alone does not affect these parameters. Finally, PFKFB4 depletion suppresses the TGFß1-driven invasion of A549 cells through Matrigel. Presented data suggest that TGFß1 induces the expression of PFKFB4 in A549 and MCF-10 cells, and PFKFB4 may be required for TGFß1-driven phenotypes such as EMT and invasion in these models.

Remote administration of BICAMS measures and the Trail-Making Test to assess cognitive impairment in multiple sclerosis.

Reliable remote cognitive testing could provide a safer assessment of cognitive impairment in multiple sclerosis (MS) during the COVID-19 pandemic and thereafter. Here we aimed to investigate the reliability and feasibility of administering Brief International Cognitive Assessment for MS (BICAMS) and the Trail-Making Test (TMT) to people with MS online. Between-group differences on BICAMS and the TMT were examined in a sample of 68 participants. Group 1 (N = 34) was tested in-person pre-pandemic. Group 2 was tested remotely. Within-group differences for in-person and virtual administrations were examined for Group 1. No significant differences between virtual and in-person administrations of the CVLT-II and SDMT were detected. BVMT-R scores were significantly higher for virtual administrations (M = 20.59, SD = 6.65) compared to in-person administrations (M = 16.35, SD = 6.05), possibly indicating inter-rater differences. Strong positive correlations were found for in-person and virtual scores within Group 1 on the CVLT-II (r = .84), SDMT (r = .85), TMT-A (r = .88), TMT-B (r = .76) and BVMT-R (r = .72). No significant differences between in-person and remote administrations of CVLT-II and SDMT in people living with MS were detected. Recommendations for future studies employing the TMT and BVMT-R online are provided.

Early Metformin in Gestational Diabetes: A Randomized Clinical Trial.

Importance: Gestational diabetes is a common complication of pregnancy and the optimal management is uncertain. Objective: To test whether early initiation of metformin reduces insulin initiation or improves fasting hyperglycemia at gestation weeks 32 or 38. Design, Setting, and Participants: Double-blind, placebo-controlled trial conducted in 2 centers in Ireland (one tertiary hospital and one smaller regional hospital). Participants were enrolled from June 2017 through September 2022 and followed up until 12 weeks' postpartum. Participants comprised 510 individuals (535 pregnancies) diagnosed with gestational diabetes based on World Health Organization 2013 criteria. Interventions: Randomized 1:1 to either placebo or metformin (maximum dose, 2500 mg) in addition to usual care. Main Outcomes And Measures: The primary outcome was a composite of insulin initiation or a fasting glucose level of 5.1 mmol/L or greater at gestation weeks 32 or 38. Results: Among 510 participants (mean age, 34.3 years), 535 pregnancies were randomized. The primary composite outcome was not significantly different between groups and occurred in 150 pregnancies (56.8%) in the metformin group and 167 pregnancies (63.7%) in the placebo group (between-group difference, -6.9% [95% CI, -15.1% to 1.4%]; relative risk, 0.89 [95% CI, 0.78-1.02]; P = .13). Of 6 prespecified secondary maternal outcomes, 3 favored the metformin group, including time to insulin initiation, self-reported capillary glycemic control, and gestational weight gain. Secondary neonatal outcomes differed by group, with smaller neonates (lower mean birth weights, a lower proportion weighing >4 kg, a lower proportion in the >90% percentile, and smaller crown-heel length) in the metformin group without differences in neonatal intensive care needs, respiratory distress requiring respiratory support, jaundice requiring phototherapy, major congenital anomalies, neonatal hypoglycemia, or proportion with 5-minute Apgar scores less than 7. Conclusion and relevance: Early treatment with metformin was not superior to placebo for the composite primary outcome. Prespecified secondary outcome data support further investigation of metformin in larger clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT02980276; EudraCT: 2016-001644-19.

Structure, function, and evolution of plant ADP-glucose pyrophosphorylase.

KEY MESSAGE: This review outlines research performed in the last two decades on the structural, kinetic, regulatory and evolutionary aspects of ADP-glucose pyrophosphorylase, the regulatory enzyme for starch biosynthesis. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the pathway of glycogen and starch synthesis in bacteria and plants, respectively. Plant ADP-Glc PPase is a heterotetramer allosterically regulated by metabolites and post-translational modifications. In this review, we focus on the three-dimensional structure of the plant enzyme, the amino acids that bind the regulatory molecules, and the regions involved in transmitting the allosteric signal to the catalytic site. We provide a model for the evolution of the small and large subunits, which produce heterotetramers with distinct catalytic and regulatory properties. Additionally, we review the various post-translational modifications observed in ADP-Glc PPases from different species and tissues. Finally, we discuss the subcellular localization of the enzyme found in grain endosperm from grasses, such as maize and rice. Overall, this work brings together research performed in the last two decades to better understand the multiple mechanisms involved in the regulation of ADP-Glc PPase. The rational modification of this enzyme could improve the yield and resilience of economically important crops, which is particularly important in the current scenario of climate change and food shortage.

Structural Determinants of Sugar Alcohol Biosynthesis in Plants: The Crystal Structures of Mannose-6-Phosphate and Aldose-6-Phosphate Reductases.

Sugar alcohols are major photosynthetic products in plant species from the Apiaceae and Plantaginaceae families. Mannose-6-phosphate reductase (Man6PRase) and aldose-6-phosphate reductase (Ald6PRase) are key enzymes for synthesizing mannitol and glucitol in celery (Apium graveolens) and peach (Prunus persica), respectively. In this work, we report the first crystal structures of dimeric plant aldo/keto reductases (AKRs), celery Man6PRase (solved in the presence of mannonic acid and NADP+) and peach Ald6PRase (obtained in the apo form). Both structures displayed the typical TIM barrel folding commonly observed in proteins from the AKR superfamily. Analysis of the Man6PRase holo form showed that residues putatively involved in the catalytic mechanism are located close to the nicotinamide ring of NADP+, where the hydride transfer to the sugar phosphate should take place. Additionally, we found that Lys48 is important for the binding of the sugar phosphate. Interestingly, the Man6PRase K48A mutant had a lower catalytic efficiency with mannose-6-phosphate but a higher catalytic efficiency with mannose than the wild type. Overall, our work sheds light on the structure-function relationships of important enzymes to synthesize sugar alcohols in plants.

Simultaneous inhibition of PFKFB3 and GLS1 selectively kills KRAS-transformed pancreatic cells.

Activating mutations of the oncogenic KRAS in pancreatic ductal adenocarcinoma (PDAC) are associated with an aberrant metabolic phenotype that may be therapeutically exploited. Increased glutamine utilization via glutaminase-1 (GLS1) is one such feature of the activated KRAS signaling that is essential to cell survival and proliferation; however, metabolic plasticity of PDAC cells allow them to adapt to GLS1 inhibition via various mechanisms including activation of glycolysis, suggesting a requirement for combinatorial anti-metabolic approaches to combat PDAC. We investigated whether targeting the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) in combination with GLS1 can selectively prevent the growth of KRAS-transformed cells. We show that KRAS-transformation of pancreatic duct cells robustly sensitizes them to the dual targeting of GLS1 and PFKFB3. We also report that this sensitivity is preserved in the PDAC cell line PANC-1 which harbors an activating KRAS mutation. We then demonstrate that GLS1 inhibition reduced fructose-2,6-bisphosphate levels, the product of PFKFB3, whereas PFKFB3 inhibition increased glutamine consumption, and these effects were augmented by the co-inhibition of GLS1 and PFKFB3, suggesting a reciprocal regulation between PFKFB3 and GLS1. In conclusion, this study identifies a novel mutant KRAS-induced metabolic vulnerability that may be targeted via combinatorial inhibition of GLS1 and PFKFB3 to suppress PDAC cell growth.

Nucleotide-sugar metabolism in plants: the legacy of Luis F. Leloir.

This review commemorates the 50th anniversary of the Nobel Prize in Chemistry awarded to Luis F. Leloir 'for his discovery of sugar-nucleotides and their role in the biosynthesis of carbohydrates'. He and his co-workers discovered that activated forms of simple sugars, such as UDP-glucose and UDP-galactose, are essential intermediates in the interconversion of sugars. They elucidated the biosynthetic pathways for sucrose and starch, which are the major end-products of photosynthesis, and for trehalose. Trehalose 6-phosphate, the intermediate of trehalose biosynthesis that they discovered, is now a molecule of great interest due to its function as a sugar signalling metabolite that regulates many aspects of plant metabolism and development. The work of the Leloir group also opened the doors to an understanding of the biosynthesis of cellulose and other structural cell wall polysaccharides (hemicelluloses and pectins), and ascorbic acid (vitamin C). Nucleotide-sugars also serve as sugar donors for a myriad of glycosyltransferases that conjugate sugars to other molecules, including lipids, phytohormones, secondary metabolites, and proteins, thereby modifying their biological activity. In this review, we highlight the diversity of nucleotide-sugars and their functions in plants, in recognition of Leloir's rich and enduring legacy to plant science.

Proteolytic cleavage of Arabidopsis thaliana phosphoenolpyruvate carboxykinase-1 modifies its allosteric regulation.

Phosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis. In this work, we analyze the proteolysis of Arabidopsis thaliana PEPCK1 (AthPEPCK1) in germinating seedlings. We found that the amount of AthPEPCK1 protein peaks at 24-48 h post-imbibition. Concomitantly, we observed shorter versions of AthPEPCK1, putatively generated by metacaspase-9 (AthMC9). To study the impact of AthMC9 cleavage on the kinetic and regulatory properties of AthPEPCK1, we produced truncated mutants based on the reported AthMC9 cleavage sites. The Δ19 and Δ101 truncated mutants of AthPEPCK1 showed similar kinetic parameters and the same quaternary structure as the wild type. However, activation by malate and inhibition by glucose 6-phosphate were abolished in the Δ101 mutant. We propose that proteolysis of AthPEPCK1 in germinating seedlings operates as a mechanism to adapt the sensitivity to allosteric regulation during the sink-to-source transition.

Gesture-Based Human Machine Interaction Using RCNNs in Limited Computation Power Devices.

The use of gestures is one of the main forms of human machine interaction (HMI) in many fields, from advanced robotics industrial setups, to multimedia devices at home. Almost every gesture detection system uses computer vision as the fundamental technology, with the already well-known problems of image processing: changes in lighting conditions, partial occlusions, variations in color, among others. To solve all these potential issues, deep learning techniques have been proven to be very effective. This research proposes a hand gesture recognition system based on convolutional neural networks and color images that is robust against environmental variations, has a real time performance in embedded systems, and solves the principal problems presented in the previous paragraph. A new CNN network has been specifically designed with a small architecture in terms of number of layers and total number of neurons to be used in computationally limited devices. The obtained results achieve a percentage of success of 96.92% on average, a better score than those obtained by previous algorithms discussed in the state of the art.

On Combining Convolutional Autoencoders and Support Vector Machines for Fault Detection in Industrial Textures.

Defects in textured materials present a great variability, usually requiring ad-hoc solutions for each specific case. This research work proposes a solution that combines two machine learning-based approaches, convolutional autoencoders, CA; one class support vector machines, SVM. Both methods are trained using only defect free textured images for each type of analyzed texture, labeling the samples for the SVMs in an automatic way. This work is based on two image processing streams using image sensors: (1) the CA first processes the incoming image from the input to the output, producing a reconstructed image, from which a measurement of correct or defective image is obtained; (2) the second process uses the latent layer information as input to the SVM to produce a measurement of classification. Both measurements are effectively combined, making an additional research contribution. The results obtained achieve a percentage of success of 92% on average, outperforming results of previous works.

Structural analysis reveals a pyruvate-binding activator site in the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

The pathways for biosynthesis of glycogen in bacteria and starch in plants are evolutionarily and biochemically related. They are regulated primarily by ADP-glucose pyrophosphorylase, which evolved to satisfy metabolic requirements of a particular organism. Despite the importance of these two pathways, little is known about the mechanism that controls pyrophosphorylase activity or the location of its allosteric sites. Here, we report pyruvate-bound crystal structures of ADP-glucose pyrophosphorylase from the bacterium Agrobacterium tumefaciens, identifying a previously elusive activator site for the enzyme. We found that the tetrameric enzyme binds two molecules of pyruvate in a planar conformation. Each binding site is located in a crevice between the C-terminal domains of two subunits where they stack via a distinct ß-helix region. Pyruvate interacts with the side chain of Lys-43 and with the peptide backbone of Ser-328 and Gly-329 from both subunits. These structural insights led to the design of two variants with altered regulatory properties. In one variant (K43A), the allosteric effect was absent, whereas in the other (G329D), the introduced Asp mimicked the presence of pyruvate. The latter generated an enzyme that was preactivated and insensitive to further activation by pyruvate. Our study furnishes a deeper understanding of how glycogen biosynthesis is regulated in bacteria and the mechanism by which transgenic plants increased their starch production. These insights will facilitate rational approaches to enzyme engineering for starch production in crops of agricultural interest and will promote further study of allosteric signal transmission and molecular evolution in this important enzyme family.

Non-inferiority randomised phase 3 trial comparing two radiation schedules (single vs. five fractions) in malignant spinal cord compression.

BACKGROUND: The optimal EBRT schedule for MSCC is undetermined. Our aim was to determine whether a single fraction (SF) was non-inferior to five daily fractions (5Fx), for functional motor outcome. METHODS: Patients not proceeding with surgical decompression in this multicentre non-inferiority, Phase 3 trial were randomised to 10 Gy/SF or 20 Gy/5Fx. A change in mobility from baseline to 5 weeks for each patient, was evaluated by a Modified Tomita score: 1 = 'Walk unaided', 2 = 'With walking aid' and 3 = 'Bed-bound'. The margin used to establish non-inferiority was a detrimental change of -0.4 in the mean difference between arms. RESULTS: One-hundred and twelve eligible patients were enrolled. Seventy-three patients aged 30-87 were evaluated for the primary analysis. The 95% CI for the difference in the mean change in mobility scores between arms was -0.12 to 0.6. Since -0.4 is not included in the interval, there is evidence that 10 Gy/SF is non-inferior to 20 Gy/5Fx. One grade 3 AE was reported in the 5Fx arm. Twelve (26%) patients in the 5Fx arm had a Grade 2-3 AE compared with six (11%) patients in the SF arm (p = 0.093). CONCLUSION: For mobility preservation, one 10-Gy fraction is non-inferior to 20 Gy in five fractions, in patients with MSCC not proceeding with surgical decompression. CLINICAL TRIAL REGISTRATION: Cancer Trials Ireland ICORG 05-03; NCT00968643; EU-20952.

PFKFB2 regulates glycolysis and proliferation in pancreatic cancer cells.

Tumor cells increase glucose metabolism through glycolysis and pentose phosphate pathways to meet the bioenergetic and biosynthetic demands of rapid cell proliferation. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are key regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent activator of glycolysis. Previous studies have reported the co-expression of PFKFB isozymes, as well as the mRNA splice variants of particular PFKFB isozymes, suggesting non-redundant functions. Majority of the evidence demonstrating a requirement for PFKFB activity in increased glycolysis and oncogenic properties in tumor cells comes from studies on PFKFB3 and PFKFB4 isozymes. In this study, we show that the PFKFB2 isozyme is expressed in tumor cell lines of various origin, overexpressed and localizes to the nucleus in pancreatic adenocarcinoma, relative to normal pancreatic tissue. We then demonstrate the differential intracellular localization of two PFKFB2 mRNA splice variants and that, when ectopically expressed, cytoplasmically localized mRNA splice variant causes a greater increase in F2,6BP which coincides with an increased glucose uptake, as compared with the mRNA splice variant localizing to the nucleus. We then show that PFKFB2 expression is required for steady-state F2,6BP levels, glycolytic activity, and proliferation of pancreatic adenocarcinoma cells. In conclusion, this study may provide a rationale for detailed investigation of PFKFB2's requirement for the glycolytic and oncogenic phenotype of pancreatic adenocarcinoma cells.

Biochemical characterization of phosphoenolpyruvate carboxykinases from Arabidopsis thaliana.

ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.