Linear Pathway Research Articles

Particle-Based Simulation Reveals Macromolecular Crowding Effects on the Michaelis-Menten Mechanism

Many computational models for analyzing and predicting cell physiology rely on in vitro data collected in dilute and controlled buffer solutions. However, this can mislead models because up to 40% of the intracellular volume—depending on the organism, the physiology, and the cellular compartment—is occupied by a dense mixture of proteins, lipids, polysaccharides, RNA, and DNA. These intracellular macromolecules interfere with the interactions of enzymes and their reactants and thus affect the kinetics of biochemical reactions, making in vivo reactions considerably more complex than the in vitro data indicates. In this work, we present a new, to our knowledge, type of kinetics that captures and quantifies the effect of volume exclusion and other spatial phenomena on the kinetics of elementary reactions. We further developed a framework that allows for the efficient parameterization of these kinetics using particle simulations. Our formulation, entitled generalized elementary kinetics, can be used to analyze and predict the effect of intracellular crowding on enzymatic reactions and was herein applied to investigate the influence of crowding on phosphoglycerate mutase in Escherichia coli, which exhibits prototypical reversible Michaelis-Menten kinetics. Current research indicates that many enzymes are reaction limited and not diffusion limited, and our results suggest that the influence of fractal diffusion is minimal for these reaction-limited enzymes. Instead, increased association rates and decreased dissociation rates lead to a strong decrease in the effective maximal velocities Vmax and the effective Michaelis-Menten constants KM under physiologically relevant volume occupancies. Finally, the effects of crowding were explored in the context of a linear pathway, with the finding that crowding can have a redistributing effect on the effective flux responses in the case of twofold enzyme overexpression. We suggest that this framework, in combination with detailed kinetics models, will improve our understanding of enzyme reaction networks under nonideal conditions.

Biological conversion of methane to putrescine using genome-scale model-guided metabolic engineering of a methanotrophic bacterium Methylomicrobium alcaliphilum 20Z

BackgroundMethane is the primary component of natural gas and biogas. The huge abundance of methane makes it a promising alternative carbon source for industrial biotechnology. Herein, we report diamine compound, putrescine, production from methane by an industrially promising methanotroph Methylomicrobium alcaliphilum 20Z.ResultsWe conducted adaptive evolution to improve putrescine tolerance of M. alcaliphilum 20Z because putrescine highly inhibits the cell growth. The evolved strain 20ZE was able to grow in the presence of 400 mM of putrescine dihydrochloride. The expression of linear pathway ornithine decarboxylase genes from Escherichia coli and Methylosinus trichosporium OB3b allowed the engineered strain to produce putrescine. A higher putrescine titer of 12.44 mg/L was obtained in the strain 20ZE-pACO with ornithine decarboxylase from M. trichosporium OB3b. For elimination of the putrescine utilization pathway, spermidine synthase (MEALZ_3408) was knocked out, resulting in no spermidine formation in the strain 20ZES1-pACO with a putrescine titer of 18.43 mg/L. Next, a genome-scale metabolic model was applied to identify gene knockout strategies. Acetate kinase (MEALZ_2853) and subsequently lactate dehydrogenase (MEALZ_0534) were selected as knockout targets, and the deletion of these genes resulted in an improvement of the putrescine titer to 26.69 mg/L. Furthermore, the putrescine titer was improved to 39.04 mg/L by overexpression of key genes in the ornithine biosynthesis pathway under control of the pTac promoter. Finally, suitable nitrogen sources for growth of M. alcaliphilum 20Z and putrescine production were optimized with the supplement of 2 mM ammonium chloride to nitrate mineral salt medium, and this led to the production of 98.08 mg/L putrescine, almost eightfold higher than that from the initial strain. Transcriptome analysis of the engineered strains showed upregulation of most genes involved in methane assimilation, citric acid cycle, and ammonia assimilation in ammonia nitrate mineral salt medium, compared to nitrate mineral salt medium.ConclusionsThe engineered M. alcaliphilum 20ZE4-pACO strain was able to produce putrescine up to 98.08 mg/L, almost eightfold higher than the initial strain. This study represents the bioconversion of methane to putrescine—a high value-added diamine compound.

Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma

Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC (i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.

Population Pharmacokinetics of Alemtuzumab (Campath) in Pediatric Hematopoietic Cell Transplantation: Towards Individualized Dosing to Improve Outcome.

Background and ObjectiveAlemtuzumab (Campath®) is used to prevent graft-versus-host disease and graft failure following pediatric allogeneic hematopoietic cell transplantation. The main toxicity includes delayed immune reconstitution, subsequent viral reactivations, and leukemia relapse. Exposure to alemtuzumab is highly variable upon empirical milligram/kilogram dosing.MethodsA population pharmacokinetic (PK) model for alemtuzumab was developed based on a total of 1146 concentration samples from 206 patients, aged 0.2–19 years, receiving a cumulative intravenous dose of 0.2–1.5 mg/kg, and treated between 2003 and 2015 in two centers.ResultsAlemtuzumab PK were best described using a two-compartment model with a parallel saturable and linear elimination pathway. The linear clearance pathway, central volume of distribution, and intercompartmental distribution increased with body weight. Blood lymphocyte counts, a potential substrate for alemtuzumab, did not impact clearance.ConclusionThe current practice with uniform milligram/kilogram doses leads to highly variable exposures in children due to the non-linear relationship between body weight and alemtuzumab PK. This model may be used for individualized dosing of alemtuzumab.Electronic supplementary materialThe online version of this article (10.1007/s40262-019-00782-0) contains supplementary material, which is available to authorized users.

Tuning stress in bacteria

Signaling In Gram-positive bacteria, protein arginine phosphorylation plays a role in the stress response by regulating transcription and marking aberrant proteins for degradation. Through structural and functional studies of the arginine kinase McsB, Suskiewicz et al. show that the bacterial phospho-signaling system goes beyond linear on-off pathways. The McsB active site is related to that in phosphagen kinases, which are animal enzymes that catalyze phosphorylation of guanido compounds, but the catalytic domain is modified so that MscB binds arginine residues that are part of a polypeptide chain rather than in small compounds. McsB acts more efficiently as a dimer, and phosphoarginine binding to the dimerization domain further stimulates enzyme activity. This additional control, superimposed on the slower transcriptional control, is likely to be important for a fast and robust stress response. Nat. Chem. Biol. 15 , 510 (2019).

Longitudinal Analysis Between Maternal Feeding Practices and Body Mass Index (BMI): A Study in Asian Singaporean Preschoolers.

Bidirectional studies between maternal feeding practices with subsequent child weight are limited, with no studies in Asian populations. In longitudinal analyses, we assessed the directionality of the associations between maternal feeding practices and body mass index (BMI) in preschoolers. Participants were 428 mother child dyads from the GUSTO (Growing Up in Singapore Toward healthy Outcomes) cohort. Feeding practices were assessed using the Comprehensive Feeding Practices Questionnaire (CFPQ) at age 5 y. Child BMI was measured at ages 4 and 6 y. BMI and maternal feeding practices subscales were transformed to SD scores and both directions of their associations examined with multivariable linear regression and pathway modeling. Higher BMI at age 4 was associated with lower encouragement of balance and variety (β = −0.33; 95%CI: −0.53, −0.13), lower pressure to eat (β = −0.49; −0.68, −0.29) and higher restriction (β = 1.10; 0.67, 1.52) at age 5, adjusting for confounders and baseline feeding practices at 3 years. In the reverse direction, only pressure and restriction at age 5 were associated with lower and higher child BMI at age 6 years, respectively. After the adjustment for baseline BMI at age 5, the association with pressure was attenuated to non-significance (β = 0.01 (−0.01, 0.03), while the association with restriction remained significant (β = 0.02; 0.002, 0.03). Overall, associations from child BMI to maternal restriction for weight control and pressure feeding practices was stronger than the association from these maternal feeding practices to child BMI (Wald's statistics = 24.3 and 19.5, respectively; p < 0.001). The strength and directionality suggests that the mothers in the Asian population were likely to adopt these feeding practices in response to their child's BMI, rather than the converse.Clinical Trial Registry Number and Website This study was registered at clinicaltrials.gov as NCT01174875 (www.clinicaltrials.gov, NCT01174875).

What is the rate-limiting step towards aging? Chemical reaction kinetics might reconcile contradictory observations in experimental aging research.

Modern geroscience is divided as regards the validity of the free radical theory of aging. Thermodynamic arguments and observations from comparative zoology support it, whereas results from experimental manipulations in representative animal species sometimes strongly contradict it. From a comparison of the multi-step aging process with a linear metabolic pathway (glycolysis), we here argue that the identification of the rate-limiting kinetic steps of the aging cascade is essential to understand the overall flux through the cascade, i.e., the rate of aging. Examining free radical reactions as a case in point, these reactions usually occur as chain reactions with three kinetically independent steps: initiation, propagation, and termination, each of which can be rate-limiting. Revisiting the major arguments in favor and against a role of free radicals in aging, we find that the majority of arguments in favor point to radical propagation as relevant and rate-limiting, whereas almost all arguments in disfavor are based on experimental manipulations of radical initiation or radical termination which turned out to be ineffective. We conclude that the overall lack of efficacy of antioxidant supplementation (which fosters termination) and antioxidant enzyme overexpression (which inhibits initiation) in longevity studies is attributable to the fact that initiation and termination are not the rate-limiting steps of the aging cascade. The biological and evolutionary plausibility of this interpretation is discussed. In summary, radical propagation is predicted to be rate-limiting for aging and should be explored in more detail.

Self-regulated learning strategies in world's first MOOC in implant dentistry.

Massive Open Online Courses (MOOCs) have been often described as a major innovation Higher Education, but their application in the teaching of clinical disciplines is still very limited, while there is a lack of scientific evaluations in this domain. The aim of this study was to investigate learners' behaviours and correlate patterns of Self-Regulated Learning (SRL) with performance and achievement during a MOOC in Implant Dentistry. The clickstream data of learners from the first run of the MOOC Implant dentistry by The University of Hong Kong were modelled and quantified based on Zimmerman's SRL model. The data were quantitatively analysed by means of k-means clustering for evidence of five SRL behavioural indicators of student activity. The clusters identified were then correlated with student engagement and learning performance. A total of 7608 individuals enrolled, 5014 engaged (active learners 65.90%), 1277 of them (25.47%) completed the course and 1232 purchased a certificate. Two major groups of learners emerged: Attentive (n=1433) who were more likely to follow the prescribed pathway in the MOOC and Auditors (n=3581) who accessed content selectively. There was significantly higher engagement, achievement and completion rates among Attentive than Auditors. Both groups included subcategories (Browser, Digger, Test-driven, Sampler, Persistent) which might reflect different SRL strategies. A MOOC in a clinical discipline can achieve high enrolment and completion rates as compared to current benchmarks. There appears to be a wide diversity of learning behaviours among learners, with two however dominant patterns. Learners with a linear learning pathway achieved significantly higher grades and completion rates than those who accessed content irregularly and selectively. Such differences however might be influenced by the demographic and professional background of the learner, as well as their motivation to attending the MOOC. Certain learning behaviours, in particular how learners access content in relation to assessments might be closer related to SRL.

Metaproteomics reveals persistent and phylum-redundant metabolic functional stability in adult human gut microbiomes of Crohn\u2019s remission patients despite temporal variations in microbial taxa, genomes, and proteomes

BackgroundThe gut microbiome plays a fundamental role in the human host’s overall health by contributing key biological functions such as expanded metabolism and pathogen defense/immune control. In a healthy individual, the gut microbiome co-exists within the human host in a symbiotic, non-inflammatory relationship that enables mutual benefits, such as microbial degradation of indigestible food products into small molecules that the host can utilize, and enhanced pathogen defense. In abnormal conditions, such as Crohn’s disease, this favorable metabolic relationship breaks down and a variety of undesirable activities result, including chronic inflammation and other health-related issues. It has been difficult, however, to elucidate the overall functional characteristics of this relationship because the microbiota can vary substantially in composition for healthy humans and possibly even more in individuals with gut disease conditions such as Crohn’s disease. Overall, this suggests that microbial membership composition may not be the best way to characterize a phenotype. Alternatively, it seems to be more informative to examine and characterize the functional composition of a gut microbiome. Towards that end, this study examines 25 metaproteomes measured in several Crohn’s disease patients’ post-resection surgery across the course of 1 year, in order to examine persistence of microbial taxa, genes, proteins, and metabolic functional distributions across time in individuals whose microbiome might be more variable due to the gut disease condition.ResultsThe measured metaproteomes were highly personalized, with all the temporally-related metaproteomes clustering most closely by individual. In general, the metaproteomes were remarkably distinct between individuals and to a lesser extent within individuals. This prompted a need to characterize the metaproteome at a higher functional level, which was achieved by annotating identified protein groups with KEGG orthologous groups to infer metabolic modules. At this level, similar and redundant metabolic functions across multiple phyla were observed across time and between individuals. Tracking through these various metabolic modules revealed a clear path from carbohydrate, lipid, and amino acid degradation to central metabolism and finally the production of fermentation products.ConclusionsThe human gut metaproteome can vary quite substantially across time and individuals. However, despite substantial intra-individual variation in the metaproteomes, there is a clear persistence of conserved metabolic functions across time and individuals. Additionally, the persistence of these core functions is redundant across multiple phyla but is not always observable in the same sample. Finally, the gut microbiome’s metabolism is not driven by a set of discrete linear pathways but a web of interconnected reactions facilitated by a network of enzymes that connect multiple molecules across multiple pathways.

PAKT pathway activation is associated with PIK3CA mutations and good prognosis in luminal breast cancer in contrast to p-mTOR pathway activation

Numerous studies have focused on the PI3K/AKT/mTOR pathway in estrogen receptor positive (ER) breast cancer (BC), as a linear signal transduction pathway and reported its association with worse clinical outcomes. We developed gene signatures that reflect the level of expression of phosphorylated-Serine473-AKT (pAKT) and phosphorylated-Serine2448-mTOR (p-mTOR) separately, capturing their corresponding level of pathway activation. Our analysis revealed that the pAKT pathway activation was associated with luminal A BC while the p-mTOR pathway activation was more associated with luminal B BC (Kruskal–Wallis test p < 10−10). pAKT pathway activation was significantly associated with better outcomes (multivariable HR, 0.79; 95%CI, 0.74–0.85; p = 2.5 × 10−10) and PIK3CA mutations (p = 0.0001) whereas p-mTOR pathway activation showed worse outcomes (multivariable HR,1.1; 95%CI, 1.1–1.2; p = 9.9 × 10−4) and associated with p53 mutations (p = 0.04). in conclusion, our data show that pAKT and p-mTOR pathway activation have differing impact on prognosis and suggest that they are not linearly connected in luminal breast cancers.

A linear pathway for mevalonate production supports growth of Thermococcus kodakarensis.

The sole unifying feature of Archaea is the use of isoprenoid-based glycerol lipid ethers to compose cellular membranes. The branched hydrocarbon tails of archaeal lipids are synthesized via the polymerization of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but many questions still surround the pathway(s) that result in production of IPP and DMAPP in archaeal species. Isotopic-labeling strategies argue for multiple biological routes for production of mevalonate, but biochemical and bioinformatic studies support only a linear pathway for mevalonate production. Here, we use a combination of genetic and biochemical assays to detail the production of mevalonate in the model archaeon Thermococcus kodakarensis. We demonstrate that a single, linear pathway to mevalonate biosynthesis is essential and that alternative routes of mevalonate production, if present, are not biologically sufficient to support growth in the absence of the classical mevalonate pathway resulting in IPP production from acetyl-CoA. Archaeal species provide an ideal platform for production of high-value isoprenoids in large quantities, and the results obtained provide avenues to further increase the production of mevalonate to drive isoprenoid production in archaeal hosts.

The Plant microRNA Pathway: The Production and Action Stages.

Plant microRNAs are an endogenous class of small regulatory RNA central to the posttranscriptional regulation of gene expression in plant development and environmental stress adaptation or in response to pathogen challenge. The plant microRNA pathway is readily separated into two distinct stages: (1) the production stage, which is localized to the plant cell nucleus and where the microRNA small RNA is processed from a double-stranded RNA precursor transcript, and (2) the action stage, which is localized to the plant cell cytoplasm and where the mature microRNA small RNA is loaded into an effector complex and is used by the complex as a sequence specificity guide to direct expression repression of target genes harboring highly complementary microRNA target sequences. Historical research indicated that the plant microRNA pathway was a highly structured, almost linear pathway requiring a small set of core machinery proteins. However, contemporary research has demonstrated that the plant microRNA pathway is highly dynamic, and to allow for this flexibility, a large and highly functionally diverse set of machinery proteins is now known to be required. For example, recent research has shown that plant microRNAs can regulate target gene expression via a translational repression mechanism of RNA silencing in addition to the standard messenger RNA cleavage-based mechanism of RNA silencing: a mode of RNA silencing originally assigned to all plant microRNAs. Using Arabidopsis thaliana as our model system, here we report on both the core and auxiliary sets of machinery proteins now known to be required for both microRNA production and microRNA action in plants.

Big Grain3, encoding a purine permease, regulates grain size via modulating cytokinin transport in rice.

Grain size is an important agronomic trait affecting grain yield, but the underlying molecular mechanisms remain to be elucidated. Here, we isolated a dominant mutant, big grain3 (bg3-D), which exhibits a remarkable increase of grain size caused by activation of the PURINE PERMEASE gene, OsPUP4. BG3/OsPUP4 is predominantly expressed in vascular tissues and is specifically suppressed by exogenous cytokinin application. Hormone profiling revealed that the distribution of different cytokinin forms, in roots and shoots of the bg3-D mutant, is altered. Quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that expression of rice cytokinin type-A RESPONSE REGULATOR (OsRR) genes is enhanced in the roots of the bg3-D mutant. These results suggest that OsPUP4 might contribute to the long-distance transport of cytokinin, by reinforcing cytokinin loading into vascular bundle cells. Furthermore, plants overexpressing OsPUP7, the closest homolog of OsPUP4, also exhibited a similar phenotype to the bg3-D mutant. Interestingly, subcellular localization demonstrated that OsPUP4 was localized on the plasma membrane, whereas OsPUP7 was localized to the endoplasmic reticulum. Based on these findings, we propose that OsPUP4 and OsPUP7 function in a linear pathway to direct cytokinin cell-to-cell transport, affecting both its long-distance movement and local allocation.

Agri-food systems in international research for development: Ten theses regarding impact pathways, partnerships, program design, and priority-setting for rural prosperity

Drawn from numerous sources, including papers in this special issue, this concluding paper synthesizes evidence on the relationship between agricultural research for development and poverty reduction, with particular emphasis on agri-food systems perspectives in shaping programs aimed at rural prosperity. Following our introduction in section 1, we revisit the ex ante set of 18 pathways in section 2 (which were laid out in our introductory paper for this SI), posing some critical questions: Can a manageable set of impact pathways be identified? How are they inter-related? Rather than independent linear pathways, is it better (both conceptually and for clarity of communication) to represent these as impact networks rather than linear pathways? These insights lead to very different and more inclusive partnerships and contain their own implications for program design in section 3. The challenges facing the world today are complex, and no single organization or sector can hope to effectively confront these issues alone. Not only is partnership increasingly seen as a multi-stakeholder phenomenon rather than a bilateral one, but there also is a discernible move towards a network framing (e.g., as “innovation systems” or “boundary spanning”). This change is driven by the progressive inclusion of agricultural research goals as part of the wider development agenda, where complexity and systemic change are central. In turn, this requires more appropriate strategies for knowledge creation, innovation, and partnership. Section 4 presents implications for program design and priority-setting that follow from foregoing insights on the interplay of pathways and partnerships.

Newly Identified Electrically Coupled Neurons Support Development of the Drosophila Giant Fiber Model Circuit.

The Drosophila giant fiber (GF) escape circuit is an extensively studied model for neuron connectivity and function. Researchers have long taken advantage of the simple linear neuronal pathway, which begins at peripheral sensory modalities, travels through the central GF interneuron (GFI) to motor neurons, and terminates on wing/leg muscles. This circuit is more complex than it seems, however, as there exists a complex web of coupled neurons connected to the GFI that widely innervates the thoracic ganglion. Here, we define four new neuron clusters dye coupled to the central GFI, which we name GF coupled (GFC) 1–4. We identify new transgenic Gal4 drivers that express specifically in these neurons, and map both neuronal architecture and synaptic polarity. GFC1–4 share a central site of GFI connectivity, the inframedial bridge, where the neurons each form electrical synapses. Targeted apoptotic ablation of GFC1 reveals a key role for the proper development of the GF circuit, including the maintenance of GFI connectivity with upstream and downstream synaptic partners. GFC1 ablation frequently results in the loss of one GFI, which is always compensated for by contralateral innervation from a branch of the persisting GFI axon. Overall, this work reveals extensively coupled interconnectivity within the GF circuit, and the requirement of coupled neurons for circuit development. Identification of this large population of electrically coupled neurons in this classic model, and the ability to genetically manipulate these electrically synapsed neurons, expands the GF system capabilities for the nuanced, sophisticated circuit dissection necessary for deeper investigations into brain formation.

Systematic Analysis of Bottlenecks in a Multibranched and Multilevel Regulated Pathway: The Molecular Fundamentals of l-Methionine Biosynthesis in Escherichia coli.

To produce chemicals and fuels from renewable resources, various strategies and genetic tools have been developed to redesign pathways and optimize the metabolic flux in microorganisms. However, in most successful cases, the target chemicals are synthesized through a linear pathway, and regular methodologies for the identification of bottlenecks and metabolic flux optimization in multibranched and multilevel regulated pathways, such as the l-methionine biosynthetic pathway, have rarely been reported. In the present study, a systematic analysis strategy was employed to gradually reveal and remove the potential bottlenecks limiting the l-methionine biosynthesis in E.coli. 80 genes in central metabolism and selected amino acids biosynthetic pathways were first repressed or upregulated to probe their effects on l-methionine accumulation. The l-methionine biosynthetic pathway was then modularized and iteratively genetic modifications were performed to uncover the multiple layers of limitations and stepwise improve the l-methionine titer. The metabolomics data further revealed a more evenly distributed metabolic flux in l-methionine biosynthesis pathway of the optimal strain and provided valuable suggestions for further optimization. The optimal strain produced 16.86 g/L of l-methionine in 48 h by fed-batch fermentation. This work is the first to our knowledge to systematically elucidate the molecular fundamentals of multilevel regulation of l-methionine biosynthesis. It also demonstrated that the systematic analysis strategy can boost our ability to identify the potential bottlenecks and optimize the metabolic flux in multibranched and multilevel regulated pathways for the production of corresponding chemicals.

Stops, Starts, and Detours: Transfer Students’ College Choice Process

ABSTRACTWith the number of transfer students increasing and graduation still an elusive goal, the researchers conducted four focus groups and four individual interviews with transfer students attending universities in the Chicago and Dallas-Fort Worth area to better understand and explore the factors that influenced their college choice decisions at their first and subsequent institutions. The specific research questions were: (a) When, why, and how do transfer students select their postsecondary institutions? (b) How do students who have transferred more than once describe their decision-making process and has it changed from their initial college choice decision? The study found many students did not pursue planned, linear pathways to transfer. Rather, they often made spontaneous decisions affected by a range of personal as well as academic issues. The article includes implications for student affairs professionals and high school counselors to facilitate the college choice process.

Utilization of glycerol for endogenous glucose and glycogen synthesis in seabass (Dicentrarchus labrax): A potential mechanism for sparing amino acid catabolism in carnivorous fish

Glycerol is a well-tolerated additive in aquafeeds. We hypothesized that in seabass, a carnivorous fish whose endogenous carbohydrate demands are mostly met by hepatic amino acid gluconeogenesis, glycerol effectively competes with these substrates for endogenous glucose and glycogen synthesis. To test this hypothesis, we injected fasted and fed juvenile seabass with an intraperitoneal 2 g.kg−1 bolus of glycerol enriched to 25% with [U-13C3]glycerol and monitored the appearance of blood glucose and hepatic glycogen 13C-isotopomers 48-h thereafter by 13C NMR spectroscopy. This information was used to determine the fractional contribution of the glycerol load to systemic glucose appearance and liver glycogen synthesis. In 21-day fasted fish, the glycerol bolus contributed 47 ± 3% of circulating glucose appearance and 40 ± 3% of hepatic glycogen at 48-h post-bolus. In fed fish, the glycerol bolus accounted for 17 ± 3% of circulating glucose and 3 ± 1% of hepatic glycogen at 48-h post-polus. While the majority of glycerol was metabolized to glucose and glycogen via the canonical linear gluconeogenic pathway, a significant minority was converted to glucose and glycogen via the hepatic Krebs cycle. In fasted fish, linear gluconeogenesis accounted for 40 ± 3% of glucose appearance while the Krebs cycle route accounted for 7 ± 2%. In fed fish, linear and Krebs cycle-mediated contributions were 10 ± 2% and 6 ± 2%, respectively. These data indicate that glycerol effectively competes with endogenous precursors for hepatic gluconeogenesis in carnivorous fish thereby representing a novel mechanism for reducing the catabolic utilization of amino acids.

A speculation on the tandem fasciclin 1 repeat of FLA4 proteins in angiosperms

ABSTRACTThe Arabidopsis thaliana Fasciclin like arabinogalactan protein 4 (FLA4) locus is required for normal root growth in a linear genetic pathway with the FEI1 and FEI2 loci coding for receptor-like kinases. The two Fas1 domains of FLA4 are conserved among angiosperms but only the C-terminal Fas1 domain is required for genetic function. We show that at low salt deletion of the N-terminal Fas1 domain of transgenic FLA4 leads to enhanced root elongation compared to the tandem Fas1 wild type version. Modeling the hypothetical interaction between FLA4 and FEI1 we show that the predicted interaction is predominantly involving the C-terminal Fas1 domain. Relative conformational mobility between the two FLA4 Fas1 domains might regulate the interaction with the FEI receptor kinases. We therefore speculate that the FLA4 FEI complex might be a sensor for environmental conditions in the apoplast.

Amidic Coupling in the Synthesis of Pyrroloquinoline 4‐ and 9‐Carboxamides as Potential STAT3 Inhibitors

AbstractThe work describes the synthesis and characterization of a small series of pyrroloquinoline carboxamide derivatives considered on the STX‐0119 model, a known inhibitor of STAT3 (Signal transducers and activators of transcription 3) by interacting with SH2 domain. The scope was to learn SARs and possibly obtain active new compounds for cancer therapy. To this aim, the quinoline scaffold of the lead compound STX‐0119 was modified into the pyrroloquinoline tricycle to obtain two subsets of carboxamide derivatives showing different angular geometry 3H‐pyrrolo[3, 2‐f] and 7H‐pyrrolo[2, 3‐h]quinolines. Successful synthesis of thirty compounds was carried out, adopting methods using both convergent and linear pathways. Amidic coupling chemistry was applied to pyrroloquinoline carboxylic acids to generate the desired carboxamides of aromatic and aliphatic primary and secondary amines. Full physico‐chemical characterization including predicted LogP and LogD of all compounds was accomplished. The biological screening of antiproliferative activity against three human tumor cell lines by MTT test revealed low or no activity.