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Modeling of the pyruvate production with Escherichia coli: comparison of mechanistic and neural networks-based models.

Three different models: the unstructured mechanistic black-box model, the input-output neural network-based model and the externally recurrent neural network model were used to describe the pyruvate production process from glucose and acetate using the genetically modified Escherichia coli YYC202 ldhA::Kan strain. The experimental data were used from the recently described batch and fed-batch experiments [ Zelić B, Study of the process development for Escherichia coli-based pyruvate production. PhD Thesis, University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia, July 2003. (In English); Zelić et al. Bioproc Biosyst Eng 26:249-258 (2004); Zelić et al. Eng Life Sci 3:299-305 (2003); Zelić et al Biotechnol Bioeng 85:638-646 (2004)]. The neural networks were built out of the experimental data obtained in the fed-batch pyruvate production experiments with the constant glucose feed rate. The model validation was performed using the experimental results obtained from the batch and fed-batch pyruvate production experiments with the constant acetate feed rate. Dynamics of the substrate and product concentration changes was estimated using two neural network-based models for biomass and pyruvate. It was shown that neural networks could be used for the modeling of complex microbial fermentation processes, even in conditions in which mechanistic unstructured models cannot be applied.

Acetates↗

An assessment of the role of proton leaks in the mechanistic stoichiometry of oxidative phosphorylation.

Rat liver mitochondria were incubated in the presence of varying concentrations of ATP, followed by ADP to initiate phosphorylation. Analysis of phosphorylation to oxygen ratios (P/O) was carried out with varied initial phosphorylation potentials (or ATP/ADP ratios). Rates of phosphorylation and respiration and magnitude of membrane potential (delta psi) were measured. The results are discussed in the framework of P/total O and P/"extra" O ratios in determination of the mechanistic P/O ratio. It is concluded that the former underestimates, and the latter overestimates the mechanistic P/O ratio.

Adenosine Diphosphate↗

Mechanistic studies of ion-selective electrodes.

A knowledge of the chemical composition of blood, urine and other body fluids is a daily requirement for departments of biochemistry. Electronic circuitry and computers to process the data are readily available, but satisfactory transducers to convert chemical composition into electrical signals are frequently the weakest link in the chain of measurement. This review is concerned with one group of transducers: ion-selective electrodes. Since the commercial success of the calcium and fluoride ions-selective electrodes in the mid-sixties, a range of other electrodes has become available. Their use has already conferred considerable benefits upon medicine, e.g. rapid, low-cost, multiple assay of major blood components; diagnostic surveys of chloride sweat levels relating to cystic fibrosis; and monitoring blood fluorides during and after halothane administration. Their adoption for indirectly sensing enzymes and associated substrates is particularly noteworthy. Recent advances in electronics, coupled with flow injection schemes based on ion-selective electrodes, have facilitated the management of hundreds of samples daily. However, developments in the mechanistic knowledge of these sensors have not matched the increase in their application, although definite progress can be reported; for example with regard to the origin of the potential signals induced by ion activities in solution. Numerous techniques have been devised to unravel mechanistic problems, among which radioisotope tracer and impedance measurements may be cited as especially valuable. Selectivity performance, particularly in complex biological media, and undesirable features such as protein poisoning, need further research. Organic chemists are now better placed to synthesize new designs of acyclic and cyclic molecules as mobile site, ion-selective, sensor materials which, with appropriate mediator solvents, provide improved sensor cocktails. This design feature is well illustrated by the continuing guest for a lithium ion-selective electrode compatible with the high levels of sodium interference in blood.

Biomedical Engineering↗

Isolation and prevalidation of an Escherichia coli tester strain for the use in mechanistic and metabolic studies of genotoxins.

We have isolated an Escherichia coli tester strain for the use in mechanistic and metabolic studies of genotoxins. We started with one of the more used and better characterized E. coli K-12 laboratory strains, AB1157. We isolated a lipopolysaccharide defective mutant of strain AB1886 which is an excision repair deficient derivative of AB1157 and introduced a newly constructed plasmid pKR11, encoding mucAB, resulting in strain MR2101/pKR11. A genotoxicity assay was designed, monitoring the reversion to arginine prototrophy and a preliminary validation was carried out against Ames tester strain TA100 with a set of diagnostic compounds. The results seem to indicate that strain MR2101/pKR11 is an adequate tester strain which can be a useful tool in mechanistic studies. Moreover, this strain can serve as mother strain to isolate improved and more specialized tester strains.

Dose-Response Relationship, Drug↗

Description of a mechanistic approach to pain management in advanced cancer. Preliminary report.

A mechanistic approach to advanced cancer pain management is proposed, based on the clinically perceived anatomical and pathophysiological mechanisms of pain generation. It is an extension of the World Health Organisation (WHO) analgesic ladder in which severity of pain is the principal determinant of analgesic choice. The mechanistic categories are: superficial somatic, deep somatic, visceral and neurogenic (mixed or pure, i.e., nociceptive component present or absent). Allocation of pain to the different categories is based on clinical history, physical findings and investigations to establish the site and extent of active primary or metastatic tumor deposits, and evidence of previous response to medication. Drug choice sequence is determined by the dominant pain mechanism judged to be present and not the severity of the pain. In order to describe this approach, mechanisms of pain, disease distribution and drug treatment have been analysed in the first 20 consecutive patients who consented to enter a longitudinal pain description and evaluation study on admission to an inpatient hospice unit. Despite a high exclusion rate from research standard monitoring due to severity of illness and related factors, the majority of eligible patients approached to enter the study did so, and the pain scoring was well tolerated. The implications of this for future research and clinical practice are discussed. In 6 patients only 1 pain mechanism was identified (visceral 4, deep somatic 2). Two mechanisms were present in 8 patients and 3 mechanisms in 6 patients. The deep somatic mechanism was identified in 15 patients, visceral mechanism in 13, neurogenic in 10 and superficial somatic in 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Risk, susceptibility and the epidemiology of proliferative neoplastic disease: descriptive vs. mechanistic approaches.

Recently there has been a confusing and somewhat frustrating difference of opinion in epidemiology concerning the relative merit and utility of descriptive versus mechanistic knowledge in approaching the comprehension and control of human cancer. This distinction has both historical and evolutionary rationale; it generally being necessary to know something of WHAT is happening before one can be legitimately concerned with HOW it is happening. As outlined below, however, there is little justification for making value judgements based on this distinction, and the situation is more one in which descriptive approaches and mechanistic approaches are equally essential in the development of a systematic viewpoint, a viewpoint providing guidance to meaningful intervention.

Disease Susceptibility↗

Use of mechanistic information in risk assessment for toxic chemicals.

Risk assessment (RA) for toxic chemicals is assumed to be a scientific activity providing a framework of principles for the complication and evaluation of all available scientific information and the rational extrapolation to human health effects in as quantitative terms as possible and with a high degree of certainty. Sensible public health decisions are made more certain through the use of mechanistic information throughout the 4 steps in RA: hazard identification, dose-response assessment, exposure (dose) assessment and risk characterisation. Examples of the use of mechanistic information to assess risks of systemic, developmental/reproductive and neurotoxic effects show how to move away from the presently used threshold/no observable adverse effect/uncertainty factor default methodology towards an evaluation based on all available scientific data. The experience gained in cancer RA in the use of metabolic and tissue binding (receptor) models as well as physiologically based pharmacokinetic (PBPK) and pharmacodynamic (PBPD) models can be transferred to non-cancer RA. A good example is the use of a PBPK model for the hepatoxicity of chloroform. As in cancer RA, as default positions are replaced by biological data the risk assessments become less uncertain when extrapolating between species. Combining information on tissue dosimetry and response data can also provide an estimate of variability within populations, which is impossible with present default type methodology but essential for adequate risk characterisation. Unlike the cancer field there is no single hypothesis for the mechanism of action for the multitude of non-cancer end-points studied.

Animals↗

Use of mechanistic and pharmacokinetic data for risk assessment at the National Institute of Environmental Health Sciences (NIEHS).

In summary, the National Institute of Environmental Health Sciences (NIEHS) and the National Toxicology Program (NTP) have been important contributors of data for hazard identification, including toxicological data as well as mechanistic and pharmacokinetic information. One of the factors that limits the use of knowledge is our lack of understanding of the animal test models currently in use. The underlying bases for regulatory controls that account for normal physiological functions are often not well understood. As a result, toxicological data tend to be used in an empirical manner rather than a manner based on mechanistic understanding. Continued testing of chemicals and random generation of data have their limits in improving our predictive abilities. Attention must be given to prioritizing studies on the basis of critical gaps in understanding that are needed to build knowledge bases in the future.

Databases, Factual↗

Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural studies of the 1,3-dichloropropene catabolic enzymes.

The use of the soil fumigant Telone II, which contains a mixture of cis- and trans-1,3-dichloropropene, to control plant-parasitic nematodes is a common agricultural practice for maximizing yields of various crops. The effectiveness of Telone II is limited by the rapid turnover of the dichloropropenes in the soil due to the presence of bacterial catabolic pathways, which may be of recent origin. The characterization of three enzymes in these pathways, trans-3-chloroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), and malonate semialdehyde decarboxylase (MSAD), has uncovered intriguing catalytic mechanisms as well as a fascinating evolutionary lineage for these proteins. Sequence comparisons and mutagenesis studies revealed that all three enzymes belong to the tautomerase superfamily. Tautomerase superfamily members with known structures are characterized by a beta-alpha-beta structural fold. Moreover, they have a conserved N-terminal proline, which plays an important catalytic role. Mechanistic, NMR, and pH rate studies of the two dehalogenases, coupled with a crystal structure of CaaD inactivated by 3-bromopropiolate, indicate that they use a general acid/base mechanism to catalyze the conversion of their respective isomer of 3-chloroacrylate to malonate semialdehyde. The reaction is initiated by the conjugate addition of water to the C-2, C-3 double bond and is followed by the loss of HCl. MSAD processes malonate semialdehyde to acetaldehyde, and is the first identified decarboxylase in the tautomerase superfamily. The catalytic mechanism is not well defined but the N-terminal proline plays a prominent role and may function as a general acid catalyst, similar to its role in CaaD and cis-CaaD. These are the first structural and mechanistic details for tautomerase superfamily members that catalyze either a hydration or a decarboxylation reaction, rather than a tautomerization reaction, in which Pro-1 serves as a general acid catalyst rather than as a general base catalyst. The available information on the 1,3-dichloropropene catabolic enzymes allows speculation on the possible evolutionary origins of their activities.

Allyl Compounds↗

Mechanistic explanation for the unique pharmacologic properties of receptor partial agonists.

Simulation data provided in this paper revealed that when a receptor full agonist and a competitive or noncompetitive antagonist for the same type of receptor are covalently linked together in some proper ways, the resulting bifunctional compound would have a reduced efficacy, and their dose-response curve patterns would look exactly like the curve patterns for many known partial agonists. It is also shown that all known pharmacologic characteristics for receptor partial agonists matched precisely the projected properties of the agonist-antagonist bifunctional compounds. The novel mechanistic explanation developed in this paper not only reveals the structural requirements for receptor partial agonists, but it also negates the long-held mechanistic explanation that the reduced efficacy of receptor partial agonists is the result of partial activation of each individual receptor molecule being occupied by the partial agonist because of its "low intrinsic activity".

Binding, Competitive↗

Structural and mechanistic insights into the action of Plasmodium falciparum spermidine synthase.

Spermidine synthase is currently considered as a promising drug target in the malaria parasite, Plasmodium falciparum, due to the vital role of spermidine in the activation of the eukaryotic translation initiation factor (eIF5A) and cell proliferation. However, very limited information was available regarding the structure and mechanism of action of the protein at the start of this study. Structural and mechanistic insights of the P. falciparum spermidine synthase (PfSpdSyn) were obtained utilizing molecular dynamics simulations of a homology model based on the crystal structures of the Arabidopsis thaliana and Thermotoga maritima homologues. Our data are supported by in vitro site-directed mutagenesis of essential residues as well as by a crystal structure of the protein that became available recently. We provide, for the first time, dynamic evidence for the mechanism of the aminopropyltransferase action of PfSpdSyn. This characterization of the structural and mechanistic properties of the PfSpdSyn as well as the elucidation of the active site residues involved in substrate, product, and inhibitor interactions paves the way toward inhibitor selection or design of parasite-specific inhibitors.

Animals↗

Lipid peroxidation and renal cell carcinoma: further supportive evidence and new mechanistic insights.

We have recently proposed lipid peroxidation as a unifying mechanistic pathway by which several seemingly unrelated risk/protective factors (obesity, hypertension, diabetes, smoking, oophorectomy/hysterectomy, parity, antioxidants) affect renal cell carcinoma development. In experimental studies, increased lipid peroxidation is a principal mechanistic pathway in renal carcinogenesis induced by different chemicals. In this communication, we provide additional lines of evidence that further support a role for lipid peroxidation on renal cell cancer development. (1) Lipid peroxidation may explain the role of other risk (analgesic use, pre-eclampsia) or protective (alcohol intake, oral contraceptives) factors for renal cell carcinoma. (2) Additional experimental evidence supports lipid peroxidation as an important mechanism in renal carcinogenesis, and (3) Existing evidence support a cross-talk between the lipid peroxidation pathway and other pathways that are relevant to renal carcinogenesis, such as apoptosis, VHL, and possibly other pathways.

Animals↗

Mechanistic insights into Claudin-14 dysfunction implicated in veins of Galen malformation.

Claudin-14 (CLDN14) is a key component of tight junctions (TJs) critical for maintaining paracellular barrier function. Variants of CLDN14 have been linked to Vein of Galen malformations (VOGMs), a rare cerebrovascular disorder; however, the molecular mechanisms underlying their pathogenicity remain unknown. Here, we investigate the mechanistic effects of two VOGM-associated mutations, A113P and V143M, using reinforcement-learning driven enhanced sampling molecular dynamics simulations combined with DiffNets-based deep learning and independent trajectory-wide structural analyses. Our analysis reveals that A113P induces broader structural disruption of CLDN14, perturbing paracellular sealing, pore symmetry, and inter-protomer communication, whereas V143M induces structural rearrangements centred around TM3 and the TM3-ECL2 region. In both cases, mutation-specific alterations are observed in structural stability and interfacial organization across oligomeric assemblies. Notably, these effects are qualitatively consistent across different modelled architectures, despite variability in local responses. In the absence of experimentally resolved structures, the structural perturbations reported here provide a mechanistic understanding of how VOGM-associated variants may influence CLDN14 structure and dynamics.

Aneurysm↗

Antibody diversification in cartilaginous fishes: Mechanistic insights from the nurse shark and comparative perspectives across jawed vertebrates.

Antibody diversity in vertebrates arises through the coordinated actions of V(D)J recombination and somatic hypermutation (SHM). Cartilaginous fishes occupy a key phylogenetic position as the sister lineage to bony vertebrates and therefore provide important comparative insights into the evolution of adaptive immunity. This review focuses on the nurse shark (Ginglymostoma cirratum) as a representative model for examining antibody-diversification mechanisms in cartilaginous fishes. Shark immunoglobulin genes exhibit a multicluster organization, while immunoglobulin new antigen receptor (IgNAR), a heavy-chain-only isotype, contains a single variable domain with an extended complementarity-determining region 3 (CDR3) that can be stabilized by non-canonical disulfide bonds. These structural features, together with intracluster multi-D V(D)J recombination and distinctive SHM characterized by single and tandem substitutions and insertions/deletions, contribute to antibody diversification in sharks. By comparing cartilaginous fishes, ray-finned fishes, and mammals, this review highlights lineage-specific combinations of immunoglobulin gene organization, recombination, mutational processing, and affinity maturation. Within the heuristic framework proposed here, shark and mammalian systems are described as emphasizing "breadth-first" repertoire generation and "precision-first" affinity optimization, respectively. These terms indicate relative mechanistic emphases rather than mutually exclusive categories or sequential evolutionary stages, while ray-finned fishes exhibit a distinct combination of genomic organization and mutational features. Investigating antibody diversification in cartilaginous fishes not only advances our understanding of vertebrate immune evolution but also provides structural and mechanistic insights that may inform the development of engineered antibodies based on the IgNAR scaffold.

Antibody diversity↗

On the mechanistic underpinning of discrete-time population models with complex dynamics.

We present a mechanistic underpinning for various discrete-time population models that can produce limit cycles and chaotic dynamics. Specific examples include the discrete-time logistic model and the Hassell model, which for a long time eluded convincing mechanistic interpretations, and also the Ricker- and Beverton-Holt models. We first formulate a continuous-time resource consumption model for the dynamics within a year, and from that we derive a discrete-time model for the between-year dynamics. Without influx of resources from the outside into the system, the resulting between-year dynamics is always overcompensating and hence may produce complex dynamics as well as extinction in finite time. We recover a connection between various standard types of continuous-time models for the resource dynamics within a year on the one hand and various standard types of discrete-time models for the population dynamics between years on the other. The model readily generalizes to several resource and consumer species as well as to more than two trophic levels for the within-year dynamics.

Animals↗

Biochemical characterization of Toxoplasma gondii 1-Cys peroxiredoxin 2 with mechanistic similarities to typical 2-Cys Prx.

TgPrx2 represents a recently discovered cytosolic 1-Cys peroxiredoxin (Prx) from the intracellular parasite Toxoplasma gondii. Over-expression of the respective gene confers protection against H(2)O(2), suggesting that the protein possesses peroxidase activity. According to the current nomenclature eukaryotic typical and atypical 2-Cys Prx contain a second conserved resolving cysteine residue whereas 1-Cys Prx work on the basis of a monothiol mechanism. Only a few 1-Cys peroxiredoxins have been biochemically characterized to date. Here we describe the mechanistic characterization of TgPrx2 in vitro, including site directed mutagenesis studies, gel filtration chromatography, and molecular modeling. TgPrx2 has general antioxidant properties as indicated by its ability to protect glutamine synthetase against a dithiothreitol Fe(3+)-catalyzed oxidation system. However, TgPrx2 does not reduce H(2)O(2) nor tert-butyl hydroperoxide at the expense of glutaredoxin, thioredoxin or glutathione. Cys(47) was identified as the active site cysteine residue. Most interestingly, Cys(47) was found to form an intermolecular disulfide with Cys(209) from the C-terminal domain of a second subunit which acts as the resolving cysteine. This is a mechanism analogous to typical peroxiredoxins. In contrast to the latter, however, dimeric TgPrx2 does not oligomerize to decamers but is able to form tetramers and hexamers which are non-covalently associated. To our knowledge, TgPrx2 is the first eukaryotic 'so called' 1-Cys peroxiredoxin shown to act on the basis of a 2-Cys mechanism. Our data indicate that mechanistic studies are essential for classifying peroxiredoxins.

Amino Acid Sequence↗

Structural complexity and mechanistic diversity of MECOM rearrangements in myeloid neoplasms.

Rearrangements involving MECOM at chromosome 3q26.2 are recurrent in myeloid neoplasms, classically represented by inv(3)(q21q26.2) and t(3;3)(q21;q26.2), which reposition the GATA2-distal haematopoietic enhancer and drive aberrant EVI1 overexpression. However, the full structural and mechanistic diversity of MECOM rearrangements (MECOM-r) is yet to be explored. We retrospectively analysed 97 cases with cytogenetically defined MECOM-r and identified 12 with complex rearrangements using GTG-banded karyotyping and tri-colour interphase/metaphase fluorescence in situ hybridisation analyses. These 12 cases demonstrated remarkable structural heterogeneity. The abnormalities encompassed translocations, inversions, insertions, duplications, and deletions, which often coexisted within the same specimen as multiple rearranged subclones. Insertional events emerged as a distinct mechanism of MECOM activation. These encompassed insertions of MYNN and/or MECOM into chromosomes 1 and 6, insertion of chromosome 8 segment into MECOM, and inverted insertions between homologous chromosome 3 segments. Recurrent breakpoints at 3q21 across multiple cases, together with localised copy number imbalances frequently involving the MYNN and GOLIM4 loci at 3q26.2, underscore the architectural fragility of these two regions. Co-occurring abnormalities such as -5/del(5q), -7/del(7q), and TP53 loss were common, reflecting a permissive genomic background for chromosomal reassembly. Our findings expand the mechanistic landscape of MECOM-r beyond canonical inv(3)/t(3;3), establishing 3q21 and 3q26.2 as structural 'hotspots' and genomic instability hubs. Distinct from fusion-driven oncogenes such as KMT2A, MECOM activation results from enhancer hijacking and regional structural remodelling, leading to EVI1 overexpression and clonal evolution in myeloid malignancies.

Humans↗

Application of pharmacogenomics in the prevention of diabetic cardiovascular disease: mechanistic basis and clinical evidence for utilization of the haptoglobin genotype in determining benefit from antioxidant therapy.

Atherosclerotic cardiovascular disease (CVD) and diabetic vascular disease have been associated with an increase in oxidative stress. Mechanistic studies in vitro and in animals have demonstrated a direct role for oxidatively modified protein and lipid molecules in the pathophysiology of these diseases. As a result of this oxidation hypothesis numerous studies have been carried out over the past 5-10 years testing the ability of antioxidant vitamins to decrease the incidence of these diseases. The general consensus from these studies, involving over 200,000 individuals, has been that antioxidant vitamins do not provide any vascular protection. Moreover, several of these studies have demonstrated that antioxidant supplementation may be associated with an increased incidence of disease and mortality. One reason why these antioxidant vitamins may have failed to demonstrate benefit may have been due to inappropriate patient selection. In this review we provide supporting clinical evidence and a mechanistic basis for utilizing a genetic marker, the haptoglobin (Hp) genotype, in determining whether antioxidant vitamin therapy may or may not be beneficial for a given patient with diabetes.

Animals↗