Linear Pathway Research Articles

Do cognitive aids reduce error rates in resuscitation team performance? Trial of emergency medicine protocols in simulation training (TEMPIST) in Australia

BackgroundResuscitation of patients with time-critical and life-threatening illness represents a cognitive challenge for emergency room (ER) clinicians. We designed a cognitive aid, the Emergency Protocols Handbook, to simplify clinical management and team processes. Resuscitation guidelines were reformatted into simple, single step-by-step pathways. This Australian randomised controlled trial tested the effectiveness of this cognitive aid in a simulated ER environment by observing team error rates when current resuscitation guidelines were followed, with and without the handbook.MethodsResuscitation teams were randomised to manage two scenarios with the handbook and two without in a high-fidelity simulation centre. Each scenario was video-recorded. The primary outcome measure was error rates (the number of errors made out of 15 key tasks per scenario). Key tasks varied by scenario. Each team completed four scenarios and was measured on 60 key tasks. Participants were surveyed regarding their perception of the usefulness of the handbook.ResultsTwenty-one groups performed 84 ER crisis simulations. The unadjusted error rate in the handbook group was 18.8% (121/645) versus 38.9% (239/615) in the non-handbook group. There was a statistically significant reduction of 54.0% (95% CI 49.9–57.9) in the estimated percentage error rate when the handbook was available across all scenarios 17.9% (95% CI 14.4–22.0%) versus 38.9% (95% CI 34.2–43.9%). Almost all (97%) participants said they would want to use this cognitive aid during a real medical crisis situation.ConclusionThis trial showed that by following the step-by-step, linear pathways in the handbook, clinicians more than halved their teams’ rate of error, across four simulated medical crises. The handbook improves team performance and enables healthcare teams to reduce clinical error rates and thus reduce harm for patients.Trial registrationACTRN12616001456448 registered: www.anzctr.org.au. Trial site: http://emergencyprotocols.org.au/

No-oscillation theorem for the transient dynamics of the linear signal transduction pathway and beyond

Understanding the connection between the topology of a biochemical reaction network and its dynamical behavior is an important topic in systems biology. We proved a no-oscillation theorem for the transient dynamics of the linear signal transduction pathway, that is, there are no dynamical oscillations for each species if the considered system is a simple linear transduction chain equipped with an initial stimulation. In the nonlinear case, we showed that the no-oscillation property still holds for the starting and ending species, but oscillations generally exist in the dynamics of intermediate species. We also discussed different generalizations on the system setup. The established theorem will provide insights on the understanding of network motifs and the choice of mathematical models when dealing with biological data.

A hypothetical MEK1-MIP1-SMEK multiprotein signaling complex may function in Dictyostelium and mammalian cells.

In a previous study, we characterized Dictyostelium SUMO targeted ubiquitin ligase (StUbL) MIP1 that associates with protein kinase MEK1 and targets SUMOylated MEK1 to ubiquitination (Sobko et al., 2002). These modifications happen in response to activation of MEK1 by the chemoattractant cAMP. Second site genetic suppressor of mek1- null phenotype (SMEK) was also identified in Dictyostelium. MEK1 and SMEK belong to the same linear pathway, in which MEK1 negatively regulates SMEK, which then negatively regulates chemotaxis and aggregation. RNF4 is mammalian homologue of MIP. RNF4 interacts with hSMEK2, the human homologue of Dictyostelium SMEK. We propose the existence of an evolutionarily conserved MEK1-SMEK signaling complex that upon MEK1 activation and SUMOylation, recruits ubiqutin ligase MIP1/RNF4, which, in turn, ubiquitinates SMEK and targets this protein for proteasomal degradation. This could be a mechanism for negative regulation of SMEK by MEK1 signaling.

Chelated Fischer carbene complexes of annulated thiophenes: synthesis, structure and electrochemistry.

Two (thieno[3,2-b]thiophene) and three annulated thiophenes (dithieno[2,3-b;3',2'-d]thiophene and dithieno[3,2-b;2',3'-d]thiophene) were employed as building blocks to synthesize linear or semi-circular chelated mononuclear biscarbene and dinuclear tetracarbene complexes. The electronic properties of the annulated thienylene chelated carbene complexes were investigated by cyclic voltammetry experiments and compared to non-chelated Fischer-type monocarbene complexes. Density functional theory (DFT) calculations were used to assign the redox events and to probe the extent of electron delocalisation as well as the possibility of electronic (intramolecular metal-metal) communication as a result of intervalence. The differences of these electronic properties in the conjugated chelated carbene complexes are compared to chelated carbene compounds without a linear conjugated pathway.

A Drug Repurposing and Protein-Protein Interaction Network Study of Ribosomopathies Using Yeast as a Model System.

Ribosomopathies result in various cancers, neurodegenerative and viral diseases, and other pathologies such as Diamond-Blackfan anemia and Shwachman-Diamond syndrome. Their pathophysiology at a proteome and functional level remains to be determined. Protein networks and highly connected hub proteins for ribosome biogenesis in Saccharomyces cerevisiae offer a potential as a model system to inform future therapeutic innovation in ribosomopathies. In this context, we report a ribosome biogenesis protein-protein interaction network in S. cerevisiae, created with 1772 proteins and 22,185 physical interactions connecting them. Moreover, by network decomposition analysis, we determined the linear pathways between the transcription factors and target proteins with a view to drug repurposing. While considering only the paths containing the three C/D box proteins (Nop56, Nop58, and Nop1), the most frequently encountered proteins were Aft1, Htz1, Ssa1, Ssb1, Ssb2, Gcn5, Cka1, Tef1, Nop1, Cdc28, Act1, Krr1, Rpl8B, and Tor1, which were then identified as potential drug targets. For drug repurposing, these candidate proteins were further searched in the DrugBank to find other diseases associated with them, as well as the drugs used to treat these diseases. To support the computational results, an experimental study was conducted using in-house manufactured microfluidic bioreactor platform, while the effect of the drug temsirolimus, Tor1 inhibitor, on yeast cells was investigated by following Nop56 protein expression. In conclusion, these results inform the ways in which ribosomopathies and associated common complex human diseases materialize and how drug repurposing might accelerate therapeutic innovation through bioinformatic studies of yeast.

Affinity maturation in a human humoral response to influenza hemagglutinin

Affinity maturation of the B cell antigen receptor (BCR) is a conserved and crucial component of the adaptive immune response. BCR lineages, inferred from paired heavy- and light-chain sequences of rearranged Ig genes from multiple descendants of the same naive B cell precursor (the lineages' unmutated common ancestor, "UCA"), make it possible to reconstruct the underlying somatic evolutionary history. We present here an extensive structural and biophysical analysis of a lineage of BCRs directed against the receptor binding site (RBS) of subtype H1 influenza virus hemagglutinin (HA). The lineage includes 8 antibodies detected directly by sequencing, 3 in 1 principal branch and 5 in the other. When bound to HA, the heavy-chain third complementarity determining region (HCDR3) fits with an invariant pose into the RBS, but in each of the 2 branches, the rest of the Fab reorients specifically, from its position in the HA-bound UCA, about a hinge at the base of HCDR3. New contacts generated by the reorientation compensate for contacts lost as the H1 HA mutated during the time between the donor's initial exposure and the vaccination that preceded sampling. Our data indicate that a "pluripotent" naive response differentiated, in each branch, into 1 of its possible alternatives. This property of naive BCRs and persistence of multiple branches of their progeny lineages can offer broader protection from evolving pathogens than can a single, linear pathway of somatic mutation.

Light Emission in Betalains: From Fluorescent Flowers to Biotechnological Applications.

The discovery of visible fluorescence in the plant pigments betalains revealed the existence of fluorescent patterns in flowers of plants of the order Caryophyllales, where betalains substitute anthocyanins. The serendipitous initial discovery led to a systemized characterization of the role of different substructures on the photophysical phenomenon. Strong fluorescence is general to all members of the family of betaxanthins linked to the structural property that the betalamic acid moiety is connected to an amine group. This property has led to bioinspired tailor-made probes and to the development of novel biotechnological applications in screening techniques or microscopy labeling. Here, we comprehensively review the photophysics, photochemistry, and photobiology of betalain fluorescence and describe all current applications.

Stoichiometry of protein complexes in plant photosynthetic membranes

Hetero-oligomeric membrane protein complexes form the electron transport chain (ETC) of oxygenic photosynthesis. The ETC complexes undertake the light-driven vectorial electron and proton transport reactions, which generate energy-rich ATP and electron-rich NADPH molecules for carbon fixation. The rate of photosynthetic electron transport depends on the availability of photons and the relative abundance of electron transport complexes. The relative abundance of the two photosystems, critical for the quantum efficiency of photosynthesis in changing light quality conditions, has been determined successfully by optical methods. Due to the lack of spectroscopic signatures, however, relatively little is known about the stoichiometry of other non-photosystem complexes in plant photosynthetic membrane. Here we determine the ratios of all major thylakoid-bound ETC complexes in Arabidopsis by a label-free quantitative mass spectrometry technique. The calculated stoichiometries are consistent with known subunit composition of complexes and current estimates of photosystem and cytochrome b6f concentrations. The implications of these stoichiometries for photosynthetic light harvesting and the partitioning of electrons between the linear and cyclic electron transport pathways of photosynthesis are discussed.

Delineating Human B Cell Precursor Development With Genetically Identified PID Cases as a Model.

B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints.

Irreversible linear pathways in enzymatic reactions: analytical solution using the homotopy perturbation method

In this work, the Homotopy Perturbation method is used for the first time to solve an irreversible linear pathway with enzyme kinetics. The enzymatic system has Michaelis–Menten kinetics and is modeled by a system of nonlinear ordinary differential equations. The analytical solution obtained with the method allow us to optimize several objectives: minimal time to reach a certain percent of final product, minimal amount of enzymes employed in the process, or even multiple objective optimization via Pareto front. We present an example to demonstrate the results.

Overexpression of pink1 or parkin in indirect flight muscles promotes mitochondrial proteostasis and extends lifespan in Drosophila melanogaster.

Dysfunctional mitochondria have been implicated in aging and age-related disorders such as Parkinson’s diseases (PD). We previously showed that pink1 and parkin, two familial PD genes, function in a linear pathway to maintain mitochondrial integrity and function. Studies of mammalian cell lines also suggest that these genes regulate mitochondrial autophagy(mitophagy). Overexpressing Parkin promotes proteostasis and function of aged muscles both in fruit flies and mice, and recent studies also indicated that mitochondrial ubiquitination are accumulated in aged muscles. However, the underlying mechanisms for pink1 and parkin mediated mitophagy on longevity is not fully understood. Here, we found that mitochondrial ubiquitination increased in indirect flight muscles (IFMs) in an age-dependent manner. Overexpression of pink1 or parkin in IFMs can abolish mitochondrial ubiquitination, restore ATP level and extend lifespan, while blocking autophagy via ATG1 knock-down suppress these effects in aged IFMs. Taken together, these results show that pink1/parkin promotes mitophagy of mitochondrial ubiquitination in aged muscles and extend lifespan in an Atg1-dependent manner. Our study provides physiological evidence that mitophagy of mitochondrial ubiquitination mediated by PINK1/ Parkin is crucial for muscle function and highlights the role of mitophagy in the pathogenesis of chronic diseases like PD.

GENE-52. ANALYSIS OF RECURRENT MUTATIONS WITHIN CANCERS REVEALS PATTERNS OF DIRECTIONAL EVOLUTION IN BRAIN TUMORS

Abstract INTRODUCTION Intratumor genetic heterogeneity is commonly caused by a stochastic evolution, but can also show remarkable selectivity resulting in directional evolution. We hypothesized that directional evolution is driven by its genetic onset-stage. This process could enforce already existing traits resulting in an enhancement of linear pathways rather than the complementary/parallel evolution. METHODS We generated a prediction model for directional evolution based on its genetic onset-stage. We performed a comprehensive analysis of 16 different tumor types of ~10,000 patients of whole-exome sequencing and copy number variation, obtained from The Cancer Genome Atlas. RESULTS We found in 5% of the patients that directional evolution occurs on a fixed genetic onset stage. There is a strong relationship between the frequencies of directional evolution and the frequencies of commonly co-occurring mutations in the onset stage. These co-occurring mutations are frequently observed in the same chromosomal region and occasionally in the same pathway. Brain tumors show a frequent directional evolution towards EGFR mutations and to a lesser extent PIK3CA mutations. CONCLUSION Given these strong correlations and since directional evolution is apparently a highly selective process, our prediction model could predict more effective therapies by choosing therapies targeting both the mutated gene that is affected by directional evolution as well as the commonly occurring co-mutated genes.

A Continuum of Innovation: Curricular Renewal Strategies in Undergraduate Medical Education, 2010-2018.

Since 2010, medical schools across the United States have engaged in a new cycle of curricular revision and renewal for their undergraduate medical curricula. But what structures, features, and trends have emerged in U.S. medical schools as a result of deliberate curricular redesign efforts? An analysis of the ways that medical schools have approached the reorganization of their curricula to prepare their students for the growing complexity of medical practice is presented. This study drew a total pool of 40 U.S. MD-granting programs, of which 25 met the inclusion criteria for the study. The authors used a qualitative coding approach to materials from the undergraduate medical education (UME) program websites to identify 4 dimensions of strategies that these programs used to renew their curricula. The analysis of the curricular maps and website content of the UME programs provided evidence for a continuum approach to the description of innovation strategies: 96% of schools employed a cohort-based linear pathway, 80% of schools used thematic basic science blocks, 47% placed their Step 1 exams outside of the second year, and 68% moved their clerkships to the second year. The Continuum of Innovation strategies will enable programs to renew their curricula in ways that promote deliberate curricular changes that are consistent with emerging needs in the field. This study and future research may be useful for UME programs with limited resources by providing consensus practices that enable them to plan curricular changes in ways that best serve their institutions.

Soriano award lecture: Light, circadian rhythms and sleep: Mechanisms to new therapeutics

Until recently, it seemed inconceivable to most vision researchers and ophthalmologists that there could be an unrecognised class of light sensor (photoreceptor) within the eye. However, by studying how circadian rhythms and sleep are regulated by the dawn/dusk cycle we demonstrated that there exists a “3rd class” of photoreceptor within the eye based upon a small number of photosensitive retinal ganglion cells (pRGCs) that utilise the blue light sensitive photopigment melanopsin (OPN4). The first part of the presentation will consider the discovery and clinical importance of the “third” photoreceptor system of the eye. The second part of the talk will address how these photoreceptors signal light to the molecular clockwork and by what means this pathway is modified by sleep history. There has been remarkable progress in understanding the complex intracellular mechanisms that generate circadian rhythms, the molecular pathways whereby the pRGCs entrain circadian biology and sleep has remained poorly understood. The suprachiasmatic nuclei (SCN) are the site of the primary circadian pacemakers within the mammalian brain. Until recently, the model for entrainment involved a simple linear pathway whereby glutamate release from the pRGCs resulted in Ca2+ influx and raised intracellular cAMP in SCN neurones, which in turn resulted in CREB phosphorylation leading to increased transcription of two key clock genes, Per1 and Per2. This signal then advanced or delayed the molecular clockwork. However, an important feature of entrainment is that circadian responses to light are limited – as typified by jet-lag. Full recovery from jet-lag requires a day for every time-zone crossed. We addressed this issue and have identified and characterized a key role for Salt Inducible Kinase 1 (SIK1) and the CREB-regulated transcription co-activator 1 (CRTC1) in clock re-setting. However, our most recent findings have shown that light entrainment also involves the parallel activation of a Ca2+-ERK1/2-AP-1 signalling pathway. Thus both CRE and AP-1 regulatory elements drive light-induced clock gene expression. In addition, whilst light activation of the Ca2+-ERK1/2-AP-1 signalling pathway increases Per1 and Per2 expression, sleep/wake behaviour alters the effects of light on the clock. Our proposed mechanism suggests that adenosine acts as a signalling molecule that encodes wake duration. Adenosine acts via inhibitory A1 receptors on the SCN to inhibit the Ca2+-ERK1/2-AP-1 signalling pathway, which in turn, reduces the expression of Per1 and Per2. Thus sleep/wake history, encoded by adenosine, reduces the phase shifting effects of light upon the circadian system, altering sleep/wake timing. Finally, the talk will explore how such signalling pathways provide a new target for the regulation of circadian rhythms and the “pharmacological” replacement of light for sleep/wake re-setting in individuals lacking eyes or other individuals with severe circadian rhythm disruption.

Linear pathways

Linear pathways

How the mechanobiome drives cell behavior, viewed through the lens of control theory.

Cells have evolved sophisticated systems that integrate internal and external inputs to coordinate cell shape changes during processes, such as development, cell identity determination, and cell and tissue homeostasis. Cellular shape-change events are driven by the mechanobiome, the network of macromolecules that allows cells to generate, sense and respond to externally imposed and internally generated forces. Together, these components build the cellular contractility network, which is governed by a control system. Proteins, such as non-muscle myosin II, function as both sensors and actuators, which then link to scaffolding proteins, transcription factors and metabolic proteins to create feedback loops that generate the foundational mechanical properties of the cell and modulate cellular behaviors. In this Review, we highlight proteins that establish and maintain the setpoint, or baseline, for the control system and explore the feedback loops that integrate different cellular processes with cell mechanics. Uncovering the genetic, biophysical and biochemical interactions between these molecular components allows us to apply concepts from control theory to provide a systems-level understanding of cellular processes. Importantly, the actomyosin network has emerged as more than simply a 'downstream' effector of linear signaling pathways. Instead, it is also a significant driver of cellular processes traditionally considered to be 'upstream'.

Multilayered Control of Protein Turnover by TORC1 and Atg1

The target of rapamycin complex 1 (TORC1) is a master regulator of cell homeostasis, which promotes anabolic reactions and synchronously inhibits catabolic processes such as autophagy-mediated protein degradation. Its prime autophagy target is Atg13, a subunit of the Atg1 kinase complex that acts as the gatekeeper of canonical autophagy. To study whether the activities of TORC1 and Atg1 are coupled through additional, more intricate control mechanisms than simply this linear pathway, we analyzed the epistatic relationship between TORC1 and Atg1 by using quantitative phosphoproteomics. Our invivo data, combined with targeted invitro TORC1 and Atg1 kinase assays, not only uncover numerous TORC1 and Atg1 effectors, but also suggest distinct bi-directional regulatory feedback loops and characterize Atg29 as a commonly regulated downstream target of both TORC1 and Atg1. Thus, an exquisitely multilayered regulatory network appears to coordinate TORC1 and Atg1 activities to robustly tune autophagy in response to nutritional cues.

Enabling Fast Charging Lithium-Ion Batteries through the Rational Design of 3-D Anode Architectures

Today’s Li-ion technology is highly optimized for performance at relatively slow charging operation. However, significant challenges still present for fast charging conditions (> 4C). These challenges include large kinetic polarizations, concentration gradients, heat generation, and Li metal plating on the surface. In state-of-the-art Li-ion batteries with high energy densities, the electrodes are relatively thick (> 100 μm), which leads to a tradeoff between energy density and high-power performance. This is because thicker electrodes with a tortuous pathway naturally limit Li-ion transport through the electrode thickness, resulting in large electrolyte concentration gradients during cycling. This leads to large cell polarization, which reduces the accessible capacity of the battery under fast-charging conditions. In addition, the electrochemical potential of the anode can easily become more negative than the thermodynamic potential of Li metal during fast charging, resulting in Li plating. Therefore, to simultaneously achieve fast charging and maintain energy density of Li-ion batteries, new approaches are required to address Li ionic transport limitations through the thick negative electrodes. In this work, we demonstrate a structural modification of conventional graphite anodes to improve their fast charge capability. This is achieved by introducing laser-patterned vertical channels into post-calendared electrodes.[1] The patterned anode architecture consists of a hexagonal close-packed array of vertical channels that serve as linear pathways for rapid ionic diffusion through the electrode thickness. This allows for a more homogeneous flux of Li throughout the volume of the electrode and decreased ionic concentration gradients, in comparison to slow/tortuous diffusion paths in the conventional anode, thus improving the accessible capacity of the electrode during fast charging. Through rationally designing and tuning the channel pore diameter and spacing, an optimal geometry can be determined to achieve optimal high-rate cycling performance for a given anode loading and porosity. In addition, we develop an electrochemical dynamics simulation framework to capture the experimentally-observed voltage changes during charge/discharge in both conventional and patterned electrodes. Through accounting for the anode physical properties and structural details, our simulation tool can predict/provide valuable information such as concentration profile and electrode polarization during fast charging. This further helps us pinpoint the design rules and tradeoffs associated with the patterned anode architecture. Utilizing the model-informed design of 3-D hierarchical anode structures, we demonstrate significant improvements (> 100%) on the accessible capacity and minimal capacity fade during fast charging (up to 6C) with anode loading of > 3 mAh/cm2. This work thus demonstrates the viability of realizing high energy density Li-ion batteries with fast charge capability based on thick electrodes. [1] Y. Kim, A. Drews, R. Chandrasekaran, T. Miller, J. Sakamoto, Ionics 24, 2935 (2018).

Disrupting the dominance of ‘linear pathways’: how institutional assumptions create ‘stuck places’ for refugee students’ transitions into higher education

ABSTRACT Although journeys into and through higher education can be challenging for all learners, students from refugee backgrounds (SfRBs) face particular difficulties due to their culturally and linguistically diverse (CALD) backgrounds, and pre-settlement experiences of instability, insecurity, likely trauma and interrupted education. Drawing on a longitudinal, ethnographic exploration of the educational trajectories of a group adult SfRBs living in a regional Australian city, this article questions how ‘traditional’ understandings of transition – as linear, ritualised and predictable – can account for the complex lived experiences of SfRBs trying to access and participate in higher education studies in a resettlement context. Working with the anthropological notion of liminality (Turner 1969; Van Gennep 1960) and the notion of ‘stuck places’ (Lather 1998), we offer a more nuanced understanding of how ‘messy’ educational transitions are experienced, and how normative notions of what transition ‘should be’ act to mask these experiences, leaving SfRBs ‘stuck’ in the liminal space of becoming a student in higher education.

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.