Step In Mechanism Research Articles

Understanding Hydrogenation Chemistry at MgB2 Reactive Edges from Ab Initio Molecular Dynamics.

Solid-state hydrogen storage materials often operate via transient, multistep chemical reactions at complex interfaces that are difficult to capture. Here, we use direct ab initio molecular dynamics simulations at accelerated temperatures and hydrogen pressures to probe the hydrogenation chemistry of the candidate material MgB2 without a priori assumption of reaction pathways. Focusing on highly reactive (101̅0) edge planes where initial hydrogen attack is likely to occur, we track mechanistic steps toward the formation of hydrogen-saturated BH4- units and key chemical intermediates, involving H2 dissociation, generation of functionalities and molecular complexes containing BH2 and BH3 motifs, and B-B bond breaking. The genesis of higher-order boron clustering is also observed. Different charge states and chemical environments at the B-rich and Mg-rich edge planes are found to produce different chemical pathways and preferred speciation, with implications for overall hydrogenation kinetics. The reaction processes rely on B-H bond polarization and fluctuations between ionic and covalent character, which are critically enabled by the presence of Mg2+ cations in the nearby interphase region. Our results provide guidance for devising kinetic improvement strategies for MgB2-based hydrogen storage materials, while also providing a template for exploring chemical pathways in other solid-state energy storage reactions.

Direct mass spectrometric observation and reaction mechanism of gas-phase initial intermediates during CL-20 decomposition

The formation and decay of initial intermediates of energetic materials (EMs) such as polynitropolyaza-caged CL-20 are responsible for the initiation of decomposition reaction network, which acts as a key mechanistic step during combustion and detonation. However, direct in situ measurements of these species have not been reported previously, and their formation mechanism remains unclear. Herein, we present direct probing of gas phase decomposition intermediates, particularly reactive initial products with parent molecule's caged skeletons from CL-20, by using in situ atmospheric pressure photoionization high-resolution mass spectrometry. The detected intermediates mainly include NO2-elimination products (C6H5O10N11, C6H6O8N10, etc.), O-elimination product (C6H6O11N12), and NO-elimination products (C6H6O11N11, etc.). Bimolecular reaction pathways involving initial intermediate C6H6O10N11 and NO2/NO are proposed and evaluated to account for the formation of initial products.

Ca2+ regulation of constitutive vesicle trafficking.

Constitutive vesicle trafficking is the default pathway used by all cells for movement of intracellular cargoes between subcellular compartments and in and out of the cell. Classically, constitutive trafficking was thought to be continuous and unregulated, in contrast to regulated secretion, wherein vesicles are stored intracellularly until undergoing synchronous membrane fusion following a Ca2+ signal. However, as shown in the literature reviewed here, many continuous trafficking steps can be up- or down-regulated by Ca2+, including several steps associated with human pathologies. Notably, we describe a series of Ca2+ pumps, channels, Ca2+-binding effector proteins, and their trafficking machinery targets that together regulate the flux of cargo in response to genetic alterations as well as baseline and agonist-dependent Ca2+ signals. Here, we review the most recent advances, organized by organellar location, that establish the importance of these components in trafficking steps. Ultimately, we conclude that Ca2+ regulates an expanding series of distinct mechanistic steps. Furthermore, the involvement of Ca2+ in trafficking is complex. For example, in some cases, the same Ca2+ effectors regulate surprisingly distinct trafficking steps, or even the same trafficking step with opposing influences, through binding to different target proteins.

Squeezing Out Interfacial Solvation: The Role of Hydrogen-Bonding in the Structural and Orientational Freedom of Molecular Self-Assembly.

Bioinspired membrane molecules with improved physical properties and enhanced stability can serve as functional models for conventional lipid or amphiphilic species. Importantly, these molecules can also provide new insights into emergent phenomena that manifest during self-assembly at interfaces. Here, we elucidate the structural response and mechanistic steps underlying the self-assembly of the amphiphilic, charged oligodimethylsiloxane imidazolium cation (ODMS-MIM+) at the air-aqueous interface using Langmuir trough methods with coincident surface-specific vibrational sum-frequency generation (SFG) spectroscopy. We find evidence for a new compression-induced desolvation step that precedes commonly known disordered-to-ordered phase transitions to form nanoscopic assemblies. The experimental data was supported by atomistic molecular dynamics (MD) simulations to provide a detailed mechanistic picture underlying the assembly and the role of water in these phase transitions. The sensitivity of the hydrophobic ODMS tail conformations to compression─owing to distinct water-ODMS interactions and tail-tail solvation properties─offers new strategies for the design of interfaces that can be further used to develop soft-matter electronics and low-dimensional materials using physical and chemical controls.

Selenate Se(VI) reduction to elemental selenium on heterojunctioned rutile/brookite nano-photocatalysts with enhanced charge utilization

Newly synthesized nanocrystalline TiO2 mixed-phase photocatalysts are shown to surpass in Selenium (VI) reduction effectiveness the commercial P25 nanoTiO2 photocatalyst Heterojunctioned rutile (63 %)/brookite (37 %) (designated as R/b) nanoTiO2 is found to be the most effective photocatalyst achieving 95 % photo-assisted reductive deposition vs. 25 % only by the benchmark P25 composed of anatase (80 %) and rutile (A/r). This is attained at very high photocharge utilization with at least 5-fold less formic acid as hole scavenger. The superior performance of R/b is attributed to a favorable band alignment at the heterojunction and its 1D-dominated nanocrystal orientation enabling efficient co-adsorption and enhanced charge separation. The deposition of elemental Se further “auto-catalyzes” the photocatalytic process by enabling visible light activity with simultaneous suppression of charge recombination. In-depth probing of the R/b photocatalyst after selenium removal provides evidence of a 2-step mechanism involving (a) reductive adsorption of Se(VI)aq → Se(IV)surf with the participation of co-adsorbed formate and Ti3+ defects, and photoreduction of Se(IV)surf → Se(0)surf.

On the controlling parameters of the thermal decomposition of inhibiting particles: A theoretical and numerical study

Alkali metal compounds, such as (NaHCO3)s and (Na2CO3)s, are widely used fire suppressants that can be spread as powders or aqueous solutions. Their efficiency depends strongly on the capacity of the alkali metal particles to decompose inside the flame front. The paper aims at providing a theoretical basis for: (1) manufacturing efficient powders; (2) selecting inhibitor candidates that could be efficient against a given reactive mixture. To do so, analytical expressions for the decomposition position of these inhibiting particles are sought to unveil the influence of flame and powder properties on the thermal decomposition problem. The latter is first solved analytically using a simple model for the thermal decomposition of alkali metals combined with a 2-step chain-branching mechanism for the flame chemistry. The analytical solutions are then confronted to numerical simulations using detailed chemistry for both CH4/air and H2/air flames. The validity of the analytical derivations is discussed in terms of combustion regime, characterized in this paper by the ratio of chain-termination to chain-branching time scales. Based on these analytical derivations, a model is proposed for the critical particle size above which mild to no inhibition may be observed. This model accounts for the thermo-chemical properties of the flame/particle system, while being perfectly suited for fine-tuning using either numerical or experimental data. Finally, the paper opens a discussion on the capacity of alkali metal powders to efficiently mitigate highly reactive mixtures, e.g.,C2H2/air and H2/air flames, given the predicted particule sizes needed for fast decomposition in such cases.

A photoredox-catalyzed approach for formal hydride abstraction to enable Csp3–H functionalization with nucleophilic partners (F, C, O, N, and Br/Cl)

A catalytic Csp3–H functionalization manifold for accessing reactive carbocation intermediates directly from a range of Csp3–H bonds is reported. The catalytic design relies on a strategy driven by visible-light photoredox catalysis that combines two mechanistic steps in one catalytic cycle: hydrogen-atom transfer (HAT) and radical-polar crossover (RPC), representing a net hydride abstraction. We herein demonstrate a mild and robust [HAT + RPC] platform that can efficiently enable a Csp3–H fluorination with nucleophilic fluoride—a classically poor nucleophile. Notably, the [HAT + RPC] protocol was successful in the late-stage fluorination of several commercial drugs. Difluorination of methylene groups is also demonstrated, and represents the first C–H difluorination with nucleophilic fluoride. Importantly, long-term potential of the design was demonstrated with the engagement of other nucleophile classes including halides, alcohols, water, N-heterocycle, carboxylic acids, azide, and arenes, thus showcasing its potential as a unified C–H functionalization method.

Stepwise disassembly of GABAergic synapses during pathogenic excitotoxicity.

SUMMARYGABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABAARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABAAR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.

Gold N-Heterocyclic Carbene Catalysts for the Hydrofluorination of Alkynes Using Hydrofluoric Acid: Reaction Scope, Mechanistic Studies and the Tracking of Elusive Intermediates.

An efficient and chemoselective methodology deploying gold-N-heterocyclic carbene (NHC) complexes as catalysts in the hydrofluorination of terminal alkynes using aqueous HF has been developed. Mechanistic studies shed light on an in situ generated catalyst, formed by the reaction of Brønsted basic gold pre-catalysts with HF in water, which exhibits the highest reactivity and chemoselectivity. The catalytic system has a wide alkyl substituted-substrate scope, and stoichiometric as well as catalytic reactions with tailor-designed gold pre-catalysts enable the identification of various gold species involved along the catalytic cycle. Computational studies aid in understanding the chemoselectivity observed through examination of key mechanistic steps for phosphine- and NHC-coordinated gold species bearing the triflate counterion and the elusive key complex bearing a bifluoride counterion.

Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions.

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

Modelling co-combustion of bituminous coal and pine sawdust: Thermal behaviour

This study focused on the effects of introducing pine sawdust and bituminous coal in a down fired combustion reactor. Co-combustion of coal and biomass waste provides an alternative to biomass waste management as well as efficiency improvement with regards to boiler optimisation if correctly applied. A Computational Fluid Dynamics model, using ANSYS Fluent, was employed alongside experimental data to study the behaviour of this co-combustion process. The co-combustion model employed was based on the discrete phase submodel which tracks discrete solid fuel particles in a fluid continuum comprising of the gaseous oxidant, intermediate species, and products. The other important submodels used in this study comprised of the single kinetic devolatilisation submodel and the multiple surface heterogenous char reaction submodel. Two homogenous volatile combustion mechanisms were tested which were the refined Westbrook and Dryer 2-step reaction mechanism as well as the refined Jones and Lindstedt 4-step reaction mechanism. The effect of particle size was monitored in detail by employing a shape factor of 0.87 for biomass particles towards the drag law and the radiative heat transfer tested the effect of using the Discrete Ordinate and P1 radiation submodels. The results showed an increase in burnout for 0.2 s residence time from 37% to 72% when sawdust was introduced in the combustion chamber whilst the temperature profiles showed a general decrease in maximum temperatures attainable as the sawdust proportion increased.

Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore.

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (-150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (-5) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPP cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

Kinetics and mechanistics steps to the electron transfer reaction of peroxo-bridged binuclear cobalt(III) complex of succinimide by glycine in aqueous acidic medium

ABSTRACT. The kinetics and mechanistic steps to the electron transfer reaction of the peroxo-bridged binuclear cobalt(III) complex of succinimide [(suc)(en)2Co(O2)Co(en)2(suc)2+] hereafter called peroxo-bridged dicobalt(III) complex ‘[Co(O2)Co2+]’ by glycine have been carried out spectrophotometrically at λ = 420 nm and T = 26 ± 1 oC, [H+] = 1 x 10-3 M and ᶙ = 0.5 M (NaCl) in aqueous acidic medium. The reaction was found to be first order with respect to [Co(O2)Co2+] and [(Gly] and experimental data indicates a second-order overall. The reactions obeyed the general rate law: (d[Co(O2)Co2+]/dt) = (a +b)[H+])[Co(O2)Co2+][Gly]. Varying hydrogen ion concentration accelerated the reaction rate and shows first-order dependence while the reactions also affected by changes in the ionic strength of the reaction medium by giving a non-negative salt effect in the course of the reaction. Free radicals were not detected in the reactions. Spectroscopic investigation and Michaelis-Menten plots suggest the absence of intermediate complex formation. The experimental result obtained in this system is concluded in favor of the outer-sphere mechanism.
 
 KEY WORDS: Cobalt(III) complex, Kinetic, Electron transfer, Spectroscopic, Mechanistic steps, Michaelis-Menten, Glycine
 
 Bull. Chem. Soc. Ethiop. 2021, 35(2), 425-434.
 DOI: https://dx.doi.org/10.4314/bcse.v35i2.15

Carollia perspicillata: A Small Bat with Tremendous Translational Potential for Studies of Brain Aging and Neurodegeneration.

As the average human lifespan lengthens, the impact of neurodegenerative disease increases, both on the individual suffering neurodegeneration and on the community that supports those individuals. Studies aimed at understanding the mechanisms of neurodegeneration have relied heavily on observational studies of humans and experimental studies in animals, such as mice, in which aspects of brain structure and function can be manipulated to target mechanistic steps. An animal model whose brain is structurally closer to the human brain, that lives much longer than rodents, and whose husbandry is practical may be valuable for mechanistic studies that cannot readily be conducted in rodents. To demonstrate that the long-lived Seba’s short-tailed fruit bat, Carollia perspicillata, may fit this role, we used immunohistochemical labeling for NeuN and three calcium-binding proteins, calretinin, parvalbumin, and calbindin, to define hippocampal formation anatomy. Our findings demonstrate patterns of principal neuron organization that resemble primate and human hippocampal formation and patterns of calcium-binding protein distribution that help to define subregional boundaries. Importantly, we present evidence for a clear prosubiculum in the bat brain that resembles primate prosubiculum. Based on the similarities between bat and human hippocampal formation anatomy, we suggest that Carollia has unique advantages for the study of brain aging and neurodegeneration. A captive colony of Carollia allows age tracking, diet and environment control, pharmacological manipulation, and access to behavioral, physiological, anatomical, and molecular evaluation.

Anthropogenic Volatile Organic Compound (AVOC) Autoxidation as a Source of Highly Oxygenated Organic Molecules (HOM).

Gas-phase hydrocarbon autoxidation is a rapid pathway for the production of in situ aerosol precursor compounds. It is a highway to molecular growth and lowering of vapor pressure, and it produces hydrogen-bonding functional groups that allow a molecule to bind into a substrate. It is the crucial process in the formation and growth of atmospheric secondary organic aerosol (SOA). Recently, the rapid gas-phase autoxidation of several volatile organic compounds (VOC) has been shown to yield highly oxygenated organic molecules (HOM). Most of the details on HOM formation have been obtained from biogenic monoterpenes and their surrogates, with cyclic structures and double bonds both found to strongly facilitate HOM formation, especially in ozonolysis reactions. Similar structural features in common aromatic compounds have been observed to facilitate high HOM formation yields, despite the lack of appreciable O3 reaction rates. Similarly, the recently observed autoxidation and subsequent HOM formation in the oxidation of saturated hydrocarbons cannot be initiated by O3 and require different mechanistic steps for initiating and propagating the autoxidation sequence. This Perspective reflects on these recent findings in the context of the direct aerosol precursor formation in urban atmospheres.

Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia

AbstractSARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Importance of the gas-phase error correction for O2 when using DFT to model the oxygen reduction and evolution reactions

DFT modelling of the oxygen reduction and evolution reactions (ORR and OER) habitually makes use of semiempirical corrections to oxygen in the gas phase. Although such corrections are tacit in the model, they should not be overlooked. In this article, we calculate the errors in the total energy of oxygen for commonly used exchange-correlation functionals, PW91, RPBE, PBE, and BEEF-vdW, to show that, for all functionals tested, the error is at least 0.3 eV. We discuss the impact this sizeable error in oxygen has on the modelling of the ORR and the OER. The error due to oxygen affects not only the overall equilibrium potential of the reaction, but also the energies of individual mechanistic steps. This illustrates that understanding the reasoning behind the semiempirical corrections for oxygen is important for researching new catalysts which may have different potential limiting steps.

The Promoter Role of Amines in the Condensation of Silicic Acid: A First-Principles Investigation.

Though well-recognized, the molecular-level understanding of the multifunctional roles of amines in the condensation of polysilicic acids, which is one of the key processes in hydrothermal synthesis of zeolites, is still limited. Taking ethylamine as a prototype, we investigated the mechanism of polysilicic acid condensation in the existence of organic amines in aqueous solution with extensive first-principles-based calculations. Because of the high proton affinity, ethylamine exists as amine silicates and alters the subsequent condensation mechanisms from a 1-step lateral attack mechanism accompanied with simultaneous intermolecular proton transfer in neutral aqueous solution to a 2-step SN2-like mechanism. Specifically, the 5-coordinated Si species that were not observed on pathways of condensation in neutral solution are effectively stabilized by the ethylamine cations as intermediates, and the barriers for condensation of ortho-silicic acid are significantly reduced from 133 kJ/mol in neutral solution to 58 and 63 kJ/mol for formation of the 5-coordinated Si intermediate and proton transfer for water release, respectively. Similar variations of mechanisms and barriers for condensation were also observed in the formation of cyclic trimers as well as linear and cyclic tetramers of ortho-silicic acids. Based on these, it was proposed that apart from acting as structure-directing agents, pore fillers, and pH adjusters, organic amines can also function as promoters in the condensation of polysilicic acids.

Kynurenic Acid and Its Synthetic Derivatives Protect Against Sepsis-Associated Neutrophil Activation and Brain Mitochondrial Dysfunction in Rats.

Background and AimsThe systemic host response in sepsis is frequently accompanied by central nervous system (CNS) dysfunction. Evidence suggests that excessive formation of neutrophil extracellular traps (NETs) can increase the permeability of the blood–brain barrier (BBB) and that the evolving mitochondrial damage may contribute to the pathogenesis of sepsis-associated encephalopathy. Kynurenic acid (KYNA), a metabolite of tryptophan catabolism, exerts pleiotropic cell-protective effects under pro-inflammatory conditions. Our aim was to investigate whether exogenous KYNA or its synthetic analogues SZR-72 and SZR-104 affect BBB permeability secondary to NET formation and influence cerebral mitochondrial disturbances in a clinically relevant rodent model of intraabdominal sepsis.MethodsSprague–Dawley rats were subjected to fecal peritonitis (0.6 g kg-1 ip) or a sham operation. Septic animals were treated with saline or KYNA, SZR-72 or SZR-104 (160 µmol kg-1 each ip) 16h and 22h after induction. Invasive monitoring was performed on anesthetized animals to evaluate respiratory, cardiovascular, renal, hepatic and metabolic parameters to calculate rat organ failure assessment (ROFA) scores. NET components (citrullinated histone H3 (CitH3); myeloperoxidase (MPO)) and the NET inducer IL-1β, as well as IL-6 and a brain injury marker (S100B) were detected from plasma samples. After 24h, leukocyte infiltration (tissue MPO) and mitochondrial complex I- and II-linked (CI–CII) oxidative phosphorylation (OXPHOS) were evaluated. In a separate series, Evans Blue extravasation and the edema index were used to assess BBB permeability in the same regions.ResultsSepsis was characterized by significantly elevated ROFA scores, while the increased BBB permeability and plasma S100B levels demonstrated brain damage. Plasma levels of CitH3, MPO and IL-1β were elevated in sepsis but were ameliorated by KYNA and its synthetic analogues. The sepsis-induced deterioration in tissue CI–CII-linked OXPHOS and BBB parameters as well as the increase in tissue MPO content were positively affected by KYNA/KYNA analogues.ConclusionThis study is the first to report that KYNA and KYNA analogues are potential neuroprotective agents in experimental sepsis. The proposed mechanistic steps involve reduced peripheral NET formation, lowered BBB permeability changes and alleviation of mitochondrial dysfunction in the CNS.

Relocation of phosphofructokinases within epithelial cells is a novel event preceding breast cancer recurrence that accurately predicts patient outcomes

Although recurrent cancers can become life threatening, little is known about the intracellular events required for cancer recurrences. Due to this lack of mechanistic information, there is no test to predict cancer recurrences or nonrecurrences during early stages of disease. In this retrospective study, we use ductal carcinoma in situ of the breast as a framework to better understand the mechanism of cancer recurrences using patient outcomes as the physiological observable. Conventional pathology slides were labeled with anti-phosphofructokinase type L (PFKL) and anti-phosphofructokinase/fructose-2,6-bisphosphatase type 4 (PFKFB4) reagents. PFKL and PFKFB4 were found in ductal epithelial cell nucleoli from DCIS samples of women who did not experience a cancer recurrence. In contrast, PFKL and PFKFB4 may be found near the plasma membrane in samples from patients who will develop recurrent cancer. With the use of machine learning to predict patient outcomes, holdout studies of individual patient micrographs for the three biomarkers PFKL, PFKFB4, and phosphorylated glucose transporter 1 (GLUT1) demonstrated 38.6% true negatives, 49.5% true positives, 11.9% false positives, and 0% false negatives ( n = 101). A subpopulation of recurrent samples demonstrated PFKL, PFKFB4, and phosphorylated GLUT1 accumulation at the apical surface of epithelial cells, suggesting that carbohydrates can be harvested from the ducts’ luminal spaces as an energy source. We suggest that PFK isotype patterns are metabolic switches representing key mechanistic steps of recurrences. Furthermore, PFK enzyme patterns within epithelial cells contribute to an accurate diagnostic test to classify DCIS patients as high or low recurrence risk.