Mechanistic Inquiry Research Articles

FLI1 promotes IFN-γ-induced kynurenine production to impair anti-tumor immunity

Nasopharyngeal carcinoma (NPC)-mediated immunosuppression within the tumor microenvironment (TME) frequently culminates in the failure of otherwise promising immunotherapies. In this study, we identify tumor-intrinsic FLI1 as a critical mediator in impairing T cell anti-tumor immunity. A mechanistic inquiry reveals that FLI1 orchestrates the expression of CBP and STAT1, facilitating chromatin accessibility and transcriptional activation of IDO1 in response to T cell-released IFN-γ. This regulatory cascade ultimately leads to augmented IDO1 expression, resulting in heightened synthesis of kynurenine (Kyn) in tumor cells. This, in turn, fosters CD8+ T cell exhaustion and regulatory T cell (Treg) differentiation. Intriguingly, we find that pharmacological inhibition of FLI1 effectively obstructs the CBP/STAT1-IDO1-Kyn axis, thereby invigorating both spontaneous and checkpoint therapy-induced immune responses, culminating in enhanced tumor eradication. In conclusion, our findings delineate FLI1-mediated Kyn metabolism as an immune evasion mechanism in NPC, furnishing valuable insights into potential therapeutic interventions.

Top-down and bottom-up constraints in mechanistic inquiry

Mechanisms play a crucial role in scientific research across various disciplines, and philosophers of science have devoted significant effort into understanding their ontology and epistemology. This paper examines the relationship between mechanisms and phenomena, highlighting the inherent dependence of mechanistic delineation on the characterization of phenomena. By acknowledging that characterizing phenomena is influenced by pragmatic considerations and research interests, the paper argues that mechanistic inquiry is inherently shaped by researchers’ perspectives. This dependence raises concerns about the possibility of a realist view of mechanisms. To address these concerns, the paper explores how top-down constraints, rooted in researchers’ interests and pragmatic concerns, can be balanced by bottom-up constraints derived from empirical considerations. In conclusion, I argue that the interplay between these constraints forms an empirical and realist counterweight to the perspectival nature of top-down constraints.

A mechanistic inquiry into the applicability of permanganate oxidizable carbon as a soil health indicator

AbstractWe determined the characteristics that render organic matter responsive to dilute permanganate oxidation to find a mechanistic explanation for the frequently observed responsiveness of permanganate oxidizable carbon (PoxC) to changes in soil health. We conclude that PoxC is primarily a measurement of phenolic and polyphenolic structures rather than of compounds traditionally viewed as preferred microbial food. Upon subjecting pure compounds to the PoxC method, we found that the susceptibility of organic molecules to permanganate oxidation was determined by the presence of specific structural features abundant in phenolic compounds, which were highly reactive (3–98 mg PoxC·g−1 C), and in a subset of low‐molecular‐weight organic acids (0–180 mg PoxC·g−1 C). In comparison, proteins were minimally reactive (0–13 mg PoxC·g−1 C) and carbohydrates were unreactive when subjected to the PoxC method. Permanganate reactivity was not found to be a predictor of decomposability in biosolids through the mirroring of C:N ratio. However, permanganate reactivity increased up to 27% throughout the decomposition process. We suggest a reinterpretation of the mechanisms that render PoxC useful as a soil health indicator. Rather than representing a preferred microbial food source, PoxC measures a carbon fraction with unique molecular properties that may be an indicator of plant‐mediated soil processes. Our research corroborates the usefulness of PoxC as a soil health indicator, albeit for entirely different reasons than previously assumed. This opens exciting avenues of further inquiry and sets the stage for intensified investigations into the general role of polyphenolic compounds in soil health.

Redox-Neutral Decarboxylative and Desulfonylative C(sp3) Trifluoromethylation: Method Development and Mechanistic Inquiry.

Sodium triflinate (CF3SO2Na) is an inexpensive bench-stable radical CF3 source that is often activated by external oxidants such as peroxides. However, despite the commercial accessibility of CF3SO2Na, the salt has never been applied to decarboxylative trifluoromethylation due to challenges in controlled cross-radical coupling. We report a redox-neutral approach to decarboxylative C(sp3) trifluoromethylation of carboxylic acid derivatives. Mechanistic inquiry is performed to address the limitations in scope.

Abstract TMP116: A Novel Microfluidic Model Of Ischemic Stroke Recapitulating Arterial Occlusion And Prolonged Retraction

To our knowledge, an ex vivo model of thrombotic occlusion that (1) mimics arterial setting, (2) allows true occlusion, and (3) drug delivery without disturbing the thrombus does not exist. A platform with these capabilities would allow for high-throughput mechanistic study of thrombosis and thrombolysis. Here we present a microfluidic system that achieves these goals, providing a biofidelic ex vivo platform to study ischemic stroke. A mold with an upstream Y and a region of stenosis was fabricated and used to manufacture microfluidic chambers. Channels were coated with collagen at the stenosis. Heparinized blood was collected from healthy human donors (N=3) and platelets were labelled with fluorescent anti-CD41. Samples were perfused at 10000/s and outflow mass collected on a scale. Occlusion was indicated by mass plateau, and thrombi were left to retract in situ for 0 or 6 hrs. Kinetic immunofluorescent images were captured and used to derive surface area and the image profile by averaging MFI in each pixel column. Sample from the same donor dosed with 0.7 or 7nM alteplase (ALT) was perfused using the Y inlet using a constant pressure head (1000/s; mimicking middle cerebral artery) for 120 min. After 6 hours of post-occlusion retraction, thrombi substantially remodeled. The MFI curves (Fig 1) demonstrate even distribution of MFI post-occlusion that shifts to a central peak after 6 hr. Dosed sample from the same donor was able to be delivered upstream of the thrombus without disruption. Immediate reperfusion resulted in a reduction in thrombus surface area of (mean (SD)) 2% (10%) with 0.7nM ALT, 23% (7%) with 7nM, and 15% (1%) with vehicle. Reperfusion after 6 hrs resulted in a reduction of 14% (14%) with 0.7nM, 45% (12%) with 7nM, and 17% (8%) with vehicle. We have established an ex vivo high throughput system capable of recapitulating arterial occlusion, thrombus retraction in situ , drug delivery, and thrombolysis. This system can be used for mechanistic inquiry and drug discovery.

Metal ions: the unfading stars of bone regeneration-from bone metabolism regulation to biomaterial applications.

In recent years, bone regeneration has emerged as a remarkable field that offers promising guidance for treating bone-related diseases, such as bone defects, bone infections, and osteosarcoma. Among various bone regeneration approaches, the metal ion-based strategy has surfaced as a prospective candidate approach owing to the extensive regulatory role of metal ions in bone metabolism and the diversity of corresponding delivery strategies. Various metal ions can promote bone regeneration through three primary strategies: balancing the effects of osteoblasts and osteoclasts, regulating the immune microenvironment, and promoting bone angiogenesis. In the meantime, the complex molecular mechanisms behind these strategies are being consistently explored. Moreover, the accelerated development of biomaterials broadens the prospect of metal ions applied to bone regeneration. This review highlights the potential of metal ions for bone regeneration and their underlying mechanisms. We propose that future investigations focus on refining the clinical utilization of metal ions using both mechanistic inquiry and materials engineering to bolster the clinical effectiveness of metal ion-based approaches for bone regeneration.

The role of agostic interaction in the mechanism of ethylene polymerisation using Cr(III) half-sandwich complexes: What dictates the reactivity?

Chromium-based catalysts play a significant role in the production of ultra-high molecular weight polyethylene, and half-sandwich functionalised-metallocene complexes were proven to be one of the most suitable candidates as catalysts for generating such large polymeric-length olefins. Earlier experimental studies on olefin polymerisation using a series of catalysts such as [L1-2CrCl2] (where L1 = 1-((pyridin-2-yl)methyl)indenyl (1) and L2 = 2-methyl-1-{[4-(yridinene-1-yl)yridine-2-yl]methyl}-1H-indenyl (2)) reveal significant variation where peripheral substitution on the ligand was found to influence the reactivity significantly. However, the specific ligand position that affects the reactivity has not been established. As these reactions are fast and robust, it is challenging to establish reactive intermediates via experiments, and therefore, mechanistic clues for such reactions are elusive. Here we have undertaken a detailed computational study by employing an array of DFT (uB3LYP-D3/def2-TZVP, CASSCF/NEVPT2, and DLPNO-CCSD(T) methods to explore the substituted and non-substituted pyridine-cyclo-pentadienyl chromium complexes and their influence on the catalytic activity in ethylene polymerisation. Our study not only unravels the catalytic pathway for olefin polymerisation for such Cr(III)-half-sandwich complexes but also reveals that the energetics of the formation of pseudo-three-coordinate alkyl intermediates is key to the variation in the reactivity observed. A detailed examination using MO and NBO analysis unveils the presence of a C-H⋯Cr agostic interaction that is found to significantly stabilise this intermediate when the pyridine ligand has strong electron-donating groups at its para position. The other substitutions, such as on the cyclopentadienyl ligand, neither yield the desired stability nor the desired interaction. Further studies on models support this proposal. In order to improve the efficiency and selectivity of catalytic systems in olefin polymerisation, we strongly advocate for the integration of agostic interactions as a crucial criterion in the design of future catalysts. Considering the prevalence of electron-deficient metal centres in successful olefin polymerisation catalysts, this research prompts a broader mechanistic inquiry to propose a unified approach for this industrially crucial reaction.

Application of CRISPR-Cas9 Gene Editing for HIV Host Factor Discovery and Validation.

Human Immunodeficiency Virus (HIV) interacts with a wide array of host factors at each stage of its lifecycle to facilitate replication and circumvent the immune response. Identification and characterization of these host factors is critical for elucidating the mechanism of viral replication and for developing next-generation HIV-1 therapeutic and curative strategies. Recent advances in CRISPR-Cas9-based genome engineering approaches have provided researchers with an assortment of new, valuable tools for host factor discovery and interrogation. Genome-wide screening in a variety of in vitro cell models has helped define the critical host factors that play a role in various cellular and biological contexts. Targeted manipulation of specific host factors by CRISPR-Cas9-mediated gene knock-out, overexpression, and/or directed repair have furthermore allowed for target validation in primary cell models and mechanistic inquiry through hypothesis-based testing. In this review, we summarize several CRISPR-based screening strategies for the identification of HIV-1 host factors and highlight how CRISPR-Cas9 approaches have been used to elucidate the molecular mechanisms of viral replication and host response. Finally, we examine promising new technologies in the CRISPR field and how these may be applied to address critical questions in HIV-1 biology going forward.

Site‐Selective Functionalization of Sila‐Adamantane and Its Ensuing Optical Effects

AbstractThe first syntheses of functionalized sila‐adamantanes via site‐selective reactions are described. Mechanistic inquiry into the isomerization of sila‐adamantane revealed new approaches for installing halides at the 2‐position of the cluster. Meanwhile, isomerization via Lewis acid catalysts with non‐nucleophilic counteranions provided access to sila‐adamantane on the gram‐scale, enabling us to discover strategies for substituting its 1‐, 3‐, 5‐, and 7‐positions with identical or distinct functional groups. Optical absorbance and density functional theory studies show that σ‐withdrawing substituents at the 1‐position strongly perturb optical absorbance in sila‐adamantane, whereas substituents at the exocyclic and 2‐position are optically inert. As silicon diamondoids are atomically precise models for silicon nanocrystals, our findings suggest that passivation at tertiary surface sites carries an outsized impact on the optical properties of surface‐functionalized Si nanocrystals.

Site‐Selective Functionalization of Sila‐Adamantane and Its Ensuing Optical Effects

The first syntheses of functionalized sila-adamantanes via site-selective reactions are described. Mechanistic inquiry into the isomerization of sila-adamantane revealed new approaches for installing halides at the 2-position of the cluster. Meanwhile, isomerization via Lewis acid catalysts with non-nucleophilic counteranions provided access to sila-adamantane on the gram-scale, enabling us to discover strategies for substituting its 1-, 3-, 5-, and 7-positions with identical or distinct functional groups. Optical absorbance and density functional theory studies show that σ-withdrawing substituents at the 1-position strongly perturb optical absorbance in sila-adamantane, whereas substituents at the exocyclic and 2-position are optically inert. As silicon diamondoids are atomically precise models for silicon nanocrystals, our findings suggest that passivation at tertiary surface sites carries an outsized impact on the optical properties of surface-functionalized Si nanocrystals.

Mechanistic inquiry and scientific pursuit: The case of visual processing

Why is it rational for scientists to pursue multiple models of a phenomenon at the same time? The literatures on mechanistic inquiry and scientific pursuit each develop answers to a version of this question which is rarely discussed by the other. The mechanistic literature suggests that scientists pursue different complementary models because each model provides detailed insights into different aspects of the phenomenon under investigation. The pursuit literature suggests that scientists pursue competing models because alternative models promise to solve outstanding empirical and conceptual problems. Looking into research on visual processing as a case study, we suggest an integrated account of why it is rational for scientists to pursue both complementary and competing models of the same mechanism in scientific practice.

Microglial Activation Damages Dopaminergic Neurons through MMP-2/-9-Mediated Increase of Blood-Brain Barrier Permeability in a Parkinson's Disease Mouse Model.

Chronic neuroinflammation has been considered to be involved in the progressive dopaminergic neurodegeneration in Parkinson’s disease (PD). However, the mechanisms remain unknown. Accumulating evidence indicated a key role of the blood–brain barrier (BBB) dysfunction in neurological disorders. This study is designed to elucidate whether chronic neuroinflammation damages dopaminergic neurons through BBB dysfunction by using a rotenone-induced mouse PD model. Results showed that rotenone dose-dependently induced nigral dopaminergic neurodegeneration, which was associated with increased Evans blue content and fibrinogen accumulation as well as reduced expressions of zonula occludens-1 (ZO-1), claudin-5 and occludin, three tight junction proteins for maintaining BBB permeability, in mice, indicating BBB disruption. Rotenone also induced nigral microglial activation. Depletion of microglia or inhibition of microglial activation by PLX3397 or minocycline, respectively, greatly attenuated BBB dysfunction in rotenone-lesioned mice. Mechanistic inquiry revealed that microglia-mediated activation of matrix metalloproteinases-2 and 9 (MMP-2/-9) contributed to rotenone-induced BBB disruption and dopaminergic neurodegeneration. Rotenone-induced activation of MMP-2/-9 was significantly attenuated by microglial depletion and inactivation. Furthermore, inhibition of MMP-2/-9 by a wide-range inhibitor, SB-3CT, abrogated elevation of BBB permeability and simultaneously increased tight junctions expression. Finally, we found that microglial depletion and inactivation as well as inhibition of MMP-2/-9 significantly ameliorated rotenone-elicited nigrostriatal dopaminergic neurodegeneration and motor dysfunction in mice. Altogether, our findings suggested that microglial MMP-2/-9 activation-mediated BBB dysfunction contributed to dopaminergic neurodegeneration in rotenone-induced mouse PD model, providing a novel view for the mechanisms of Parkinsonism.

Utilizing preclinical models to develop targeted therapies for rare central nervous system cancers.

Patients with rare central nervous system (CNS) tumors typically have a poor prognosis and limited therapeutic options. Historically, these cancers have been difficult to study due to small number of patients. Recent technological advances have identified molecular drivers of some of these rare cancers which we can now use to generate representative preclinical models of these diseases. In this review, we outline the advantages and disadvantages of different models, emphasizing the utility of various in vitro and ex vivo models for target discovery and mechanistic inquiry and multiple in vivo models for therapeutic validation. We also highlight recent literature on preclinical model generation and screening approaches for ependymomas, histone mutated high-grade gliomas, and atypical teratoid rhabdoid tumors, all of which are rare CNS cancers that have recently established genetic or epigenetic drivers. These preclinical models are critical to advancing targeted therapeutics for these rare CNS cancers that currently rely on conventional treatments.

Cortical overgrowth in a preclinical forebrain organoid model of CNTNAP2-associated autism spectrum disorder

We utilized forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of Autism Spectrum Disorder (ASD) with a homozygous protein-truncating mutation in CNTNAP2, to study its effects on embryonic cortical development. Patients with this mutation present with clinical characteristics of brain overgrowth. Patient-derived forebrain organoids displayed an increase in volume and total cell number that is driven by increased neural progenitor proliferation. Single-cell RNA sequencing revealed PFC-excitatory neurons to be the key cell types expressing CNTNAP2. Gene ontology analysis of differentially expressed genes (DEgenes) corroborates aberrant cellular proliferation. Moreover, the DEgenes are enriched for ASD-associated genes. The cell-type-specific signature genes of the CNTNAP2-expressing neurons are associated with clinical phenotypes previously described in patients. The organoid overgrowth phenotypes were largely rescued after correction of the mutation using CRISPR-Cas9. This CNTNAP2-organoid model provides opportunity for further mechanistic inquiry and development of new therapeutic strategies for ASD.

B-PO05-007 RAPID AND ROBUST MAPPING OF CLINICAL DATA ON PERSONALIZED VOLUMETRIC ATRIAL GEOMETRIES

Quantification and visualization of clinical data on patient-derived volumetric atrial geometries is important for mechanistic inquiry and investigation of optimal guidance of atrial ablation. However, available methodologies lack efficiency, robustness and require execution of manual tasks. To develop a fast and robust methodology to map multi-modal clinical data on personalized volumetric atrial geometries. A custom tool, easy to use in clinic, enables close alignment of anatomically relevant structures and generates body-conformal coordinates on atrial geometry for accurate mapping. Efficient interpolation techniques compute the intramural data, drastically reducing mapping time. The performance was assessed on 60 atrial geometries. Electroanatomic map (EAM) and fibrosis mapping accuracy was measured by identity test (mapping data back onto original geometry) and visual comparisons by trained experts. Mapped fiber orientations were compared against published methodology of atrial registration by performing personalized electrophysiological simulations to predict locations of re-entrant drivers (RDs) that sustain AF. (Figure) The time for atrial registration of volumetric meshes was reduced from 8-10hrs to 30min. Visual comparisons and identity tests (avg. rel. error<=5%) verified the accuracy of EAM and fibrosis maps. Fiber orientations were successfully mapped, and RD locations remained unchanged when compared with published data. A fast and robust methodology to map clinical data on personalized volumetric atrial geometries could contribute significantly to clinical decision making.

Cell Division and Rearrangement Dynamics Coordinated by Planar Cell Polarity Pathway in Growth Plate Cartilage

Chondrodysplasias, resulting in dwarfism and altered skeletal growth, are heritable disorders affecting 1 in 4000 to 5000 births. Long bone growth is tightly controlled by sequential chondrocyte maturation through spatially distinct zones in growth plate cartilage. Discoid proliferative chondrocytes form a division plane parallel to the longitudinal axis of the bone and rotate 90 degrees to stack the cells in a unique columnar architecture, which potentiates further limb growth. Noncanonical, β-catenin-independent, Wnt signaling is needed to align division planes and promote organized column formation, which makes the planar cell polarity (PCP) pathway a strong candidate in determining column architecture. Viral manipulation of PCP pathway proteins in chicken embryo growth plates interfered with cell division orientation, but conclusions were limited by fixed tissue sections. To expand our depth of mechanistic inquiry, we sought to develop a method to analyze genetic deletions of PCP pathway proteins and dynamically monitor chondrocyte rearrangement in living growth plate tissue. We hypothesize that Wnt-dependent PCP signaling is required for proliferative chondrocyte rearrangement. Using mice with membrane bound GFP expression and confocal microscopy, we generated time-lapse videos of chondrocytes from live cranial base growth plate explants. Genetic knockout of upstream Wnt5a ligand and inducible, cartilage-specific deletion of Vangl2 transmembrane receptor and Ror2 kinase co-receptor result in significant differences in the final division plane angle distributions compared to normal (p-value<0.0001). Vangl2 is a core PCP protein phosphorylated by Ror2 in response to Wnt5a signaling. While disrupted rearrangement could be a cause of the disorganized proliferative zone, the daughter cells also have premature separation at their interface. Normal chondrocytes separate on average 14.0±6.6 hours after division, while Ror2, Wnt5a, and Vangl2 mutants have significantly shorter separation times (5.2±4.0, 7.8±4.4, and 9.1±3.9 hours, respectively). Our results show that proper rearrangement of proliferative chondrocytes is dependent on PCP pathway activity; however, it is unclear if the premature separation is occurring through disengagement of the cadherin-dependent daughter cell interface or deposition of extracellular matrix between the cells. The focus of future studies is to determine what is acting downstream of the PCP pathway to regulate adhesion dynamics and determine the extrinsic requirements guiding polarized chondrocyte behaviors. These results will advance approaches to cartilage engineering to treat chondrodysplasias by understanding the mechanistic cues required for proper cartilage tissue architecture and growth.

Expanding the Enzyme Repertoire for Sugar Nucleotide Epimerization: The CDP-Tyvelose 2-Epimerase from Thermodesulfatator atlanticus for Glucose/Mannose Interconversion.

Epimerization of sugar nucleotides is central to the structural diversification of monosaccharide building blocks for cellular biosynthesis. Epimerase applicability to carbohydrate synthesis can be limited, however, by the high degree of substrate specificity exhibited by most sugar nucleotide epimerases. Here, we discovered a promiscuous type of CDP-tyvelose 2-epimerase (TyvE)-like enzyme that promotes C2-epimerization in all nucleotide (CDP, UDP, GDP, ADP, TDP)-activated forms of d-glucose. This new epimerase, originating from Thermodesulfatator atlanticus, is a functional homodimer that contains one tightly bound NAD+/subunit and shows optimum activity at 70°C and pH 9.5. The enzyme exhibits a k cat with CDP-dglucose of ∼1.0 min-1 (pH 7.5, 60°C). To characterize the epimerase kinetically and probe its substrate specificity, we developed chemo-enzymatic syntheses for CDP-dmannose, CDP-6-deoxy-dglucose, CDP-3-deoxy-dglucose and CDP-6-deoxy-dxylo-hexopyranos-4-ulose. Attempts to obtain CDP-dparatose and CDP-dtyvelose were not successful. Using high-resolution carbohydrate analytics and in situ NMR to monitor the enzymatic conversions (60°C, pH 7.5), we show that the CDP-dmannose/CDP-dglucose ratio at equilibrium is 0.67 (± 0.1), determined from the kinetic Haldane relationship and directly from the reaction. We further show that deoxygenation at sugar C6 enhances the enzyme activity 5-fold compared to CDP-dglucose whereas deoxygenation at C3 renders the substrate inactive. Phylogenetic analysis places the T. atlanticus epimerase into a distinct subgroup within the sugar nucleotide epimerase family of SDR (short-chain dehydrogenases/reductases), for which the current study now provides the functional context. Collectively, our results expand an emerging toolbox of epimerase-catalyzed reactions for sugar nucleotide synthesis.IMPORTANCE Epimerases of the sugar nucleotide-modifying class of enzymes have attracted considerable interest in carbohydrate (bio)chemistry, for the mechanistic challenges and the opportunities for synthesis involved in the reactions catalyzed. Discovery of new epimerases with expanded scope of sugar nucleotide substrates used is important to promote the mechanistic inquiry and can facilitate the development of new enzyme applications. Here, a CDP-tyvelose 2-epimerase-like enzyme from Thermodesulfatator atlanticus is shown to catalyze sugar C2 epimerization in CDP-glucose and other nucleotide-activated forms of dglucose. The reactions are new to nature in the context of enzymatic sugar nucleotide modification. The current study explores the substrate scope of the discovered C2-epimerase and, based on modeling, suggests structure-function relationships that may be important for specificity and catalysis.

Dehydroxyhispolon Methyl Ether, A Hispolon Derivative, Inhibits WNT/β-Catenin Signaling to Elicit Human Colorectal Carcinoma Cell Apoptosis.

Colorectal cancer (CRC) is the fourth leading cause of cancer mortality worldwide. Aberrant activation of WNT/β-catenin signaling present in the vast majority of CRC cases is indispensable for CRC initiation and progression, and thus is a promising target for CRC therapeutics. Hispolon is a fungal-derived polyphenol with a pronounced anticancer effect. Several hispolon derivatives, including dehydroxyhispolon methyl ether (DHME), have been chemically synthesized for developing lead molecules with stronger anticancer activity. Herein, a DHME-elicited anti-CRC effect with the underlying mechanism is reported for the first time. Specifically, DHME was found to be more cytotoxic than hispolon against a panel of human CRC cell lines, while exerting limited toxicity to normal human colon cell line CCD 841 CoN. Additionally, the cytotoxic effect of DHME appeared to rely on inducing apoptosis. This notion was evidenced by DHME-elicited upregulation of poly (ADP-ribose) polymerase (PARP) cleavage and a cell population positively stained by annexin V, alongside the downregulation of antiapoptotic B-cell lymphoma 2 (BCL-2), whereas the blockade of apoptosis by the pan-caspase inhibitor z-VAD-fmk attenuated DHME-induced cytotoxicity. Further mechanistic inquiry revealed the inhibitory action of DHME on β-catenin-mediated, T-cell factor (TCF)-dependent transcription activity, suggesting that DHME thwarted the aberrantly active WNT/β-catenin signaling in CRC cells. Notably, ectopic expression of a dominant–active β-catenin mutant (∆N90-β-catenin) abolished DHME-induced apoptosis while also restoring BCL-2 expression. Collectively, we identified DHME as a selective proapoptotic agent against CRC cells, exerting more potent cytotoxicity than hispolon, and provoking CRC cell apoptosis via suppression of the WNT/β-catenin signaling axis.

Fostered Nrf2 expression antagonizes iron overload and glutathione depletion to promote resistance of neuron-like cells to ferroptosis

Neurological diseases were often characterized by progressive neuronal death, and emerging evidences suggested that ferroptosis may be an active driver of multiple neurodegenerative diseases. However, the mechanisms underlying ferroptosis in neuron cells are unclear. Here, we demonstrated that ferroptotic stimuli caused injury in neuron-like PC12 cells by modulating the expression of proteins involved in iron metabolism and lipid peroxidation at multiple levels, such as altering iron import/export, activating ferritinophagy, and decreasing glutathione (GSH) level. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates multiple genes involved in ferroptosis, however, its exact role remain elusive. Our mechanistic inquiry revealed that Nrf2 expression enhanced iron storage capacity by increasing ferritin heavy chain 1 (FTH1) expression in PC12 cells. Moreover, Nrf2 alleviated the decrease in GSH level by promoting the expression of genes related to GSH synthesis, including solute carrier family 7 member 11 (SLC7A11) and cysteine ligase (GCL). The contribution of Nrf2 on ferroptosis resistance was further verified by increasing cell tolerance to oxidative stress. Furthermore, Nfe2l2 (Nrf2) knockdown sensitized cells to ferroptotic cell death. Taken together, our findings suggested that iron accumulation caused by altering iron metabolism and the decrease of GSH content are key factors in determining ferroptosis in PC12 cells, and Nrf2 inhibits ferroptosis by combating iron-induced oxidative stress. Our present study provided new clues for the intervention and prevention against ferroptosis-associated neurological diseases.

Demonstrating More Realistic Research Outcomes to Undergraduates in the Organic Teaching Lab: Separation of Product Mixtures, Structure Analysis, and Mechanistic Inquiry

How well does the undergraduate organic chemistry laboratory curriculum prepare students for academic research, or for a bench job in industry? Running experiments that are more representative of research chemistry has the potential to better equip students for working in a research laboratory, while at the same time giving them insight into a typical outcome in a research project. We describe here an experiment that we have launched in the sophomore organic chemistry lab at UC Davis that presents challenges in chromatographic separation, structural assignment by NMR, and mechanistic interpretation. Optional exercises in computational modeling of the NMR data and chromophore analysis by UV–vis/fluorescence measurements are included.