Nucleotide Excision Repair and Transcriptional Regulation: TFIIH and Beyond.

Transcription factor IIH (TFIIH) is a multiprotein complex involved in both transcription and DNA repair, revealing a striking functional link between these two processes. Some of its subunits also belong to complexes involved in other cellular processes, such as chromosome segregation and cell cycle regulation, emphasizing the multitasking capabilities of this factor. This review aims to depict the structure of TFIIH and to dissect the roles of its subunits in different cellular mechanisms. Our understanding of the biochemistry of TFIIH has greatly benefited from studies focused on diseases related to TFIIH mutations. We address the etiology of these disorders and underline the fact that TFIIH can be considered a promising target for therapeutic strategies.

CDK7 inhibition suppresses super-enhancer-linked oncogenic transcription in MYCN-driven cancer.

The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, may be useful therapy for cancers that are driven by MYC family oncoproteins.

Structural Basis for the Modulation of Human KCNQ4 by Small-Molecule Drugs.

Among the five KCNQ channels, also known as the Kv7 voltage-gated potassium (Kv) channels, KCNQ2-KCNQ5 control neuronal excitability. Dysfunctions of KCNQ2-KCNQ5 are associated with neurological disorders such as epilepsy, deafness, and neuropathic pain. Here, we report the cryoelectron microscopy (cryo-EM) structures of human KCNQ4 and its complexes with the opener retigabine or the blocker linopirdine at overall resolutions of 2.5, 3.1, and 3.3 Å, respectively. In all structures, a phosphatidylinositol 4,5-bisphosphate (PIP2) molecule inserts its head group into a cavity within each voltage-sensing domain (VSD), revealing an unobserved binding mode for PIP2. Retigabine nestles in each fenestration, inducing local shifts. Instead of staying within the central pore, linopirdine resides in a cytosolic cavity underneath the inner gate. Electrophysiological analyses of various mutants corroborated the structural observations. Our studies reveal the molecular basis for the modulatory mechanism of neuronal KCNQ channels and provide a framework for structure-facilitated drug discovery targeting these important channels.

CDK7 regulates organ size and tumor growth by safeguarding the Hippo pathway effector Yki/Yap/Taz in the nucleus.

Hippo signaling controls organ size and tumor progression through a conserved pathway leading to nuclear translocation of the transcriptional effector Yki/Yap/Taz. Most of our understanding of Hippo signaling pertains to its cytoplasmic regulation, but how the pathway is controlled in the nucleus remains poorly understood. Here we uncover an evolutionarily conserved mechanism by which CDK7 promotes Yki/Yap/Taz stabilization in the nucleus to sustain Hippo pathway outputs. We found that a modular E3 ubiquitin ligase complex CRL4DCAF12 binds and targets Yki/Yap/Taz for ubiquitination and degradation, whereas CDK7 phosphorylates Yki/Yap/Taz at S169/S128/S90 to inhibit CRL4DCAF12 recruitment, leading to Yki/Yap/Taz stabilization. As a consequence, inactivation of CDK7 reduced organ size and inhibited tumor growth, which could be reversed by restoring Yki/Yap activity. Our study identifies an unanticipated layer of Hippo pathway regulation, defines a novel mechanism by which CDK7 regulates tissue growth, and implies CDK7 as a drug target for Yap/Taz-driven cancer.

Ferroptosis: an iron-dependent form of nonapoptotic cell death.

Nonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(-)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.

Ferrostatin-1 inhibits ferroptosis of vascular smooth muscle cells and alleviates abdominal aortic aneurysm formation through activating the SLC7A11/GPX4 axis.

Ferroptosis, a type of iron-catalyzed necrosis, is responsible for vascular smooth muscle cell (VSMC) death and serves as a potential therapeutic target for alleviating aortic aneurysm. Here, our study explored the underlying mechanism of ferroptosis affecting VSMC functions and the resultant formation of AAA using its inhibitor Ferrostatin-1 (Fer-1). Microarray-based gene expression profiling was employed to identify differentially expressed genes related to AAA and ferroptosis. An AAA model was established by angiotensin II (Ang II) induction in apolipoprotein E-knockout (ApoE-/- ) mice, followed by injection of Fer-1 and RSL-3 (ferroptosis inducer). Then, the role of Fer-1 and RSL-3 in the ferroptosis of VSMCs and AAA formation was analyzed in Ang II-induced mice. Primary mouse VSMCs were cultured in vitro and treated with Ang II, Fer-1, sh-SLC7A11, or sh-GPX4 to assess the effect of Fer-1 via the SLC7A11/GPX axis. Bioinformatics analysis revealed that GPX4 was involved in the fibrosis formation of AAA, and there was an interaction between SLC7A11 and GPX4. In vitro assays showed that Fer-1 alleviated Ang II-induced ferroptosis of VSMCs and retard the consequent AAA formation. The mechanism was associated with activation of the SLC7A11/GPX4 pathway. Silencing of SLC7A11 or GPX4 could inhibit the ameliorating effect of Fer-1 on the ferroptosis of VSMCs. In vivo animal studies further demonstrated that Fer-1 inhibited Ang II-induced ferroptosis and vessel wall structural abnormalities in AAA mouse through activation of the SLC7A11/GPX4 pathway. Fer-1 may prevent AAA formation through activation of the SLC7A11/GPX4 pathway.

Oxidized phospholipid POVPC contributes to vascular calcification by triggering ferroptosis of vascular smooth muscle cells.

Vascular calcification is an actively regulated biological process resembling bone formation, and osteogenic differentiation of vascular smooth muscle cells (VSMCs) plays a crucial role in this process. 1-Palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), an oxidized phospholipid, is found in atherosclerotic plaques and has been shown to induce oxidative stress. However, the effects of POVPC on osteogenic differentiation and calcification of VSMCs have yet to be studied. In the present study, we investigated the role of POVPC in vascular calcification using in vitro and ex vivo models. POVPC increased mineralization of VSMCs and arterial rings, as shown by alizarin red staining. In addition, POVPC treatment increased expression of osteogenic markers Runx2 and BMP2, indicating that POVPC promotes osteogenic transition of VSMCs. Moreover, POVPC increased oxidative stress and impaired mitochondria function of VSMCs, as shown by increased ROS levels, impairment of mitochondrial membrane potential, and decreased ATP levels. Notably, ferroptosis triggered by POVPC was confirmed by increased levels of intracellular ROS, lipid ROS, and MDA, which were decreased by ferrostatin-1, a ferroptosis inhibitor. Furthermore, ferrostatin-1 attenuated POVPC-induced calcification of VSMCs. Taken together, our study for the first time demonstrates that POVPC promotes vascular calcification via activation of VSMC ferroptosis. Reducing the levels of POVPC or inhibiting ferroptosis might provide a novel strategy to treat vascular calcification.

Histone deacetylase-3 regulates the expression of the amyloid precursor protein and its inhibition promotes neuroregenerative pathways in Alzheimer's disease models.

HDAC3 inhibition has been shown to improve memory and reduce amyloid-ß (Aß) in Alzheimer's disease (AD) models, but the underlying mechanisms are unclear. We investigated the molecular effects of HDAC3 inhibition on AD pathology, using in vitro and ex vivo models of AD, based on our finding that HDAC3 expression is increased in AD brains. For this purpose, N2a mouse neuroblastoma cells as well as organotypic brain cultures (OBCSs) of 5XFAD and wild-type mice were incubated with various concentrations of the HDAC3 selective inhibitor RGFP966 (0.1-10 µM) for 24 h. Treatment with RGFP966 or HDAC3 knockdown in N2a cells was associated with an increase on amyloid precursor protein (APP) and mRNA expressions, without alterations in Aß42 secretion. In vitro chromatin immunoprecipitation analysis revealed enriched HDAC3 binding at APP promoter regions. The increase in APP expression was also detected in OBCSs from 5XFAD mice incubated with 1 µM RGFP966, without changes in Aß. In addition, HDAC3 inhibition resulted in a reduction of activated Iba-1-positive microglia and astrocytes in 5XFAD slices, which was not observed in OBCSs from wild-type mice. mRNA sequencing analysis revealed that HDAC3 inhibition modulated neuronal regenerative pathways related to neurogenesis, differentiation, axonogenesis, and dendritic spine density in OBCSs. Our findings highlight the complexity and diversity of the effects of HDAC3 inhibition on AD models and suggest that HDAC3 may have multiple roles in the regulation of APP expression and processing, as well as in the modulation of neuroinflammatory and neuroprotective genes.

A novel mechanism of ferroptosis inhibition-enhanced atherosclerotic plaque stability: YAP1 suppresses vascular smooth muscle cell ferroptosis through GLS1.

Atherosclerosis is a leading cause of cardiovascular diseases (CVDs), often resulting in major adverse cardiovascular events (MACEs), such as myocardial infarction and stroke due to the rupture or erosion of vulnerable plaques. Ferroptosis, an iron-dependent form of cell death, has been implicated in the development of atherosclerosis. Despite its involvement in CVDs, the specific role of ferroptosis in atherosclerotic plaque stability remains unclear. In this study, we confirmed the presence of ferroptosis in unstable atherosclerotic plaques and demonstrated that the ferroptosis inhibitor ferrostatin-1 (Fer-1) stabilizes atherosclerotic plaques in apolipoprotein E knockout (Apoe-/-) mice. Using bioinformatic analysis combining RNA sequencing (RNA-seq) with single-cell RNA sequencing (scRNA-seq), we identified Yes-associated protein 1 (YAP1) as a potential key regulator of ferroptosis in vascular smooth muscle cells (VSMCs) of unstable plaques. In vitro, we found that YAP1 protects against oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in VSMCs. Mechanistically, YAP1 exerts its anti-ferroptosis effects by regulating the expression of glutaminase 1 (GLS1) to promote the synthesis of glutamate (Glu) and glutathione (GSH). These findings establish a novel mechanism where the inhibition of ferroptosis promotes the stabilization of atherosclerotic plaques through the YAP1/GLS1 axis, attenuating VSMC ferroptosis. Thus, targeting the YAP1/GLS1 axis to suppress VSMC ferroptosis may represent a novel strategy for preventing and treating unstable atherosclerotic plaques.

A novel tsRNA-5008a promotes ferroptosis in cardiomyocytes that causes atrial structural remodeling predisposed to atrial fibrillation.

Atrial fibrillation (AF) is an extremely common clinical arrhythmia disease, but whether its mechanism is associated with ferroptosis remains unclear. The tRNA-derived small RNAs (tsRNAs) are involved in a variety of cardiovascular diseases, however, their role and mechanism in atrial remodeling in AF have not been studied. We aimed to explore whether tsRNAs mediate ferroptosis in AF progression. The AF models were constructed to detect ferroptosis-related indicators, and Ferrostatin-1 (Fer-1) was introduced to clarify the relationship between ferroptosis and AF. Atrial myocardial tissue was used for small RNA sequencing to screen potential tsRNAs. tsRNA functioned on ferroptosis and AF was explored. Atrial fibrosis and changes in the cellular structures and arrangement were observed in AF mice model, and these alterations were accompanied by ferroptosis occurrence, exhibited by the accumulation of Fe2+ and MDA levels and the decrease of expression of FTH1, GPX4, and SLC7A11. Blocking above ferroptosis activation with Fer-1 resulted in a significant improvement for AF. A total of 7 tsRNAs were upregulated (including tsRNA-5008a) and 2 tsRNAs were downregulated in atrial myocardial tissue in the AF group compared with the sham group. We constructed a tsRNA-mRNA regulated network, which showed tsRNA-5008a targeted 16 ferroptosis-related genes. Knockdown of tsRNA-5008a significantly suppressed ferroptosis through targeting SLC7A11 and diminished myocardial fibrosis both in vitro and in vivo. On the contrary, tsRNA-5008a mimics promoted ferroptosis in cardiomyocytes. Collectively, tsRNA-5008a involved in AF through ferroptosis. Our study provides novel insights into the role of tsRNA-5008a mediated ferroptosis in AF progression.

Ferroptosis contributes to airway epithelial E-cadherin disruption in a mixed granulocytic asthma mouse model.

Aberrant expression of airway epithelial E-cadherin is a key feature of asthma, yet the underlying mechanisms are largely unknown. Ferroptosis is a novel form of regulated cell death involved in asthma pathogenesis. This study was aimed to evaluate the role of ferroptosis and to investigate whether ferroptosis mediates E-cadherin disruption in mixed granulocyte asthma (MGA). Two murine models of MGA were established using toluene diisocyanate (TDI) or ovalbumin with Complete Freund's Adjuvant (OVA/CFA). Specific antagonists of ferroptosis, including Liproxstatin-1 (Lip-1) and Ferrostatin-1 (Fer-1) were given to the mice. The allergen-exposed mice displayed markedly shrunk mitochondria in the airway epithelia, with decreased volume and denser staining accompanied by down-regulated GPX4 as well as up-regulated FTH1 and malondialdehyde, which are markers of ferroptosis. Decreased pulmonary expression of E-cadherin was also observed, with profound loss of membrane E-cadherin in the airway epithelia, as well as increased secretion of sE-cadherin. Treatment with Lip-1 not only showed potent protective effects against the allergen-induced airway hyperresponsiveness and inflammatory responses, but also rescued airway epithelial E-cadherin expression and inhibited the release of sE-cadherin. Taken together, our data demonstrated that ferroptosis mediates airway epithelial E-cadherin dysfunction in MGA.

The HDAC inhibitor CI-994 acts as a molecular memory aid by facilitating synaptic and intracellular communication after learning.

Long-term memory formation relies on synaptic plasticity, neuronal activity-dependent gene transcription, and epigenetic modifications. Multiple studies have shown that HDAC inhibitor (HDACi) treatments can enhance individual aspects of these processes and thereby act as putative cognitive enhancers. However, their mode of action is not fully understood. In particular, it is unclear how systemic application of HDACis, which are devoid of substrate specificity, can target pathways that promote memory formation. In this study, we explore the electrophysiological, transcriptional, and epigenetic responses that are induced by CI-994, a class I HDACi, combined with contextual fear conditioning (CFC) in mice. We show that CI-994­mediated improvement of memory formation is accompanied by enhanced long-term potentiation in the hippocampus, a brain region recruited by CFC, but not in the striatum, a brain region not primarily implicated in fear learning. Furthermore, using a combination of bulk and single-cell RNA-sequencing, we find that, when paired with CFC, HDACi treatment engages synaptic plasticity-promoting gene expression more strongly in the hippocampus, specifically in the dentate gyrus (DG). Finally, using chromatin immunoprecipitation-sequencing (ChIP-seq) of DG neurons, we show that the combined action of HDACi application and conditioning is required to elicit enhancer histone acetylation in pathways that underlie improved memory performance. Together, these results indicate that systemic HDACi administration amplifies brain region-specific processes that are naturally induced by learning.

Ferrostatin-1 specifically targets mitochondrial iron-sulfur clusters and aconitase to improve cardiac function in Sirtuin 3 cardiomyocyte knockout mice.

AIMS: Ferroptosis is a form of iron-regulated cell death implicated in ischemic heart disease. Our previous study revealed that Sirtuin 3 (SIRT3) is associated with ferroptosis and cardiac fibrosis. In this study, we tested whether the knockout of SIRT3 in cardiomyocytes (SIRT3cKO) promotes mitochondrial ferroptosis and whether the blockade of ferroptosis would ameliorate mitochondrial dysfunction. METHODS AND RESULTS: Mitochondrial and cytosolic fractions were isolated from the ventricles of mice. Cytosolic and mitochondrial ferroptosis were analyzed by comparison to SIRT3loxp mice. An echocardiography study showed that SIRT3cKO mice developed heart failure as evidenced by a reduction of EF% and FS% compared to SIRT3loxp mice. Comparison of mitochondrial and cytosolic fractions of SIRT3cKO and SIRT3loxp mice revealed that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased. Similarly, acetylated p53 was significantly upregulated only in the mitochondria. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase. Most importantly, loss of SIRT3 resulted in significant reductions of frataxin, aconitase, and glutathione peroxidase 4 (GPX4) in the mitochondria. This was accompanied by a significant increase in levels of mitochondrial 4-hydroxynonenal. Treatment of SIRT3cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) for 14 days significantly improved preexisting heart failure. Mechanistically, Fer-1 treatment significantly increased GPX4 and aconitase expression/activity, increased mitochondrial iron­sulfur clusters, and improved mitochondrial membrane potential and Complex IV activity. CONCLUSIONS: Inhibition of ferroptosis ameliorated cardiac dysfunction by specifically targeting mitochondrial aconitase and iron­sulfur clusters. Blockade of mitochondrial ferroptosis may be a novel therapeutic target for mitochondrial cardiomyopathies.

Kv7 channel activation reduces brain endothelial cell permeability and prevents kainic acid-induced blood-brain barrier damage.

Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions.NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.

The oxidized phospholipid PGPC impairs endothelial function by promoting endothelial cell ferroptosis via FABP3.

Ferroptosis is a novel cell death mechanism that is mediated by iron-dependent lipid peroxidation. It may be involved in atherosclerosis development. Products of phospholipid oxidation play a key role in atherosclerosis. 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) is a phospholipid oxidation product present in atherosclerotic lesions. It remains unclear whether PGPC causes atherosclerosis by inducing endothelial cell ferroptosis. In this study, human umbilical vein endothelial cells (HUVECs) were treated with PGPC. Intracellular levels of ferrous iron, lipid peroxidation, superoxide anions (O2•-), and glutathione were detected, and expression of fatty acid binding protein-3 (FABP3), glutathione peroxidase 4 (GPX4), and CD36 were measured. Additionally, the mitochondrial membrane potential (MMP) was determined. Aortas from C57BL6 mice were isolated for vasodilation testing. Results showed that PGPC increased ferrous iron levels, the production of lipid peroxidation and O2•-, and FABP3 expression. However, PGPC inhibited the expression of GPX4 and glutathione production and destroyed normal MMP. These effects were also blocked by ferrostatin-1, an inhibitor of ferroptosis. FABP3 silencing significantly reversed the effect of PGPC. Furthermore, PGPC stimulated CD36 expression. Conversely, CD36 silencing reversed the effects of PGPC, including PGPC-induced FABP3 expression. Importantly, E06, a direct inhibitor of the oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine IgM natural antibody, inhibited the effects of PGPC. Finally, PGPC impaired endothelium-dependent vasodilation, ferrostatin-1 or FABP3 inhibitors inhibited this impairment. Our data demonstrate that PGPC impairs endothelial function by inducing endothelial cell ferroptosis through the CD36 receptor to increase FABP3 expression. Our findings provide new insights into the mechanisms of atherosclerosis and a therapeutic target for atherosclerosis.

Kv7/M channel dysfunction produces hyperexcitability in hippocampal CA1 pyramidal cells of Fmr1 knockout mice.

Fragile X syndrome (FXS), the most frequent monogenic form of intellectual disability, is caused by transcriptional silencing of the FMR1 gene that could render neuronal hyperexcitability. Here we show that pyramidal cells (PCs) in the dorsal CA1 region of the hippocampus elicited a larger action potential (AP) number in response to suprathreshold stimulation in juvenile Fmr1 knockout (KO) than wild-type (WT) mice. Because Kv7/M channels modulate CA1 PC excitability in rats, we investigated if their dysfunction produces neuronal hyperexcitability in Fmr1 KO mice. Immunohistochemical and western blot analyses showed no differences in the expression of Kv7.2 and Kv7.3 channel subunits between genotypes; however, the current mediated by Kv7/M channels was reduced in Fmr1 KO mice. In both genotypes, bath application of XE991 (10 µM), a blocker of Kv7/M channels: produced an increased AP number, produced an increased input resistance, produced a decreased AP voltage threshold and shaped AP medium afterhyperpolarization by increasing mean velocities. Retigabine (10 µM), an opener of Kv7/M channels, produced opposite effects to XE991. Both XE991 and retigabine abolished differences in all these parameters found in control conditions between genotypes. Furthermore, a low concentration of retigabine (2.5 µM) normalized CA1 PC excitability of Fmr1 KO mice. Finally, ex vivo seizure-like events evoked by 4-aminopyiridine (200 µM) in the dorsal CA1 region were more frequent in Fmr1 KO mice, and were abolished by retigabine (5-10 µM). We conclude that CA1 PCs of Fmr1 KO mice exhibit hyperexcitability, caused by Kv7/M channel dysfunction, and increased epileptiform activity, which were abolished by retigabine. KEY POINTS: Dorsal pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice exhibit hyperexcitability. Kv7/M channel activity, but not expression, is reduced in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Kv7/M channel dysfunction causes hyperexcitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice by increasing input resistance, decreasing AP voltage threshold and shaping medium afterhyperpolarization. A Kv7/M channel opener normalizes neuronal excitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Ex vivo seizure-like events evoked in the dorsal CA1 region were more frequent in Fmr1 KO mice, and such an epileptiform activity was abolished by a Kv7/M channel opener depending on drug concentration. Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome.

Bioinspired Heterocoordination in Adaptable Cobalt Metal-Organic Framework for DNA Epigenetic Modification Detection.

Metal-organic frameworks (MOFs) show unique advantages in simulating the dynamics and fidelity of natural coordination. Inspired by zinc finger protein, a second linker was introduced to affect the homogeneous MOF system and thus facilitate the emergence of diverse functionalities. Under the systematic identification of 12 MOF species (i.e., metal ions, linkers) and 6 second linkers (trigger), a dissipative system consisting of Co-BDC-NO2 and o-phenylenediamine (oPD) was screened out, which can rapidly and in situ generate a high photothermal complex (η = 36.9%). Meanwhile, both the carboxylation of epigenetic modifications and metal ion (Fe3+, Ni2+, Cu2+, Zn2+, Co2+ and Mn2+) screening were utilized to improve the local coordination environment so that the adaptable Co-MOF growth on the DNA strand was realized. Thus, epigenetic modification information on DNA was converted to an amplified metal ion signal, and then oPD was further introduced to generate bimodal dissipative signals by which a simple, high-sensitivity detection strategy of 5-hydroxymethylcytosine (LOD = 0.02%) and 5-formylcytosine (LOD = 0.025‰) was developed. The strategy provides one low-cost method (< 0.01 $/sample) for quantifying global epigenetic modifications, which greatly promotes epigenetic modification-based early disease diagnosis. This work also proposes a general heterocoordination design concept for molecular recognition and signal transduction, opening a new MOF-based sensing paradigm.

Covalent Organic Framework-Structured Raman Probes for Ultrasensitive In Vivo Bioimaging.

Organic Raman probes, including polymers and small molecules, have attracted great attention in biomedical imaging owing to their excellent biocompatibility. However, the development of organic Raman probes is usually hindered by a mismatch between their absorption spectra and wavelength-fixed excitation, which makes it difficult to achieve resonance excitation necessary to obtain strong Raman signals. Herein, we introduce a covalent organic framework (COF) into the fine absorption spectrum regulation of organic Raman probes, resulting in their significant Raman signal enhancement. In representative examples, a polymer poly(diketopyrrolopyrrole-p-phenylenediamine) (DPP-PD) and a small molecule azobenzene are transformed into the corresponding COF-structured Raman probes. Their absorption peaks show an accurate match of less than 5 nm with the NIR excitation. As such, the COF-structured Raman probes acquire highly sensitive bioimaging capabilities compared to their precursors with negligible signals. By further mechanism studies, we discover that the crystallinity and size of COFs directly affect the π-conjugation degree of Raman probes, thus changing their bandgaps and absorption spectra. Our study offers a universal and flexible method for improving the signal performance of organic Raman probes without changing their structural units, making it more convenient to obtain the highly sensitive organic Raman probes for in vivo bioimaging.

Visual Sensor Array for Multiple Aromatic Amines via Specific Ascorbic Acid Oxidase Mimic Triggered Schiff-Base Chemistry.

Redox nanozymes have exhibited various applications in recognizing environmental pollutants but not aromatic amines (a type of typical pollutant). Herein, with Cu2+ as a node and tryptophan (Trp) as a linker, Cu-Trp as a specific ascorbic acid oxidase mimic was synthesized, which could catalyze ascorbic acid (AA) oxidation to dehydroascorbic acid (DHAA). Alternatively, with other natural amino acids as linkers to synthesize Cu-based nanozymes, such catalytic performances are also observed. The as-produced DHAA could react with o-phenylenediamine (OPD) and its derivatives (2,3-naphthalene diamine (NDA), 4-nitro-o-phenylenediamine (4-NO2-OPD), 4-fluoro-o-phenylenediamine (4-F-OPD), 4-chloro-o-phenylenediamine(4-Cl-OPD), and 4-bromo-o-phenylenediamine(4-Br-OPD)) to form a Schiff base and emit fluorescence. Based on the results, with Cu-Trp + AA and Cu-Arg (with arginine (Arg) as a linker) + AA as two sensing channels and extracted red, green, and blue (RGB) values from emitted fluorescence as read-out signals, a visual sensor array was constructed to efficiently distinguish OPD, NDA, 4-NO2-OPD, 4-F-OPD, 4-Cl-OPD, and 4-Br-OPD as low as 10 µM. Such detecting performance was further confirmed through discriminating binary, ternary, quinary, and senary mixtures with various concentration ratios, recognizing 18 unknown samples, and even quantitatively analyzing single aromatic amine. Finally, the discriminating ability was further validated in environmental waters, providing an efficient assay for large-scale scanning levels of multiple aromatic amines.

Cyclophosphamide activates ferroptosis-induced dysfunction of Leydig cells via SMAD2 pathway†.

Cyclophosphamide (CP) is a widely used chemotherapeutic drug and immunosuppressant in the clinic, and the hypoandrogenism caused by CP is receiving more attention. Some studies found that ferroptosis is a new mechanism of cell death closely related to chemotherapeutic drugs and plays a key role in regulating reproductive injuries. The purpose of this study is to explore ferroptosis' role in testicular Leydig cell dysfunction and molecular mechanisms relating to it. In this study, the level of ferroptosis in the mouse model of testicular Leydig cell dysfunction induced by CP was significantly increased and further affected testosterone synthesis. The ferroptosis inhibitors ferrostatin-1 (Fer-1) and iron chelator deferoxamine (DFO) can improve injury induced by CP. The results of immunohistochemistry showed that Fer-1 and DFO could improve the structural disorder of seminiferous tubules and the decrease of the number of Leydig cells in testicular tissue induced by CP. Immunofluorescence and western blot confirmed that Fer-1 and DFO could improve the expression of key enzymes in testosterone synthesis. The activation of SMAD family member 2 (Smad2)/cyclin-dependent kinase inhibitor 1A (Cdkn1a) pathway can improve the ferroptosis of Leydig cells induced by CP and protect the function of Leydig cells. By inhibiting the Smad2/Cdkn1a signal pathway, CP can regulate ferroptosis, resulting in testicular Leydig cell dysfunction. In this study, CP-induced hypoandrogenism is explained theoretically and a potential therapeutic strategy is provided.