The myelin sheath is an essential structure for the rapid transmission of electrical impulses through axons, and peripheral myelination is a well-programmed postnatal process of Schwann cells (SCs), the myelin-forming peripheral glia. SCs transdifferentiate into demyelinating SCs (DSCs) to remove the myelin sheath during Wallerian degeneration after axonal injury and demyelinating neuropathies, and macrophages are responsible for the degradation of myelin under both conditions. In this study, the mechanism by which DSCs acquire the ability of myelin exocytosis was investigated. Using serial ultrastructural evaluation, we found that autophagy-related gene 7-dependent formation of a "secretory phagophore (SP)" and tubular phagophore was necessary for exocytosis of large myelin chambers by DSCs. DSCs seemed to utilize myelin membranes for SP formation and employed p62/sequestosome-1 (p62) as an autophagy receptor for myelin excretion. In addition, the acquisition of the myelin exocytosis ability of DSCs was associated with the decrease in canonical autolysosomal flux and was demonstrated by p62 secretion. Finally, this SC demyelination mechanism appeared to also function in inflammatory demyelinating neuropathies. Our findings show a novel autophagy-mediated myelin clearance mechanism by DSCs in response to nerve damage.

Chemokine CXCL8 is a key facilitator of the human host immune response, mediating neutrophil migration, and activation at the site of infection and injury. The oxidative burst is an important effector mechanism which leads to the generation of reactive nitrogen species (RNS), including peroxynitrite. The current study was performed to determine the potential for nitration to alter the biological properties of CXCL8 and its detection in human disease. Here, we show peroxynitrite nitrates CXCL8 and thereby regulates neutrophil migration and activation. The nitrated chemokine was unable to induce transendothelial neutrophil migration in vitro and failed to promote leukocyte recruitment in vivo. This reduced activity is due to impairment in both G protein-coupled receptor signaling and glycosaminoglycan binding. Using a novel antibody, nitrated CXCL8 was detected in bronchoalveolar lavage samples from patients with pneumonia. These findings were validated by mass spectrometry. Our results provide the first direct evidence of chemokine nitration in human pathophysiology and suggest a natural mechanism that limits acute inflammation.

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding.

The question how proteins fold is especially pointed for large multi-domain, multi-spanning membrane proteins with complex topologies. We have uncovered the sequence of events that encompass proper folding of the ABC transporter CFTR in live cells by combining kinetic radiolabeling with protease-susceptibility assays. We found that CFTR folds in two clearly distinct stages. The first, co-translational, stage involves folding of the 2 transmembrane domains TMD1 and TMD2, plus one nucleotide-binding domain, NBD1. The second stage is a simultaneous, post-translational increase in protease resistance for both TMDs and NBD2, caused by assembly of these domains onto NBD1. Our assays probe every 2-3 residues (on average) in CFTR. This in-depth analysis at amino-acid level allows detailed analysis of domain folding and importantly also the next level: assembly of the domains into native, folded CFTR. Defects and changes brought about by medicines, chaperones, or mutations also are amenable to analysis. We here show that the well-known disease-causing mutation F508del, which established cystic fibrosis as protein-folding disease, caused co-translational misfolding of NBD1 but not TMD1 nor TMD2 in stage 1, leading to absence of stage-2 folding. Corrector drugs rescued stage 2 without rescuing NBD1. Likewise, the DxD motif in NBD1 that was identified to be required for export of CFTR from the ER we found to be required already upstream of export as CFTR mutated in this motif phenocopies F508del CFTR. The highly modular and stepwise folding process of such a large, complex protein explains the relatively high fidelity and correctability of its folding.

BACKGROUND AND AIMS: Thiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among inflammatory bowel disease patients treated with azathioprine (AZA), representing a significant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear. METHODS: To model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca2+ signaling, ductal Cl- and HCO3- secretion, as well as cystic fibrosis transmembrane conductance regulator (CFTR) expression were assessed using microscopy techniques. Ras-related C3 botulinum toxin substrate (RAC1) activity was measured with a G-LISA assay. Super-resolution microscopy was used to determine protein colocalization. RESULTS: We demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fluid and ductal HCO3- and Cl- secretion, but did not affect acinar cells. Furthermore, ex vivo AZA exposure also inhibited RAC1 activity in ductal cells leading to decreased co-localization of CFTR and the anchor protein ezrin, resulting in impaired plasma membrane localization of CFTR. CONCLUSIONS: AZA impaired the ductal HCO3- and Cl- secretion through the inhibition of RAC1 activity leading to diminished ezrin-CFTR interaction and disturbed apical plasma membrane expression of CFTR. We report a novel direct toxic effect of AZA on pancreatic ductal cells and suggest that the restoration of ductal function might help to prevent TIP in the future.

Little is known about the impact of metabolic stimuli on brain tissue at a molecular level. The ketone body beta-hydroxybutyrate (BHB) can be a signaling molecule regulating gene transcription. Thus, we assessed lysine beta-hydroxybutyrylation (K-bhb) levels in proteins extracted from the cerebral cortex of mice undergoing a ketogenic metabolic challenge (48 h fasting). We found that fasting enhanced K-bhb in a variety of proteins including histone H3. ChIP-seq experiments showed that K9 beta-hydroxybutyrylation of H3 (H3K9-bhb) was significantly enriched by fasting on more than 8000 DNA loci. Transcriptomic analysis showed that H3K9-bhb on enhancers and promoters correlated with active gene expression. One of the most enriched functional annotations both at the epigenetic and transcriptional level was "circadian rhythms''. Indeed, we found that the diurnal oscillation of specific transcripts was modulated by fasting at distinct zeitgeber times both in the cortex and suprachiasmatic nucleus. Moreover, specific changes in locomotor activity daily features were observed during re-feeding after 48-h fasting. Thus, our results suggest that fasting remarkably impinges on the cerebral cortex transcriptional and epigenetic landscape, and BHB acts as a powerful epigenetic molecule in the brain through direct and specific histone marks remodeling in neural tissue cells.

Technological advancements have facilitated the implementation of realistic, terrestrial-based complex 33-beam galactic cosmic radiation simulations (GCR Sim) to now probe central nervous system functionality. This work expands considerably on prior, simplified GCR simulations, yielding new insights into responses of male and female mice exposed to 40-50 cGy acute or chronic radiations relevant to deep space travel. Results of the object in updated location task suggested that exposure to acute or chronic GCR Sim induced persistent impairments in hippocampus-dependent memory formation and reconsolidation in female mice that did not manifest robustly in irradiated male mice. Interestingly, irradiated male mice, but not females, were impaired in novel object recognition and chronically irradiated males exhibited increased aggressive behavior on the tube dominance test. Electrophysiology studies used to evaluate synaptic plasticity in the hippocampal CA1 region revealed significant reductions in long-term potentiation after each irradiation paradigm in both sexes. Interestingly, network-level disruptions did not translate to altered intrinsic electrophysiological properties of CA1 pyramidal cells, whereas acute exposures caused modest drops in excitatory synaptic signaling in males. Ultrastructural analyses of CA1 synapses found smaller postsynaptic densities in larger spines of chronically exposed mice compared to controls and acutely exposed mice. Myelination was also affected by GCR Sim with acutely exposed mice exhibiting an increase in the percent of myelinated axons; however, the myelin sheathes on small calibur (< 0.3 mm) and larger (> 0.5 mm) axons were thinner when compared to controls. Present findings might have been predicted based on previous studies using single and mixed beam exposures and provide further evidence that space-relevant radiation exposures disrupt critical cognitive processes and underlying neuronal network-level plasticity, albeit not to the extent that might have been previously predicted.

Tumor cells have an increased demand for nutrients to sustain their growth, but how these increased metabolic needs are ensured or how this influences tumor formation and progression remains unclear. To unravel tumor metabolic dependencies, particularly from extracellular metabolites, we have analyzed the role of plasma membrane metabolic transporters in Drosophila brain tumors. Using a well-established neural stem cell-derived tumor model, caused by brat knockdown, we have found that 13 plasma membrane metabolic transporters, including amino acid, carbohydrate and monocarboxylate transporters, are upregulated in tumors and are required for tumor growth. We identified CD98hc and several of the light chains with which it can form heterodimeric amino acid transporters, as crucial players in brat RNAi (brat IR) tumor progression. Knockdown of these components of CD98 heterodimers caused a dramatic reduction in tumor growth. Our data also reveal that the oncogene dMyc is required and sufficient for the upregulation of CD98 transporter subunits in these tumors. Furthermore, tumor-upregulated dmyc and CD98 transporters orchestrate the overactivation of the growth-promoting signaling pathway TOR, forming a core growth regulatory network to support brat IR tumor progression. Our findings highlight the important link between oncogenes, metabolism, and signaling pathways in the regulation of tumor growth and allow for a better understanding of the mechanisms necessary for tumor progression.

Desmoglein 3 (Dsg3) is a desmosomal cadherin mediating cell adhesion within desmosomes and is the antigen of the autoimmune blistering skin disease pemphigus vulgaris. Therefore, understanding of the complex desmosome turnover process is of high biomedical relevance. Recently, super resolution microscopy was used to characterize desmosome composition and turnover. However, studies were limited because adhesion measurements on living cells were not possible in parallel. Before desmosomal cadherins are incorporated into nascent desmosomes, they are not bound to intermediate filaments but were suggested to be associated with the actin cytoskeleton. However, direct proof that adhesion of a pool of desmosomal cadherins is dependent on actin is missing. Here, we applied single-molecule force spectroscopy measurements with the novel single molecule hybrid-technique STED/SMFS-AFM to investigate the cytoskeletal anchorage of Dsg3 on living keratinocytes for the first time. By application of pharmacological agents we discriminated two different Dsg3 pools, only one of which is anchored to actin filaments. We applied the actin polymerization inhibitor Latrunculin B to modify the actin cytoskeleton and the PKCα activator PMA to modulate intermediate filament anchorage. On the cellular surface Dsg3 adhesion was actin-dependent. In contrast, at cell-cell contacts, Dsg3 adhesion was independent from actin but rather is regulated by PKC which is well established to control desmosome turn-over via intermediate filament anchorage. Taken together, using the novel STED/SMFS-AFM technique, we demonstrated the existence of two Dsg3 pools with different cytoskeletal anchorage mechanisms.

The proportions of the various muscle fiber types are important in the regulation of skeletal muscle metabolism, as well as animal meat production. Four-and-a-half LIM domain protein 3 (FHL3) is highly expressed in fast glycolytic muscle fibers and differentially regulates the expression of myosin heavy chain (MyHC) isoforms at the cellular level. Whether FHL3 regulates the transformation of muscle fiber types in vivo and the regulatory mechanism is unclear. In this study, muscle-specific FHL3 transgenic mice were generated by random integration, and lentivirus-mediated gene knockdown or overexpression in muscles of mice or pigs was conducted. Functional analysis showed that overexpression of FHL3 in muscles significantly increased the proportion of fast-twitch myofibers and muscle mass but decreased muscle succinate dehydrogenase (SDH) activity and whole-body oxygen consumption. Lentivirus-mediated FHL3 knockdown in muscles significantly decreased muscle mass and the proportion of fast-twitch myofibers. Mechanistically, FHL3 directly interacted with the Yin yang 1 (YY1) DNA-binding domain, repressed the binding of YY1 to the fast glycolytic MyHC2b gene regulatory region, and thereby promoted MyHC2b expression. FHL3 also competed with EZH2 to bind the repression domain of YY1 and reduced H3K27me3 enrichment in the MyHC2b regulatory region. Moreover, FHL3 overexpression reduced glucose tolerance by affecting muscle glycolytic metabolism, and its mRNA expression in muscle was positively associated with hemoglobin A1c (HbA1c) in patients with type 2 diabetes. Therefore, FHL3 is a novel potential target gene for the treatment of muscle metabolism-related diseases and improvement of animal meat production.

Adequate endometrial growth is a critical factor for successful embryo implantation and pregnancy maintenance. We previously reported the efficacy of intrauterine administration of botulinum toxin A (BoTA) in improving the endometrial angiogenesis and the rates of embryo implantation. Here, we further evaluated its potent therapeutic effects on the uterine structural and functional repair and elucidated underlying molecular regulatory mechanisms. This study demonstrated that a murine model of thin endometrium was successfully established by displaying dramatically decreased endometrial thickness and the rates of embryo implantation compared to normal endometrium. Interestingly, the expressions of insulin-like growth factor binding protein-3 (IGFBP3) and an active 35 kDa-form of osteopontin (OPN) were significantly reduced in thin endometrium, which were almost fully restored by intrauterine BoTA administration. Neutralization of BoTA-induced IGFBP3 subsequently suppressed proteolytic cleavage of OPN, exhibiting un-recovered endometrial thickness even in the presence of BoTA administration, suggesting that BoTA-induced endometrial regeneration might be mediated by IGFBP3-dependent OPN proteolytic cleavage. Our findings suggest that intrauterine BoTA administration improves the endometrial environment in our murine model with thin endometrium by increasing endometrial receptivity and angiogenesis in a manner dependent on the regulatory effect of IGFBP3 on OPN proteolytic cleavage, proposing BoTA as an efficient therapeutic strategy for the patients with thin endometrium.

At the onset of Drosophila metamorphosis, plenty of secretory glue granules are released from salivary gland cells and the glue is deposited on the ventral side of the forming (pre)pupa to attach it to a dry surface. Prior to this, a poorly understood maturation process takes place during which secretory granules gradually grow via homotypic fusions, and their contents are reorganized. Here we show that the small GTPase Rab26 localizes to immature (smaller, non-acidic) glue granules and its presence prevents vesicle acidification. Rab26 mutation accelerates the maturation, acidification and release of these secretory vesicles as well as the lysosomal breakdown (crinophagy) of residual, non-released glue granules. Strikingly, loss of Mon1, an activator of the late endosomal and lysosomal fusion factor Rab7, results in Rab26 remaining associated even with the large glue granules and a concomitant defect in glue release, similar to the effects of Rab26 overexpression. Our data thus identify Rab26 as a key regulator of secretory vesicle maturation that promotes early steps (vesicle growth) and inhibits later steps (lysosomal transport, acidification, content reorganization, release, and breakdown), which is counteracted by Mon1.

Histone lysine-specific methyltransferase 2 (KMT2A-D) proteins, alternatively called mixed lineage leukemia (MLL1-4) proteins, mediate positive transcriptional memory. Acting as the catalytic subunits of human COMPASS-like complexes, KMT2A-D methylate H3K4 at promoters and enhancers. KMT2A-D contain understudied highly conserved triplets and a quartet of plant homeodomains (PHDs). Here, we show that all clustered (multiple) PHDs localize to the well-defined loci of H3K4me3 and H3 acetylation-rich active promoters and enhancers. Surprisingly, we observe little difference in binding pattern between PHDs from promoter-specific KMT2A-B and enhancer-specific KMT2C-D. Fusion of the KMT2A CXXC domain to the PHDs drastically enhances their preference for promoters over enhancers. Hence, the presence of CXXC domains in KMT2A-B, but not KMT2C-D, may explain the promoter/enhancer preferences of the full-length proteins. Importantly, targets of PHDs overlap with KMT2A targets and are enriched in genes involved in the cancer pathways. We also observe that PHDs of KMT2A-D are mutated in cancer, especially within conserved folding motifs (Cys4HisCys2Cys/His). The mutations cause a domain loss-of-function. Taken together, our data suggest that PHDs of KMT2A-D guide the full-length proteins to active promoters and enhancers, and thus play a role in positive transcriptional memory.

Neural stem cells reside in the subgranular zone, a specialized neurogenic niche of the hippocampus. Throughout adulthood, these cells give rise to neurons in the dentate gyrus, playing an important role in learning and memory. Given that these core cognitive processes are disrupted in numerous disease states, understanding the underlying mechanisms of neural stem cell proliferation in the subgranular zone is of direct practical interest. Here, we report that mature neurons, neural stem cells and neural precursor cells each secrete the neurovascular protein epidermal growth factor-like protein 7 (EGFL7) to shape this hippocampal niche. We further demonstrate that EGFL7 knock-out in a Nestin-CreERT2-based mouse model produces a pronounced upregulation of neurogenesis within the subgranular zone. RNA sequencing identified that the increased expression of the cytokine VEGF-D correlates significantly with the ablation of EGFL7. We substantiate this finding with intraventricular infusion of VEGF-D upregulating neurogenesis in vivo and further show that VEGF-D knock-out produces a downregulation of neurogenesis. Finally, behavioral studies in EGFL7 knock-out mice demonstrate greater maintenance of spatial memory and improved memory consolidation in the hippocampus by modulation of pattern separation. Taken together, our findings demonstrate that both EGFL7 and VEGF-D affect neurogenesis in the adult hippocampus, with the ablation of EGFL7 upregulating neurogenesis, increasing spatial learning and memory, and correlating with increased VEGF-D expression.

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder caused by biallelic mutations in the lysosomal trafficking regulator (LYST) gene. Even though enlarged lysosomes and/or lysosome-related organelles (LROs) are the typical cellular hallmarks of CHS, they have not been investigated in human neuronal models. Moreover, how and why the loss of LYST function causes a lysosome phenotype in cells has not been elucidated. We report that the LYST-deficient human neuronal model exhibits lysosome depletion accompanied by hyperelongated tubules extruding from enlarged autolysosomes. These results have also been recapitulated in neurons differentiated from CHS patients' induced pluripotent stem cells (iPSCs), validating our model system. We propose that LYST ensures the correct fission/scission of the autolysosome tubules during autophagic lysosome reformation (ALR), a crucial process to restore the number of free lysosomes after autophagy. We further demonstrate that LYST is recruited to the lysosome membrane, likely to facilitate the fission of autolysosome tubules. Together, our results highlight the key role of LYST in maintaining lysosomal homeostasis following autophagy and suggest that ALR dysregulation is likely associated with the neurodegenerative CHS phenotype.

One of the major mysteries in science is how it is possible to pack the cellular chromatin with a total length of over 1 m, into a small sphere with a diameter of 5 mm "the nucleus", and even more difficult to envisage how to make it functional. Although we know that compaction is achieved through the histones, however, the DNA needs to be accessible to the transcription machinery and this is allowed thanks to a variety of very complex epigenetic mechanisms. Either DNA (methylation) or post-translational modifications of histone proteins (acetylation, methylation, ubiquitination and sumoylation) play a crucial role in chromatin remodelling and consequently on gene expression. Recently the serotonylation and dopaminylation of the histone 3, catalyzed by the Transglutaminase type 2 (TG2), has been reported. These novel post-translational modifications catalyzed by a predominantly cytoplasmic enzyme opens a new avenue for future investigations on the enzyme function itself and for the possibility that other biological amines, substrate of TG2, can influence the genome regulation under peculiar cellular conditions. In this review we analyzed the nuclear TG2's biology by discussing both its post-translational modification of various transcription factors and the implications of its epigenetic new face. Finally, we will focus on the potential impact of these events in human diseases.

The recent elucidation of atomistic structures of Cl- channel CFTR provides opportunities for understanding the molecular basis of cystic fibrosis. Despite having been activated through phosphorylation and provided with ATP ligands, several near-atomistic cryo-EM structures of CFTR are in a closed state, as inferred from the lack of a continuous passage through a hydrophobic bottleneck region located in the extracellular portion of the pore. Here, we present repeated, microsecond-long molecular dynamics simulations of human CFTR solvated in a lipid bilayer and aqueous NaCl. At equilibrium, Cl- ions enter the channel through a lateral intracellular portal and bind to two distinct cationic sites inside the channel pore but do not traverse the narrow, de-wetted bottleneck. Simulations conducted in the presence of a strong hyperpolarizing electric field led to spontaneous Cl- translocation events through the bottleneck region of the channel, suggesting that the protein relaxed to a functionally open state. Conformational changes of small magnitude involving transmembrane helices 1 and 6 preceded ion permeation through diverging exit routes at the extracellular end of the pore. The pore bottleneck undergoes wetting prior to Cl- translocation, suggesting that it acts as a hydrophobic gate. Although permeating Cl- ions remain mostly hydrated, partial dehydration occurs at the binding sites and in the bottleneck. The observed Cl- pathway is largely consistent with the loci of mutations that alter channel conductance, anion binding, and ion selectivity, supporting the model of the open state of CFTR obtained in the present study.

The transdifferentiation from cardiac fibroblasts to myofibroblasts is an important event in the initiation of cardiac fibrosis. However, the underlying mechanism is not fully understood. Circ-sh3rf3 (circular RNA SH3 domain containing Ring Finger 3) is a novel circular RNA which was induced in hypertrophied ventricles by isoproterenol hydrochloride, and our work has established that it is a potential regulator in cardiac hypertrophy, but whether circ-sh3rf3 plays a role in cardiac fibrosis remains unclear, especially in the conversion of cardiac fibroblasts into myofibroblasts. Here, we found that circ-sh3rf3 was down-regulated in isoproterenol-treated rat cardiac fibroblasts and cardiomyocytes as well as during fibroblast differentiation into myofibroblasts. We further confirmed that circ-sh3rf3 could interact with GATA-4 proteins and reduce the expression of GATA-4, which in turn abolishes GATA-4 repression of miR-29a expression and thus up-regulates miR-29a expression, thereby inhibiting fibroblast-myofibroblast differentiation and myocardial fibrosis. Our work has established a novel Circ-sh3rf3/GATA-4/miR-29a regulatory cascade in fibroblast-myofibroblast differentiation and myocardial fibrosis, which provides a new therapeutic target for myocardial fibrosis.

Haematopoietic Stem cells (HSCs) have the potential for self-renewal and multilineage differentiation, and their behaviours are finely tuned by the microenvironment. HSC transplantation (HSCT) is widely used in the treatment of haematologic malignancies while limited by the quantity of available HSCs. With the development of tissue engineering, hydrogels have been deployed to mimic the HSC microenvironment in vitro. Engineered hydrogels influence HSC behaviour by regulating mechanical strength, extracellular matrix microstructure, cellular ligands and cytokines, cell-cell interaction, and oxygen concentration, which ultimately facilitate the acquisition of sufficient HSCs. Here, we review recent advances in the application of hydrogel-based microenvironment engineering of HSCs, and provide future perspectives on challenges in basic research and clinical practice.

Dysregulated cell migration and invasion are hallmarks of many disease states. This dysregulated migratory behavior is influenced by the changes in expression of aquaporins (AQPs) that occur during pathogenesis, including conditions such as cancer, endometriosis, and arthritis. The ubiquitous function of AQPs in migration of diseased cells makes them a crucial target for potential therapeutics; this possibility has led to extensive research into the specific mechanisms underlying AQP-mediated diseased cell migration. The functions of AQPs depend on a diverse set of variables including cell type, AQP isoform, disease state, cell microenvironments, and even the subcellular localization of AQPs. To consolidate the considerable work that has been conducted across these numerous variables, here we summarize and review the last decade's research covering the role of AQPs in the migration and invasion of cells in diseased states.

Histone H3 trimethylation on lysine 9 (H3K9me3) is a defining feature of mammalian pericentromeres, loss of which results in genome instability. Here we show that CDYL2 is recruited to pericentromeres in an H3K9me3-dependent manner and is required for faithful mitosis and genome stability. CDYL2 RNAi in MCF-7 breast cancer cells and Hela cervical cancer cells inhibited their growth, induced apoptosis, and provoked both nuclear and mitotic aberrations. Mass spectrometry analysis of CDYL2-interacting proteins identified the neurodevelopmental disease-linked mitotic regulators CHAMP1 and POGZ, which are associated with a central non-conserved region of CDYL2. RNAi rescue assays identified both the CDYL2 chromodomain and the CHAMP1-POGZ interacting region as required and, together, sufficient for CDYL2 regulation of mitosis and genome stability. CDYL2 RNAi caused loss of CHAMP1 localization at pericentromeres. We propose that CDYL2 functions as an adaptor protein that connects pericentromeric H3K9me3 with CHAMP1 and POGZ to ensure mitotic fidelity and genome stability.

Nanoceria or cerium oxide nanoparticles characterised by the co-existing of Ce3+ and Ce4+ that allows self-regenerative, redox-responsive dual-catalytic activities, have attracted interest as an innovative approach to treating cancer. Depending on surface characteristics and immediate environment, nanoceria exerts either anti- or pro-oxidative effects which regulate reactive oxygen species (ROS) levels in biological systems. Nanoceria mimics ROS-related enzymes that protect normal cells at physiological pH from oxidative stress and induce ROS production in the slightly acidic tumour microenvironment to trigger cancer cell death. Nanoceria as nanozymes also generates molecular oxygen that relieves tumour hypoxia, leading to tumour cell sensitisation to improve therapeutic outcomes of photodynamic (PDT), photothermal (PTT) and radiation (RT), targeted and chemotherapies. Nanoceria has been engineered as a nanocarrier to improve drug delivery or in combination with other drugs to produce synergistic anti-cancer effects. Despite reported preclinical successes, there are still knowledge gaps arising from the inadequate number of studies reporting findings based on physiologically relevant disease models that accurately represent the complexities of cancer. This review discusses the dual-catalytic activities of nanoceria responding to pH and oxygen tension gradient in tumour microenvironment, highlights the recent nanoceria-based platforms reported to be feasible direct and indirect anti-cancer agents with protective effects on healthy tissues, and finally addresses the challenges in clinical translation of nanoceria based therapeutics.

Preeclampsia is a pregnancy-specific cardiovascular disorder, involving significant maternal endothelial dysfunction. Although inappropriate placentation due to aberrant angiogenesis, inflammation and shallow trophoblast invasion are the root causes of preeclampsia, pathogenic mechanisms are poorly understood, particularly in early pregnancy. Here, we first confirm the abnormal expression of important vascular and inflammatory proteins, FK506-binding protein-like (FKBPL) and galectin-3 (Gal-3), in human plasma and placental tissues from women with preeclampsia and normotensive controls. We then employ a three-dimensional microfluidic placental model incorporating human umbilical vein endothelial cells (HUVECs) and a first trimester trophoblast cell line (ACH-3P) to investigate FKBPL and Gal-3 signaling in inflammatory conditions. In human samples, both circulating (n = 17 controls; n = 30 preeclampsia) and placental (n ≥ 6) FKBPL and Gal-3 levels were increased in preeclampsia compared to controls (plasma: FKBPL, p < 0.0001; Gal-3, p < 0.01; placenta: FKBPL, p < 0.05; Gal-3, p < 0.01), indicative of vascular dysfunction in preeclampsia. In our placenta-on-a-chip model, we show that endothelial cells are critical for trophoblast-mediated migration and that trophoblasts effectively remodel endothelial vascular networks. Inflammatory cytokine tumour necrosis factor-α (10 ng/mL) modulates both FKBPL and Gal-3 signaling in conjunction with trophoblast migration and impairs vascular network formation (p < 0.005). Our placenta-on-a-chip recapitulates aspects of inappropriate placental development and vascular dysfunction in preeclampsia.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder resulting from a CAG expansion in the huntingtin (HTT) gene, which leads to the production and accumulation of mutant huntingtin (mHTT). While primarily considered a disorder of the central nervous system, multiple changes have been described to occur throughout the body, including activation of the immune system. In other neurodegenerative disorders, activation of the immune system has been shown to include the production of antibodies against disease-associated pathological proteins. However, the existence of mHTT-targeted antibodies has never been reported. In this study, we assessed the presence and titer of antibodies recognizing HTT/mHTT in patients with HD (n = 66) and age- and gender-matched healthy controls (n = 66) using a combination of Western blotting and ELISA. Together, these analyses revealed that antibodies capable of recognizing HTT/mHTT were detectable in the plasma samples of all participants, including healthy controls. When antibody levels were monitored at different disease stages, it was observed that antibodies against full-length mHTT were highest in patients with severe disease while antibodies against HTTExon1 were elevated in patients with mild disease. Combined, these results suggest that antibodies detecting different forms of mHTT peak at different disease stages.

Ubiquitin-specific protease (USP)19 is a deubiquitinating enzyme that regulates the stability and function of multiple proteins, thereby controlling various biological responses. The alternative splicing of USP19 results in the expression of two major encoded variants that are localized to the endoplasmic reticulum (ER) (USP19-ER) and cytoplasm (USP19-CY). The importance of alternative splicing for the function of USP19 remains unclear. Here, we demonstrated that USP19-CY promotes TGF-β signaling by directly interacting with TGF-β type I receptor (TβRI) and protecting it from degradation at the plasma membrane. In contrast, USP19-ER binds to and sequesters TβRI in the ER. By decreasing cell surface TβRI levels, USP19-ER inhibits TGF-β/SMAD signaling in a deubiquitination-independent manner. Moreover, USP19-ER inhibits TGF-β-induced epithelial-mesenchymal transition (EMT), whereas USP19-CY enhances EMT, as well as the migration and extravasation of breast cancer cells. Furthermore, USP19-CY expression is correlated with poor prognosis and is higher in breast cancer tissues than in adjacent normal tissues. Notably, the splicing modulator herboxidiene inhibits USP19-CY, increases USP19-ER expression and suppresses breast cancer cell migration. Targeting USP19 splicing or its deubiquitinating activity may have potential therapeutic effects on breast cancer.

N-methyl-D-aspartate receptors (NMDARs) play vital roles in normal brain functions (i.e., learning, memory, and neuronal development) and various neuropathological conditions, such as epilepsy, autism, Parkinson's disease, Alzheimer's disease, and traumatic brain injury. Endogenous neuroactive steroids such as 24(S)-hydroxycholesterol (24(S)-HC) have been shown to influence NMDAR activity, and positive allosteric modulators (PAMs) derived from 24(S)-hydroxycholesterol scaffold can also enhance NMDAR function. This study describes the structural determinants and mechanism of action for 24(S)-hydroxycholesterol and two novel synthetic analogs (SGE-550 and SGE-301) on NMDAR function. We also show that these agents can mitigate the altered function caused by a set of loss-of-function missense variants in NMDAR GluN subunit-encoding GRIN genes associated with neurological and neuropsychiatric disorders. We anticipate that the evaluation of novel neuroactive steroid NMDAR PAMs may catalyze the development of new treatment strategies for GRIN-related neuropsychiatric conditions.

SRC is the first identified oncogene, and its aberrant activation has been implicated as a driving event in tumor initiation and progression. However, its role in cancer stemness regulation and the underlying regulatory mechanism are still elusive. Here, we identified a YAP1 tyrosine phosphorylation-dependent YAP1-KLF5 oncogenic module, as the key downstream mediator of SRC kinase regulating cancer stemness and metastasis in triple-negative breast cancer (TNBC). SRC was overexpressed in TNBC patient tissues and its expression level was highly correlated with the tumor malignancy. SRC activation induced, while inhibition of SRC kinase reduced the cancer stemness, tumor cell growth and metastasis in vitro and in vivo. Transcriptomic and proteomic analysis revealed that SRC-mediated YAP1 tyrosine phosphorylation induced its interaction with Kruppel-like factor 5 (KLF5) to form a YAP1/TEAD-KLF5 complex in TNBC cells. YAP1-KLF5 association further promoted TEAD-mediated transcriptional program independently of canonical Hippo kinases, which eventually gave rise to the enhanced cancer stemness and metastasis. Disruption of YAP1-KLF5 module in TNBC cells dramatically attenuated the SRC-induced cancer stemness and metastasis in vitro and in vivo. Accordingly, co-upregulations of SRC and YAP1-KLF5 module in TNBC tissues were significantly positively correlated with the tumor malignance. Altogether, our work presents a novel tyrosine phosphorylation-dependent YAP1-KLF5 oncogenic module governing SRC-induced cancer stemness and metastasis in TNBC. Therefore, targeting YAP1/KLF5-mediated transcription may provide a promising strategy for TNBC treatment with SRC aberrantly activation.

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.

Since the turn of the century, advances in targeted therapy and immunotherapy have revolutionized the treatment of cancer. Although these approaches have far outperformed traditional therapies in various clinical settings, both remain plagued by mechanisms of innate and acquired resistance that limit therapeutic efficacy in many patients. With a focus on tumor-intrinsic resistance to immunotherapy, this review highlights our current understanding of the immunologic and oncogenic pathways whose genetic dysregulation in cancer cells enables immune escape. Emphasis is placed on genomic, epigenomic, transcriptomic, and proteomic aberrations that influence the activity of these pathways in the context of immune resistance. Specifically, the role of pathways that govern interferon signaling, antigen processing and presentation, and immunologic cell death as determinants of tumor immune susceptibility are discussed. Likewise, mechanisms of tumor immune resistance mediated by dysregulated RAS-MAPK, WNT, PI3K-AKT-mTOR, and cell cycle pathways are described. Finally, this review highlights the ways in which recent insight into genetic dysregulation of these immunologic and oncogenic signaling pathways is informing the design of combination targeted therapy-immunotherapy regimens that aim to restore immune susceptibility of cancer cells by overcoming resistance mechanisms that often limit the success of monotherapies.

Non-alcoholic steatohepatitis (NASH) is associated with obesity and increased expression of hepatic peroxisome proliferator-activated receptor γ (PPARγ). However, the relevance of hepatocyte PPARγ in NASH associated with obesity is still poorly understood. In this study, hepatocyte PPARγ was knocked out (PpargΔHep) in male and female mice after the development of high-fat diet-induced obesity. The diet-induced obese mice were then maintained on their original diet or switched to a high fat, cholesterol, and fructose (HFCF) diet to induce NASH. Hepatic PPARγ expression was mostly derived from hepatocytes and increased by high fat diets. PpargΔHep reduced HFCF-induced NASH progression without altering steatosis, reduced the expression of key genes involved in hepatic fibrosis in HFCF-fed male and female mice, and decreased the area of collagen-stained fibrosis in the liver of HFCF-fed male mice. Moreover, transcriptomic and metabolomic data suggested that HFCF-diet regulated hepatic amino acid metabolism in a hepatocyte PPARγ-dependent manner. PpargΔHep increased betaine-homocysteine s-methyltransferase expression and reduced homocysteine levels in HFCF-fed male mice. In addition, in a cohort of 102 obese patients undergoing bariatric surgery with liver biopsies, 16 cases were scored with NASH and were associated with increased insulin resistance and hepatic PPARγ expression. Our study shows that hepatocyte PPARγ expression is associated with NASH in mice and humans. In male mice, hepatocyte PPARγ negatively regulates methionine metabolism and contributes to the progression of fibrosis.

BACKGROUND: Vascular endothelial dysfunction is regarded as an early event of hypertension. Galectin-3 (Gal-3) is known to participate in various pathological processes. Whilst previous studies showed that inhibition of Gal-3 effectively ameliorates angiotensin II (Ang II)-induced atherosclerosis or hypertension, it remains unclear whether Ang II regulates Gal-3 expression and actions in vascular endothelium. METHODS: Using techniques of molecular biology and myograph, we investigated Ang II-mediated changes in Gal-3 expression and activity in thoracic aortas and mesenteric arteries from wild-type and Gal-3 gene deleted (Gal-3-/-) mice and cultured endothelial cells. RESULTS: The serum level of Gal-3 was significantly higher in hypertensive patients or in mice with chronic Ang II-infusion. Ang II infusion to wild-type mice enhanced Gal-3 expression in the aortic and mesenteric arteries, elevated systolic blood pressure and impaired endothelium-dependent relaxation of the thoracic aortas and mesenteric arteries, changes that were abolished in Gal-3-/- mice. In human umbilical vein endothelial cells, Ang II significantly upregulated Gal-3 expression by promoting nuclear localization of Yes-associated protein (YAP) and its interaction with transcription factor Tead1 with enhanced YAP/Tead1 binding to Gal-3 gene promoter region. Furthermore, Gal-3 deletion augmented the bioavailability of nitric oxide, suppressed oxidative stress, and alleviated inflammation in the thoracic aorta of Ang II-infused mice or endothelial cells exposed to Ang II. CONCLUSIONS: Our results demonstrate for the first time that Ang II upregulates Gal-3 expression via increment in YAP nuclear localization in vascular endothelium, and that Gal-3 mediates endothelial dysfunction contributing to the development of hypertension.