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Objective Poly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer. Methods We assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model. Results Entinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability. Conclusion Entinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.

Accurate DNA replication is constantly threatened by DNA lesions arising from endogenous and exogenous sources. Specialized DNA replication stress response pathways ensure replication fork progression in the presence of DNA lesions with minimal delay in fork elongation. These pathways broadly include translesion DNA synthesis, template switching, and replication fork repriming. Here, we discuss recent advances toward our understanding of the mechanisms that regulate the fine-tuned balance between these different replication stress response pathways. We also discuss the molecular pathways required to fill single-stranded DNA gaps that accumulate throughout the genome after repriming, and the biological consequences of using repriming instead of other DNA damage tolerance pathways on genome integrity and cell fitness.

Correction for ‘Spatiotemporal distribution and speciation of silver nanoparticles in the healing wound’ by Marco Roman et al., Analyst, 2020, 145, 6456–6469, DOI: 10.1039/D0AN00607F.

Chen et al. show that XLF functions to limit fork reversal during DNA replication. H2AX prevents MRE11-dependent replication stress in XLF-deficient cells, suggesting that H2AX prevents the resection of regressed arms at reversed forks., XRCC4-like factor (XLF) is a non-homologous end joining (NHEJ) DNA double strand break repair protein. However, XLF deficiency leads to phenotypes in mice and humans that are not necessarily consistent with an isolated defect in NHEJ. Here we show that XLF functions during DNA replication. XLF undergoes cell division cycle 7–dependent phosphorylation; associates with the replication factor C complex, a critical component of the replisome; and is found at replication forks. XLF deficiency leads to defects in replication fork progression and an increase in fork reversal. The additional loss of H2AX, which protects DNA ends from resection, leads to a requirement for ATR to prevent an MRE11-dependent loss of newly synthesized DNA and activation of DNA damage response. Moreover, H2ax−/−:Xlf−/− cells exhibit a marked dependence on the ATR kinase for survival. We propose that XLF and H2AX function in series to prevent replication stress induced by the MRE11-dependent resection of regressed arms at reversed replication forks.

MicroRNAs play an important role in tumorigenesis and, among them, miR-21 is found to be aberrantly up-regulated in various tumors. The tumor-associated antigen, folate receptor alpha is a GPI-membrane protein overexpressed in many malignant tumors of epithelial origin, including ovarian and cervical cancers. Covalently bound octahedral DNA nanocages were functionalized with folate molecules and utilized as scaffolds to engineer four sequestering units with a miR-21 complementary sequence for obtaining biocompatible Fol-miR21-NC non-toxic nanostructures, to be able to selectively recognize folate receptor alpha-overexpressing cancer cells and sequester the oncogenic miR-21. qPCR assays showed that Fol-miR21-NCs reduce the miR-21 expression up to 80% in cancer cells in the first 2 days of treatment. Functional assays demonstrated that miR-21 sequestering leads to up-regulation of miR-21 tumor suppressor targets (i.e., PTEN and Pdcd4), reduction in cancer cell migration, reduction in proliferation, and increase in cell death. Fol-miR21-NCs can be efficiently loaded with the chemotherapeutic agent doxorubicin. Co-delivery of anti-miR-21 and doxorubicin showed additive cytotoxic effects on tumor cells, paving the way for their use as selective nucleic acid drugs.

The discovery of several unexpected complex biological roles of hyaluronic acid (HA) has promoted new research impetus for biologists and, the clinical interest in several fields of medicine, such as ophthalmology, articular pathologies, cutaneous repair, skin remodeling, vascular prosthesis, adipose tissue engineering, nerve reconstruction and cancer therapy. In addition, the great potential of HA in medicine has stimulated the interest of pharmaceutical companies which, by means of new technologies can produce HA and several new derivatives in order to increase both the residence time in a variety of human tissues and the anti-inflammatory properties. Minor chemical modifications of the molecule, such as the esterification with benzyl alcohol (Hyaff-11® biomaterials), have made possible the production of water-insoluble polymers that have been manufactured in various forms: membranes, gauzes, nonwoven meshes, gels, tubes. All these biomaterials are used as wound-covering, anti-adhesive devices and as scaffolds for tissue engineering, such as epidermis, dermis, micro-vascularized skin, cartilage and bone. In this review, the essential biological functions of HA and the applications of its derivatives for pharmaceutical and tissue regeneration purposes are reviewed.

Summary DNA replication forks use multiple mechanisms to deal with replication stress, but how the choice of mechanisms is made is still poorly understood. Here, we show that CARM1 associates with replication forks and reduces fork speed independently of its methyltransferase activity. The speeding of replication forks in CARM1-deficient cells requires RECQ1, which resolves reversed forks, and RAD18, which promotes translesion synthesis. Loss of CARM1 reduces fork reversal and increases single-stranded DNA (ssDNA) gaps but allows cells to tolerate higher replication stress. Mechanistically, CARM1 interacts with PARP1 and promotes PARylation at replication forks. In vitro, CARM1 stimulates PARP1 activity by enhancing its DNA binding and acts jointly with HPF1 to activate PARP1. Thus, by stimulating PARP1, CARM1 slows replication forks and promotes the use of fork reversal in the stress response, revealing that CARM1 and PARP1 function as a regulatory module at forks to control fork speed and the choice of stress response mechanisms.

Selective targeting is a crucial property of nanocarriers used for drug delivery in cancer therapy. We generated biotinylated octahedral DNA nanocages functionalized with folic acid through bio-orthogonal conjugation chemistry. Molecular modelling indicated that a distance of about 2.5 nm between folic acid and DNA nanocage avoids steric hindrance with the folate receptor. HeLa cells, a folate receptor positive tumour cell line, internalize folate-DNA nanocages with efficiency greater than 40 times compared to cells not expressing the folate receptors. Functionalized DNA nanocages are highly stable, not cytotoxic and can be efficiently loaded with the chemotherapeutic agent doxorubicin. After entry into cells, doxorubicin-loaded nanoparticles are confined in vesicular structures, indicating that DNA nanocages traffic through the endocytic pathway. Doxorubicin release from loaded DNA cages, facilitated by low pH of endocytic vesicles, induces toxic pathways that, besides selectively killing folate receptor-positive cancer cells, leads to cage degradation avoiding nanoparticles accumulation inside cells.

The nucleolin-binding G-quadruplex AS1411 aptamer has been widely used for cancer therapy and diagnosis and linked to nanoparticles for its selective targeting activity. We applied a computational and experimental integrated approach to study the effect of engineering AS1411 aptamer on an octahedral truncated DNA nanocage to obtain a nanostructure able to combine selective cancer-targeting and anti-tumor activity. The nanocages functionalized with one aptamer molecule (Apt-NC) displayed high stability in serum, were rapidly and selectively internalized in cancer cells through an AS1411-dependent mechanism, and showed over 200-fold increase in anti-cancer activity when compared with the free aptamer. Comparison of Apt-NCs and free AS1411 intracellular distribution showed that they traffic differently inside cells: Apt-NCs distributed through the endo-lysosomal pathway and were never found in the nuclei, while the free AS1411 was mostly found in the perinuclear region and in nucleoli. Molecular dynamics simulations indicated that the aptamer, when linked to the nanocage, sampled a limited conformational space, more confined than in the free state, which is characterized by a large number of metastable conformations. A different intracellular trafficking of Apt-NCs compared with free aptamer and the confined aptamer conformations induced by the nanocage were likely correlated with the high cytotoxic enhancement, suggesting a structure–function relationship for the AS1411 aptamer activity.

PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2 conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.

Objectives: More than 80% of women with high grade serous ovarian carcinoma (HGSOC) ultimately develop platinum resistance. There are no FDA approved agents to improve sensitivity to carboplatin. On candidate target is growth arrest-specific 6 (GAS6) which has been associated with prognosis in HGSOC. We hypothesized that GAS6 inhibition with AVB-500 (AVB) sensitizes platinum-resistant cells to platinum chemotherapy by stalling replication forks and increasing replication stress. We also hypothesized that GAS6 expression would predict response to neoadjuvant chemotherapy. Methods: AVB was supplied by Aravive Biologics. Both homologous recombination (HR)-proficient (OVCAR3-TPMES, CAOV3, COV362) and HR-deficient (OVCAR8) cells were used for all experiments. In vitro clonogenic assays were done on chemoresistant ovarian tumor cells treated with carbo±AVB. The effect of carbo + AVB on intraperitoneal tumor burden was evaluated in mouse models. For DNA fiber assays, cells were labeled with the thymidine analog IdU for 20 minutes followed by CldU for 60 minutes and treatment with carbo, cisplatin (cis), or AVB. Immunofluorescent (IF) assays targeting γH2AX for DNA damage, RAD51, BRCA1, and BRCA2 for HR and 53BP1 for non-homologous end joining (NHEJ) were performed. Human HGSOC samples were obtained pre- and post-neoadjuvant chemotherapy. GAS6 expression was measured by tissue immunohistochemistry (IHC) and serum ELISA. Results: Carbo + AVB decreased survival of platinum-resistant cells as measured by clonogenic colonies than carbo alone (p 80%) (n=7) or serum GAS6 concentrations (>25ng/mL) (n=13) were more likely to have a poor response to neoadjuvant chemotherapy than those with low GAS6 (P=0.002). Additionally, high GAS6 concentration was associated with decreased overall survival (24.4 months versus median not reached, P=0.009). Conclusions: Inhibition of the GAS6 pathway with AVB improves sensitivity of platinum-resistant cells to platinum chemotherapy by increasing replication stress and DNA damage and decreasing HR. GAS6 is a potential biomarker predictive of poor response to platinum-based neoadjuvant chemotherapy and might identify patients who would benefit from treatment with AVB.

DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.

SUMMARYThe replication stress response which serves as an anti-cancer barrier is activated not only by DNA damage and replication obstacles, but also oncogenes, mystifying how cancer evolves. Here, we identify that oncogene expression, similar to cancer therapies, induces single stranded DNA (ssDNA) gaps that reduce cell fitness, unless suppressed by translesion synthesis (TLS). DNA fiber analysis and electron microscopy reveal that TLS restricts replication fork slowing, reversal, and fork degradation without inducing replication fork gaps. Evidence that TLS gap suppression is fundamental to cancer, a small molecule inhibitor targeting the TLS factor, REV1, not only disrupts DNA replication and cancer cell fitness, but also synergizes with gap-inducing therapies. This work illuminates that gap suppression during replication is critical for cancer cell fitness and therefore a targetable vulnerability.

It is known that users of psychotropic drugs often have weight gain, adverse effects on bone mineral density and osteoporosis, but the molecular basis for these side effects is poorly understood. The aim of this study is to evaluate the effects in vitro of duloxetine (a serotonin and norepinephrine reuptake inhibitor) and fluoxetine (a selective serotonin reuptake inhibitor) on the physiology of human adult stem cells. Adipose-derived stem cells (ADSCs) were isolated and characterized investigating phenotype morphology, expression and frequency of surface markers. Then, a non-toxic concentration of duloxetine and fluoxetine was selected to treat cells during adipogenic and osteogenic differentiation. Stemness properties and the differentiation potential of drug-treated cells were investigated by the quantification of adipogenic and osteogenic markers gene expression and histological staining. The collected data showed that the administration of a daily non-toxic dose of duloxetine and fluoxetine has not directly influenced ADSCs proliferation and their stemness properties. The treatment with duloxetine or fluoxetine did not lead to morphological alterations during adipogenic or osteogenic commitment. However, treatments with the antidepressant showed a slight difference in adipogenic gene expression timing. Furthermore, duloxetine treatment caused an advance in gene expression of early and late osteogenic markers. Fluoxetine instead caused an increase in expression of osteogenic genes compared to untreated cells. In contrast, in pre-differentiated cells, the daily treatment with duloxetine or fluoxetine did not alter the expression profile of adipogenic and osteogenic differentiation. In conclusion, a non-toxic concentration of duloxetine and fluoxetine does not alter the stemness properties of ADSCs and does not prevent the commitment of pre-differentiated ADSCs in adipocytes or osteocyte. Probably, the weight gain and osteoporotic effects associated with the use of psychotropic drugs could be closely related to the direct action of serotonin.

RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks.

A strong influence of the amount of the Co3O4 promoter on the catalytic performance in methanol oxidation of different gold catalysts supported on ceria was observed. The activity followed the order: Au/10 wt% Co3O4-doped CeO2 > Au/5 wt% Co3O4-doped CeO2 > Au/15 wt% Co3O4-doped CeO2 > Au/CeO2 ≫ Au/Co3O4. FTIR measurements of adsorbed CO indicate that oxidized gold sites are initially present on the activated samples and that such species are involved in the methanol reaction. Methanol oxidation performed under static conditions gave rise at 75 °C to mainly formate species on Au/CeO2 and to a large variety of different carbonate species on Au/10 wt% Co3O4-doped CeO2. FTIR and EXAFS analyses revealed that the active sites present on the best performing Au/CeO2 catalyst added with 10 wt% Co3O4 are oxidized gold species, close to Co sites, at the interface with the support, which are reduced under reaction conditions. These species are able to activate and to react with oxygen giving rise to formate and carbonate species.

The development and progression of osteoarthritis (OA) is associated with macrophage-mediated inflammation that generates a broad spectrum of cytokines and reactive oxygen species (ROS). This study investigates the effects of mid-MW hyaluronic acid (HA) in combination with a lactose-modified chitosan (CTL), on pro-inflammatory molecules and metalloproteinases (MMPs) expression, using an in vitro model of macrophage-mediated inflammation. Methods. To assess chondrocyte response to HA and CTL in the presence of macrophage derived inflammatory mediators, cells were exposed to the conditioned medium (CM) of U937 activated monocytes and changes in cell viability, pro-inflammatory mediators and MMPs expression or ROS generation were analysed. Results. CTL induced changes in chondrocyte viability that are reduced by the presence of HA. The CM of activated U937 monocytes (macrophages) significantly increased gene expression of pro-inflammatory molecules and MMPs and intracellular ROS generation in human chondrocyte cultures. HA, CTL and their combinations counteracted the oxidative damage and restored gene transcription for IL-1&beta, TNF-&alpha, Gal-1, MMP-3 and MMP-13 to near baseline values. Conclusions. This study suggests that HA-CTL mixture attenuated macrophage-induced inflammation, inhibited MMPs expression and exhibited anti-oxidative effects. This evidence provides an initial step toward the development of an early stage OA therapeutic treatment

The capacity of inducing angiogenesis is a recognized hallmark of cancer cells. The cancer microenvironment, characterized by hypoxia and inflammatory signals, promotes proliferation, migration and activation of quiescent endothelial cells (EC) from surrounding vascular network. Current anti-angiogenic drugs present side effects, temporary efficacy, and issues of primary resistance, thereby calling for the identification of new therapeutic targets. MICALs are a unique family of redox enzymes that destabilize F-actin in cytoskeletal dynamics. MICAL2 mediates Semaphorin3A-NRP2 response to VEGFR1 in rat ECs. MICAL2 also enters the p130Cas interactome in response to VEGF in HUVEC. Previously, we showed that MICAL2 is overexpressed in metastatic cancer. A small-molecule inhibitor of MICAL2 exists (CCG-1423). Here we report that 1) MICAL2 is expressed in neo-angiogenic ECs in human solid tumors (kidney and breast carcinoma, glioblastoma and cardiac myxoma, n = 67, were analyzed with immunohistochemistry) and in animal models of ischemia/inflammation neo-angiogenesis, but not in normal capillary bed; 2) MICAL2 protein pharmacological inhibition (CCG-1423) or gene KD reduce EC viability and functional performance; 3) MICAL2 KD disables ECs response to VEGF in vitro. Whole-genome gene expression profiling reveals MICAL2 involvement in angiogenesis and vascular development pathways. Based on these results, we propose that MICAL2 expression in ECs participates to inflammation-induced neo-angiogenesis and that MICAL2 inhibition should be tested in cancer- and noncancer-associated neo-angiogenesis, where chronic inflammation represents a relevant pathophysiological mechanism.

Acellular matrices are widespread biomaterials used in surgical practice as tissue reinforcement and anatomical support to favor tissue regeneration. It is clear that a fundamental role in the regeneration of tissue is played by cell-material interaction. In this work, the interaction between a bovine pericardium membrane and human adult stem cells was investigated by microscopy analysis and gene expression analysis. Parallel cell cultures were prepared on the pericardium membrane or tissue culture plate. They were incubated in basal growth medium or in adipogenic differentiation medium to perform experiments on the seventh and the 14th day of culture. Results demonstrated that the membrane allows cell viability, adhesion, and proliferation of human stem cells. During adipogenic commitment on the membrane, the accumulation of cytoplasmatic lipid droplets and the expression of adipogenic gene PPARG, CEBPA, GLUT4, FABP4, and ADIPOQ were detected. Concurrently, a downregulation of mesenchymal stem cell gene CD29, CD90, and CD105 was detected. In basal medium, the adipogenic gene expression was upregulated, whereas the mesenchymal markers were indifferently expressed. These findings suggest that the bovine pericardium membrane is a biocompatible matrix and that their rough surface allows cell adhesion, spreading, and proliferation. The surface morphology activates mechanochemical signals that stimulate the adipogenic commitment of stem cells in basal medium and potentiate their commitment in adipogenic differentiation medium.

DNA has been used to build nanostructures with potential biomedical applications. However, their use is limited by the lack of information on the mechanism of entry, intracellular fate and degradation rate of nanostructures inside cells. We generated octahedral DNA nanocages functionalized with folic acid and investigated the cellular uptake mediated by two distinctive internalization pathways, using two cellular systems expressing the oxidized low-density lipoprotein receptor-1 (LOX-1) and the α isoform of the folate receptor (αFR), respectively. Here, we report that DNA nanocages are very efficiently and selectively internalized by both receptors with an efficiency at least 30 times higher than that observed in cells not expressing the receptors. When internalized by LOX-1, nanocages traffic to lysosomes within 4 hours and are rapidly degraded. When the uptake is mediated by αFR, DNA nanocages are highly stable (>48 hours) and accumulate inside cells in a time-dependent way. These data demonstrate that the selection of the cellular receptor is crucial for targeting specific sub-cellular compartments and for modulating the DNA nanocage intracellular half-life, indicating that vitamin-mediated uptake may constitute a protected pathway for intracellular drug delivery.

Pds5 is required for sister chromatid cohesion, and somewhat paradoxically, to remove cohesin from chromosomes. We found that Pds5 plays a critical role during DNA replication that is distinct from its previously known functions. Loss of Pds5 hinders replication fork progression in unperturbed human and mouse cells. Inhibition of MRE11 nuclease activity restores fork progression, suggesting that Pds5 protects forks from MRE11-activity. Loss of Pds5 also leads to double-strand breaks, which are again reduced by MRE11 inhibition. The replication function of Pds5 is independent of its previously reported interaction with BRCA2. Unlike Pds5, BRCA2 protects forks from nucleolytic degradation only in the presence of genotoxic stress. Moreover, our iPOND analysis shows that the loading of Pds5 and other cohesion factors on replication forks is not affected by the BRCA2 status. Pds5 role in DNA replication is shared by the other cohesin-removal factor Wapl, but not by the cohesin complex component Rad21. Interestingly, depletion of Rad21 in a Pds5-deficient background rescues the phenotype observed upon Pds5 depletion alone. These findings support a model where loss of either component of the cohesin releasin complex perturbs cohesin dynamics on replication forks, hindering fork progression and promoting MRE11-dependent fork slowing.

The TPX2/Aurora A heterodimeric kinase canonically orchestrates mitotic events. Byrum et al. identify two new roles for this complex in regulating DNA double-stranded break repair and the protection of DNA forks during replication stress., 53BP1 is a chromatin-associated protein that regulates the DNA damage response. In this study, we identify the TPX2/Aurora A heterodimer, nominally considered a mitotic kinase complex, as a novel binding partner of 53BP1. We find that TPX2/Aurora A plays a previously unrecognized role in DNA damage repair and replication fork stability by counteracting 53BP1 function. Loss of TPX2 or Aurora A compromises DNA end resection, BRCA1 and Rad51 recruitment, and homologous recombination. Furthermore, loss of TPX2 or Aurora A causes deprotection of stalled replication forks upon replication stress induction. This fork protection pathway counteracts MRE11 nuclease activity but functions in parallel to BRCA1. Strikingly, concurrent loss of 53BP1 rescues not only BRCA1/Rad51 recruitment but also the fork instability induced upon TPX2 loss. Our work suggests the presence of a feedback mechanism by which 53BP1 is regulated by a novel binding partner and uncovers a unique role for 53BP1 in replication fork stability.

A study of secondary, benzylic, and primary alcohols oxidation on a reference Au/TiO2 catalyst is reported. Reactions gave rise to ketones, aldehydes, or esters in good yields. The FT-IR results and catalytic tests data provide to elucidate the reaction mechanism and the specific role of the active sites on titania.

Replication stress is a crucial driver of genomic instability. Understanding the mechanisms of replication stress response is instrumental to improve diagnosis and treatment of human disease. Electron microscopy (EM) is currently the only technique that allows to directly visualize a high number of replication intermediates and to monitor their remodeling upon stress. At the same time, DNA fiber analysis is useful to gain mechanistic insight on how genotoxic agents perturb replication fork dynamics genome-wide at single-molecule resolution. Combining these techniques has proven invaluable to achieve a comprehensive view of the mechanisms that ensure error-free processing of damaged replication forks. Here, we review how EM and single-molecule DNA fiber approaches can be used together to shed light into the mechanisms of replication stress response and discuss important cautions to be taken into account when comparing results obtained by EM and DNA fiber.

Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lower motor neurons, with unknown aetiology. Lipid rafts, cholesterol enriched microdomains of the plasma membrane, have been linked to neurodegenerative disorders like ALS. The NMDA-receptor subcellular localization in lipid rafts is known to play many roles, from modulating memory strength to neurotoxicity. In this study, performed on the widely used G93A mouse model of ALS, we have shown an equal content of total membrane cholesterol in Control and G93A cortical cultures. Moreover, by electrophysiological studies, we have recorded NMDA- and AMPA-evoked currents which were not significantly different between the two neuronal populations. To study the role of membrane cholesterol on glutamate receptor functionality, we have analysed NMDA and AMPA receptors following cholesterol membrane depletion by methyl-β-cyclodextrin (MβCD). Interestingly, MβCD chronic treatment has provoked a significant reduction of NMDA-evoked currents in both cellular populations which was dose- and time-dependent but significantly higher in ALS neurons compared to Control. The different MβCD effect on NMDA-evoked currents was not due to a different membrane receptor subunit composition but seemed to cause in both neuronal populations a NMDA receptor membrane redistribution. MβCD treatment effect was receptor-specific since no alterations in the two neuronal populations were detected on AMPA receptors. These results lead us to speculate for an altered proteomic composition of lipid rafts in cortical mutated neurons and suggest the need for further studies on the lipid rafts composition and on their interaction with membrane receptors in ALS cortices.

Insulin plays a major role in glucose metabolism and insulin-signaling defects are present in obesity and diabetes. CK2 is a pleiotropic protein kinase implicated in fundamental cellular pathways and abnormally elevated in tumors. Here we report that in human and murine adipocytes CK2-inhibition decreases the insulin-induced glucose-uptake by counteracting Akt-signaling and GLUT4-translocation to the plasma membrane. In mice CK2 acts on insulin-signaling in adipose tissue, liver and skeletal muscle and its acute inhibition impairs glucose tolerance. Notably, CK2 protein-level and activity are greatly up-regulated in white adipose tissue from ob/ob and db/db mice as well as from obese patients, regardless the severity of their insulin-resistance and the presence of pre-diabetes or overt type 2 diabetes. Weight loss obtained by both bariatric surgery or hypocaloric diet reverts CK2 hyper-activation to normal level. Our data suggest a central role of CK2 in insulin-sensitivity, glucose homeostasis and adipose tissue remodeling. CK2 up-regulation is identified as a hallmark of adipose tissue pathological expansion, suggesting a new potential therapeutic target for human obesity.

In a lifetime, a human being synthesizes approximately 2×1016 meters of DNA, a distance that corresponds to 130,000 times the distance between the Earth and the Sun. This daunting task is executed by thousands of replication forks, which progress along the chromosomes and frequently stall when they encounter DNA lesions, unusual DNA structures, RNA polymerases, or tightly-bound protein complexes. To complete DNA synthesis before the onset of mitosis, eukaryotic cells have evolved complex mechanisms to process and restart arrested forks through the coordinated action of multiple nucleases, topoisomerases, and helicases. In this review, we discuss recent advances in understanding the role and regulation of nucleases acting at stalled forks with a focus on the nucleolytic degradation of nascent DNA, a process commonly referred to as fork resection. We also discuss the effects of deregulated fork resection on genomic instability and on the unscheduled activation of the interferon response under replication stress conditions.

Break-induced replication (BIR) is a DNA double-strand break repair pathway that leads to genomic instabilities similar to those observed in cancer. BIR proceeds by a migrating bubble where asynchrony between leading and lagging strand synthesis leads to accumulation of long single-stranded DNA (ssDNA). It remains unknown how this ssDNA is prevented from unscheduled pairing with the template, which can lead to genomic instability. Here, we propose that uncontrolled Rad51 binding to this ssDNA promotes formation of toxic joint molecules that are counteracted by Srs2. First, Srs2 dislodges Rad51 from ssDNA preventing promiscuous strand invasions. Second, it dismantles toxic intermediates that have already formed. Rare survivors in the absence of Srs2 rely on structure-specific endonucleases, Mus81 and Yen1, that resolve toxic joint-molecules. Overall, we uncover a new feature of BIR and propose that tight control of ssDNA accumulated during this process is essential to prevent its channeling into toxic structures threatening cell viability., Break-induced replication (BIR) is a double-strand break repair pathway that can lead to genomic instability. Here the authors show that the absence of Srs2 helicase during BIR leads to uncontrolled binding of Rad51 to single-stranded DNA, which promotes the formation of toxic intermediates that need to be resolved by Mus81 or Yen1.

Background Celiac disease (CD) is an immune-mediated intolerance to dietary gluten, affecting genetically predisposed individuals. ELISA-based serological tests help to decide if further duodenal biopsy is necessary, for this the diagnostic kits have to be accurate, specific, and sensible. In this study, we investigate the performance of an ELISA that uses the purified cross-linked complex of tissue transglutaminase and gliadin, referred as the “neoepitope” (AESKULISA® tTG New Generation), as antigen. Methods We evaluated 41 newly diagnosed celiac patients, 18 celiac patients on gluten-free diet, and 169 controls, comprising healthy subjects, patients affected by other autoimmune diseases, and patients affected by several non-autoimmune diseases. Results and Conclusion The assay has an excellent performance. Due to its high level of diagnostic accuracy, this assay constitutes a new approach for the screening of celiac patients not only for the diagnosis of CD, but also for monitoring patients on gluten-free diet and their compliance. Moreover, cases of neoepitope-positive subjects who were tested negative with “classical” serological markers could have a predictive value for this pathology. This aspect will require further studies of elaboration.

The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways. In this work, we provide evidences linking HMGA1 with Non-Homologous End Joining DNA repair. We show that HMGA1 is in complex with and is a substrate for DNA-PK. HMGA1 enhances Ligase IV activity and it counteracts the repressive histone H1 activity towards DNA ends ligation. Moreover, breast cancer cells overexpressing HMGA1 show a faster recovery upon induction of DNA double-strand breaks, which is associated with a higher survival. These data suggest that resistance to DNA-damaging agents in cancer cells could be partially attributed to HMGA1 overexpression thus highlighting the relevance of considering HMGA1 expression levels in the selection of valuable and effective pharmacological regimens.

RecQ helicases play essential roles in the maintenance of genome stability and contain a highly conserved helicase region generally followed by a characteristic RecQ-C-terminal (RQC) domain, plus a number of variable associated domains. Notable exceptions are the RecQ4 helicases, where none of these additional regions have been described. Particularly striking was the fact that no RQC domain had been reported, considering that the RQC domain had been shown to play an essential role in the catalytic mechanism of most RecQ family members. Here we present the results of detailed bioinformatic analyses of RecQ4 proteins that identify, for the first time, the presence of a putative RQC domain, including some of the key residues involved in DNA binding and unwinding. We also describe the presence of a novel “Zn knuckle” domain, as well as an additional Sld2-homology region, providing new insights into the architecture, function and evolution of these enzymes.

The catalytic performance in the total oxidation of CO and methanol over gold catalysts supported on ceria doped by different metal oxides (Me = Fe, Mn and Co) was studied and a strong influence of the nature of dopant was observed. The activity towards the oxidation of CO and CH3OH was in the order: AuCeCo > AuCe > AuCeFe > AuCeMn. The characterization by XRD and HRTEM evidenced differences in the average size and the distribution of gold particles. AuCeCo catalyst exhibited superior low-temperature CO oxidation activity (100% conversion degree was obtained at 25 °C) and almost 100% total oxidation of CH3OH at about 40 °C. Higher hydrogen consumption was estimated by means of TPR over this catalyst. The effect of modification with Co3O4 of Au/CeO2 catalysts on their CO oxidation activity was further studied by varying of the dopant content (5, 10 and 15 wt.% Co3O4).

Supported gold nanoparticles are very effective catalysts for the selective oxidation of glycerol which represents an important bio-derived feedstock. In this paper we report that the acid/base properties, especially the acid site density, of these catalysts are the key factor in tuning the selectivity. A range of supported AuPt catalysts have been prepared by sol immobilization using acidic (H-mordenite, SiO2, MCM-41, and sulfated ZrO2) and basic (NiO and MgO) oxides as supports. In particular, using MCM-41 as the support, a high selectivity to glyceraldehyde, an important labile intermediate, was found.

Understanding the mechanisms of replication stress response following genotoxic stress induction is rapidly emerging as a central theme in cell survival and human disease. The DNA fiber assay is one of the most powerful tools to study alterations in replication fork dynamics genome-wide at single-molecule resolution. This approach relies on the ability of many organisms to incorporate thymidine analogs into replicating DNA and is widely used to study how genotoxic agents perturb DNA replication. Here, we review different approaches available to prepare DNA fibers and discuss important limitations of each approach. We also review how DNA fiber analysis can be used to shed light upon several replication parameters including fork progression, restart, termination, and new origin firing. Next, we discuss a modified DNA fiber protocol to monitor the presence of single-stranded DNA (ssDNA) gaps on ongoing replication forks. ssDNA gaps are very common intermediates of several replication stress response mechanisms, but they cannot be detected by standard DNA fiber approaches due to the resolution limits of this technique. We discuss a novel strategy that relies on the use of an ssDNA-specific endonuclease to nick the ssDNA gaps and generate shorter DNA fibers that can be used as readout for the presence of ssDNA gaps. Finally, we describe a follow-up DNA fiber approach that can be used to study how ssDNA gaps are repaired postreplicatively.

Antipsychotic drugs are currently used in clinical practice for a variety of mental disorders. Among them, clozapine is the most effective medication for treatment-resistant schizophrenia and is most helpful in controlling aggression and the suicidal behavior in schizophrenia and schizoaffective disorder. Although clozapine is associated with a low likelihood of extrapyramidal symptoms and other neurological side effects, it is well known for the weight gain and metabolic side effects, which expose the patient to a greater risk of cardiovascular disorders and premature death, as well as psychosocial issues, leading to non-adherence to therapy. The mechanisms underlying these iatrogenic metabolic disorders are still controversial. We have therefore investigated the in vivo effects of the selective PKCβ inhibitor, ruboxistaurin (LY-333531), in a preclinical model of long-term clozapine-induced weight gain. Cell biology, biochemistry, and behavioral tests have been performed in wild-type and PKCβ knockout mice to investigate the contribution of endogenous PKCβ and its pharmacological inhibition to the psychomotor effects of clozapine. Finally, we also shed light on a novel aspect of the mechanism underlying the clozapine-induced weight gain, demonstrating that the clozapine-dependent PKCβ activation promotes the inhibition of the lipid droplet-selective autophagy process. This paves the way to new therapeutic approaches to this serious complication of clozapine therapy.

A series of 1 wt% AuPt (6:4) catalysts were prepared by sol immobilization using acidic [TiO2, H-Mordenite, SiO2, MCM-41, sulphated ZrO2 (S-ZrO2)] and one basic (MgO) oxide supports. A careful characterization of the catalyst was performed by HRTEM and FTIR spectroscopy. EDX analysis showed that on all catalysts only alloyed AuPt nanoparticles are present, but the final size of AuPt particles is significantly affected by the support. Indeed, on TiO2 the mean AuPt nanoparticles diameter is 3.7 nm, whereas for all the remaining supports, larger AuPt nanoparticles with diameter of 6–7.5 nm were obtained. AuPt catalysts result very active in catalyzing the liquid phase hydrogenolysis of glycerol to 1,2-propandiol (68% conversion after 16 h) and 90% selectivity with ethylene glycol, 1-propanol and 2-propanol as main by-products. The role of the support has been highlighted in terms of acidic properties. The medium strength of Lewis acid sites of TiO2 leads to the best performance in terms of activity, selectivity and stability of the catalytic system.

Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of ageing. Here we address this question by taking advantage of the highly specific role of DNA ligase IV in the repair of DNA double-strand breaks by non-homologous end-joining, and by the discovery of a unique mouse strain with a hypomorphic Lig4(Y288C) mutation. The Lig4(Y288C) mouse, identified by means of a mutagenesis screening programme, is a mouse model for human LIG4 syndrome, showing immunodeficiency and growth retardation. Diminished DNA double-strand break repair in the Lig4(Y288C) strain causes a progressive loss of haematopoietic stem cells and bone marrow cellularity during ageing, and severely impairs stem cell function in tissue culture and transplantation. The sensitivity of haematopoietic stem cells to non-homologous end-joining deficiency is therefore a key determinant of their ability to maintain themselves against physiological stress over time and to withstand culture and transplantation.

A study of secondary, benzylic, and primary alcohols oxidation on a reference Au/TiO2 catalyst is reported. Reactions gave rise to ketones, aldehydes, or esters in good yields. The FT-IR results and catalytic tests data provide to elucidate the reaction mechanism and the specific role of the active sites on titania.

DNA offers excellent programming properties for the generation of nanometer-scaled polyhedral structures with a broad variety of potential applications. Translation to biomedical applications requires improving stability in biological fluids, efficient and selective cell binding, and/or internalization of the assembled DNA nanostructures. Here, we report an investigation on the selective mechanism of cellular uptake of pristine DNA nanocages in cells expressing the receptor "oxidized low-density lipoprotein receptor-1" (LOX-1), a scavenger receptor associated with cardiovascular diseases and, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin-streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological fluids, including human serum, and are selectively bound and very efficiently internalized in vesicles only in LOX-1-expressing cells. The amount of internalized cages is 30 times higher in LOX-1-expressing cells than in normal fibroblasts, indicating that the receptor-mediated uptake of pristine DNA nanocages can be pursued for a selective cellular internalization. These results open the route for a therapeutic use of pristine DNA cages targeting LOX-1-overexpressing tumor cells.

In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

The major goal of regenerative medicine is to determine experimental techniques that take maximal advantage of reparative processes that occur naturally in the animal body. Injection of mesenchymal stem cells into the core of a damaged tendon represents such an approach. Decellularization of native tendons as potential targets and seeding protocols are currently under investigation. The aim of our study was to manufacture a recellularized biocompatible scaffold from cadaveric tissue for use in total or partial tendon injuries. Results showed that it was possible to introduce proliferating cells into the core of a decellularized tendon to treat the scaffold with a collagen gel. The method was effective in maintaining scaffold extracellular matrix and for expressing collagen type I and cartilage oligomeric matrix protein by injecting mesenchymal stem cells.