Nucleosomes represent a physical barrier for cleavage activity of DNA topoisomerase I in vivo

DNA topoisomerase I together with the other cellular DNA topoisomerases releases the torsional stress from DNA caused by processes such as replication, transcription and recombination. Despite the well-defined knowledge of its mechanism of action, DNA topoisomerase I in vivo activity has been only partially characterized. In fact the basic question concerning the capability of the enzyme to cleave and rejoin DNA wrapped around a histone octamer remains still unanswered. By studying both in vivo and in vitro the cleavage activity of DNA topoisomerase I in the presence of camptothecin on a repeated trinucleotide sequence, (TTA)35, lying in chromosome XIII of Saccharomyces cerevisiae, we can conclude that nucleosomes represent a physical barrier for the enzyme activity. © The Authors

A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function

Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a "leaky" character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro. Altogether, these results indicate abEC1.1 is a promising tool for further translational studies

Mouse Panx1 Is Dispensable for Hearing Acquisition and Auditory Function

Panx1 forms plasma membrane channels in brain and several other organs, including the inner ear. Biophysical properties, activation mechanisms and modulators of Panx1 channels have been characterized in detail, however the impact of Panx1 on auditory function is unclear due to conflicts in published results. To address this issue, hearing performance and cochlear function of the Panx1−/− mouse strain, the first with a reported global ablation of Panx1, were scrutinized. Male and female homozygous (Panx1−/−), hemizygous (Panx1+/−) and their wild type (WT) siblings (Panx1+/+) were used for this study. Successful ablation of Panx1 was confirmed by RT-PCR and Western immunoblotting in the cochlea and brain of Panx1−/− mice. Furthermore, a previously validated Panx1-selective antibody revealed strong immunoreactivity in WT but not in Panx1−/− cochleae. Hearing sensitivity, outer hair cell-based “cochlear amplifier” and cochlear nerve function, analyzed by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recordings, were normal in Panx1+/− and Panx1−/− mice. In addition, we determined that global deletion of Panx1 impacts neither on connexin expression, nor on gap-junction coupling in the developing organ of Corti. Finally, spontaneous intercellular Ca2+ signal (ICS) activity in organotypic cochlear cultures, which is key to postnatal development of the organ of Corti and essential for hearing acquisition, was not affected by Panx1 ablation. Therefore, our results provide strong evidence that, in mice, Panx1 is dispensable for hearing acquisition and auditory function

A novel glyco-conjugate vaccine against fungal pathogens

To generate a vaccine to protect against a variety of human pathogenic fungi, we conjugated laminarin (Lam), a well-characterized but poorly immunogenic β-glucan preparation from the brown alga Laminaria digitata, with the diphtheria toxoid CRM197, a carrier protein used in some glyco-conjugate bacterial vaccines. This Lam-CRM conjugate proved to be immunogenic and protective as immunoprophylactic vaccine against both systemic and mucosal (vaginal) infections by Candida albicans. Protection probably was mediated by anti-β-glucan antibodies as demonstrated by passive transfer of protection to naive mice by the whole immune serum, the immune vaginal fluid, and the affinity-purified anti-β-glucan IgG fractions, as well as by administration of a β-glucan–directed IgG2b mAb. Passive protection was prevented by adsorption of antibodies on Candida cells or β-glucan particles before transfer. Anti-β-glucan antibodies bound to C. albicans hyphae and inhibited their growth in vitro in the absence of immune-effector cells. Remarkably, Lam-CRM–vaccinated mice also were protected from a lethal challenge with conidia of Aspergillus fumigatus, and their serum also bound to and markedly inhibited the growth of A. fumigatus hyphae. Thus, this novel conjugate vaccine can efficiently immunize and protect against two major fungal pathogens by mechanisms that may include direct antifungal properties of anti-β-glucan antibodies

Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing

In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT

Radiation Genes: a database devoted to microarrays screenings revealing transcriptome alterations induced by ionizing radiation in mammalian cells

The analysis of the great extent of data generated by using DNA microarrays technologies has shown that the transcriptional response to radiation can be considerably different depending on the quality, the dose range and dose rate of radiation, as well as the timing selected for the analysis. At present, it is very difficult to integrate data obtained under several experimental conditions in different biological systems to reach overall conclusions or build regulatory models which may be tested and validated. In fact, most available data is buried in different websites, public or private, in general or local repositories or in files included in published papers; it is often in various formats, which makes a wide comparison even more difficult. The Radiation Genes Database (http://www.caspur.it/RadiationGenes) collects microarrays data from various local and public repositories or from published papers and supplementary materials. The database classifies it in terms of significant variables, such as radiation quality, dose, dose rate and sampling timing, as to provide user-friendly tools to facilitate data integration and comparison

Folate-Targeted Curcumin-Loaded Niosomes for Site-Specific Delivery in Breast Cancer Treatment: In Silico and In Vitro Study

As the most common cancer in women, efforts have been made to develop novel nanomedicine-based therapeutics for breast cancer. In the present study, the in silico curcumin (Cur) properties were investigated, and we found some important drawbacks of Cur. To enhance cancer therapeutics of Cur, three different nonionic surfactants (span 20, 60, and 80) were used to prepare various Cur-loaded niosomes (Nio-Cur). Then, fabricated Nio-Cur were decorated with folic acid (FA) and polyethylene glycol (PEG) for breast cancer suppression. For PEG-FA@Nio-Cur, the gene expression levels of Bax and p53 were higher compared to free drug and Nio-Cur. With PEG-FA-decorated Nio-Cur, levels of Bcl2 were lower than the free drug and Nio-Cur. When MCF7 and 4T1 cell uptake tests of PEG-FA@Nio-Cur and Nio-Cur were investigated, the results showed that the PEG-FA-modified niosomes exhibited the most preponderant endocytosis. In vitro experiments demonstrate that PEG-FA@Nio-Cur is a promising strategy for the delivery of Cur in breast cancer therapy. Breast cancer cells absorbed the prepared nanoformulations and exhibited sustained drug release characteristics

SIR2 modifies histone H4-K16 acetylation and affects superhelicity in the ARS region of plasmid chromatin in Saccharomyces cerevisiae

The null mutation of the SIR2 gene in Saccharomyces cerevisiae has been associated with a series of different phenotypes including loss of transcriptional silencing, genome instability and replicative aging. Thus, the SIR2 gene product is an important constituent of the yeast cell. SIR2 orthologues and paralogues have been discovered in organisms ranging from bacteria to man, underscoring the pivotal role of this protein. Here we report that a plasmid introduced into sir2Δ cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells. This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids. Our results indicate that this new phenotype is due to the lack of Sir2p histone deacetylase activity in the sir2Δ strain, because only the H4-K16 residue of the histone octamer undergoes an alteration of its acetylation state. A model proposing interference with the replication machinery is discussed

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function