Synergistic Ototoxicity of Gentamicin and Low-Dose Irradiation: Molecular Basis and Clinical Significance.

BACKGROUND: Inner ear structures may be included in the radiation fields when irradiation is used to treat patients with head and neck cancers. These patients may also have concurrent infections that require gentamicin treatment. Radiation and gentamicin are both potentially ototoxic, and their combined use has been shown to result in synergistic ototoxicity in animals. OBJECTIVE: We aimed to confirm the synergistic ototoxicity of combined gentamicin and low-dose irradiation treatment and identify the underlying molecular mechanisms using an in vitro model. METHOD: We compared the ototoxic effects of gentamicin, low-dose irradiation, and their combination in the OC-k3 mouse cochlear cell line using cell viability assay, live/dead stain, apoptosis detection assay, oxidative stress detection, and studied the molecular mechanisms involved using immunoblot analysis. RESULTS: Combined treatment led to prolonged oxidative stress, reduced cell viability, and synergistic apoptosis. Gentamicin induced the concurrent accumulation of LC3b-II and SQSTM1/p62, suggesting an impairment of autophagic flux. Low-dose irradiation induced transient p53 phosphorylation and persistent Akt phosphorylation in response to DNA damage. In combined treatment, gentamicin attenuated irradiation-induced Akt activation. CONCLUSIONS: Besides increased oxidative stress, synergistic apoptosis observed in combined treatment could be attributed to gentamicin-induced perturbation of autophagic flux and attenuation of Akt phosphorylation, which led to an impairment of radiation-induced DNA repair response.

A genome-wide screen for genetic variants that modify the recruitment of REST to its target genes.

Increasing numbers of human diseases are being linked to genetic variants, but our understanding of the mechanistic links leading from DNA sequence to disease phenotype is limited. The majority of disease-causing nucleotide variants fall within the non-protein-coding portion of the genome, making it likely that they act by altering gene regulatory sequences. We hypothesised that SNPs within the binding sites of the transcriptional repressor REST alter the degree of repression of target genes. Given that changes in the effective concentration of REST contribute to several pathologies-various cancers, Huntington's disease, cardiac hypertrophy, vascular smooth muscle proliferation-these SNPs should alter disease-susceptibility in carriers. We devised a strategy to identify SNPs that affect the recruitment of REST to target genes through the alteration of its DNA recognition element, the RE1. A multi-step screen combining genetic, genomic, and experimental filters yielded 56 polymorphic RE1 sequences with robust and statistically significant differences of affinity between alleles. These SNPs have a considerable effect on the the functional recruitment of REST to DNA in a range of in vitro, reporter gene, and in vivo analyses. Furthermore, we observe allele-specific biases in deeply sequenced chromatin immunoprecipitation data, consistent with predicted differenes in RE1 affinity. Amongst the targets of polymorphic RE1 elements are important disease genes including NPPA, PTPRT, and CDH4. Thus, considerable genetic variation exists in the DNA motifs that connect gene regulatory networks. Recently available ChIP-seq data allow the annotation of human genetic polymorphisms with regulatory information to generate prior hypotheses about their disease-causing mechanism.

An epigenetic signature of developmental potential in neural stem cells and early neurons.

A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur before any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons.

Repressor element 1 silencing transcription factor couples loss of pluripotency with neural induction and neural differentiation.

Neural differentiation of embryonic stem cells (ESCs) requires coordinated repression of the pluripotency regulatory program and reciprocal activation of the neurogenic regulatory program. Upon neural induction, ESCs rapidly repress expression of pluripotency genes followed by staged activation of neural progenitor and differentiated neuronal and glial genes. The transcriptional factors that underlie maintenance of pluripotency are partially characterized whereas those underlying neural induction are much less explored, and the factors that coordinate these two developmental programs are completely unknown. One transcription factor, REST (repressor element 1 silencing transcription factor), has been linked with terminal differentiation of neural progenitors and more recently, and controversially, with control of pluripotency. Here, we show that in the absence of REST, coordination of pluripotency and neural induction is lost and there is a resultant delay in repression of pluripotency genes and a precocious activation of both neural progenitor and differentiated neuronal and glial genes. Furthermore, we show that REST is not required for production of radial glia-like progenitors but is required for their subsequent maintenance and differentiation into neurons, oligodendrocytes, and astrocytes. We propose that REST acts as a regulatory hub that coordinates timely repression of pluripotency with neural induction and neural differentiation.

Is Electroacupuncture Treatment More Effective in Somatic Tinnitus Than in Nonsomatic Tinnitus?

Background: In recent years, evidence has been accumulating linking subjective tinnitus to the somatosensory system. Somatic tinnitus is defined as tinnitus in which forceful contractions of jaw and neck muscles modulate the psychoacoustic attributes of tinnitus, such as pitch and loudness. Being a somatosensory-based treatment modality, needling might well be more effective for treating somatic than nonsomatic tinnitus. Objective: The aim of this study was to compare the outcomes of electroacupuncture (EA) treatment between patients with somatic and nonsomatic tinnitus. Materials and Methods: A single-blinded prospective study was carried wherein 27 patients with tinnitus were divided into either a somatic or a nonsomatic group, based on whether their tinnitus could be modulated by at least one of a series of forceful jaw and neck muscular contraction maneuvers. Tinnitus responses were evaluated after a single session of EA on selected acupoints for 30 minutes. Results: Seventeen of the 27 patients (63.0%) studied were found to have somatic tinnitus. Generalized estimating equation model analysis did not find any overall statistically significant difference in EA response between patients with somatic and nonsomatic tinnitus. However, patients with somatic tinnitus who were consistent in their responses to the muscular contraction maneuvers were more likely to improve with EA than variable responders to these maneuvers (62.5% versus 22.0%). Conclusions: EA did not provide increased benefits for patients with somatic tinnitus, compared to those with nonsomatic tinnitus overall. However, within the somatic tinnitus group, a subpopulation of patients appeared to be relatively more responsive to EA treatment.

The Neurogenic Potential of Astrocytes Is Regulated by Inflammatory Signals.

Although the adult brain contains neural stem cells (NSCs) that generate new neurons throughout life, these astrocyte-like populations are restricted to two discrete niches. Despite their terminally differentiated phenotype, adult parenchymal astrocytes can re-acquire NSC-like characteristics following injury, and as such, these 'reactive' astrocytes offer an alternative source of cells for central nervous system (CNS) repair following injury or disease. At present, the mechanisms that regulate the potential of different types of astrocytes are poorly understood. We used in vitro and ex vivo astrocytes to identify candidate pathways important for regulation of astrocyte potential. Using in vitro neural progenitor cell (NPC)-derived astrocytes, we found that exposure of more lineage-restricted astrocytes to either tumor necrosis factor alpha (TNF-α) (via nuclear factor-κB (NFκB)) or the bone morphogenetic protein (BMP) inhibitor, noggin, led to re-acquisition of NPC properties accompanied by transcriptomic and epigenetic changes consistent with a more neurogenic, NPC-like state. Comparative analyses of microarray data from in vitro-derived and ex vivo postnatal parenchymal astrocytes identified several common pathways and upstream regulators associated with inflammation (including transforming growth factor (TGF)-ß1 and peroxisome proliferator-activated receptor gamma (PPARγ)) and cell cycle control (including TP53) as candidate regulators of astrocyte phenotype and potential. We propose that inflammatory signalling may control the normal, progressive restriction in potential of differentiating astrocytes as well as under reactive conditions and represent future targets for therapies to harness the latent neurogenic capacity of parenchymal astrocytes.

Molecular characterization of hepatitis B virus surface antigen mutants in Singapore patients with hepatocellular carcinoma and hepatitis B virus carriers negative for HBsAg but positive for anti-HBs and anti-HBc.

BACKGROUND AND AIMS: Mutations on the a-determinant of hepatitis B virus surface antigen (HBsAg), capable of escaping detection and vaccination, are identified in HBsAg-positive/anti-HBs-positive vaccinated infants. We studied the prevalence of these mutants in HBsAg-negative/anti-HBc-positive chronic HBV carriers and patients with hepatocellular carcinoma (HCC). METHODS: DNA sequence coding for the antigenic a-determinant of HBsAg was amplified from either HCC genomic DNA or serum samples of the selected patients and sequenced. The replicative mutant genomes were reconstituted in vitro and their reactivity to commercial kits measured. RESULTS: Mutations within and/or outside the a-determinant were identified in patients seronegative for HBsAg. They were then reconstituted in vitro and transiently transfected into HepG2 cells. Culture medium containing secreted HBV viral particles was collected and assayed for their binding to commercial kits. Drastic decrease of reactivity to these kits was seen with most of the identified mutations, including those located outside the a-determinant. CONCLUSION: The existence of a more complex antigenic structure of HBsAg is indicated by the decreased reactivity to detection of mutations, some of which are outside the a-determinant, escape vaccination and may persist in seronegative patients. The high proportion of HBsAg mutants that are integrated in HCC genomes suggests a role of these mutants in hepatocarcinogenesis, possibly leading to mutant HBV-related HCC.