Month: July 2020

Based on current evidence, the majority of cancers have aberrant hypermethylation in the promoters of TSGs, although the whole genome may appear to be hypomethylated. Our study was limited to 24 promoters, and the results could only identify methylation changes in a small number of the potential targets relevant to lung cancer. Ongoing efforts in our laboratory are focused on whole genome methylation screening using additional animal models to look at spontaneously occurring lung tumors as well as inhaled carcinogen-induced lung cancer. These studies are expected to yield much more Sorafenib valuable information on the biology of lung carcinogenesis, which should guide the development of improved therapeutic strategies. Reed et al. have recently reported a similar study using aerosol Aza for the treatment of an orthotopic lung cancer model in nude rats. They concluded that their aerosol and systemic deliveries are equally effective in terms of antitumor efficacy as well as gene demethylation. Although the methodologies, tumor models, and endpoints used in Reed study and our study are significantly different, the fact that both studies lead to some similar conclusions using aerosol Aza represents strong proof of concept of the potential of aerosol demethylation therapy as a novel strategy for the management of lung cancer and bronchial premalignancy. In our study, we found that the effective dose was much lower than the optimal IV dose, indicating that targeted aerosol delivery of the minimal effective dose rather than the maximum tolerated dose of an epigenetic agent is a potentially effective therapeutic strategy. In addition, our aerosol formulation showed a very good aerodynamic size range that makes it suitable for use in humans. We have recently completed preclinical toxicity studies of aerosol Vidaza in mice and FDA approved us to initiate a Phase I clinical study with aerosol Vidaza in patients with advanced lung cancer with disease mostly confined to the lung parenchyma that are not candidates for therapies of a higher priority. In this study, in addition to determining the toxicity profile of aerosol Vidaza, we will investigate by sequential bronchoscopy whether aerosol Vidaza can lead to TSG reexpression in the bronchial epithelium and or/tumor tissue. We expect that the results of this study will determine whether inhaled demethylation therapy should be further developed both as an early intervention or even prevention strategy. If that is the case, significant effort will have to be devoted to explore the use of other candidate agents as Aza is potentially carcinogenic and therefore not an ideal candidate as a preventive agent. It has been hypothesized in the literature that exposure to extremely low frequency electromagnetic fields may lead to human health effects such as childhood leukemia or brain tumors. However, this hypothesis was derived from epidemiological studies which per se do not implicate causal relationships. The latter can only be addressed with experiments carried out under carefully controlled conditions. Among the experiments on rats and mice listed in the ‘BioInitiative Report’
, the following results related to brain cells seem to be of particular importance: Lai and Singh ; found nuclear DNA singlestrand breaks and double-strand breaks from 0.01 mT magnetic field exposure onwards in a dosedependent manner in rats. It is of note that these effects could be blocked by pretreating rats with a vitamin E analog, a nitric oxide synthase inhibitor, or an iron chelator. From these data, the authors concluded that MF exposure might lead to increased generation of free radicals via the so-called Fenton reaction within mitochondria which, thereafter, cause nuclear DNA damage.

S. Typhimurium and E. coli harboring pRST98 formed thicker BF in vitro, compared with the isogenic strains not carrying pRST98. It was also observed that S. Typhimurium x3306 and x3337 had similar abilities to form BFs, which is inconsistent with the study of Teodo´ sio JS et al. We speculated the different plasmids and BF-producing systems may contribute to this inconsistency. We noticed that E. coli K12W1485/pRST98 had a weak ability to form BFs compared with Salmonella strains harboring pRST98. This heterogeneity in BF formation may arise because the synthesis of extracellular polymeric substances in Salmonella outcompetes that in E. coli in medium, as reported by Rong Wang et al. Regarding the heterogeneity in the promotion of BFs by conjugative plasmids, Røder HL et al. proposed that the different genetic backgrounds of the plasmidharboring hosts may account for different BF formation when the same plasmid was used. Our previous study demonstrated that in different genera, the conjugal transfer conditions of the pRST98 plasmid were different in vitro or in mice, and the resistance markers encoded by the same plasmid varied in different strains, which showed the diversity and complexity of the gene expression from the plasmid. Thus, the effects of BF formation by different plasmids in various hosts may demand specific analysis. In animal experiments, a tumor bearing mouse model was used to study the effects of pRST98 on BF formation in S. Typhimurium, which was used as a surrogate of S. Typhi because no animal model is available for S. Typhi infection. In the tumor-bearing mouse model, x3337lux/pRST98 was found preferentially in tumors with a considerably larger amount than x3337lux. The observation that solid tumors are treatable via bacterial infection was made previously. Colonization of bacteria on solid tumors could cause growth retardation or even the complete elimination of the tumors. pRST98 promoting host bacterial BF formation may have a therapeutic potential in fighting against tumors. Furthermore, our invasion study in vitro proved that bacteria in BFs showed a lower invasion ability compared with the corresponding planktonic form, which is consistent with the finding by Katja Crull et al. that BF-forming bacteria did not invade intracellularly in vivo after they established BFs. The intracellular invasion by Salmonella may be due to the differential expression of invasive genes on Salmonella pathogenicity island 1 induced by BF formation. Another animal model, a mouse urethral catheter model, was established to study the effects of pRST98 in E. coli on BF formation in vivo. E. coli K12W1485/pRST98 was found to form only discrete patchy BFs at 3 d post-implantation, while E. coli K12W1485 was not detected in tubes until 5 d post-implantation. E. coli K12W1485/pRST98 developed denser BFs at 5 d post-implantation, in line with bacterial titers recovered from established BFs on tubes. No histological changes were observed in the livers and kidneys of either group. When the implantation with tubes pre-incubated with E. coli was extended to 8 d or beyond, more BKM120 severe inflammation was observed. Significantly, S. Typhimurium x3337lux/pRST98 caused more severe inflammation in organs than x3337lux did. A similar phenomenon was observed for E. coli K12W1485/pRST98 and K12W1485. These results indicate that pRST98 aggravates the infection by promoting BF formation. Recently Rong Wang and Victoria J. Savage et al. demonstrated that the BF increases horizontal transfer of multi-resistant conjugative plasmids to plasmid-free bacteria compared to planktonic bacteria. Therefore, it seems that conjugative plasmids facilitate BF formation, and vice versa.

Therefore, several clinical trials are currently investigating the efficacy and safety of MSCs as a treatment option for various pathologies. We have previously shown in a mouse model of neonatal HI brain damage that intranasal administration of murine MSCs significantly improves motor and cognitive behavior and reduces cerebral lesion volume. In contrast to current pharmacological therapies for neonatal HI, we found that MSC treatment has a long therapeutic window of 10 days after the insult. Studies from our group and others have shown that intracranial and intravenous injection of murine MSCs actively promote proliferation and differentiation of neuronal and glial precursor cells as well as axonal regeneration. Moreover, MSCs have been shown to exert strong anti-inflammatory properties and to modulate immune responses, for example by suppressing the proliferation of T cells and B cells in various disease models such as graft-versus-host disease. Before MSCs can be used in the clinic for the treatment of neonatal brain damage, the neuroregenerative potential of human MSCs has to be determined. A few studies in the adult rodent MCAO model for Wortmannin stroke have investigated the efficacy of hMSCs to repair stroke induced brain lesion and behavioral deficits, but none have studied the effects of hMSCs on neonatal encephalopathy. The results from these studies show that hMSCs improve motor behavior, decrease lesion size and enhance angiogenesis. In our study, we used an in vitro assay to assess the capacity of hMSCs to induce mouse neural stem cell to differentiate towards neuronal and glial cell fates. Moreover, we determined in vivo whether hMSCs are able to migrate towards the injury site in our mouse model of neonatal HI brain injury and which chemotactic factors may mediate MSC migration to the lesion. Most importantly, we investigated whether treatment with hMSCs improves motor behavior and decreases lesion size and gliosis following HI injury in the neonatal mice. Our study shows that human MSCs have the capacity to promote neuroregeneration. This finding is reflected by our results showing that intranasal administration of hMSCs significantly improves motor behavior, and decreases lesion size and scar formation at 28 days after HI brain damage in neonatal mice. Furthermore, our in vitro results demonstrate that human MSCs are capable of inducing mNCSs to differentiate towards astrocytic and neuronal cell fate. This suggests that hMSCs do not need cell to cell contact with neural stem cells, but rather promote endogenous neurogenesis and lesion repair by the secretion of neurotrophic factors. We also show that hMSCs reach the damaged brain region in the mouse within 24 hours after intranasal administration. Importantly, our work also provides new insight into the chemotactic factors that may regulate MSC migration towards the lesion site. Our results show that the chemokine CXCL10 is strongly upregulated at 10 days following HI.

For example, recent studies suggested that one protein family, called the mitochondrial transcription termination factors, plays important roles in regulating the organellar transcription machinery. The mTERF proteins were first identified two decades ago as regulators of transcription termination in human mitochondria. Phylogenetic analyses of mTERF homologs in metazoans and plants revealed the presence of 4 subfamilies, mTERF1 to mTERF4. These proteins share a common 30-amino-acid repeat module called the mTERF motif. The proteins within this family possess diverse numbers and arrangements of these motifs, yet the folding patterns of these proteins are similar. Moreover, crystal structure studies of mTERF1, mTERF3 and mTERF4 suggest that the helical structure of the mTERF motifs may be essential for their nucleic acid-binding activities. Human mTERF1 binds specific sites located at the 39-end of the 16S rRNA and tRNALer genes to terminate mitochondrial transcription. Additionally, mTERF1 binds to the mitochondrial transcription initiation site to create a DNA loop that allows for the recycling of the transcriptional machinery. This simultaneous link between mitochondrial transcriptional initiation and termination sites may explain the high rate of mitochondrial rRNA biogenesis. mTERF2 binds to mitochondrial DNA in a non-specific manner and associates with nucleoids. mTERF2 loss-of-function mice exhibit myopathies, memory deficits, and impaired respiratory function due to a reduction in the number of mitochondrial transcripts. mTERF3 is essential, and mTERF3-knockout mice die during the early stages of embryogenesis; mTERF3 non-specifically interacts with mtDNA promoter regions and mediates the repression of mitochondrial transcription initiation. Mouse NSUN4 is a methyltransferase involved in the methylation of ribosomes. Mouse mTERF4 first binds 16S and 12S rRNAs, then forms a stoichiometric complex with NSUN4, which is essential for proper mitochondrial ribosomal LDK378 1032900-25-6 assembly and translation. Our genetic, molecular and biochemical results indicate that mTERF15 is a unique, mitochondria-localized protein with RNA-binding activity. This protein is critical for post-transcriptional modification in the RNA splicing of mitochondrial nad2 intron 3. Mutating mTERF15 impaired the normal activity of mitochondrial respiratory chain complex I, thereby resulting in abnormal mitochondrial development and the widespread retardation of plant growth and development. mTERFs constitute a broad family of eukaryotic proteins that are essential for the initiation and termination of organellar transcription and the translation and replication of the organellar transcription machinery ; however, mounting evidence suggests an emerging role for these proteins in RNA splicing in plants. The human genome contains only 4 genes encoding mTERF paralogs, whereas the Arabidopsis genome contains at least 35 genes for mTERF proteins with a diverse arrangement and number of mTERF motifs.

Growing axons also require a steady delivery of membrane and microtubules to the migrating growth cone. Thus, proper positioning of the centrosome is crucial for membrane trafficking and polarized microtubule-based delivery to the axon. Here, we have tested our hypothesis that NA14 plays a role in axonal development. We found that, like spastin, NA14 enhances the formation of axons. Endogenous spastin and NA14 proteins in HeLa cells and rat cortical neurons in primary culture show a clear distribution to centrosomes, with NA14 specifically at centrioles. Stable knockdown of NA14 dramatically affects cell division, in particular cytokinesis. Furthermore, overexpression of NA14 in neurons significantly increases axon outgrowth and branching; it also enhances neuronal differentiation without modifying the number of centrosomes. Taken together, our data suggest that NA14 may act as adaptor protein, regulating spastin localization to centrosomes and possibly contributing to the spatial and temporal regulation of its microtubule-severing activity. In this study, we have demonstrated that the spastin M87 isoform co-localizes with its interacting partner NA14 at the centrosome, a non-membranous organelle composed of centrioles and pericentriolar material. An association between NA14 and the spastin M1 isoform cannot be formally ruled out, however, as the lower expression of Myc-tagged spastin M1 could have affected the ability of our immunoprecipitation experiments to detect an interaction. Evaluation of HeLa cell lines stably expressing epitope-tagged NA14 showed that NA14 is enriched at centrioles. Over the years, several groups have reported the localization of NA14 and its orthologs to the centrosome, first in the biflagellated unicellular organism C. reinhardtii. The C. reinhardtii NA14 ortholog DIP13 localizes to cytoplasmic microtubules and basal bodies, which are structurally and functionally very similar to mammalian centrioles. Several other studies using immunostaining and mass spectrometry-based proteomic analyses have supported the localization of NA14 to the centrosome. Even so, the function of NA14 at the centrosome remains unknown. A number of studies have suggested that DIP13/NA14 is involved in cell division by stabilizing microtubules or linking microtubule structures to the division machinery. Indeed, knockdown of C. reinhardtii DIP13 results in multinucleated and multiflagellate cells. Our data suggest that the depletion of NA14 does not affect the structure of the centrosome per se but might stabilize microtubules, in particular during axon development. Also, our data AG-013736 indicate that NA14 is directly involved in cell division and could be important for abscission, the final phase of cytokinesis. In concert with previous studies, these observations suggest that NA14 is a molecular adaptor involved in targeting proteins to the centrosome and midbodies. In agreement with previous observations, we found that NA14 localizes to midbodies during cytokinesis.