The Ctf18 RFC-like complex positions yeast telomeres but does not specify their replication time

Peer reviewedPreprin

FLT3 mutations in canine acute lymphocytic leukemia

<p>Abstract</p> <p>Background</p> <p>FMS-like tyrosine kinase 3 (FLT3) is a commonly mutated protein in a variety of human acute leukemias. Mutations leading to constitutively active FLT3, including internal tandem duplications of the juxtamembrane domain (ITD), result in continuous cellular proliferation, resistance to apoptotic cell death, and a poorer prognosis. A better understanding of the molecular consequences of FLT3 activation would allow improved therapeutic strategies in these patients. Canine lymphoproliferative diseases, including lymphoma and acute leukemias, share evolutionarily conserved chromosomal aberrations and exhibit conserved mutations within key oncogenes when compared to their human counterparts. A small percentage of canine acute lymphocytic leukemias (ALL) also exhibit <it>FLT3 </it>ITD mutations.</p> <p>Methods</p> <p>We molecularly characterized <it>FLT3 </it>mutations in two dogs and one cell line, by DNA sequencing, gene expression analysis via quantitative real-time PCR, and sensitivity to the FLT3 inhibitor lestaurtinib via <it>in vitro </it>proliferation assays. FLT 3 and downstream mediators of FLT3 activation were assessed by Western blotting.</p> <p>Results</p> <p>The canine B-cell leukemia cell line, GL-1, and neoplastic cells from 2/7 dogs diagnosed cytologically with ALL were found to have <it>FLT3 </it>ITD mutations and <it>FLT3 </it>mRNA up-regulation. Lestaurtinib, a small molecule FLT3 inhibitor, significantly inhibited the growth of GL-1 cells, while not affecting the growth of two other canine lymphoid cell lines without the <it>FLT3 </it>mutation. Finally, western blots were used to confirm the conserved downstream mediators of <it>FLT3 </it>activating mutations.</p> <p>Conclusions</p> <p>These results show that ALL and FLT3 biology is conserved between canine and human patients, supporting the notion that canine ALL, in conjunction with the GL-1 cell line, will be useful in the development of a relevant large animal model to aid in the study of human FLT3 mutant leukemias.</p

Replicating viral vector platform exploits alarmin signals for potent CD8⁺ T cell-mediated tumour immunotherapy.

Viral infections lead to alarmin release and elicit potent cytotoxic effector T lymphocyte (CTL &lt;sup&gt;eff&lt;/sup&gt; ) responses. Conversely, the induction of protective tumour-specific CTL &lt;sup&gt;eff&lt;/sup&gt; and their recruitment into the tumour remain challenging tasks. Here we show that lymphocytic choriomeningitis virus (LCMV) can be engineered to serve as a replication competent, stably-attenuated immunotherapy vector (artLCMV). artLCMV delivers tumour-associated antigens to dendritic cells for efficient CTL priming. Unlike replication-deficient vectors, artLCMV targets also lymphoid tissue stroma cells expressing the alarmin interleukin-33. By triggering interleukin-33 signals, artLCMV elicits CTL &lt;sup&gt;eff&lt;/sup&gt; responses of higher magnitude and functionality than those induced by replication-deficient vectors. Superior anti-tumour efficacy of artLCMV immunotherapy depends on interleukin-33 signalling, and a massive CTL &lt;sup&gt;eff&lt;/sup&gt; influx triggers an inflammatory conversion of the tumour microenvironment. Our observations suggest that replicating viral delivery systems can release alarmins for improved anti-tumour efficacy. These mechanistic insights may outweigh safety concerns around replicating viral vectors in cancer immunotherapy

Global DNA Hypomethylation in Peripheral Blood Leukocytes as a Biomarker for Cancer Risk: A Meta-Analysis

BACKGROUND: Good biomarkers for early detection of cancer lead to better prognosis. However, harvesting tumor tissue is invasive and cannot be routinely performed. Global DNA methylation of peripheral blood leukocyte DNA was evaluated as a biomarker for cancer risk. METHODS: We performed a meta-analysis to estimate overall cancer risk according to global DNA hypomethylation levels among studies with various cancer types and analytical methods used to measure DNA methylation. Studies were systemically searched via PubMed with no language limitation up to July 2011. Summary estimates were calculated using a fixed effects model. RESULTS: The subgroup analyses by experimental methods to determine DNA methylation level were performed due to heterogeneity within the selected studies (p<0.001, I(2): 80%). Heterogeneity was not found in the subgroup of %5-mC (p = 0.393, I(2): 0%) and LINE-1 used same target sequence (p = 0.097, I(2): 49%), whereas considerable variance remained in LINE-1 (p<0.001, I(2): 80%) and bladder cancer studies (p = 0.016, I(2): 76%). These results suggest that experimental methods used to quantify global DNA methylation levels are important factors in the association study between hypomethylation levels and cancer risk. Overall, cancer risks of the group with the lowest DNA methylation levels were significantly higher compared to the group with the highest methylation levels [OR (95% CI): 1.48 (1.28-1.70)]. CONCLUSIONS: Global DNA hypomethylation in peripheral blood leukocytes may be a suitable biomarker for cancer risk. However, the association between global DNA methylation and cancer risk may be different based on experimental methods, and region of DNA targeted for measuring global hypomethylation levels as well as the cancer type. Therefore, it is important to select a precise and accurate surrogate marker for global DNA methylation levels in the association studies between global DNA methylation levels in peripheral leukocyte and cancer risk

Cell-Cycle Dependence of Transcription Dominates Noise in Gene Expression

The large variability in mRNA and protein levels found from both static and dynamic measurements in single cells has been largely attributed to random periods of transcription, often occurring in bursts. The cell cycle has a pronounced global role in affecting transcriptional and translational output, but how this influences transcriptional statistics from noisy promoters is unknown and generally ignored by current stochastic models. Here we show that variable transcription from the synthetic tetO promoter in S. cerevisiae is dominated by its dependence on the cell cycle. Real-time measurements of fluorescent protein at high expression levels indicate tetO promoters increase transcription rate ~2-fold in S/G2/M similar to constitutive genes. At low expression levels, where tetO promoters are thought to generate infrequent bursts of transcription, we observe random pulses of expression restricted to S/G2/M, which are correlated between homologous promoters present in the same cell. The analysis of static, single-cell mRNA measurements at different points along the cell cycle corroborates these findings. Our results demonstrate that highly variable mRNA distributions in yeast are not solely the result of randomly switching between periods of active and inactive gene expression, but instead largely driven by differences in transcriptional activity between G1 and S/G2/M.GM095733BBBE 103316MIT Startup Fun

Early onset MSI-H colon cancer with MLH1 promoter methylation, is there a genetic predisposition?

<p>Abstract</p> <p>Background</p> <p>To investigate the etiology of <it>MLH1 </it>promoter methylation in mismatch repair (MMR) mutation-negative early onset MSI-H colon cancer. As this type of colon cancer is associated with high ages, young patients bearing this type of malignancy are rare and could provide additional insight into the etiology of sporadic MSI-H colon cancer.</p> <p>Methods</p> <p>We studied a set of 46 MSI-H colon tumors cases with <it>MLH1 </it>promoter methylation which was enriched for patients with an age of onset below 50 years (n = 13). Tumors were tested for CIMP marker methylation and mutations linked to methylation: <it>BRAF, KRAS</it>, <it>GADD45A </it>and the <it>MLH1 </it>-93G>A polymorphism. When available, normal colon and leukocyte DNA was tested for <it>GADD45A </it>mutations and germline <it>MLH1 </it>methylation. SNP array analysis was performed on a subset of tumors.</p> <p>Results</p> <p>We identified two cases (33 and 60 years) with <it>MLH1 </it>germline promoter methylation. <it>BRAF </it>mutations were less frequent in colon cancer patients below 50 years relative to patients above 50 years (p-value: 0.044). CIMP-high was infrequent and related to <it>BRAF </it>mutations in patients below 50 years. In comparison with published controls the G>A polymorphism was associated with our cohort. Although similar distribution of the pathogenic A allele was observed in the patients with an age of onset above and below 50 years, the significance for the association was lost for the group under 50 years. <it>GADD45A </it>sequencing yielded an unclassified variant. Tumors from both age groups showed infrequent copy number changes and loss-of-heterozygosity.</p> <p>Conclusion</p> <p>Somatic or germline <it>GADD45A </it>mutations did not explain sporadic MSI-H colon cancer. Although germline <it>MLH1 </it>methylation was found in two individuals, locus-specific somatic <it>MLH1 </it>hypermethylation explained the majority of sporadic early onset MSI-H colon cancer cases. Our data do not suggest an intrinsic tendency for CpG island hypermethylation in these early onset MSI-H tumors other than through somatic mutation of <it>BRAF</it>.</p

Age-Related Changes of Myelin Basic Protein in Mouse and Human Auditory Nerve

Age-related hearing loss (presbyacusis) is the most common type of hearing impairment. One of the most consistent pathological changes seen in presbyacusis is the loss of spiral ganglion neurons (SGNs). Defining the cellular and molecular basis of SGN degeneration in the human inner ear is critical to gaining a better understanding of the pathophysiology of presbyacusis. However, information on age-related cellular and molecular alterations in the human spiral ganglion remains scant, owing to the very limited availably of human specimens suitable for high resolution morphological and molecular analysis. This study aimed at defining age-related alterations in the auditory nerve in human temporal bones and determining if immunostaining for myelin basic protein (MBP) can be used as an alternative approach to electron microscopy for evaluating myelin degeneration. For comparative purposes, we evaluated ultrastructural alternations and changes in MBP immunostaining in aging CBA/CaJ mice. We then examined 13 temporal bones from 10 human donors, including 4 adults aged 38–46 years (middle-aged group) and 6 adults aged 63–91 years (older group). Similar to the mouse, intense immunostaining of MBP was present throughout the auditory nerve of the middle-aged human donors. Significant declines in MBP immunoreactivity and losses of MBP+ auditory nerve fibers were observed in the spiral ganglia of both the older human and aged mouse ears. This study demonstrates that immunostaining for MBP in combination with confocal microscopy provides a sensitive, reliable, and efficient method for assessing alterations of myelin sheaths in the auditory nerve. The results also suggest that myelin degeneration may play a critical role in the SGN loss and the subsequent decline of the auditory nerve function in presbyacusis

Painful and painless mutations of SCN9A and SCN11A voltage-gated sodium channels

Chronic pain is a global problem affecting up to 20% of the world’s population and has a significant economic, social and personal cost to society. Sensory neurons of the dorsal root ganglia (DRG) detect noxious stimuli and transmit this sensory information to regions of the central nervous system (CNS) where activity is perceived as pain. DRG neurons express multiple voltage-gated sodium channels that underlie their excitability. Research over the last 20 years has provided valuable insights into the critical roles that two channels, NaV1.7 and NaV1.9, play in pain signalling in man. Gain of function mutations in NaV1.7 cause painful conditions while loss of function mutations cause complete insensitivity to pain. Only gain of function mutations have been reported for NaV1.9. However, while most NaV1.9 mutations lead to painful conditions, a few are reported to cause insensitivity to pain. The critical roles these channels play in pain along with their low expression in the CNS and heart muscle suggest they are valid targets for novel analgesic drugs

Defective Membrane Remodeling in Neuromuscular Diseases: Insights from Animal Models

Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches