Epigenetic loss of heterozygosity of Apc and an inflammation-associated mutational signature detected in Lrig1+/--driven murine colonic adenomas.

BACKGROUND: The loss of a single copy of adenomatous polyposis coli (Apc) in leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1)-expressing colonic progenitor cells induces rapid growth of adenomas in mice with high penetrance and multiplicity. The tumors lack functional APC, and a genetic loss of heterozygosity of Apc was previously observed. METHODS: To identify genomic features of early tumorigenesis, and to profile intertumoral genetic heterogeneity, tumor exome DNA (n = 9 tumors) and mRNA (n = 5 tumors) sequences were compared with matched nontumoral colon tissue. Putative somatic mutations were called after stringent variant filtering. Somatic signatures of mutational processes were determined and splicing patterns were observed. RESULTS: The adenomas were found to be genetically heterogeneous and unexpectedly hypermutated, displaying a strong bias toward G:C > A:T mutations. A genetic loss of heterozygosity of Apc was not observed, however, an epigenetic loss of heterozygosity was apparent in the tumor transcriptomes. Complex splicing patterns characterized by a loss of intron retention were observed uniformly across tumors. CONCLUSION: This study demonstrates that early tumors originating from intestinal stem cells with reduced Lrig1 and Apc expression are highly mutated and genetically heterogeneous, with an inflammation-associated mutational signature and complex splicing patterns that are uniform across tumors.

High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq).

BACKGROUND: Polymorphic loci exist throughout the genomes of a population and provide the raw genetic material needed for a species to adapt to changes in the environment. The minor allele frequencies of rare Single Nucleotide Polymorphisms (SNPs) within a population have been difficult to track with Next-Generation Sequencing (NGS), due to the high error rate of standard methods such as Illumina sequencing. RESULTS: We have developed a wet-lab protocol and variant-calling method that identifies both sequencing and PCR errors, called Paired-End Low Error Sequencing (PELE-Seq). To test the specificity and sensitivity of the PELE-Seq method, we sequenced control E. coli DNA libraries containing known rare alleles present at frequencies ranging from 0.2-0.4 % of the total reads. PELE-Seq had higher specificity and sensitivity than standard libraries. We then used PELE-Seq to characterize rare alleles in a Caenorhabditis remanei nematode worm population before and after laboratory adaptation, and found that minor and rare alleles can undergo large changes in frequency during lab-adaptation. CONCLUSION: We have developed a method of rare allele detection that mitigates both sequencing and PCR errors, called PELE-Seq. PELE-Seq was evaluated using control E. coli populations and was then used to compare a wild C. remanei population to a lab-adapted population. The PELE-Seq method is ideal for investigating the dynamics of rare alleles in a broad range of reduced-representation sequencing methods, including targeted amplicon sequencing, RAD-Seq, ddRAD, and GBS. PELE-Seq is also well-suited for whole genome sequencing of mitochondria and viruses, and for high-throughput rare mutation screens.

ZBP1 and TRIF trigger lethal necroptosis in mice lacking caspase-8 and TNFR1.

Necroptosis is a lytic form of cell death that is mediated by the kinase RIPK3 and the pseudokinase MLKL when caspase-8 is inhibited downstream of death receptors, toll-like receptor 3 (TLR3), TLR4, and the intracellular Z-form nucleic acid sensor ZBP1. Oligomerization and activation of RIPK3 is driven by interactions with the kinase RIPK1, the TLR adaptor TRIF, or ZBP1. In this study, we use immunohistochemistry (IHC) and in situ hybridization (ISH) assays to generate a tissue atlas characterizing RIPK1, RIPK3, Mlkl, and ZBP1 expression in mouse tissues. RIPK1, RIPK3, and Mlkl were co-expressed in most immune cell populations, endothelial cells, and many barrier epithelia. ZBP1 was expressed in many immune populations, but had more variable expression in epithelia compared to RIPK1, RIPK3, and Mlkl. Intriguingly, expression of ZBP1 was elevated in Casp8-/- Tnfr1-/- embryos prior to their succumbing to aberrant necroptosis around embryonic day 15 (E15). ZBP1 contributed to this embryonic lethality because rare Casp8-/- Tnfr1-/- Zbp1-/- mice survived until after birth. Necroptosis mediated by TRIF contributed to the demise of Casp8-/- Tnfr1-/- Zbp1-/- pups in the perinatal period. Of note, Casp8-/- Tnfr1-/- Trif-/- Zbp1-/- mice exhibited autoinflammation and morbidity, typically within 5-7 weeks of being born, which is not seen in Casp8-/- Ripk1-/- Trif-/- Zbp1-/-, Casp8-/- Ripk3-/-, or Casp8-/- Mlkl-/- mice. Therefore, after birth, loss of caspase-8 probably unleashes RIPK1-dependent necroptosis driven by death receptors other than TNFR1.

A consensus process on the use of exercises and after action reports to assess and improve public health emergency preparedness and response.

INTRODUCTION: The objective of disaster preparedness is to ensure that appropriate systems, procedures, and resources are in place to provide prompt, effective assistance to disaster victims, thus facilitating relief measures and rehabilitation of services. Disaster preparedness efforts include the identification of possible health scenarios based on the probability of hazards and vulnerability of the population as a basis for creating a disaster plan. Exercises that simulate emergency response, involving the health and other sectors, have been suggested as useful tools to test the plans on a regular basis and measure preparedness efforts; the absence of actual testing is likely to negate even the best of abstract plans. Problem Exercises and after action reports (AARs) are used to document preparedness activities. However, to date, limited analysis has been performed on what makes an exercise an effective tool to assess public health emergency preparedness (PHEP), and how AARs can be developed and used to support PHEP improvement efforts. The scope of this project was to achieve consensus on: (1) what makes an exercise an effective tool to assess PHEP; and (2) what makes an AAR an effective tool to guide PHEP improvement efforts. METHODS: Sixty-one PHEP experts were convened by the use of Nominal Group Techniques to achieve consensus on a series of characteristics that exercises should have when designed to assess PHEP and on the recommendations for developing high-quality AARs. RESULTS: The panelists achieved consensus on a list of recommendations to improve the use of exercises and AARs in PHEP improvement efforts. Such recommendations ranged from the characteristics of the exercise audience to the evaluation methodology being used and the characteristics of the produced AAR such as its structure and content. CONCLUSIONS: The characteristics of the exercise audience, scenario and scope are among the most important attributes to the effectiveness of an exercise conducted for PHEP evaluation purposes. The evaluation instruments used to gather observations need an appropriate matching between exercise objectives and the response capabilities tested during the exercise, to build the base for the production of a good AAR. Improvements in the design and creation of exercises and AARs could facilitate better reporting and measurement of preparedness outcomes.

Eribulin: a novel cytotoxic chemotherapy agent.

OBJECTIVE: To review the chemistry, pharmacology, pharmacokinetics, safety, and efficacy of eribulin (Halaven). DATA SOURCES: A literature search (up to December 2011) using the terms eribulin, Halaven, ER-086526, and E7389 was performed with PubMed, Google Scholar, selected Ovid bibliography searches, and the abstract search tool from the American Society of Clinical Oncology Annual Meetings and the San Antonio Breast Cancer Symposia. Additional references from the bibliographies of these articles were also assessed. DATA EXTRACTION: English-language preclinical and clinical studies on the chemistry, pharmacology, pharmacokinetics, safety, and efficacy of eribulin were reviewed. DATA SYNTHESIS: Eribulin is a novel microtubule inhibitor with a unique mechanism of action, which involves interaction with a distinct binding site on ß-tubulin leading to G(2)/M phase cell-cycle arrest and apoptosis. Eribulin has been approved by the Food and Drug Administration for the treatment of metastatic breast cancer in patients who have been previously treated with an anthracycline and a taxane. In a pivotal Phase 3 study conducted in patients with metastatic breast cancer, eribulin 1.4 mg/m(2), administered over 2-5 minutes as an intravenous infusion on days 1 and 8 of 21-day cycles, was associated with a significantly increased median overall survival of 13.1 months compared to the median overall survival of 10.6 months in the therapy of physician's choice. Eribulin has also shown activity in Phase 2 studies in other types of cancers, such as non-small cell lung cancer, prostate cancer, urothelial cancer, soft tissue sarcomas, and platinum-susceptible ovarian, fallopian tube, or peritoneal cancers. The most severe (grade 3/4) adverse effects associated with eribulin include neutropenia, leukopenia, and peripheral neuropathy. Common toxicities include fatigue, neutropenia, alopecia, anemia, and peripheral neuropathy. CONCLUSIONS: Eribulin is a promising new cytotoxic chemotherapy agent due to its ability to treat cancers that are refractory or resistant to other drugs as well as its manageable toxicity profile.

Public health emergency preparedness exercises: lessons learned.

The Harvard School of Public Health Center for Public Health Preparedness exercise program has two aims: (1) educating the public health workforce on key public health system emergency preparedness issues, and (2) identifying specific systems-level challenges in the public health response to large-scale events. Rigorous evaluation of 38 public health emergency preparedness (PHEP) exercises employing realistic scenarios and reliable and accurate outcome measures has demonstrated the usefulness of PHEP exercises in clarifying public health workers' roles and responsibilities, facilitating knowledge transfer among these individuals and organizations, and identifying specific public health systems-level challenges.

Cochlear Implant Mapping Through Telemedicine-A Feasibility Study.

OBJECTIVE: Access to postoperative aural rehabilitation limits cochlear implant (CI) penetration to the candidate population. The purpose of this study was to evaluate the effectiveness of remote CI programming and aural rehabilitation via telehealth. STUDY DESIGN AND SETTING: Retrospective study of one cochlear implant center. PATIENTS AND INTERVENTION: Patients undergoing cochlear implantation from 2015 to 2018 undergoing remote programming as part of routine audiologic follow up. MAIN OUTCOME MEASURES: AzBio scores, impedances, comfort and threshold levels, and responses to the International Outcome Inventory for Hearing Aids questionnaire modified for CIs (IOI-CI). RESULTS: A total of 22 CIs in 20 patients were included during the study period. Threshold, comfort, and impedance levels were readily obtained via telehealth and were not significantly different between telehealth and live sessions. AzBio scores and warble tone pure tone averages were also similar and acceptable in both session modalities. Based on IOI-CI scores, patients were very satisfied with their hearing outcomes. CONCLUSIONS: Using telemedicine, reliable measurements were readily obtained and hearing outcomes after remote programming were comparable to those expected after in-person programming sessions. Patients were overall satisfied with their remote programming sessions. Telehealth is a cost-effective and safe way to deliver post-CI audiologic care, particularly to patients with limited mobility or those in remote locations.

RIP1 kinase activity is critical for skin inflammation but not for viral propagation.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.

Genome-wide identification of hypoxia-induced enhancer regions.

Here we present a genome-wide method for de novo identification of enhancer regions. This approach enables massively parallel empirical investigation of DNA sequences that mediate transcriptional activation and provides a platform for discovery of regulatory modules capable of driving context-specific gene expression. The method links fragmented genomic DNA to the transcription of randomer molecule identifiers and measures the functional enhancer activity of the library by massively parallel sequencing. We transfected a Drosophila melanogaster library into S2 cells in normoxia and hypoxia, and assayed 4,599,881 genomic DNA fragments in parallel. The locations of the enhancer regions strongly correlate with genes up-regulated after hypoxia and previously described enhancers. Novel enhancer regions were identified and integrated with RNAseq data and transcription factor motifs to describe the hypoxic response on a genome-wide basis as a complex regulatory network involving multiple stress-response pathways. This work provides a novel method for high-throughput assay of enhancer activity and the genome-scale identification of 31 hypoxia-activated enhancers in Drosophila.

Local de novo assembly of RAD paired-end contigs using short sequencing reads.

Despite the power of massively parallel sequencing platforms, a drawback is the short length of the sequence reads produced. We demonstrate that short reads can be locally assembled into longer contigs using paired-end sequencing of restriction-site associated DNA (RAD-PE) fragments. We use this RAD-PE contig approach to identify single nucleotide polymorphisms (SNPs) and determine haplotype structure in threespine stickleback and to sequence E. coli and stickleback genomic DNA with overlapping contigs of several hundred nucleotides. We also demonstrate that adding a circularization step allows the local assembly of contigs up to 5 kilobases (kb) in length. The ease of assembly and accuracy of the individual contigs produced from each RAD site sequence suggests RAD-PE sequencing is a useful way to convert genome-wide short reads into individually-assembled sequences hundreds or thousands of nucleotides long.