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The mononegaviral family Filoviridae has eight members assigned to three genera and seven species. Until now, genus and species demarcation were based on arbitrarily chosen filovirus genome sequence divergence values (≈50% for genera, ≈30% for species) and arbitrarily chosen phenotypic virus or virion characteristics. Here we report filovirus genome sequence-based taxon demarcation criteria using the publicly accessible PAirwise Sequencing Comparison (PASC) tool of the US National Center for Biotechnology Information (Bethesda, MD, USA). Comparison of all available filovirus genomes in GenBank using PASC revealed optimal genus demarcation at the 55–58% sequence diversity threshold range for genera and at the 23–36% sequence diversity threshold range for species. Because these thresholds do not change the current official filovirus classification, these values are now implemented as filovirus taxon demarcation criteria that may solely be used for filovirus classification in case additional data are absent. A near-complete, coding-complete, or complete filovirus genome sequence will now be required to allow official classification of any novel “filovirus.” Classification of filoviruses into existing taxa or determining the need for novel taxa is now straightforward and could even become automated using a presented algorithm/flowchart rooted in RefSeq (type) sequences.

Study Design: Multicenter retrospective study. Objectives: To investigate the risk of symptomatic recurrent laryngeal nerve palsy (RLNP) following cervical spine surgery, to examine risk factors for its development, and to report its treatment and outcomes. Methods: A multicenter study from 21 high-volume surgical centers from the AOSpine North America Clinical Research Network was performed. Each center screened for rare complications following cervical spine surgery, including RLNP. Patients were included if they underwent cervical spine surgery (levels from C2 to C7) between January 1, 2005 and December 31, 2011. Data were analyzed with regard to complication treatment and outcome. Cases were compared to a control group from the AOSpine CSM and CSM-I studies. Results: Three centers reported 19 cases of RLNP from a cohort of 1345 patients. The reported incidence of RLNP ranged from 0.6% to 2.9% between these 3 centers. Fifteen patients (79%) in the RLNP group were approached from the left side. Ten patients (52.6%) required treatment for RLNP—6 required medical therapy (steroids), 1 interventional treatment (injection laryngoplasty), and 3 conservative therapy (speech therapy). When examining outcomes, 73.7% (14/19) of cases resolved completely, 15.8% (3/19) resolved with residual effects, and in 10.5% (2/19) of cases this could not be determined. Conclusions: In this multicenter study examining rare complications following cervical spine surgery, the risk of RLNP after cervical spine surgery ranged from 0.6% to 2.9% between centers. Though rare, it was found that 16% of patients may experience partial resolution with residual effects, and 74% resolve completely.

Objectives To examine the association between vascular pathology and rate of cognitive decline in older adults independent of Alzheimer's disease ( AD ) pathology. Design Prospective cohort study. Setting Community sample. Participants Individuals from the Einstein Aging Study autopsy series (N = 62). Measurements The Blessed Information‐Memory‐Concentration ( BIMC ) test was used to assess global cognitive status. AD pathology was quantified according to Braak stage (<3 vs ≥ 3). Vascular pathology was quantified using a previously reported macrovascular lesion ( MVL ) score. The association between vascular pathology and antemortem rates of cognitive decline adjusted for level of AD pathology was assessed using linear mixed‐effects models. Results Mean age was 81.8 at enrollment and 89.0 at death. Participants with more than two MVL s had faster cognitive decline than those with no MVL s (difference in annual rate of change in BIMC 0.74 points/yr, P = .03). Braak stage was also associated with cognitive decline (difference 0.57 points/yr, P = .03). The difference in rate of cognitive decline between those with more than two MVL s and those free of vascular lesions persisted after adjustment for AD pathology (difference in rate of change in BIMC 0.68 points/yr, P = .04). The effect of vascular pathology on cognitive decline was not significantly different according to AD pathology. Conclusion Vascular brain pathology is associated with rate of cognitive decline after adjusting for level of AD pathology.

AbstractObjectiveTo determine the financial and psycho‐social impact of participation in clinical trials of patients with BCR/ABL‐negative myeloproliferative neoplasms (MPN).MethodsAn international, observational cross‐sectional study was performed in 143 consecutive MPN patients (51% myelofibrosis, 36% polycythemia vera, 13% essential thrombocythemia), 68% from Italy, 17% from USA, and 15% from Spain.ResultsThirty‐five percent of patients reported having spent more money during the trial than in previous treatments and 21% having missed more workdays. Twelve percent replied that they would not have participated in the trial if the financial consequences would have been known beforehand. In 10% of the patients, the interpersonal relationships were more affected during the trial than in previous treatment but, overall, 91% subjects believed that participating in the clinical trial was worth the financial or emotional suffering. Concerning patients’ suggestions, 54% of them indicated that the number of visits required for the trial should be clearly specified in the informed consent, 60% recommended travel cost reimbursement, and 23% hotel cost reimbursement.ConclusionsPhysicians and pharmaceutical companies involved in clinical trials with patients with hematological diseases should be aware of these problems and make efforts to attenuate the socioeconomic burden of participation in the trials.

479 Background: We and others have previously shown that the ccRCC epigenome is characterized by widespread DNA hypermethylation (CCR 2014, Nature 2014). Various important tumor suppressor genes (TSGs), such as SMAD7 (inhibitor of oncogenic TGF-β signaling), are under-expressed due to aberrantly methylated promoters or enhancers. We hypothesized that the hypermethylation in ccRCC could be due to low activity of the TET enzymes, which convert methylcytosine (5-mc) to hydroxymethylcytosine (5-hmc). Loss of function of TET enzymes can occur with an inactivating mutation (TET-2 is mutated in about 6% of ccRCC (Science Signaling 2013) or hypoactivity of normal TET enzymes, through inhibition by metabolic intermediates. AA is an essential co-factor for TET enzymes (JACS 2015). We hypothesized that high dose AA treatment of ccRCC could potentially increase the functional activity of TET enzymes leading to demethylation of the RCC genome, and enhance expression of TSGs. Methods: In vitro TET activity was performed on ccRCC cell line 769P with increasing doses of AA (L-AA). Genome wide quantitative 5-mc and 5-hmc was evaluated using mass spectrometry. SMAD7 expression was determined using qRT-PCR. Proliferation assay (MTT) was performed with AA in combination with pazopanib. Cell cycle and apoptosis assays were performed. Results: AA, at doses achieved only by the intravenous route (1-10mM), increased TET activity in ccRCC cell line 769P. Genome wide 5-mc was significantly reduced and 5-hmc was increased, correlating with increase in TET activity. SMAD7 expression was increased with AA treatment. High dose AA led to proliferation inhibition with a cell cycle arrest in the G1 phase, and had a synergistic effect with pazopanib. Since AA leads to the generation of H2O2 in-vitro, catalase was used as control. This did not reverse the effect of AA on epigenetic changes; and proliferation inhibition was seen despite catalase control. Conclusions: High dose AA causes TET mediated DNA demethylation of the hypermethylated RCC genome, resulting in the re-expression of TSGs, and proliferation inhibition. Sequencing and xenograft studies are underway.

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.