The Epstein-Barr Virus Enhancer Interaction Landscapes in Virus-Associated Cancer Cell Lines.

Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARF1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. IMPORTANCE EBV is the first human DNA tumor virus identified, discovered over 50 years ago. EBV causes ~200,000 cases of various cancers each year. EBV-encoded oncogenes, noncoding RNAs, and microRNAs (miRNAs) can promote cell growth and survival and suppress senescence. Regulation of EBV gene expression is very complex. The viral C promoter regulates the expression of all EBV nuclear antigens (EBNAs), some of which are very far away from the C promoter. Another way by which the virus activates remote gene expression is through DNA looping. In this study, we describe the viral genome looping patterns in various EBV-associated cancer cell lines and identify important EBV enhancers in these cells. This study also identified novel opportunities to perturb and eventually control EBV gene expression in these cancer cells.

Molecular determinants of phase separation for Drosophila DNA replication licensing factors.

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using Drosophila melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.

Clinico-pathological predictors of mismatch repair deficiency in sebaceous neoplasia: A large case series from a single Australian private pathology service.

BACKGROUND/OBJECTIVES: Loss of expression of mismatch repair (MMR) proteins is frequently observed in sebaceous skin lesions and can be a herald for Lynch syndrome. The aim of this study was to identify clinico-pathological predictors of MMR deficiency in sebaceous neoplasia that could aid dermatologists and pathologists in determining which sebaceous lesions should undergo MMR immunohistochemistry (IHC). METHODS: An audit of sebaceous skin lesions (excluding hyperplasia) where pathologist-initiated MMR IHC was performed between January 2009 to December 2016 was undertaken from a single pathology practice identifying 928 lesions from 882 individuals. Lesions were further analysed for differences in gender, age at diagnosis, lesion type and anatomic location, stratified by MMR status. RESULTS: The 882 individuals (67.7% male) had a mean (SD) age of diagnosis of 68.4 ± 13.3 years. Nearly two-thirds of the lesions were sebaceous adenomas, with 82.6% of all lesions occurring on the head and neck. MMR deficiency, observed in 282 of the 919 lesions (30.7%), was most common in sebaceous adenomas (210/282; 74.5%). MMR-deficient lesions occurred predominantly on the trunk or limbs (64.7%), compared with 23.2% in head or neck (P < 0.001). Loss of MSH2 and MSH6 protein expression was most frequent pattern of loss (187/281; 66.5%). The highest AUC for discriminating MMR-deficient sebaceous lesions from MMR-proficient lesions was observed for the ROC curve based on subgroups defined by type and anatomic location of the sebaceous lesion (AUC = 0.68). CONCLUSION: The best combination of measured clinico-pathological features achieved only modest positive predictive values, sensitivity and specificity for identifying MMR-deficient sebaceous skin lesions.

Somatic mutations of the coding microsatellites within the beta-2-microglobulin gene in mismatch repair-deficient colorectal cancers and adenomas.

In colorectal cancers (CRCs) with tumour mismatch repair (MMR) deficiency, genes involved in the host immune response that contain microsatellites in their coding regions, including beta-2-microglobulin (B2M), can acquire mutations that may alter the immune response, tumour progression and prognosis. We screened the coding microsatellites within B2M for somatic mutations in MMR-deficient CRCs and adenomas to determine associations with tumour subtypes, clinicopathological features and survival. Incident MMR-deficient CRCs from Australasian Colorectal Cancer Family Registry (ACCFR) and the Melbourne Collaborative Cohort Study participants (n = 144) and 63 adenomas from 41 MMR gene mutation carriers from the ACCFR were screened for somatic mutations within five coding microsatellites of B2M. Hazard ratios (HR) and 95% confidence intervals (CI) for overall survival by B2M mutation status were estimated using Cox regression, adjusting for age at CRC diagnosis, sex, AJCC stage and grade. B2M mutations occurred in 30 (20.8%) of the 144 MMR-deficient CRCs (29% of the MLH1-methylated, 17% of the Lynch syndrome and 9% of the suspected Lynch CRCs). No B2M mutations were identified in the 63 adenomas tested. B2M mutations differed by site, stage, grade and lymphocytic infiltration although none reached statistical significance (p > 0.05). The HR for overall survival for B2M mutated CRC was 0.65 (95% CI 0.29-1.48) compared with B2M wild-type. We observed differences in B2M mutation status in MMR-deficient CRC by tumour subtypes, site, stage, grade, immune infiltrate and for overall survival that warrant further investigation in larger studies before B2M mutation status can be considered to have clinical utility.

Carotenoid and vitamin content of Karat and other Micronesian banana cultivars.

We previously found high carotenoid levels in Karat and other Micronesian bananas, indicating potential importance for alleviating vitamin A deficiency and other nutritionally related health problems in the Federated States of Micronesia. Past work focused on carotenoid and mineral analyses, whereas here we investigated 16 cultivars (most not previously analysed) for a broader micronutrient profile, including seven vitamins. Karat carotenoid levels were higher than in previous analyses, confirming Karat as exceptionally carotenoid-rich. We identified an additional 10 carotenoid-rich cultivars, expanding the range having potential for alleviating vitamin A deficiency. A striking finding is the high riboflavin level in Karat, including high levels of uncharacterized flavonoids. Niacin and alpha-tocopherol are at levels that may contribute importantly to dietary intake within normal patterns of consumption. These data present a more complete basis for promoting the nutritional benefits of these banana cultivars where they are consumed in the Pacific, and potential benefits for promoting elsewhere.

High-salt stabilization of Laponite clay particles.

Colloidal radioactive transuranic wastes are currently buried in large tanks in the form of dense colloids in high salt, high pH aqueous media. These facilities are beginning to fail, so it is necessary to transport and "package" them for more permanent disposal, processes requiring understanding of the microstructure that develops under such conditions. Laponite RD clay is believed to be a good simulant for the colloids in the waste tanks, and the present study addresses their behavior under high salt conditions, where previous studies have frequently observed the phenomenon of "restabilization," i.e., the attainment of aggregation stability at high electrolyte conditions. Specifically, the aggregation kinetics and the resulting cluster structure (fractal dimension) of Laponite RD clay colloids at high concentrations of BaCl2 (an electrolyte previously shown to lead to restabilization) are investigated. At low-to-intermediate electrolyte concentrations, the clay is found to behave in accord with DLVO theory, i.e., low salt conditions yield slow aggregation into densely-packed aggregates, whereas intermediate salt concentrations, sufficient to cause double layer collapse, produce rapid aggregation into open aggregates. High salt concentrations, however, show slow rates of aggregation. The aggregate structure under these conditions is found to mimic that found for very low electrolyte concentrations, i.e., high fractal dimension. Further experiments show that a sudden increase in salt concentration in a system containing young open aggregates produced under intermediate salt concentrations causes them to reform into more compact structures.

Media communication center using brain computer interface.

This paper attempts to make use of brain computer interface (BCI) in implementing an application called the media communication center for the paralyzed people. The application is based on the event-related potential called P300 to perform button selections on media and communication programs such as the mp3 player, video player, photo gallery and e-book. One of the key issues in such system is the usability. We study how various tasks affect the application operation, in particular, how typical mental activities cause false trigger during the operation of the application. We study the false acceptance rate under the conditions of closing eyes, reading a book, listening to music and watching a video. Data from 5 subjects is used to obtain the false rejection rate and false acceptance rate of the BCI system. Our study shows that different mental activities show different impacts on the false acceptance performances.

Transient aggregation during dry-down of solvent-borne dispersions.

The present work investigates the role of changing solvency conditions on the stability of sterically stabilized colloids in evaporating solvent-borne dispersions and the recovery time for the redispersion of aggregates if destabilization occurs. Process conditions during the conversion of the coated fluid dispersion to a solid film must be carefully controlled to ensure that aggregation, leading to uneven pigment distribution, does not occur. Although a polymeric binder serves as a retention aid in a solvent-borne coating, it also act as a steric stabilizer during dry-down or curing of the coating. Because the dispersion medium in the coatings often is a mixture of solvents and nonsolvents, the binder's interactions with the dispersion medium govern whether aggregation is likely to occur. Tracking such an occurrence may be difficult due to rapidly changing solvent compositions. Light scattering is utilized herein to indicate if the conditions of a high speed coating and drying process (in this case, the production of magnetic data storage media) may lead to aggregation and, if aggregation is detected, the ability of aggregates to fully redisperse. Conformational changes of free polymeric binder chains in solution are followed to gauge the polymer-solvent interactions and, therefore, the binder's ability to prevent aggregation. A "danger zone" (conditions where aggregation is probable) is constructed from these measurements, and further dynamic light scattering measurements on binder-grafted-oxide particles detect aggregation in this zone. The aggregates are redispersed in a series of "good" solvents, and redispersion is found not to be instantaneous, suggesting that high-speed drying processes may not allow the flocculated colloids enough recovery time.

Thiazolidinediones and insulin: rationale for use and role of combination therapy in type 2 diabetes mellitus.

The range of therapeutic modalities to treat type 2 diabetes mellitus has broadened in recent years. Biguanides and thiazolidinediones are the two currently available classes of anti-hyperglycemic agents with insulin-sensitizing properties. Thiazolidinediones, in particular, have received much attention, not only for the well documented hepatotoxicity of troglitazone that led to its removal from the market in 2000, but also for the emerging data that support the beneficial effects of the thiazolidinedione class of drugs on beta-cell rejuvenation and cardiovascular risk reduction. In the US, thiazolidinediones are indicated either as monotherapy or in combination with a sulfonylurea, metformin, or insulin in cases where diet, exercise, and a single drug fail. In contrast, the UK National Institute for Clinical Excellence included in its re-appraisal of 'glitazones' in August 2003 the continued exclusion from licensed use in the UK of combination therapy with thiazolidinediones and insulin. When added to insulin therapy, thiazolidinediones appear to effectively lower glucose levels and reduce insulin dosage in clinical trials involving individuals with poorly controlled type 2 diabetes. However, weight gain, hypoglycemia, and fluid retention pose problems in certain patients. The fluid retention may exacerbate or even precipitate congestive heart failure, which usually necessitates discontinuation of the drug. Risk stratification and careful management of patients at risk for heart failure, including those taking insulin concomitantly, allow healthcare providers to safely administer combination therapy with thiazolidinediones in patients with type 2 diabetes. Hepatic toxicity with currently available thiazolidinediones has been found to be minimal overall.

A non-dairy probiotic's (poi) influence on changing the gastrointestinal tract's microflora environment.

JUSTIFICATION: Yogurt has been historically used to restore gut microflora adversely affected by antibiotic treatment. Certain fermented dairy products are probiotics; "live microorganisms which when administered in adequate amounts confer a health benefit to the host." Microorganisms in foods may benefit certain health conditions such as diarrhea, gastroenteritis, irritable bowel syndrome, inflammatory bowel disease, and cancer. A potential new probiotic from a Polynesian traditional food is poi; a starchy paste made from the corm of taro plants. OBJECTIVE: The purpose of this study was to determine if consumption of poi, a potential non-dairy probiotic, altered the microflora in the gastrointestinal tract of healthy adults. METHODS: A cross-over clinical study included 18 subjects (19-64 years of age) divided into a poi group (n = 10) and control group (n = 8). The study duration of 14 weeks consisted of a 2-week washout, 4-week treatment or control, a subsequent 2-week washout, cross-over of 4-week treatment or control, and a final 2-week washout. Subjects thus served as their own controls. While receiving the poi treatment, participants consumed fresh poi (1-2 days old) three times a day (130 g/meal or approximately 1/2 cup/meal); the control group did not. Both groups filled out 3-day dietary records to ensure compliance. Measurable outcomes included pre- and post-treatment microbiological fecal culture analyses. RESULTS: We found no significant differences in total bacterial counts following a poi diet versus following a control diet, nor were significant differences found in counts of specific bacterial species. Lactococcus tends to be higher in poi when it is analyzed for specific bacteria, but the poi consumption in our study did not alter the mean concentration of individual bacterial species (log10 CFU/g wet feces) for Escherichia coli, Enterobacter, Klebsiella, Lactobacillus, Lactococcus, and Bifidobacterium. No significant differences in stool frequency or consistency were observed between the treatment and control group periods. CONCLUSION: Poi consumption did not significantly alter total or individual bacterial counts in the human gastrointestinal tract. Further research might determine if "sour poi" (3-4 days old) has a greater affect than "fresh poi" (1-2 days old) as a potential probiotic, and a larger trial with longer diet durations may detect more subtle effects of poi consumption on bacterial counts.

Aggregate restructuring by polymer solvency effects.

This study investigates the aggregation in cyclohexane of silica particles initially stabilized by grafted polystyrene and destabilized by temperature reduction. It complements an earlier study by Zhu and Napper (P.W. Zhu, D.H. Napper, Phys. Rev. E 50 (1994) 1360) in which the aggregation of polystyrene latex particles with tethered poly(N-isopropyl acrylamide) (PNIPAM) in water was investigated. Their dynamic light scattering results showed that both the rate of aggregation and the aggregate fractal dimension increased with a sufficient decrease in the PNIPAM adlayer solvency, achieved by means of either salt (NaNO3) addition or temperature rise. This result stands in contrast to those obtained when an electrostatically stabilized colloid is destabilized, i.e., that the more rapidly aggregates are formed, the lower the resulting fractal dimension. The authors explained their results in terms of the effects of both salt effects and increased temperature on the extent of the hydrophobic interactions between the adlayer-covered surfaces in the water. The present study examines a sterically-stabilized colloid in a nonaqueous solvent, where neither salt effects nor hydrophobic effects play a role. Temperature is decreased to bring the system from better-than-theta-conditions to worse-than-theta-conditions. Power-law aggregation kinetics are observed at 15.7 degrees C by dynamic light scattering. The particles first undergo reduced rate aggregation, producing low-fractal-dimension aggregates, which after some time, restructure into more compact aged clusters. The fractal dimension of these aged clusters increases with increasing initial aggregation rate, consistent with results seen by Zhu and Napper, but without the presence of hydrophobic effects. The ability of the polymer-grafted particles to rearrange suggests aggregation into a secondary minimum, with the ability to slide over one another to achieve a more energetically favorable, denser configuration. The reversible nature of the aggregation is verified by additional experiments gradually bringing the system from worse-than-theta-conditions back to better-than-theta-conditions, with an attendant decrease in aggregate fractal dimension, and ultimately full redispersion.