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Background Bladder cancer (BC) is the 5th most common cancer in the USA. Non-muscle invasive bladder cancer represents about 70% of all cases and has generally a favorable outcome. However, recurrence rates as high as 60 to 70% and progression rates of 10 to 20% necessitate intensive surveillance with cystoscopy. The invasiveness and high cost of cystoscopy poses significant burden on BC patients as well as on the healthcare system. In this study we test the feasibility of a simple, sensitive, and non-invasive detection of BC using Bladder CARE test in urine samples. Results Urine from 136 healthy and 77 BC subjects was collected using the at-home Bladder CARE Urine Collection Kit and analyzed with Bladder CARE test. The test measures the methylation level of three BC-specific biomarkers and two internal controls using methylation-sensitive restriction enzymes coupled with qPCR. Bladder CARE showed an overall sensitivity of 93.5%, a specificity of 92.6%, and a PPV and NPV of 87.8% and 96.2%, respectively. Bladder CARE has an LOD as low as 0.046%, which equates to detecting 1 cancer cell for every 2,200 cells analyzed. We also provided evidence that bisulfite-free methods to assess DNA methylation, like Bladder CARE, are advantageous compared to conventional methods that rely on bisulfite conversion of the DNA. Conclusion Highly sensitive detection of BC in urine samples is possible using Bladder CARE. The low LOD of the test and the measurement of epigenetic biomarkers make Bladder CARE a good candidate for the early detection of BC and possibly for the routine screening and surveillance of BC patients. Bladder CARE and the at-home urine sample collection system have the potential to (1) reduce unnecessary invasive testing for BC (2) reduce the burden of surveillance on patients and on the healthcare system, (3) improve the detection of early stage BC, and (4) allow physicians to streamline the monitoring of patients.

Introduction: Bladder cancer (BC) is the 5th most common cancer in the USA, with 81,190 new cases and 17,240 deaths in 2018. The high recurrence rate of BC requires a lifelong patient follow-up with cystoscopy. However, the invasiveness of cystoscopy and the high costs associated to this procedure, poses a significant burden on patients as well as on the healthcare system. Several tests for the non-invasive detection of BC have been developed in the past years however, because of relatively low sensitivities and/or specificities (especially for low-grade tumors), these tests have not been adopted for the routine clinical use. Therefore, a non-invasive, simple and sensitive test for detection and routine monitoring of BC is needed. Methods: Bladder CARE is a new non-invasive and quantitative test that measures the methylation levels of three BC specific biomarkers and two internal controls in urine samples. Contrary to other epigenetic tests, Bladder CARE analyzes DNA methylation without converting the DNA with sodium bisulfite, thus minimizing the degradation of the target DNA. In this clinical study, we evaluated the performance of this test using voided urine samples collected from 136 healthy individuals and 77 BC patients with no prior history of bladder cancer. Urine specimens were collected and stabilized using Bladder CARE at-home urine sample collection kit and BC detection was performed by a CLIA-certified and CAP-accredited laboratory (Pangea Laboratory LLC) using Bladder CARE. The results were then compared to the clinical information (cystoscopy). Additionally, the test linearity and limit of detection (LOD) were determined by analyzing artificial samples with Bladder CARE test. Results: Bladder CARE was able to discriminate BC from healthy control samples with a sensitivity of 93.5%, a specificity of 92.6%, and a positive and negative predictive value of 87.8% and 96.2%, respectively. Remarkably, low-grade tumors were detected with a sensitivity of 90.0%. We also found that Bladder CARE test has a LOD of 0.046% (the equivalent of detecting 1 cancer cell in a sample containing 2,200 normal cells). Conclusions: While cystoscopy remains the gold standard to confirm the presence of BC, the superior sensitivity of Bladder CARE test, together with its low LOD, make this test an effective early indicator of low-grade tumors and for routine recurrence monitoring of BC patients. In addition, Bladder CARE urine collection kit allows to collect and stabilize the urine specimen comfortably at-home and mail it to the testing laboratory at room temperature, streamlining the patients' detection and monitoring process. Citation Format: Paolo Piatti, Michiko Suwoto, Taikun Jamada, Yap Ching Chew, Gangning Liang, Saum Ghodossipour, Siamak Daneshmand, Xi-Yu Jia. Bladder care epigenetic test allows the sensitive early detection of bladder cancer from urine samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2295.

Background: DNA methylation clocks have been developed recently to estimate epigenetic age which is associated with chronological age in mouse and human. Changes in epigenetic age were reported in patients with health conditions including cancer. Different from genetic variations, epigenetic properties can be modified by environmental factors. Epigenetic age tests using blood samples have been offered directly to customers recently. We sought to analyze relationships between epigenetic aging and lifestyle factors, medication and aging interventions in the hope of assisting healthier living. Methods: Epigenetic age, called DNAge, is estimated using linear regression of DNA methylation levels at 206 loci containing 1344 CpGs and chronological age. Delta age is defined as the difference of epigenetic age minus chronological age. Correlations between lifestyle factors or medication and delta age of hundreds of whole blood samples were evaluated using biweight midcorrelation. Statistical significance is set at 0.05. Results: Our results showed positive correlations between accelerated epigenetic aging and BMI (bicor=0.148, p=0.009, n=311), current smoker (bicor=0.143, p=0.011, n=315), or stress (bicor=0.088, p=0.128, n=302). Factors with positive correlation but not reaching statistical significance included total cholesterol, alcohol consumption and Keto diet. Factors with negative correlation but not statistically significant included exercise, balanced diet, mostly meat, mostly vegetables, and education. Conclusions: The epigenetic age analysis of real customer data confirmed beneficial and adversarial health effects of certain lifestyle factors. The DNAge test offered proves to be useful as a biomarker for biological age and as a practical tool for monitoring and evaluation of health status which may be used in cancer epidemiology study and help cancer prevention. Citation Format: Wei Guo, Yap Ching Chew, Xiaojing Yang, Michiko Suwoto, Taikun Yamada, Xi Yu Jia. Correlation of lifestyle factors with epigenetic aging unveiled in the practice of a DNA methylation-based age test [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2332.

Aging represents the most important risk factor for many chronic diseases including cardiovascular diseases, diabetes, and cancer, therefore understanding the mechanisms of aging is a fundamental s...

Aging represents the most important risk factor for many chronic diseases including cardiovascular diseases, diabetes, and cancer, therefore understanding the mechanisms of aging is a fundamental step for designing new treatments for chronic diseases. DNA methylation is the most reliable and accurate molecular marker for aging quantification, however, genome-wide DNA methylation profiling techniques, such as reduced representative bisulfite sequencing and Illumina Bead Array that are widely used in aging research are prohibitively expensive and have poor data quality at low-read coverage sites. Here we report a robust targeted bisulfite sequencing approach, called SWARM® (Simplified Whole-panel Amplification Reaction Method), for the accurate biological age determination. SWARM™ is flexible and low cost, requires relatively low DNA starting material, allows the simultaneous amplification and sequencing of hundreds of loci, and has shown to increase sample throughput. Using the SWARM® approach, we were able to analyze the methylation level of several hundreds of age-associated loci including the published Horvath Clock sites. Gender-specific age-predictive models were built using the elastic net regression of DNA methylation levels of the loci and chronological age of urine DNA samples of over 300 healthy subjects of 18 to 88 years old. Urine samples from bladder cancer patients exhibit significant age acceleration, with an average of >10 years. In brief, our gender-specific urine DNAge® analysis is a tool for the precise biological aging quantification and can be used to address questions in aging and urinary track cancers. Citation Format: Yap Ching Chew, Wei Guo, Xiaojing Yang, Paolo Piatti, Mingda Jin, Keith Booher, Benjamin Jara, Xi-Yu Jia. Accelerated epigenetic aging in bladder cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 828.

In this investigation we report on the influence of volumetric flow rate, flow velocity, complementary DNA concentration, height of a microfluidic flow channel and time on DNA hybridization kinetics. A syringe pump was used to drive Cy3-labeled target DNA through a polydimethylsiloxane (PDMS) microfluidic flow channel to hybridize with immobilized DNA from the West Nile Virus. We demonstrate that a reduction of channel height, while keeping a fixed volumetric flow rate or a fixed flow velocity, enhances mass transport of target DNA to the capture probes. Compared to a passive hybridization, the DNA hybridization in the microfluidic flow channel generates higher fluorescence intensities for lower concentration of target DNA during the same fixed period of time. Within a fixed 2min time period the fastest DNA hybridization at a 50pM concentration of target DNA is achieved with a continuous flow of target DNA at the highest flow rate and the lowest channel height.

The relationship between oxidative stress and neuronal cell death has been suggested for many years. To understand the influence of oxidative stress on neuronal cell death, we investigated the influence of oxidative stress on DEV cells, a human glial cell line. Using enterovirus infection and/or malnutrition to induce oxidative stress, our results demonstrate that those stressors severely influence the antioxidant defense system in DEV cells. Although the expression of mitochondrial manganese superoxide dismutase (MnSOD) in DEV cells was significantly increased in acute infection with viral and nutritional stress, in persistent infection and nutritional stress, the expression of the MnSOD was drastically downregulated. We believe that this downregulation of MnSOD expression in the chronic stress model is due to repression of antioxidant defense. The downregulation of the MnSOD expression may lead to an increase of free-radical production and thus explain why the cells in the chronic stress model were more vulnerable to other oxidative stress influences. The vulnerability of DEV cells to additional stress factors resulted in progressive cell death, which may be analogous to the cell death in neurodegenerative diseases.

Current methods for genomic mapping of 5-hydroxymethylcytosine (5hmC) have been limited by either costly sequencing depth, high DNA input, or lack of single-base resolution. We present an approach called Reduced Representation 5-Hydroxymethylcytosine Profiling (RRHP) to map 5hmC sites at single-base resolution by exploiting the use of beta-glucosyltransferase to inhibit enzymatic digestion at the junction where adapters are ligated to a genomic library. Therefore, only library fragments presenting glucosylated 5hmC residues at the junction are sequenced. RRHP can detect sites with low 5hmC abundance, and when combined with RRBS data, 5-methylcytosine and 5-hydroxymethylcytosine can be compared at a specific site. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0456-5) contains supplementary material, which is available to authorized users.

A trans -encapsidation assay was established to study the specificity of picornavirus RNA encapsidation. A poliovirus replicon with the luciferase gene replacing the capsid protein-coding region was coexpressed in transfected HeLa cells with capsid proteins from homologous or heterologous virus. Successful trans -encapsidation resulted in assembly and production of virions whose replication, upon subsequent infection of HeLa cells, was accompanied by expression of luciferase activity. The amount of luciferase activity was proportional to the amount of trans -encapsidated virus produced from the cotransfection. When poliovirus capsid proteins were supplied in trans , >2 × 10 6 infectious particles/ml were produced. When coxsackievirus B3, human rhinovirus 14, mengovirus, or hepatitis A virus (HAV) capsid proteins were supplied in trans , all but HAV showed some encapsidation of the replicon. The overall encapsidation efficiency of the replicon RNA by heterologous capsid proteins was significantly lower than when poliovirus capsid was used. trans -encapsidated particles could be completely neutralized with specific antisera against each of the donor virus capsids. The results indicate that encapsidation is regulated by specific viral nucleic acid and protein sequences.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.

e18199Background: Urine represents an extremely important body fluid for the sensitive and non-invasive detections of multiple diseases including urinary tract infections and cancers. Recently, uri...

Hepatitis A virus (HAV) cDNAs encoding the P3 region proteins were expressed in vivo and in vitro to characterize the HAV 3D protein and to identify the cleavage site between 30 and 3D. Protein coding sequences were placed under control of a T7 promoter and an EMCV translational initiation signal. T7 RNA polymerase was provided by simultaneous infection of transfected BS-C-1 cells with a recombinant vaccinia virus vTF7-3 (T. R. Fuerst et al., Proc. Natl. Acad. Sci. USA 83, 8122-8126, 1986). Efficient synthesis and processing of P3 proteins occurred to yield 3CD (78 kDa), 3D (54 kDa), 3ABC (33 kDa), 3BC (25 kDa), and 3C (23 kDa). Similar products were produced by translation of T7 transcripts in a rabbit reticulocyte lysate in vitro. The 3C/3D cleavage site was mapped by comparing the mobility of 3D in SDS-PAGE with 3D proteins engineered to begin at each of the two proposed cleavage sites; in addition, direct N-terminal sequencing of radiolabeled 3D protein from translation in vitro was performed. The results showed that 3D was formed by cleavage at the glutamine-arginine (Q/R) pair at position 1738 and 1739 of the HAV polyprotein. HAV 3D protein produced by autocatalytic cleavage of P3 precursor proteins in BS-C-1 cells is virtually completely insoluble and sediments after low-speed centrifugation. This is in contrast to the poliovirus 3D protein, produced from a similar construct, a significant portion of which remains soluble. Extracts containing the poliovirus 3D protein manifested high levels of RNA-dependent RNA polymerase activity, whereas those containing the HAV 3D protein showed no detectable activity by the same assay.

The nonstructural proteins of hepatitis A virus (HAV), produced during active virus replication, are alternative antigens that could be used to differentiate disease from inactivated vaccine-induced antibodies. An assay based on immune precipitation of proteins translated from transcripts of the P2 region of viral cDNA was used to evaluate the development of antibodies after natural infection or vaccination. Antibodies against P2 proteins were found in all sera from clinical cases of hepatitis A following the acute phase. Chimpanzees vaccinated with inactivated or cell-adapted HAV had no detectable antibodies against P2 products, either before or after wild type virus challenge. A serosurvey of sera positive for total anti-HAV (HAVAB, Abbott Laboratories, North Chicago) suggested that some individuals had no detectable antibodies to the P2 antigen by immune precipitation. These results were attributed to the lower sensitivity of the immunoprecipitation assay, since antibodies to capsid proteins, as measured by immunoprecipitation, were also not detected in most of these sera. © 1993 Wiley-Liss, Inc.

Hepatitis A virus (HAV), a RNA virus of positive polarity, contains a long 5' noncoding region (5'NCR) that lacks the characteristic m7GpppN cap group of most eukaryotic messages. By creating bicistronic constructs that contain the bacterial chloramphenicol acetyltransferase gene followed by the HAV 5'NCR and the luciferase gene we have demonstrated by assaying in vitro and in vivo that ribosome entry for translation initiation occurs via binding to sequences within the HAV 5'NCR. Using mutations created within this region we have identified that the HAV internal ribosome entry site (IRES) is located downstream of nucleotide 45 and including sequences up to nucleotide 734 of the HAV 5'NCR. Translation of a number of mutant constructs both in vitro in a rabbit reticulocyte lysate and in vivo by transfection of the cDNAs into BS-C-1 cells in the presence of the recombinant vaccinia virus, vTF7-3, gave similar results. However, a 4-nucleotide insertion at base 628 showed an increased activity over wild-type when transfected into BS-C-1 cells that was not seen in vitro. This increase in activity correlated with an increase in luciferase gene product as assayed by immunoprecipitations of [35S]methionine radiolabeled cells. Comparison of mono- and bicistronic RNAs that were synthesized with or without a m7GpppG cap group showed a competition for ribosome binding when translated in a rabbit reticulocyte lysate system. The presence of the cap group on the RNA 5'terminus of the RNA led to a greater ability of this RNA to translate than the RNA containing the HAV IRES.

Subgenomic hepatitis A virus (HAV) RNA sequences were translated in vitro to produce proteins representing the structural (P1) and nonstructural (P2 and P3) domains of the viral polyprotein. These proteins were used as antigens to detect the presence of antibodies in sera from acute and convalescent humans and an experimentally infected chimpanzee. All infected individuals tested had antibodies that recognized uncleaved P1 proteins as well as nonstructural proteins. Antibodies in sera from infected individuals recognized conformation-dependent epitopes that were sensitive to SDS and heat treatment. Time-course studies of the experimentally infected chimpanzee showed that antibodies to the HAV proteins were detectable between 24 and 31 days after infection and persisted for greater than 6 months. Human sera remained positive for antibodies to both structural and nonstructural antigens for at least 2 1/2 years. The data suggest that HAV nonstructural proteins could be used as serologic markers for HAV diagnosis and for evaluating field trials of inactivated vaccines.

Currently, the immune status of an individual exposed to hepatitis A virus (HAV) is determined by assays which measure antibodies against the capsid proteins. These assays indicate exposure to the viral capsid that could result from either infection or from vaccination. Recent data indicate that proteins from the non-structural genome region of the virus (P2 or P3), which are only produced during active virus replication, generate antibodies after clinical disease. A sub-genomic cDNA segment of HAV corresponding to the P2 region was used for in vitro transcription-translation followed by immune precipitation of the translated products under non-denaturing conditions. Serial serum specimens from experimentally infected chimpanzees and humans naturally infected with hepatitis A verified the development of antibodies to P2 proteins following infection. A serosurvey of individuals positive for antibodies to the HAV capsid (HAV AB assay, Abbott Laboratories) revealed that 50-60% of children and 16-32% of adults had no detectable antibodies to the P2 antigen by immune precipitation. These results may reflect subclinical infections resulting in a lower level of antibodies against the non-structural antigens or may represent a greater sensitivity of the competitive assay (HAV AB) used to detect capsid antibodies compared to the immunoprecipitation assay used to detect non-structural antigens.

DNA methylation plays an important role in normal organismal development and in cellular differentiation in higher organisms. Changes in DNA methylation have been shown to correlate with disease risk, response to therapy and survival in a wide range of clinical conditions such as cancer and autoinflammatory diseases. Recent advances in next-generation sequencing and microarray technology have made it possible to map DNA methylation genome-wide, at a high resolution. However, these genomic assays tend to be costly, labor-intensive and impractical in the clinic. Thus, DNA methylation assays that measure a small number of genomic regions in large cohorts are needed for validating biomarker candidates discovered from genomic studies. For this reason, we have developed a targeted bisulfite sequencing platform for simultaneously measuring DNA methylation in multiple loci with multiple samples by combining microfluidics-based technology with next generation sequencing. First, post-bisulfite PCR primers for each target region were designed using our proprietary program Bisulfite Primer Seeker, and then experimentally validated using a standard genomic control. Primers which pass the validation were used for the subsequent target amplification. Target amplification were performed using Fluidigm Access Array 48.48 which allows parallel PCR reactions for 48 samples by 48 single-plex assays. An attractive aspect of this platform is that relatively small quantities of template are required (∼50 ng/sample). Assays can also be multiplexed to improve throughput. Furthermore, as with many targeted approaches, index or barcoding tags can be incorporated into the universal adapter regions of the PCR product enabling the pooling of samples before direct sequencing. Using this platform we have evaluated 9 genomic loci with 9 artificial mixed samples containing different methylation levels. Results showed the target bisulfite sequencing platform to be robust and reproducible. The methylation value between the technical replicates were highly correlated. Each of the amplicons was covered by a thousand or more reads, making the detection sensitivity reach down to the low methylation levels. Compared with other target bisulfite sequencing methods, our assay has several advantages. First, it uses relatively small amounts of gDNA as input, making it practical for most clinical samples. Secondly the workflow is simple, straightforward and can be automated. Finally, it avoids the complexity and difficulties of multiplex PCR by utilizing microfluidics-based single-plex PCR. All these features make it an ideal platform for DNA methylation biomarker validation. Citation Format: Emily Putnam, Lam Nguyen, Hunter Chung, Peisheng Shi, Xueguang Sun, Marc E. Van Eden, Xi-Yu Jia. A targeted bisulfite sequencing method combining microfluidics-based PCR with Next-Gen sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2956. doi:10.1158/1538-7445.AM2015-2956

The distribution of 5-methylcytosine (5-mC) in DNA within the eukaryotic genome is known to greatly affect gene regulation and is currently a major topic of research. Studies on DNA methylation have been aided by advancements in bisulfite conversion and Next-Gen sequencing technologies which can provide single-base resolution of 5-mC across the entire genome. Many whole-genome bisulfite sequencing (WGBS) library preparation protocols designed for analysis of 5-mC distribution employ bisulfite to chemically convert unmethylated cytosine bases into uracil following the library preparation step. While these protocols produce reliable results, degradation of DNA is always an inherent issue when dealing with bisulfite conversion, and a large proportion of the adapterized library is often fragmented and can no longer be amplified. This requires high levels of DNA to serve as the input material. However, by rearranging the order of library preparation and bisulfite conversion, we developed a streamlined protocol that necessitates less input material for accurate whole-genome methylation analysis at single-base resolution. This unique workflow accommodates picogram quantities of starting material, making it ideal for analysis of precious samples that are of limited availability (e.g., FFPE). Comparison of sequencing data generated using this new library preparation method and data generated with established Reduced Representation Bisulfite Sequencing demonstrated a correlation of 0.95 in CpG sites with > 10X coverage in human DNA. Also, with only slight modification, this protocol is versatile enough to be used in the preparation of libraries for ChIP-Seq and RNA-Seq analyses. Citation Format: Karolyn Giang, TzuHung Chung, Xueguang Sun, Marc E. Van Eden, Xi Yu Jia. A fast and simple method for whole-genome bisulfite library preparation from ultra-low DNA inputs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2293. doi:10.1158/1538-7445.AM2014-2293

5-hydroxymethylcytosine (5hmC) is an epigenetic mark abundant in embryo stem cells and brain tissues. The exact biological functions of 5hmC are still under close investigation although several lines of evidence have indicated it could be involved in active DNA demethylation. Meanwhile, extensive studies have been carried out to determine its genomic distribution. A number of approaches have been developed using either affinity based enrichment, such as hMeDIP, that rely on antibody and other specific binding proteins to target 5hmC, or modified bisulfite sequencing, namely oxidative bisulfite sequencing (OxBS) and TET assisted bisulfite sequencing (TAB-sequencing). However, all those methods have limitations which hamper their application. For example, affinity based methods lack single base resolution while modified bisulfite sequencing methods require efficient chemical or enzymatic oxidation which cannot be easily achieved or guaranteed. As an alternative, we have developed a novel genome-wide sequencing method that utilizes an enzyme based modification approach coupled with bisulfite-sequencing for detecting 5hmC. This methodology allows quantification of 5hmC levels with single CpG resolution and can also be employed for locus-specific assays. Using this method, we were able to map and quantify 5hmC sites at the genomic scale for several different biological samples. This novel method can determine the exact location and abundance of 5hmC, which will facilitate our understanding of 5hmC in regulating gene expression in different biological contexts. Citation Format: Xueguang Sun, Tzu-Hung Chung, Yap Ching Chew, Darany Tan, Xi-Yu Jia. A novel sequencing method for genome-wide profiling of 5-hydroxymethylcytosine with single-base resolution. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 374. doi:10.1158/1538-7445.AM2014-374

5-Hydroxymethylcytosine (5-hmC) is a new epigenetic hallmark rapidly getting much interest as the subject of mapping and sequencing work. While the exact function of 5-hmC is not fully understood, it is likely to regulate gene expression via active DNA de-methylation. Previous studies have shown it may play an important role in cell differentiation and carcinogenesis. Cells that are more stem- and progenitor-like have greatly reduced levels of 5-hmC compared with more differentiated cells. Similarly, tumor cells display less 5-hmC than their normal counterparts. This was not associated with either grade or stage, suggesting that global loss of 5-hmC may be an early event in carcinogenesis. To date, several methods have been developed to profile 5-hmC at the genomic level: most are enrichment-based and utilize antibodies to 5-hmC or its modified forms or make use of bioorthogonal labeling and pull-down of glycosylated 5-hmC. The caveat is that these approaches are low resolution and that they require large amounts of input genomic DNA. Initial efforts for detection of 5-hmC at single-base resolution require several micrograms of DNA, require parallel or subtractive sequencing, and employ successive chemical treatments that can degrade DNA and hinder sequencing. Here we report on a new method that by combining modification-sensitive restriction enzymes with massively parallel (“next-generation”) sequencing approaches, genome-wide 5-hmC can be mapped at single site resolution from low (100 ng) DNA inputs. Importantly, this method can be used for strand-specific localization of 5-hmC as well as direct identification of single nucleotide polymorphisms (SNPs) within the sequencing reads. Data can also be compared directly with single-base resolution DNA methylation data from reduced-representation bisulfite sequencing (RRBS) for simultaneous 5-mC and 5-hmC profiling from the same sample. 5-mC and 5-hmC of human brain DNA using this combined method indicates unique distributions of the 5-hmC modification. This new method should provide a unique tool in enhancing our understanding of the interplay of genetic and epigenetic regulations in carcinogenesis. Citation Format: XueGuang Sun, Adam Petterson, Hunter Chung, Xi Yu Jia, Marc E. Van Eden. A novel method for sequence- and strand-specific, genome-wide 5-hmC profiling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4230. doi:10.1158/1538-7445.AM2013-4230

Sera from recent and past cases of poliomyelitis as well as from healthy subjects vaccinated with live attenuated poliovirus, all contain antibodies to poliovirus nonstructural proteins. Reactivity of these antisera with nonstructural protein antigens present in virus-infected HeLa cells or translated from cDNA transcripts in rabbit reticulocyte lysates was sometimes sensitive to denaturation of the antigens. The finding of antibodies to nonstructural proteins of poliovirus and other viruses should have applications in the development of diagnostic assays for virus infection and may have implications for future vaccine design.

Background: Colorectal adenoma (CRA) to cancer (CRC) progression involves in epigenetic changes including DNA methylation. Here we performed whole genome Bisulfite Next-Generation sequencing (WGBSNGS) on a normal, a tubular adenoma and a tumor patients’ tissue DNA to elucidate epigenetic drivers in normal to cancer progression. Patients and Methods: Genomic DNA was isolated from fresh frozen tissues from a patient with normal colon, from a tubular adenoma lesion (20X depth was performed on these DNA samples., Alignment, mapping and CpG methylation analyses were done. Pyrosequencing and immunohistochemistry (IHC) was used to confirm selected CpG methylation. Results: We identified 94 unique CpG sites hypermethylated in 4 novel genes in adenoma compared to normal and 180 unique CpG sites in 7 genes in cancer. Ingenuity pathway analysis (IPA) showed that the methylated genes including a novel gene ATXN7L1 were involved in WNT pathway, ubiquotlation process and cross-talk with B-catenin, in the adenoma. Three of the hypermethylated genes in cancer were, Loc100506436: a novel gene with 17 unique CpG sites (EGFR pathway, cRNA CpG site hypermethylated is part of MEK1/2), GPNMB with 7 unique CpG sites (a G protein) and TNFAIP2 with 8 unique CpG sites (its role was noted in Head and neck cancer). IHC confirmed the lack of expression in cancer. Conclusion: This work provides insight into differential CpG island methylation profiles in CRA, CRC vs. normal colon tissue and provides a window into the more complex factors, including alteration of known and novel genes as a multiple CpG methylation drivers in colorectal carcinogenesis. Investigations into the possible roles of the novel gene targets in the context of early and prognostic methylation biomarker are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4521. doi:1538-7445.AM2012-4521

Hepatocellular carcinoma (HCC) is one of the most common and lethal malignancies worldwide, accounting for approximately half a million annual deaths globally. HCC is completely asymptomatic in the early stages of the disease; therefore, early detection of HCC in afflicted patients is vital to receive therapeutic benefits from curative surgery. The standard diagnosis of HCC relies upon detection of the serum alpha-fetoprotein (AFP) level in at-risk subjects followed by hepatic ultrasonography to identify suspicious nodules. Accurate levels of AFP are often difficult to detect, and the imaging method to identify nodules is both operator-sensitive and subject to a high false-negative rate. While it is known that hepatocellular carcinoma is a multi-step process that requires altered expression of multiple genes, recent evidence has indicated that epigenetic abnormalities also play an important role in hepatocarcinogenesis. The discovery of reliable and accurate epigenetic biomarkers may open up new avenues for the development of novel diagnostic tools and provide new target for therapeutic interventions. Combining Next Generation Sequencing methods with well-established bisulfite conversation chemistry, a unique genome-wide epigenetic profile for HCC was done in liver tumor and normal tissues. Hypermethylated and hypomethylated gene regions from the genome-wide epigenetic profile were then validated for percent methylation in liver tumor and normal tissues using a streamlined system of methylation-sensitive restriction enzyme (MSRE) digestion combined with real-time PCR. Serum circulated genomic DNA from liver cancer patients were also examined to look for potential epigenetic markers for clinical diagnostic applications. More than one hundred gene loci were identified at differentially methylated regions (DMRs) by comparing several different stage liver tumors with their correspondent normal tissue by the genome wide epigenetic profile. Calculated CT values from combined MSRE digestion and real-time PCR quantitation indicated that percent methylation of the CpG island in the gene ADAM8 was highest in stage IV liver tumor tissue, while percent methylation of the CpG island in CSMD3 was lowest in stage IV tissue compared to normal adjacent tissue. Percent methylation of the CpG island in ZNF783 was high in all stages of HCC tumor liver tissue compared to normal adjacent tissue. Epigenomic profiling and screening with MSRE digestion and real-time PCR identified and validated differences in the CpG island methylation status of three gene loci in different stages of HCC liver tissue and normal tissue. Extensive validation of these gene specific loci in larger numbers of HCC samples is necessary before they can be considered possible epigenetic biomarkers for novel diagnostic tools and therapeutic interventions for HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4026. doi:1538-7445.AM2012-4026

Epigenetic mis-regulation leading to diseases such as cancer is classically understood in terms of aberrant DNA methylation patterns. For decades, 5-methylcytosine (5-mC) has been the focus of epigenetic DNA modification studies. But recently, a novel epigenetic DNA modification, 5-hydroxymethylcytosine (5-hmC), has been shown to be present in mammalian brain and ES cells. Current tools available to map DNA methylation (e.g. bisulfite conversion) cannot distinguish between 5-mC and 5-hmC. Here we describe a new method for locus specific interrogation of 5-hmC within DNA. Treatment of DNA with a 5-hmC glucosyltransferase enzyme specifically adds a glucose moiety onto preexisting 5-hmC residues, yielding glucosyl-5-hydroxymethylcytosine (glu-5-hmC). Subsequent digestion of DNA with glu-5-hmC sensitive restriction endonucleases effectively cleaves DNA with cytosine, 5-mC, or 5-hmC within their recognition site. But when glu-5-hmC is present, DNA is not able to be cleaved. Exploiting this difference in “cleavability”, 5-hmC can be detected at loci of interest using qPCR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3006. doi:10.1158/1538-7445.AM2011-3006

The VP4 capsid protein of several picornaviruses has been shown to be myristoylated at an N-terminal glycine residue. Myristoylation occurs after removal of an initial methionine residue or a leader peptide, resulting in the exposure of an N-terminal eight amino acid myristoylation signal including a consensus G-x-x-x-T/S motif. Analysis of the amino acid sequence of hepatitis A virus (HAV) capsid protein reveals a potential myristoylation site beginning at position 5 of the VP4 sequence. To assess the significance of this apparent myristoylation signal, mutations were engineered (G to A; T to N) to alter the consensus sequence as well as the potential cleavage site that would be required to remove the short leader. An additional mutant was constructed in which the proposed leader was deleted. Expression of these HAV sequences in BS-C-1 cells showed that leader cleavage did not occur in the wild-type or mutant proteins, although the threonine to asparagine mutation resulted in reduced translation and processing efficiency. Transfection of BS-C-1 or FRhK-4 cells with transcripts derived from the wild-type or mutagenized cDNA clones gave rise to infectious virus, with no detectable incorporation of myristate. The results indicate that HAV does not require leader cleavage and myristoylation of VP4 for growth in cultured cells.

Portions of the P1 and P2 hepatitis A virus (HAV) polyprotein were generated by in vitro translation of cDNA transcripts and analyzed for a primary cleavage reaction that would release the capsid protein precursor. No autocatalytic activity was observed on either side of the 2A coding sequence. Incubation of these proteins with an extract containing active HAV 3C protease, however, resulted in cleavage at a position approximately 40 amino acids downstream of the previously proposed P1-2A junction. This cleavage site likely accounts for the VP1-containing proteins of approximately 38-40 kDa observed in HAV-infected cells.

Hepatitis A virus (HAV) RNAs were translated in vitro in rabbit reticulocyte lysates. The pattern of proteins synthesized from full-length HAV RNA was highly complex, consisting of a continuous spectrum of polypeptides ranging from less than 20,000 to greater than 200,000 Da. The pattern was not significantly altered by varying incubation times, ion, or other reaction parameters, or by the addition of HeLa or BS-C-1 cell extracts to the translation reactions. Plasmids engineered with mutations in the 3C coding region produced transcripts which directed the synthesis of the same overall pattern of polypeptide products as those transcribed from wild-type sequences, suggesting that protein processing by 3C did not generate the complex set of protein products. Translation of RNA containing only the P3 coding region of HAV, directly adjacent to the HAV 5' noncoding region, generated a set of protein products which precisely matched a subset of those synthesized from full-length HAV RNA. The translation products of P3 RNA, full-length RNA, and mutant 3C-containing RNAs were analyzed by immunoprecipitation with antisera specific for 3D, VP1, and 2C sequences; several products were subjected to N-terminal sequence analysis. All together, the results demonstrate that translation of HAV RNA in rabbit reticulocyte lysates initiates predominantly at a large number of internal AUG codons, especially those in the P3 coding region. A minor population of products is initiated from sites in the P1 and P2 regions. The latter proteins undergo some proteolytic processing, at unidentified sites, catalyzed by 3C protein sequences. Replacement of the HAV 5' noncoding region with encephalomyocarditis virus 5' end sequences increased initiation at the correct polyprotein start site and both reduced and altered the products generated by internal initiation.

Although the genome organization and overall structure of hepatitis A virus are similar to those of other picornaviruses, nothing is known about the protein-processing pathways used by this virus to generate its capsid and nonstructural proteins from the polyprotein precursor. RNA transcripts of cloned hepatitis A virus cDNAs representing parts of the P2 and P3 regions of the genome were translated in rabbit reticulocyte lysates in vitro, and the translation products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after immunoprecipitation with specific antisera. Pulse-chase experiments demonstrated rapid cleavage at the P2-P3 junction, followed by further but incomplete processing at the 3C-3D junction. Mutation of the 3C coding sequence eliminated all cleavages. Efforts to demonstrate intermolecular cutting of the P2-P3 cleavage site by active 3C or 3CD sequences were unsuccessful; thus, it is likely that this cleavage occurs by intramolecular reaction, in cis.

Summary Analysis of the genome of the flavivirus responsible for the 1999 New York City encephalitis epidemic cloned from human brain by reverse-transcription polymerase chain reaction indicates its identity as a lineage I West Nile virus (WNV; WNV-NY 1999) closely related to WNVs previously isolated in the Middle East.