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ObjectiveColonoscopy surveillance aims to reduce colorectal cancer (CRC) incidence after polypectomy. The 2020 UK guidelines recommend surveillance at 3 years for ‘high-risk’ patients with ≥2 premalignant polyps (PMPs), of which ≥1 is ‘advanced’ (serrated polyp (or adenoma) ≥10 mm or with (high-grade) dysplasia); ≥5 PMPs; or ≥1 non-pedunculated polyp ≥20 mm; ‘low-risk’ patients without these findings are instead encouraged to participate in population-based CRC screening. We examined the appropriateness of these risk classification criteria and recommendations.DesignRetrospective analysis of patients who underwent colonoscopy and polypectomy mostly between 2000 and 2010 at 17 UK hospitals, followed-up through 2017. We examined CRC incidence by baseline characteristics, risk group and number of surveillance visits using Cox regression, and compared incidence with that in the general population using standardised incidence ratios (SIRs).ResultsAmong 21 318 patients, 368 CRCs occurred during follow-up (median: 10.1 years). Baseline CRC risk factors included age ≥55 years, ≥2 PMPs, adenomas with tubulovillous/villous/unknown histology or high-grade dysplasia, proximal polyps and a baseline visit spanning 2–90 days. Compared with the general population, CRC incidence without surveillance was higher among those with adenomas with high-grade dysplasia (SIR 1.74, 95% CI 1.21 to 2.42) or ≥2 PMPs, of which ≥1 was advanced (1.39, 1.09 to 1.75). For low-risk (71%) and high-risk (29%) patients, SIRs without surveillance were 0.75 (95% CI 0.63 to 0.88) and 1.30 (1.03 to 1.62), respectively; for high-risk patients after first surveillance, the SIR was 1.22 (0.91 to 1.60).ConclusionThese guidelines accurately classify post-polypectomy patients into those at high risk, for whom one surveillance colonoscopy appears appropriate, and those at low risk who can be managed by non-invasive screening.

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5' end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5'-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5' end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5'-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5' end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks.

ObjectivePostpolypectomy colonoscopy surveillance aims to prevent colorectal cancer (CRC). The 2002 UK surveillance guidelines define low-risk, intermediate-risk and high-risk groups, recommending different strategies for each. Evidence supporting the guidelines is limited. We examined CRC incidence and effects of surveillance on incidence among each risk group.DesignRetrospective study of 33 011 patients who underwent colonoscopy with adenoma removal at 17 UK hospitals, mostly (87%) from 2000 to 2010. Patients were followed up through 2016. Cox regression with time-varying covariates was used to estimate effects of surveillance on CRC incidence adjusted for patient, procedural and polyp characteristics. Standardised incidence ratios (SIRs) compared incidence with that in the general population.ResultsAfter exclusions, 28 972 patients were available for analysis; 14 401 (50%) were classed as low-risk, 11 852 (41%) as intermediate-risk and 2719 (9%) as high-risk. Median follow-up was 9.3 years. In the low-risk, intermediate-risk and high-risk groups, CRC incidence per 100 000 person-years was 140 (95% CI 122 to 162), 221 (195 to 251) and 366 (295 to 453), respectively. CRC incidence was 40%–50% lower with a single surveillance visit than with none: hazard ratios (HRs) were 0.56 (95% CI 0.39 to 0.80), 0.59 (0.43 to 0.81) and 0.49 (0.29 to 0.82) in the low-risk, intermediate-risk and high-risk groups, respectively. Compared with the general population, CRC incidence without surveillance was similar among low-risk (SIR 0.86, 95% CI 0.73 to 1.02) and intermediate-risk (1.16, 0.97 to 1.37) patients, but higher among high-risk patients (1.91, 1.39 to 2.56).ConclusionPostpolypectomy surveillance reduces CRC risk. However, even without surveillance, CRC risk in some low-risk and intermediate-risk patients is no higher than in the general population. These patients could be managed by screening rather than surveillance.

1. Rebecca Butterfield, MD* 2. Bhavita Patel, MD* 1. *Department of Pediatrics, Albany Medical Center, Albany, NY. An otherwise healthy 9-month-old boy is brought to the emergency department by his foster parents with a chief complaint of unusual bruises and marks on his skin. Two days ago, the foster parents had noticed left eye redness and swelling, which has subsequently progressed to a large, swollen blue-black lesion on his left cheek and new scattered bluish lesions on his thighs. The child had been diagnosed with a viral upper respiratory tract infection 3 weeks ago and has had some lingering nasal congestion with tactile temperatures for the past week but is otherwise acting very well, eating normally, and not fussy. He has had no recent immunizations or medications. His past medical history includes a preterm birth at 31 weeks’ gestation via vaginal delivery. The biological mother’s pregnancy was complicated by prolonged premature rupture of membranes and polysubstance abuse. The boy did receive parenteral vitamin K at birth. On physical examination in the emergency department, the child’s vital signs are within normal limits for age. He appears well, in no distress, and well nourished. Results of head and neck examinations are within normal limits, with a soft and open anterior fontanelle. Cardiovascular, pulmonary, and abdominal examinations also yield no abnormalities. Skin findings include a large targetoid ecchymosis with underlying edema on the left cheek, a right posterior auricular ecchymosis, and several scattered annular ecchymoses on the bilateral posterior thighs (Figs 1-4). There are no lesions on the trunk. Mucus membranes are intact and without lesions. The rest of his physical examination findings …

The English Bowel Cancer Screening Programme (BCSP) recommends 3 yearly colonoscopy surveillance for patients at intermediate risk of colorectal cancer (CRC) postpolypectomy (those with three to four small adenomas or one ≥10 mm). We investigated whether faecal immunochemical tests (FITs) could reduce surveillance burden on patients and endoscopy services.Intermediate-risk patients (60-72 years) recommended 3 yearly surveillance were recruited within the BCSP (January 2012-December 2013). FITs were offered at 1, 2 and 3 years postpolypectomy. Invitees consenting and returning a year 1 FIT were included. Participants testing positive (haemoglobin ≥40 µg/g) at years one or two were offered colonoscopy early; all others were offered colonoscopy at 3 years. Diagnostic accuracy for CRC and advanced adenomas (AAs) was estimated considering multiple tests and thresholds. We calculated incremental costs per additional AA and CRC detected by colonoscopy versus FIT surveillance.74% (5938/8009) of invitees were included in our study having participated at year 1. Of these, 97% returned FITs at years 2 and 3. Three-year cumulative positivity was 13% at the 40 µg/g haemoglobin threshold and 29% at 10 µg/g. 29 participants were diagnosed with CRC and 446 with AAs. Three-year programme sensitivities for CRC and AAs were, respectively, 59% and 33% at 40 µg/g, and 72% and 57% at 10 µg/g. Incremental costs per additional AA and CRC detected by colonoscopy versus FIT (40 µg/g) surveillance were £7354 and £180 778, respectively.Replacing 3 yearly colonoscopy surveillance in intermediate-risk patients with annual FIT could reduce colonoscopies by 71%, significantly cut costs but could miss 30%-40% of CRCs and 40%-70% of AAs.ISRCTN18040196; Results.

Introduction Individuals at intermediate-risk for colorectal cancer (CRC) following adenoma removal within the English Bowel Cancer Screening Programme (BCSP) are invited for three-yearly surveillance colonoscopy. Given the invasive nature of colonoscopy and scarcity of endoscopy resources, there is a need for an alternative surveillance method. We aimed to determine whether annual testing with the faecal immunochemical test (FIT) is an effective alternative. Methods Individuals aged 60–72 years and scheduled for surveillance following removal of intermediate-risk adenomas were recruited within the BCSP from January 2012 to December 2013. Quantitative FIT (OC-Sensor, Eiken) was offered at one, two, and three years post-polypectomy. Invitees who returned a completed consent form and an analysable FIT at Round 1 were included. Participants testing positive (≥40 µg haemoglobin (Hb)/g faeces) at Rounds 1 or 2 were offered early colonoscopy and were not invited to further FIT rounds. All other participants were offered the routine three-year surveillance colonoscopy. Diagnostic accuracy for CRC and advanced adenomas (AAs: adenomas≥10 mm, with tubulovillous or villous histology, or high grade dysplasia) was calculated at each round, using colonoscopy as the reference standard. We estimated diagnostic accuracy with lower haemoglobin thresholds and multiple rounds. Results Of 8008 invitees, 5946 (74%) consented and returned an analysable FIT at Round 1. Uptake of FIT was higher (97%) in Rounds 2 and 3. FIT positivity decreased by round, from 6% to 4% in Rounds 1 to 3. In total, 26 participants were diagnosed with CRC and 443 with AAs. At 40 µg/g, sensitivity and specificity of the first FIT were, respectively, 31% and 94% for CRC and 18% and 95% for AAs. Sensitivities for CRC and AAs were higher, and specificities lower, with lower thresholds and multiple rounds. At 10 µg/g, the programme sensitivity and specificity of three rounds were, respectively, 85% and 71% for CRC and 57% and 73% for AAs. Conclusions Annual low threshold FIT achieved relatively high sensitivity for CRC over three years. If this strategy replaced three-yearly surveillance colonoscopy, the number of colonoscopies could potentially be reduced by 70%. However, sensitivity for AAs was limited. Further research is needed to consider the implications for clinical practice of missing CRCs and AAs with FIT-based surveillance.

Background In the UK, patients with one or two adenomas, of which at least one is ≥ 10 mm in size, or three or four small adenomas, are deemed to be at intermediate risk of colorectal cancer (CRC) and referred for surveillance colonoscopy 3 years post polypectomy. However, colonoscopy is costly, can cause discomfort and carries a small risk of complications. Objectives To determine whether or not annual faecal immunochemical tests (FITs) are effective, acceptable and cost saving compared with colonoscopy surveillance for detecting CRC and advanced adenomas (AAs). Design Diagnostic accuracy study with health psychology assessment and economic evaluation. Setting Participants were recruited from 30 January 2012 to 30 December 2013 within the Bowel Cancer Screening Programme in England. Participants Men and women, aged 60–72 years, deemed to be at intermediate risk of CRC following adenoma removal after a positive guaiac faecal occult blood test were invited to participate. Invitees who consented and returned an analysable FIT were included. Intervention We offered participants quantitative FITs at 1, 2 and 3 years post polypectomy. Participants testing positive with any FIT were referred for colonoscopy and not offered further FITs. Participants testing negative were offered colonoscopy at 3 years post polypectomy. Acceptibility of FIT was assessed using discussion groups, questionnaires and interviews. Main outcome measures The primary outcome was 3-year sensitivity of an annual FIT versus colonoscopy at 3 years for detecting advanced colorectal neoplasia (ACN) (CRC and/or AA). Secondary outcomes included participants’ surveillance preferences, and the incremental costs and cost-effectiveness of FIT versus colonoscopy surveillance. Results Of 8008 invitees, 5946 (74.3%) consented and returned a round 1 FIT. FIT uptake in rounds 2 and 3 was 97.2% and 96.9%, respectively. With a threshold of 40 µg of haemoglobin (Hb)/g faeces (hereafter referred to as µg/g), positivity was 5.8% in round 1, declining to 4.1% in round 3. Over three rounds, 69.2% (18/26) of participants with CRC, 34.3% (152/443) with AAs and 35.6% (165/463) with ACN tested positive at 40 µg/g. Sensitivity for CRC and AAs increased, whereas specificity decreased, with lower thresholds and multiple rounds. At 40 µg/g, sensitivity and specificity of the first FIT for CRC were 30.8% and 93.9%, respectively. The programme sensitivity and specificity of three rounds at 10 µg/g were 84.6% and 70.8%, respectively. Participants’ preferred surveillance strategy was 3-yearly colonoscopy plus annual FITs (57.9%), followed by annual FITs with colonoscopy in positive cases (31.5%). FIT with colonoscopy in positive cases was cheaper than 3-yearly colonoscopy (£2,633,382), varying from £485,236 (40 µg/g) to £956,602 (10 µg/g). Over 3 years, FIT surveillance could miss 291 AAs and eight CRCs using a threshold of 40 µg/g, or 189 AAs and four CRCs using a threshold of 10 µg/g. Conclusions Annual low-threshold FIT with colonoscopy in positive cases achieved high sensitivity for CRC and would be cost saving compared with 3-yearly colonoscopy. However, at higher thresholds, this strategy could miss 15–30% of CRCs and 40–70% of AAs. Most participants preferred annual FITs plus 3-yearly colonoscopy. Further research is needed to define a clear role for FITs in surveillance. Future work Evaluate the impact of ACN missed by FITs on quality-adjusted life-years. Trial registration Current Controlled Trials ISRCTN18040196. Funding National Institute for Health Research (NIHR) Health Technology Assessment programme, NIHR Imperial Biomedical Research Centre and the Bobby Moore Fund for Cancer Research UK. MAST Group Ltd provided FIT kits.

NTRODUCTION: Evidence - based medicine (EBM) is defined as the „conscientious, explicit, and judicious use of current best evidence‟. It i s an important tool for lifelong learning in medicine, and medical students can develop the skills necessary to understand and use EBM. The teaching of EBM in Sumandeep Vidyapeeth is as part of Evidence Based Education System (EBES). The university has imp lemented the 16 hours of teaching with project work on Evidence Based Medicine in 1st MBBS and 2nd MBBS curriculum in addition to MBBS syllabus. AIMS & OBJECTIVES: This study was planned to take feedback from all the faculties those who are involved in Evi dence based Medicine teaching to evaluate their attitude and perception towards this innovative teaching method and to recommend improvements. MATERIAL & METHODS: A Descriptive, self - structured , pilot pretested questionnaire based cross sectional study was conducted in the year 2013 - 2014 among 40 faculties from 7 Departments like Anatomy, Physiology, Biochemistry, Microbiology, Pharmacology, Pathology and Forensic Medicine teaching Evidence Base d Medicine to students at S.B.K.S MI & RC, Sumandeep Vidyapeeth. Data was expressed as percentage. RESULTS: The response rate for the study was 75%. Almost 87% of faculties agreed that teaching EBM is a welcoming development during pre and para clinical ye ars. About 80% faculties agreed that it will help them in future clinical learning. 87% faculties agreed that literature and research searching improves their day to day teaching. About 77% of faculties have attended workshop and training held in Universit y and 83% of faculties agreed that they are interested in more learning and improving skills necessary to incorporate Evidence based medicine into their discipline. Barriers included shortage of time and need for training in teaching EBM. CONCLUSION: Facul ties of this University teaching Pre - clinical and Para - clinical medical students recognized EBM as an important component of undergraduate education. Majority have positive attitude towards teaching EBM to undergraduates and agreed that it will help them i n future clinical learning although they need more training to improve their ability to teach EBM.

Cellular genome is continuously challenged by endogenous and exogenous DNA damaging agents. In the genome, base modifications are the most common form of DNA damage. Base excision DNA repair (BER) is the most important pathway for the removal of oxidized or alkylated DNA. If these base modifications continue to persist, the replisome may stall at DNA lesions, causing the replication fork to collapse, leading to genetic instability. While the main components of the BER pathway are well defined, its regulatory mechanism remains poorly understood. We report here that apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional protein interacts with the pre-mRNA splicing factor ISY1. However, this interaction is independent of DNA or RNA based upon our pull-down experimental results. ISY1 expression is induced by oxidative or DNA alkylation damage, which would then provide an immediate up-regulation of APE1 activity in vivo and enhanced BER of oxidized bases. This was supported from our results of in vitro reconstituted experiment suggesting that ISY1 can stimulate 5’-3’ endonuclease 1 activity of APE1 in both the short- and long-patch BER pathways and increase its binding to AP site DNA. The interaction between ISY1 and APE1 also establishes a connection between DNA damage repair and pre-mRNA splicing. Based upon these results, we propose that ISY1 is an important regulator of BER and enhances the ability of APE1 to efficiently recognize abasic sites in DNA. Role of ISY1 becomes important in aging tissues where decreased APE1 activity results in the accumulation of oxidative genomic damages. Therefore, ISY1 may work as an anti-aging factor. Induction of ISY1 in malignancies could constitute a mechanism to avoid apoptotic death after treatment with alkylating agents. Citation Format: Aruna Jaiswal, Elizabeth Williamson, Bhavita Patel, Gayathri Srinivasan, Kimi Kong, Carrie Lomelino, Satya Narayan, Robert Hromas. Splicing component ISY1 interacts with APE1 and regulates base excision repair [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 1758.

Aggressive cancers often express E-cadherin in cytoplasmic vesicles rather than on the plasma membrane and this may contribute to the invasive phenotype of these tumors. Therapeutic strategies are not currently available that restore the anti-invasive function of E-cadherin in cancers. MDA-MB-231 cells are a frequently used model of invasive triple-negative breast cancer, and these cells express low levels of E-cadherin that is mislocalized to cytoplasmic vesicles. MDA-MB-231 cell lines stably expressing wild-type E-cadherin or E-cadherin fused to glutathione S-transferase or green fluorescent protein were used as experimental systems to probe the mechanisms responsible for cytoplasmic E-cadherin localization in invasive cancers. Although E-cadherin expression partly reduced cell invasion in vitro, E-cadherin was largely localized to the cytoplasm and did not block the invasiveness of the corresponding orthotopic xenograft tumors. Further studies indicated that the glucocorticoid dexamethasone and the highly potent class I histone deacetylase (HDAC) inhibitor largazole cooperated to induce E-cadherin localization to the plasma membrane in triple-negative breast cancers, and to suppress cellular invasion in vitro. Dexamethasone blocked the production of the cleaved form of the CDCP1 (that is, CUB domain-containing protein 1) protein (cCDCP1) previously implicated in the pro-invasive activities of CDCP1 by upregulating the serine protease inhibitor plasminogen activator inhibitor-1. E-cadherin preferentially associated with cCDCP1 compared with the full-length form. In contrast, largazole did not influence CDCP1 cleavage, but increased the association of E-cadherin with γ-catenin. This effect on E-cadherin/γ-catenin complexes was shared with the nonisoform selective HDAC inhibitors trichostatin A (TSA) and vorinostat (suberoylanilide hydroxamic acid, SAHA), although largazole upregulated endogenous E-cadherin levels more strongly than TSA. These results demonstrate that glucocorticoids and HDAC inhibitors, both of which are currently in clinical use, cooperate to suppress the invasiveness of breast cancer cells through novel, complementary mechanisms that converge on E-cadherin.

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ., Author Summary The cell itself damages its own DNA throughout the cell cycle as a result of oxidative metabolism, and this damage creates barriers for replication fork progression. Thus, DNA replication is not a smooth and continuous process, but rather one of stalls and restarts. Therefore, proper replication fork restart is crucial to maintain the integrity of the cell’s genome, and preventing its own death or immortalization. To restart after stalling, the replication fork subverts a DNA repair pathway termed homologous recombination. Using any other pathway for fork repair will result in an unstable genome. How the homologous recombination repair pathway is initiated at the replication fork is not well defined. In this study we demonstrate the previously uncharacterized EEPD1 protein is a novel gatekeeper for the initiation of this fork repair pathway. EEPD1 promotes 5’ end resection, the initial step of homologous recombination, which also prevents alternative fork repair pathways that lead to unstable chromosomes. Thus, EEPD1 protects the integrity of the cell genome by promoting the safe homologous recombination fork repair pathway.

The abundance and structure of denitrifying bacterial community in different activated sludge samples were assessed, where the abundance of denitrifying functional genes showed nirS in the range of 10(4)-10(5), nosZ with 10(4)-10(6) and 16S rRNA gene in the range 10(9)-10(10) copy number per ml of sludge. The culturable approach revealed Pseudomonas sp. and Alcaligenes sp. to be numerically high, whereas culture independent method showed betaproteobacteria to dominate the sludge samples. Comamonas sp. and Pseudomonas fluorescens isolates showed efficient denitrification, while Pseudomonas mendocina, Pseudomonas stutzeri and Brevundimonas diminuta accumulated nitrite during denitrification. Numerically dominant RFLP OTUs of the nosZ gene from the fertilizer factory sludge samples clustered with the known isolates of betaproteobacteria. The data also suggests the presence of different truncated denitrifiers with high numbers in sludge habitat.

A kinetic model of oxidation of aluminum has been previously established based on thermal analysis data using heating rates in the 1-40 K/min r ange. Ignition, on the other hand, involves heating rates that are many orders of magnitude higher, and experimental work under these conditions is ongoing. In this pro ject, we use thermogravimetry at heating rates up to 500 K/min (8.3 K/s) to validate and extend the k inetic model currently in use for aluminum oxidation. Oxidation studies are conducted in argon/oxygen mixtures. The stepwise aluminum oxidation process reported for lower heating rates is also observed for the higher heating rates addressed in this project. Activati on energies for individual oxidation steps are generally consistent with previous data obtaine d from low heating rate measurements. Additional oxidation process features are obse rved at the higher heating rates and interpreted in the framework of the general concept proposed earlier and involving growth of various alumina polymorphs and transformations between these polymorphs. It is expected that the wider range of heating rates covered exper imentally will improve the reliability of current ignition models, as well as establish which oxidation steps identifiable in the low-heating rate thermo-analytical oxidation experime nts cause ignition of aluminum in practical conditions.

Histone modifications play an important role in the process of transcription. However, in contrast to lysine methylation, the role of arginine methylation in chromatin structure and transcription has been underexplored. The globin genes are regulated by a highly organized chromatin structure that juxtaposes the locus control region (LCR) with downstream globin genes. We report here that the targeted recruitment of asymmetric dimethyl H4R3 catalyzed by PRMT1 (protein arginine methyltransferase 1) facilitates histone H3 acetylation on Lys9/Lys14. Dimethyl H4R3 provides a binding surface for P300/CBP-associated factor (PCAF) and directly enhances histone H3 acetylation in vitro. We show that these active modifications are essential for efficient interactions between the LCR and the βmaj-promoter as well as transcription of the β-globin gene. Furthermore, knockdown (KD) of PRMT1 by RNA interference in erythroid progenitor cells prevents histone acetylation, enhancer and promoter interaction, and recruitment of transcription complexes to the active β-globin promoter. Reintroducing rat PRMT1 into the PRMT1 KD MEL cells rescues PRMT1 binding, β-globin transcription, and erythroid differentiation. Taken together, our data suggest that PRMT1-mediated dimethyl H4R3 facilitates histone acetylation and enhancer/promoter communications, which lead to the efficient recruitment of transcription preinitiation complexes to active promoters.

Child abuse and neglect remains a major cause of morbidity and mortality among children worldwide. Over the last few decades, there has been growing research in the field of Child Abuse Pediatrics with greater recognition and research into potential diagnostic mimics of inflicted injury. This paper reviews some common skin findings and bleeding disorders that have features in common with child abuse.

Circadian rhythms govern all biological functions. Circadian misalignment has a major impact on health. Late chronotype is a risk factor for circadian misalignment which in turn can affect the control of seizures in epilepsy patients. We compared a group of 87 confirmed epilepsy patients regardless of subtypes with age- and sex-matched healthy controls. We compared generalized epilepsy patients with localization related epilepsy patients and with healthy controls. We found that primary generalized epilepsy patients were 5 times more likely to have a late chronotype than healthy controls. We did not find any significant differences between localization related epilepsy patients and healthy controls or between the overall epilepsy cohort and healthy controls. Generalized epilepsy patients are more likely to be evening types as compared to those with focal epilepsy or subjects without epilepsy. Epilepsy patients do not experience the same age related increase in morningness as do age-matched healthy controls. This is important in regard to timing of AED, identifying and preventing sleep deprivation, and integrating chronotype evaluations and chronotherapy in comprehensive epilepsy care. Further studies, using objective phase markers or the impact of chronotherapy on seizure control, are necessary.

Current technological advancements and genome-wide studies provide compelling evidence that dynamic chromatin interaction and three-dimensional genome organization in nuclei play an important role in regulating gene expression. This chapter highlights current technical advances in mapping interactions within the same chromosome (intrachromosomal) and between different chromosomes (interchromosomal), as well as outlines the roles of dynamic chromatin interactions in transcription regulation. We further explored the function of chromatin modulators, such as chromatin insulator binding protein CTCF, cohesin, SATB1, and transcription cofactors, in chromatin interactions and genome organization. Changes in higher-order organization of chromatin will alter global gene expression and promote genome rearrangements. Finally, we focused on the implications of these studies in cancer and development.

Metnase, a human SET-transposase fusion protein contains two functional domains: a SET domain and transposase domain. Transposase domain exhibit strand transfer and end joining activity while set domain is responsible for histone lysine methyltransferase activity (KMT). Metnase also increases the efficiency of double strand break repair by non-homologous end joining (NHEJ); however, its role in the excision of clustered DNA damage remains to be investigated. We hypothesize that 5’-3’ endonuclease activity of metnase could possibly substitute for APE1 in BER and serve as an alternative initiator of BER in tumor cells lacking APE1. In the present study, we examined in vitro endonuclease activity of Metnase using abasic site containing artificial DNA, and describe its activity in BER. Its unique 5’ endonuclease activity was selective only for abasic lesions, but not other base modifications such as 8-oxoguanine, uracil, hypoxanthine or xanthenes. Metnase specifically initiates removal of reduced abasic lesions from DNA, and allows completion of short-patch or long-patch BER. APE1 has difficulty cleaving 5’ of clustered abasic lesions. This endonuclease that promotes BER in clustered oxidative DNA damage is not known. Metnase also cleaves multiple abasic lesions (clustered DNA damage) and facilitates the repair of DNA if the multiple DNA damages are 3 or more nucleotides apart on the opposing strands. However, repair of multiple DNA damages remains inefficient if these are in close proximity of each other (< 3nt apart on the opposing strands) and results in DNA double strand break (DSB). These results suggest that Metnase can promote BER of oxidative nucleotides, where APE1 is unable to function. Citation Format: Aruna S. Jaiswal, Elizabeth A. Williamson, Bhavita Patel, Gayathri Srinivasan, Satya Narayan, Robert A. Hromas. The endonuclease Metnase promotes base excision repair of Clustered abasic DNA lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1420. doi:10.1158/1538-7445.AM2017-1420

Introduction: Obesity is a condition in which excess body fat accumulates, which leads to various adverse effects on health, particularly cardiovascular diseases (CVDs), which reduce life expectancy and/or increase health problems. Fast food consumption is one of the factors which have been reported as a cause of obesity. Body mass index (BMI) is used to assess obesity and overweight, which can be calculated by using the formula, weight in kg, divided by square of height in metres. Aim: This study focused on the relationship of body mass index with fast food consumption, associated soft drink consumption and physical activity. Methods: Descriptive cross-sectional study was conducted in Department of Biochemistry, SBKS MI and RC, and Sumandeep Vidyapeeth. This study was approved by the ethical review board .One hundred and forty seven medical students from 1st year MBBS course were included in this study. Self-structured questionnaire was used, which contained several data like information on age, height, weight, education level. The formula used for calculating BMI was, weight in kg, divided by square of height in metres (Kg/m2). Results: In our study, out of 147 students, a total of 138 students (more than 90%) used to have fast food. Among these, a total of 47 students (34.05%) were pre-obese and obese. Out of 147 students, 87 students (59.18%) were in normal weight range, while 13 (8.84%) students were underweight. Statistical Analysis: Data was compiled in an Excel worksheet and it was analyzed for percentages and proportions. Chi-square and Pearson’s correlation test were also applied wherever they were applicable and Alpha error was set at a 5% level. Conclusion: In our study, a significant relationship was found between BMI and fast food consumption, less physical activity, and intake of soft drinks.

Many molecular alterations are involved in the morphogenesis of T-cell acute leukemia (TALL), classified as lymphoblastic leukemia/lymphoma by the World Health Organization. TALL is a malignant disease of the thymocytes which accounts for approximately 15% of pediatric acute lymphoblastic leukemia (ALL) and 20-25% of adult ALL. Frequently, it presents with a high tumor load accompanied by rapid disease progression. About 30% of T-ALL cases relapse within the first two years following diagnosis with long term remission in 70-80% of children and 40% of adults [1]-[4]. This poor prognosis is a consequent of our insufficient knowledge of the molecular mechanisms underlying abnormal T-cell pathogenesis. Under‐ standing the abnormal molecular changes associated with T-ALL biology will provide us with the tools for better diagnosis and treatment of lymphoblastic leukemia. Recent improvements in genome-wide profiling methods have identified several genetic aberrations which are associated with T-ALL pathogenesis. For simplification these molecular changes can be separated into 4 different groups: chromosome aberrations, gene mutations, gene expression profiles, and epigenetic alterations. This chapter will discuss these molecular changes in depth.

TAL1/SCL is a hematopoietic-specific oncogene and its activity is regulated by associated transcriptional co-activators and corepressors. Dysregulation of TAL1 activity has been associated with T-cell leukemogenesis. However, it remains unclear how the interactions between TAL1 and corepressors versus co-activators are properly regulated. Here, we reported that protein kinase A (PKA)-mediated phosphorylation regulates TAL1 interaction with the lysine-specific demethylase (LSD1) that removes methyl group from methylated Lys 4 on histone H3 tails. Phosphorylation of serine 172 in TAL1 specifically destabilizes the TAL1-LSD1 interaction leading to promoter H3K4 hypermethylation and activation of target genes that have been suppressed in normal and malignant hematopoiesis. Knockdown of TAL1 or LSD1 led to a derepression of the TAL1 target genes in T-cell acute lymphoblast leukemia (T-ALL) Jurkat cells, which is accompanied by elevating promoter H3K4 methylation. Similarly, treatment of PKA activator forskolin resulted in derepression of target genes by reducing its interaction with LSD1 while PKA inhibitor H89 represses them by suppressing H3K4 methylation levels. Consistent with the dual roles of TAL1 in transcription, TAL1-associated LSD1 is decreased while recruitment of hSET1 is increased at the TAL1 targets during erythroid differentiation. This process is accompanied by a dramatic increase in H3K4 methylation. Thus, our data revealed a novel interplay between PKA phosphorylation and TAL1-mediated epigenetic regulation that regulates hematopoietic transcription and differentiation programs during hematopoiesis and leukemogenesis.

Mammalian genomes encode tens of thousands of long intergenic noncoding RNAs (lincRNAs). Accumulated evidence suggests that many of lincRNAs are expressed in a development- or tissue-specific manner. However, it remains largely unknown how lincRNAs reprogram lineage-specific gene expression and regulate lineage-specific differentiation processes during early embryonic development. It is also important to identify specific lincRNA(s) that is involved in early hematopoietic lineage formation and differentiation. Here, we reported a HoxB locus-associated lincRNA HOTSET that is conservatively expressed in both mouse and human hematopoietic stem and progenitor cells (HS/PCs). During embryonic development, HOTSET RNA is dramatically induced upon embryoid body (EB) hematopoietic differentiation. Expression of HOTSET RNA is required for development of the mesoderm-derived Flk1+ bipotential precursors of blood and endothelium lineages and CD41+/c-Kit+ hematopoietic stem and progenitor cells. We demonstrated that HOTSET RNA directly interacts with the SET domains of Setd1a and MLL1 histone methyltransferases in a sense-strand dependent manner. HOTSET RNA directly binds to the HoxB1-6 gene promoters and recruits the Setd1a/MLL1 complexes to the anterior HoxB locus upon differentiation. Furthermore, HOTSET RNA mediated H3K4 methylation is essential for long-range chromatin loops that bring anterior HoxB genes into a close proximity with HOTSET RNA locus and for subsequent activation of anterior HoxB gene expression. In addition, knockdown of HOTSET RNA disrupts the recruitment of the Setd1a/MLL1 complexes, H3K4me3 levels, the three-dimensional chromatin architecture, and HoxB gene transcription at the HoxB gene cluster. Depletion of HOTSET RNA also impairs differentiation of the CD41+/c-Kit+ HS/PC population and hematopoietic transcription factor and signaling networks required for early hematopoietic differentiation. Importantly, re-expression of HoxB2-4 genes in the HOTSET-depleted embryoid bodies rescues the expression of core hematopoietic transcription factors as well as the Flk1+ mesoderm-derived precursors and CD41+/c-Kit+ HS/PCs that undergo hematopoietic differentiation. Thus, HOTSET plays an important role in early hematopoietic lineage commitment by shaping the three-dimensional chromatin landscape in the HoxB locus to activate anterior HoxB genes that subsequently modulate lineage-specific transcription networks during hematopoietic development. Disclosures No relevant conflicts of interest to declare.

Introduction: C-Reactive protein is known as a marker of morbidity and mortality in malaria. It correlates closely with other complications in malaria and can be used to predict severity. Thus, measurement of CRP can be useful in understanding the pathogenesis of severe malaria. However, studies regarding CRP in malaria are very rare from India. Aims & objectives: The present study aimed to study CRP levels in patients with malarial infection and to find out the usefulness of CRP in assessment of disease severity. Material and Methods: The study population consisted of 50 patients with malarial infection, of them 29 were Plasmodium falciparum, 21 were P. vivax-infected and no patient found with dual infection in our study. Out of 50 patients, 17 patients required admission for various reasons and of them 5 patients died. Serum C-reactive protein concentrations were measured in all the patients. Percentage parasitemia, platelet count and Liver function tests were measured on the day of admission. Statistical tests included were Students t-test, chi-square test and Pearson correlation coefficient. Results: Serum C-reactive protein concentration was significantly elevated in all the malaria patients and it was significantly higher (P

Liver disease is leading cause of morbidity and mortality worldwide. Cholinesterase is a family of enzymes that catalyse the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid. It is an enzyme synthesized by hepatocytes and its serum levels reflect the synthetic function of liver. Objectives: To estimate serum cholinesterase in liver disease patients and to compare serum cholinesterase level with other liver functio n tests like SGOT, SGPT, ALP and Bilirubin levels. Methodology: The present cross sectional study was conducted at the Biochemistry department of tertiary care institute. Thirty patients with liver disease were included in the group A and 30 healthy patients not having liver disease were enrolled in group B as contr ol .Serum cholinesterase and Liver Function Tests were estimated in all participants. Results: The levels of cholinesterase were significantly lower in liver disease patients. Serum cholinesterase was 3424.77 ± 2149.30 i n group A vs.7320.77 ± 1577.26 in group B (P

Background: Diabetes mellitus is a metabolic characterized bydeficiency of insulin and insulin resistance. Adenosine deaminase (ADA) modulates the bioactivity of insulin and Adenosine receptorsstimulate glucogenolysis and gluconeogenesis . but its role in Type 2 diabetes mellitus is not yet characterized. Aim & Objectives : The present study aimed to evaluate the serum Adenosine Deaminase level and to correlate ADA levels with Blood Glucose and Glycated hemoglobin levels in Type-2 Diabetes Mellitus patients. Material and Method: The study population of 120 patients were divided into 3 groups: Group A which consisted of 40 normal healthy individuals without any complication or history of Diabetes. Group B included 40 patients of Type 2 Diabetes Mellitus both with HbA1c 7% (n=40). Fasting blood sugar, HbA1c, ADA, and body mass Index were estimated in all the subjects under study. Results: The study showed that mean Fasting Blood Sugar, Post prandial Blood sugar and HbA1c concentration was significantly higher in Group C & Group B compared to Group A. Compared with the well-controlled Type-2 Diabetes Mellitus patients (HbA1c 7%) showed significantly increased ADA activity (49.79 18.12 U/L vs. 90.10 32.75; P

Background: Abnormalities of lipid metabolism are common in human immunodeficiency virus (HIV) infected patients and tend to be accentuated in those receiving antiretroviral therapy, particularly with protease inhibitors (PIs). However, there is a dearth of information on serum lipid profiles and biochemical parameters among treatment-naive HIV-positive patients in our environment. Aims & Objective: To study changes in lipid profiles and other biochemical parameters in HIV-1 infected patients. Materials and Methods: A prospective, observational study was carried out on HIV- 1 infected patients attending anti-retroviral drug (ARV) clinic and who were started HAART between December 2012 and December 2013 from Gujarat state. Clinical, anthropometrical and laboratory data were recorded at the start of the study and after one years of HAART. Patients’ data were collected in a computerized database for later statistical analysis; Categorical variables were analyzed with the χ2 test, or the Fisher exact test when necessary. Results: We found that after 12 months of highly active antiretroviral therapy (HAART), there was a significant increase in serum lipids. After 12 months of HAART, renal impairment was associated with a low increase in mean HDL and a high increase in triglycerides (TG). In conclusion, abnormality of serum lipid is common and showed female preponderance among treatment-naive HIV patients in our environment. Conclusion: Patients with HIV infection on HAART should be screened for lipid disorders given their high prevalence as observed in this study, because of its potential for morbidity and mortality in patients on HAART.

Introduction: Diabetes is the leading cause of chronic kidney disease (CKD) and is associated with excessive cardiovascular morbidity and mortality. Anemia is common among those with diabetes and chronic kidney disease and greatly contributes to patient outcomes. Observational studies indicate that low hemoglobin levels in such patients may increase risk for progression of kidney disease and cardiovascular morbidity and mortality. Objective: 1. Estimation of glycosylated hemoglobin (HbA1c) and hemoglobin level in patients with type II diabetes mellitus. 2. To determine the prevalence of anaemia in type 2 diabetes mellitus with or without chronic kidney disease. Methodology: This is a descriptive analytical cross-sectional study carried out in Dhiraj Hospital, Piparia, Vadodara. A total no of 100 consecutive patients were enrolled; 25 patients having Diabetes, 25 patients having diabetes and chronic kidney disease, 25 patients having only chronic kidney disease but no diabetes and 25 patients neither having diabetes nor chronic kidney disease taken as a control group. Results: Anemia was present in 37% diabetic patients, 17% in diabetic patients with chronic kidney disease, 3% in patients with only chronic kidney disease. Anaemia was significantly higher in patients with diabetes, chronic kidney disease and diabetes with chronic kidney disease. Conclusion: Anemia was more prevalent in persons with diabetes and diabetes associated with chronic kidney disease compared to persons without diabetes. Therefore anemia may be particularly harmful in individuals with diabetes and chronic kidney disease. Correction of anemia may have a significant role in prevention of other diabetic complications.

Abstract 3507 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant disease of thymocytes that mainly affects children and has very poor prognosis with high rates of relapse. A prominent feature observed in 60% of T-ALL childhood patients is the ectopic expression of a key hematopoietic transcription factor TAL1/SCL. Although several enhancers has been identified to play an important role in normal hematopoietic differentiation, the histone modification patterns and chromatin organization over the whole TAL1 locus reveled that none of them is active in T-ALL cell lines such as Jurkat and Rex cells. It remains currently unknown how TAL1 is activated in the majority of T-ALL patients lacking the TAL1 locus rearrangements. To understand the molecular mechanism underlying regulation of the TAL1 oncogene in leukemic T-cells, we employed circularized chromosome conformation capture (4C) methodology to identify new regulatory elements that activate TAL1 specifically in T-ALL leukemia. Using the TAL1 promoter 1a as the bait, we discovered that the TAL1 promoter 1a interacts with the TIL16 element (TAL1 interacting locus in chromosome 16) that is located at ∼15 Kb downstream of T-cell specific CD2BP2 gene in T-ALL cell line Jurkat, but not in erythroid progenitor K562 cells. The CD2BP2 protein is a cellular adapter protein that was originally identified as a binding partner of the T cell adhesion protein CD2 in the context of T cell signaling. The TIL16 element contains the bind sites for several transcription factors that are important for hematopoiesis such as C-Maf, Pax5, HoxA7 and USF2. The inter-chromosomal interaction between the TIL16 and the TAL1 promoter 1a was further confirmed by chromosome conformation capture (3C) assay in three TAL1 over-expressing T-ALL cell lines, Jurkat, REX and Molt4, but not in K562 cells. Recent genome wide study has correlates H3K4 mono- or dimethyl marks with distal enhancers while trimethyl H3K4 is enriched in promoters of active genes. To further test if the TIL16 acts as T-cell specific enhancer for TAL1 activation in T-ALL cells, we carried out ChIP-seq and ChIP analysis in CD4 T cells, Jurkat, and K562 cells. We found that the TIL16 element is specifically marked by H3K4me1 in Jurkat and CD4+ T-cells but not in K562 cells. The enrichment of H3K4me1 is correlated with the binding of c-Maf, a T-cell specific transcription factor. To further test whether the TIL16 element contributes to transcription activity, a DNA fragments containing the TIL16 element were cloneed into SV40 minimal promoter driven luciferase reporter and introduced into K562 and several T-ALL cell lines. Compared to the pGL3-SV40 vector that showed only minimal luciferase activity, the 1 Kb TIL element specifically activated transcription of the luciferase reporter in T-ALL cells, but not in erythroid progenitor K562 cells suggesting that the TIL16 element functions as a T-cell specific TAL1 enhancer. Thus, our data revealed a novel epigenetic mechanism by which the TAL1 oncogene is ectopically activated in T-cell leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 281 The key hematopoietic transcription factor TAL1/SCL is essential for the specification of hematopoietic stem cells (HSCs) and differentiation along myeloid and erythroid lineages. However, ectopic activation of TAL1 gene is the most frequent gain-of-function mutation associated with childhood T-cell acute lymphoblastic leukemia (T-ALL) in approximately 60% of patients. To understand underlying epigenetic mechanisms of TAL1 activation in normal and in malignant hematopoiesis, we performed ChIP, ChIP-seq, and chromatin conformation capture assays to investigate chromatin structure profile which correlates with transcriptional activation, at the TAL1 locus comparing erythroid progenitors, K562 and CD36+ cells, with T-ALL cells, Jurkat and Rex cells. We found distinct epigenetic landscape and chromatin organization across the TAL1 locus in these two different types of hematopoietic cells. Although H3K4me3 is enriched at the TAL1 promoter in both erythroid progenitors and in T-ALL cells, the erythroid specific +51 enhancer is marked by active histone modifications only in erythroid progenitors, but not in T-ALL cells. Furthermore, we demonstrated that +51 enhancer interacts with the TAL1 promoter 1a via a long range chromatin loop in vivo in erythroid CD36+ and K562 cells. The recruitment of hSET1 HMT complex facilitates this enhancer/promoter chromatin interaction and depletion of hSET1 leads to loss of H3K4 methylation, enhancer/promoter interaction, RNA PolII loading, and TAL1 transcription at both the TAL1 promoter 1a and the +51 enhancer. In addition, loss of SET1 in CD34+ HSCs exhibited a significant reduction in the formation of CFU-E and BFU-E colonies. In contrast, neither H3K4 methylation nor enhancer/promoter interaction was detected at the +51 enhancer in T-ALL cells. Finally, we investigated the role of insulator protein CTCF in the regulation of TAL1 expression in normal and in malignant cells. We found that regardless of similar CTCF binding patterns at the TAL1 locus in erythroid progenitors and T-ALL cells, CTCF elements flanking the TAL1 locus mediate a long-range loop that encloses TAL1 promoter and enhancer in the same chromatin domain in erythroid progenitors, whereas in T-ALL Jurkat cells the CTCF site located between the promoter 1a and the +51 enhancer forms a repressive loop that expels the +51 enhancer from the same domain with the promoter 1a. Overall, our study demonstrates that a novel long range intrachromosomal interaction between the TAL1 promoter 1a and the +51 enhancer regulated and organized by hSET1 histone methyltransferase and insulator protein CTCF controls TAL1 promoter activity in normal versus malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1(+) mesoderm and then promotes hematopoietic differentiation through regulation of hoxb pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated knockdown or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1(+) precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1(+) precursors and differentiation of Flk1(+) cells into hematopoietic lineages.