Selective Enhancer Dependencies in MYC -Intact and MYC -Rearranged Germinal Center B-cell Diffuse Large B-cell Lymphoma.

High expression of MYC and its target genes define a subset of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) associated with poor outcomes. Half of these high-grade cases show chromosomal rearrangements between the MYC locus and heterologous enhancer-bearing loci, while focal deletions of the adjacent non-coding gene PVT1 are enriched in MYC -intact cases. To identify genomic drivers of MYC activation, we used high-throughput CRISPR-interference (CRISPRi) profiling of candidate enhancers in the MYC locus and rearrangement partner loci in GCB-DLBCL cell lines and mantle cell lymphoma (MCL) comparators that lacked common rearrangements between MYC and immunoglobulin (Ig) loci. Rearrangements between MYC and non-Ig loci were associated with unique dependencies on specific enhancer subunits within those partner loci. Notably, fitness dependency on enhancer modules within the BCL6 super-enhancer ( BCL6 -SE) cluster regulated by a transcription factor complex of MEF2B, POU2F2, and POU2AF1 was higher in cell lines bearing a recurrent MYC::BCL6 -SE rearrangement. In contrast, GCB-DLBCL cell lines without MYC rearrangement were highly dependent on a previously uncharacterized 3' enhancer within the MYC locus itself (GCBME-1), that is regulated in part by the same triad of factors. GCBME-1 is evolutionarily conserved and active in normal germinal center B cells in humans and mice, suggesting a key role in normal germinal center B cell biology. Finally, we show that the PVT1 promoter limits MYC activation by either native or heterologous enhancers and demonstrate that this limitation is bypassed by 3' rearrangements that remove PVT1 from its position in cis with the rearranged MYC gene. Key points: CRISPR-interference screens identify a conserved germinal center B cell MYC enhancer that is essential for GCB-DLBCL lacking MYC rearrangements. Functional profiling of MYC partner loci reveals principles of MYC enhancer-hijacking activation by non-immunoglobulin rearrangements.

Primary Hemophagocytic Lymphohistiocytosis With Prolonged Primary Neurologic Presentation.

Primary hemophagocytic lympho-histiocytosis (HLH) is a hyperinflammatory syndrome with devastating consequences. Multisystem involvement is a hallmark of HLH; however, HLH may rarely present with signs and symptoms isolated to the central nervous system (CNS). Within the brain, HLH can mimic demyelination, chronic infection, or vasculitis, leading to a diagnostic delay of months to years. We describe here a 7-year-old boy who presented with a history of prolonged fever and multiple focal neurologic deficits, which were being treated as CNS tuberculosis at the referring hospital. In view of the relapsing course with multiple areas of hemorrhagic tumefactive lesions on neuroimaging, the diagnosis was revised to acquired demyelination, and he received multiple cycles of immunotherapy. A brain biopsy was inconclusive. Subsequently, 13 months after disease onset, the child presented with features of systemic HLH in the form of fever, pancytopenia, splenomegaly, elevated ferritin, and triglycerides. Primary HLH was suspected, and genetic testing revealed a likely pathologic compound heterozygous variation in the PRF1 gene confirming the diagnosis. We planned a hematopoietic stem cell transplant as definitive therapy, but the child succumbed to an episode of sepsis and aspiration pneumonia. We infer from this case that primary HLH is a great mimicker. A high index of suspicion is required to establish a timely diagnosis. Primary HLH may stay isolated to CNS for months and should be considered in the differential diagnosis of all refractory cases of demyelination.

ETV6 Deficiency Unlocks ERG-Dependent Microsatellite Enhancers to Drive Aberrant Gene Activation in B-Lymphoblastic Leukemia.

Distal enhancers play critical roles in sustaining oncogenic gene-expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6-RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6-RUNX1+/- like B-ALL "signature" genes, including the likely leukemogenic driver EPOR. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of repeat enhancer-activated genes. Together, our findings reveal an epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene-expression program of ETV6-RUNX1+/- like B-ALL. SIGNIFICANCE: We find a unifying mechanism underlying a leukemia subtype-defining gene-expression signature that relies on repetitive elements with poor conservation between humans and rodents. The ability of ETV6 to antagonize promiscuous, nonphysiologic ERG activity may shed light on other roles of these key regulators in hematolymphoid development and human disease. See related commentary by Mercher, p. 2. This article is highlighted in the In This Issue feature, p. 1.

Identifying the Potential Determinants of Tobacco Counseling Implementation among Oral Health Professionals of India: A Cross-Sectional Survey

ABSTRACT Objective: To identify the potential determinants of tobacco counseling implementation among oral health professionals in India. Material and Methods: A cross-sectional survey was carried out among the 298 dentists of Aligarh and Gwalior. The questionnaire used in the study had sections on dentists' sociodemographic data and a 35-item questionnaire to assess the potential determinants of tobacco cessation counseling. Descriptive statistics were carried out, and a Chi-square test was utilized to determine the association. P-value <0.05 was considered statistically significant. Results: Domains "knowledge", "Professional Responsibility and Identity", and "Remembrance, awareness, and judgment" showed a statistically significant correlation with most tobacco cessation counseling behaviors. In addition, undergraduate education received in Tobacco Cessation counseling, and Continuing education received in Tobacco Cessation counseling had significantly impacted the practice of tobacco cessation counseling (p=0.02 and 0.04, respectively). Conclusion: This study suggests that "Knowledge", "Professional Responsibility and Identity" and "Remembrance, awareness, and judgment" are the potential determinants that could be used to design effective strategies to enhance tobacco counseling among dentists in India.

In vitro and in vivo NIR fluorescence lifetime imaging with a time-gated SPAD camera.

Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo. Single-photon avalanche diode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Förster resonant energy transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated intensified charge-coupled device (ICCD) camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.

Loss of FANCD2 and related proteins may predict malignant transformation in oral epithelial dysplasia.

OBJECTIVE: Predicting malignant transformation (MT) in oral epithelial dysplasia (OED) is challenging. The higher rate of MT reported in nonsmokers suggests an endogenous etiology in oncogenesis. We hypothesize that loss of FANCD2 and associated proteins could influence genomic instability and MT in the absence of environmental carcinogens. STUDY DESIGN: Longitudinal archival samples were obtained from 40 individuals with OED: from diagnosis to the most recent review in 23 patients with stable OED or until excision of the squamous cell carcinoma in 17 patients with unstable OED undergoing MT. Histopathological reassessment, immunohistochemistry for FANCD2, and Western blotting for phosphorylation/monoubiquitylation status of ATR, CHK1, FANCD2, and FANCG were undertaken on each tissue sample. RESULTS: Decreased expression of FANCD2 was observed in the diagnostic biopsies of OED lesions that later underwent MT. Combining the FANCD2 expression scores with histologic grading more accurately predicted MT (P = .005) than histology alone, and it correctly predicted MT in 10 of 17 initial biopsies. Significantly reduced expression of total FANCD2, pFANCD2, pATR, pCHK-1, and pFANCG was observed in unstable OED. CONCLUSIONS: There is preliminary evidence that defects in the DNA damage sensing/signaling/repair cascade are associated with MT in OED. Loss of expression of FANCD2 protein in association with a higher histologic grade of dysplasia offered better prediction of MT than clinicopathologic parameters alone.

Unravelling the attributes of novel cyanobacteria Jacksonvillea sp. ISTCYN1 by draft genome sequencing.

Filamentous cyanobacteria, Jacksonvillea sp. ISTCYN1 was isolated from agriculture field and cultured in BG-11 medium. This study, report the genome sequence of cyanobacteria Jacksonvillea thatto the best of our knowledgeis the firstgenome sequenceof thisgenus. The 5.7 MB draft genome sequence of this cyanobacterium contains 5134 protein-coding genes. The phylogenetic tree was constructed based on genome and Desertifilum sp. IPPAS B-1220 validated the closest relationship with Jacksonvillea sp. ISTCYN1. The growth of strain ISTCYN1 has been reported in the presence of different types of plastic when used as a sole carbon source. SEM analysis revealed biofilm formation by cyanobacterial strain ISTCYN1 on the surface of high and low-density polyethylene and polypropylene. In the presence of these plastics, EPS production has also been reported by this strain. Whole genome sequence analysis reveals the presence of many genes involved in biofilm formation. The presence of key enzymes responsible for plastic degradation laccase, esterase, lipase, thioesterase, and peroxidase have been predicted in the genome analysis. Genome analysis also provides insight into the genes involved in biotin biosynthetic pathways. Furthermore, the presence of many selenoproteins reveals the selenium acquisition by this cyanobacterium.

Integrated approach of whole-genome analysis, toxicological evaluation and life cycle assessment for pyrene biodegradation by a psychrophilic strain, Shewanella sp. ISTPL2.

Polycyclic aromatic hydrocarbons (PAHs) such as pyrene are universal contaminants existing in the environment which have known cancer-causing and mutagenic characteristics. A psychrophilic bacterial strain Shewanella sp. ISTPL2 was isolated from the sediment sample collected from the Pangong lake, Jammu & Kashmir, India. In our previous study, the pyrene degradation potential of the ISTPL2 strain was studied in both mineral salt media as well as in soil artificially spiked with different concentrations of pyrene. Whole-genome sequencing of ISTPL2 strain in the current study highlighted the key genes of pyrene metabolism, including alcohol dehydrogenase and ring hydroxylating dioxygenase alpha-subunit. Pyrene cytotoxicity was evaluated on HepG2, a human hepato-carcinoma cell line. The cytotoxicity of the organic extract decreased with the increasing duration of bacterial treatment. To develop a more sustainable biodegradation approach, the potential impacts were evaluated for human health and ecosystem using life-cycle assessment (LCA) following the ReCiPe methodology for the considered PAH. The results implemented that global warming potential (GWP) had the highest impact, whereas both ecotoxicity and human toxicity had least from this study.

Oral Health Consequences of Use of Smokeless Tobacco in North India: A Cross-Sectional Survey

ABSTRACT Objective: To evaluate the effects of commonly used smokeless tobacco forms on oral health at habitual placement sites of smokeless tobacco compared to non-placement sites among the North Indian population. Material and Methods: This cross-sectional study was conducted among 542 individuals using smokeless tobacco recruited from the outpatient wing of the Dental College. Subjects completed a questionnaire and received an oral examination. Periodontal pocket depth, gingival index, plaque index, gingival recession, and oral mucosal changes were assessed. Kendal's Tau test, paired t-test, and chi-square test were carried out to compare different variables among placement and non-placement sites. Results: Most of the subjects were male, reporting an average of 11.26 years of SLT use. Clinical inflammation of gingiva was significantly greater (p=0.01) at placement-sites (1.64 ± 0.53) of SLT in comparison to non-placement-sites (1.40 ± 0.41). The difference in the GR and PPD at placement and non-placement-sites was also statistically significant with p=0.002 and p=0.001, respectively. Clinically, the majority of subjects had mucosal changes at the placement sites, and a statistically significant association (p=0.034) was observed between the duration of the use of smokeless tobacco and the mucosal changes. Conclusion: Smokeless tobacco use predisposes to increased risk of periodontal diseases and oral mucosal changes at the placement sites in an individual due to the local irritant effect.