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BACKGROUND Acute gastroenteritis (AGE) is a common pediatric diagnosis in emergency medicine, accounting for 1.7 million visits annually. Little is known about racial/ethnic differences in care in the setting of standardized care models. METHODS We used quality improvement data for children 6 months to 18 years presenting to a large, urban pediatric emergency department (ED) treated via a clinical pathway for AGE/dehydration between 2011 and 2018. Race/ethnicity was evaluated as a single variable (non-Hispanic [NH]-White, NH-Black, Hispanic, and NH-other) related to ondansetron and intravenous fluid (IVF) administration, ED length of stay (LOS), hospital admission, and ED revisits using multivariable regression. RESULTS Of 30,849 ED visits for AGE/dehydration, 18.0% were NH-White, 57.2% NH-Black, 12.5% Hispanic, and 12.3% NH-other. Multivariable mixed-effects generalized linear regression controlling for age, sex, triage acuity, payer, and language revealed that, compared to NH-White patients, NH-other patients were more likely to receive ondansetron (adjusted odds ratio [95% CI] = 1.30 [1.17 to 1.43]). NH-Black, Hispanic, and NH-other patients were significantly less likely to receive IVF (0.59 [0.53 to 0.65]; 0.74 [0.64 to 0.84]; 0.74 [0.65 to 0.85]) or be admitted to the hospital (0.54 [0.45 to 0.64]; 0.62 [0.49 to 0.78]; 0.76 [0.61 to 0.94]), respectively. NH-Black and Hispanic patients had shorter LOS (median = 245 minutes for NH-White, 176 NH-Black, 199 Hispanic, and 203 NH-other patients) without significant differences in ED revisits. CONCLUSIONS Despite the presence of a clinical pathway to guide care, NH-Black, Hispanic, and NH-other children presenting to the ED with AGE/dehydration were less likely to receive IVF or hospital admission and had shorter LOS compared to NH-White counterparts. There was no difference in patient revisits, which suggests discretionary overtreatment of NH-White patients, even with clinical guidelines in place. Further research is needed to understand the drivers of differences in care to develop interventions promoting equity in pediatric emergency care.

We report a case of cutaneous cryptococcosis due to Cryptococcus neoformans in a pediatric patient with hyper IgM syndrome with scalp lesions that resembled tinea capitis on gross examination and mimicked juvenile xanthogranuloma on histologic examination. This case highlights the importance of considering cutaneous cryptococcosis in patients with hyper IgM syndrome.

A long-range MYC enhancer is duplicated in human T-ALL and is required for T cell development and NOTCH1-induced leukemogenesis.

Oncogenic activation of NOTCH1 signaling plays a central role in the pathogenesis of T-cell acute lymphoblastic leukemia, with mutations on this signaling pathway affecting more than 60% of patients at diagnosis. However, the transcriptional regulatory circuitries driving T-cell transformation downstream of NOTCH1 remain incompletely understood. Here we identify Hairy and Enhancer of Split 1 (HES1), a transcriptional repressor controlled by NOTCH1, as a critical mediator of NOTCH1-induced leukemogenesis strictly required for tumor cell survival. Mechanistically, we demonstrate that HES1 directly downregulates the expression of BBC3, the gene encoding the PUMA BH3-only proapoptotic factor in T-cell acute lymphoblastic leukemia. Finally, we identify perhexiline, a small-molecule inhibitor of mitochondrial carnitine palmitoyltransferase-1, as a HES1-signature antagonist drug with robust antileukemic activity against NOTCH1-induced leukemias in vitro and in vivo.

Over the last years, numerous studies have dissected the mutational landscape of T-cell acute lymphoblastic leukemia (T-ALL) resulting in the identification of numerous oncogenes and tumor suppressors implicated in T-cell transformation. However, most genetic abnormalities found in cancer are located in intergenic regions, whose role in cancer development, if any, remains poorly understood. Here we hypothesized that recurrent cancer-associated intergenic mutations, amplifications and deletions may implicate strong transcriptional regulatory sequences responsible for the activation of key oncogenic factors in the pathogenesis of T-ALL. To address this question, we first used ChIPseq analysis to map the genomic landscape of enhancer sequences controlled by NOTCH1, a critical T-ALL oncogene activated by mutations in over 60% of human T-ALLs. In addition, we performed high resolution aCGH analysis of somatic chromosomal amplifications and deletions in a comprehensive series of 160 T-ALL samples. These analyses revealed recurrent focal duplications at chromosome 8q24 in 8/160 (5%) T-ALL cases in an area devoid of protein-coding genes containing a prominent 1 kb NOTCH1-binding peak. Strikingly, this putative oncogenic element is located +1,427 kb 3’ from the MYC locus and chromatin configuration 3C analysis demonstrated its direct association with the MYC proximal promoter. Multispecies DNA sequence alignment revealed remarkable conservation of this region in mammals, birds and reptiles and local ChIP analysis revealed bona fide active enhancer features including P300 occupancy and high levels of H3K4me1 with low levels of H3K4me3. In addition, detailed analysis of epigenetic marks across 64 hematopoietic and non-hematopoietic cell lines and tissues revealed that his regulatory element is located within a major superenhancer specifically active in T-cells. Based on these results, we proposed that this regulatory sequence, hereby named N-Me for NOTCH-bound MYCenhancer, could function as an important regulatory element driving the activation of MYC downstream of NOTCH1 in T-ALL. Consistently, luciferase reporter assays showed strong, orientation-independent and NOTCH-dependent activation of reporter constructs containing N-Me in association with a -2.5 kb MYC proximal promoter in JURKAT T-ALL cells. Next and to formally test the functional relevance of this enhancer in T-cell development and transformation we generated and characterized N-Me knockout and conditional knockout mice. N-Me null animals were viable and showed a marked and selective reduction in thymus size and cellularity as their only developmental alteration. Detailed immunophenotypic analysis of N-Me knockout thymocytes demonstrated accumulation of double negative 3 (DN3) T cells and marked reductions in double positive and CD4+ and CD8+ single positive cells. Mechanistically, this phenotype was associated with a marked reduction in Myc expression in DN3, DN4 and ISP cells. Moreover, transplantation of N-Me knockout bone marrow hematopoietic progenitors demonstrated that this phenotype is cell autonomous, and can be rescued upon retroviral expression of MYC. Next, we analyzed the role of N-Me in the induction of NOTCH-driven leukemias by transplanting mice with N-Me wild-type and knockout hematopoietic progenitors infected with retroviruses expressing a mutant constitutively active form of NOTCH1 (ΔE-NOTCH1). In this context, mice transplanted with ΔE-NOTCH1-infected N-Me wild type cells developed T-ALL with 100% penetrance 6 weeks after transplant. In contrast, mice transplanted with ΔE-NOTCH1-N-Me knockout cells showed complete resistance to NOTCH1-induced transformation remaining 100% leukemia-free at 15 weeks post-transplant. In addition, secondary deletion of N-Me in established NOTCH1 induced leukemias from tamoxifen-inducible N-Me conditional knockout mice (Rosa26TMCre N-Meflox/flox) induced profound antileukemic effects with extended survival and almost complete suppression of leukemia initiating cell activity. Altogether, these results identify N-Me as the first long range oncogenic enhancer directly implicated in the pathogenesis of human leukemia. Disclosures No relevant conflicts of interest to declare.

Sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs), NAD(+)-dependent enzymes, link cellular energy status with responses to environmental stresses. Skin is frequently exposed to the DNA damaging effects of UV irradiation, a known etiology in skin cancer. Thus, understanding the defense mechanisms in response to UV, including the role of SIRTs and PARPs, may be important in developing skin cancer prevention strategies. Here, we report expression of the seven SIRT family members in human skin. SIRTs gene expressions are progressively upregulated in A431 epidermoid carcinoma cells (SIRTs1 and 3), actinic keratoses (SIRTs 2, 3, 5, 6, and 7) and squamous cell carcinoma (SIRTs 1-7). Photodamage induces dynamic changes in SIRT expression with upregulation of both SIRT1 and SIRT4 mRNAs. Specific losses of SIRT proteins occur early after photodamage followed by accumulation later, especially for SIRT4. Niacin restriction, which decreases NAD(+), the sirtuin substrate, results in an increase in acetylated proteins, upregulation of SIRTs 2 and 4, increased inherent DNA damage, alterations in SIRT responses to photodamage, abrogation of PARP activation following photodamage, and increased sensitivity to photodamage that is completely reversed by repleting niacin. These data support the hypothesis that SIRTs and PARPs play important roles in resistance to photodamage and identify specific SIRTs that respond to photodamage and may be targets for skin cancer prevention.

Dinitrotoluene (DNT) is an industrial chemical of importance in the production of urethane foams and elastomers. The technical grade material is a hepatocarcinogen in rodents but shows sex- and species-dependent differences in potency. We have studied the pathways of metabolism of 2,4-dinitrotoluene (2,4-DNT), the major component of technical material, in the cecal microflora of male rats, female rats, male mice, and in human feces and ileal contents. 2,4-DNT was not metabolized by any of these preparations in the presence of oxygen. Under anaerobic conditions an ordered sequence of reductive metabolism was observed. The 2- and 4-nitro groups were reduced to amino groups via nitroso intermediates which were identified by GC-MS. The reduction of the nitroso intermediate to the amino compound is presumed to involve a hydroxylamino intermediate which could not be isolated. The aminonitro compounds were then reduced to diaminotoluene. No intermediates in this sequence could be isolated. No sex- or species-dependent differences in the pathways of metabolism were observed and only small species-dependent differences in the rate of metabolism of 2,4-DNT were observed. It is concluded that the intestinal microflora of rodents represent the major site of reductive metabolism of 2,4-DNT and may play an important role in the carcinogenic action of DNT isomers.

Activating systems for in vitro mutagenesis assays are commonly prepared and stored at low temperature until required. The objective of the studies reported here was to determine the long-term stability of activating systems stored at - 85 degrees C. A broad range of microsomal enzymes in the postmitochondrial supernatant (PMS) and the microsomal fraction of livers from Aroclor 1254 treated rats were studied in conjunction with the ability of these fractions to catalyse the conversion of dimethylnitrosamine (DMN) and benzo(a)pyrene (B(a)P) to products mutagenic to Chinese hamster ovary (CHO) cells and Salmonella typhimurium TM677. Biphenyl-2- and biphenyl-4-hydroxylase showed a rapid decline in activity on storage, epoxide hydratase activity increased with storage and other enzyme activities studied were relatively stable for up to 32 weeks. No consistent trends in the ability of either the microsomes or the PMS to catalyze DMN or B(a)P induced mutation were observed for up to 12 weeks with CHO cells and 24 weeks with bacteria. It is concluded that low temperature storage of activating systems is an acceptable procedure. However, the results also indicate that certain enzyme activities change during storage, suggesting that aberrant results may be obtained when stored activating systems are used in in vitro tests to screen for mutagens.

The hepatic microsomal mixed function monooxygenase system (MFO) is the major enzyme system responsible for the activation and deactivation of xenobiotics. This study was designed to compare the hepatic MFO system in Fischer-344 and CD (Sprague-Dawley) rats. Hepatic microsomes were prepared from control, phenobarbital (3 × 80 mg/kg)-, and 3-methylcholanthrene (3 × 20 mg/kg)-pretreated male F-344 and CD rats (49 days old). Both control and phenobarbital-treated F-344 rats had significantly lower microsomal epoxide hydratase activity than corresponding preparations from CD rats. In addition, the pattern of benzo(a)pyrene metabolism was significantly different between the strains. Microsomes from F-344 rats produced less dihydrodiols and quinones than the corresponding preparations from CD rats. The spectral characteristics of cytochrome P-450 in hepatic microsomes from both control and phenobarbital-treated F-344 rats were significantly different from those observed in CD rat hepatic microsomes. Specifically, the λmax for the reduced cytochrome P-450 CO complex occurred at a slightly longer wavelength and the reduced ETNC 430 455 nm peak ratios were larger by about 70%. No significant strain differences were detected in control rats or rats pretreated with either phenobarbital or 3-methylcholanthrene with regard to microsomal protein, benzphetamine-N-demethylase, NADPH-cytochrome c reductase, biphenyl-4-hydroxylase, biphenyl-2-hydroxylase, arylhydrocarbon hydroxylase, ethoxycoumarin- or ethoxyresorufin-O-deethylase activities. These results suggest that differences do exist in the in vitro hepatic microsomal metabolism of xenobiotics in these two strains of rats. These differences may be of importance with respect to the susceptibility of the two strains of rats to various toxic agents.

Dinitrotoluene (DNT) is an industrial chemical used as an intermediate in the production of toluene diisocyanate for the manufacture of polyurethane foams, coatings and elastomers. Technical grade DNT is a potent hepatocarcinogen which produces a dose related incidence of hepatocellular carcinoma in Fischer 344 rats (CIIT, 1979). The technical grade material comprises approximately 75% 2,4-DNT, 19.5% 2,6-DNT and 4% other DNT isomers with small amounts of mono-and tri-nitrotoluenes and nitrocresols also present. The major component of the technical grade material, 2,4-DNT, has also been demonstrated to be a hepatocarcinogen in CD-1 rats (Ellis et al., 1979).