The origin and evolution of mutations in acute myeloid leukemia.

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

Pathogen and Antibody Identification in Children with Encephalitis in Myanmar.

OBJECTIVE: Prospective studies of encephalitis are rare in regions where encephalitis is prevalent, such as low middle-income Southeast Asian countries. We compared the diagnostic yield of local and advanced tests in cases of pediatric encephalitis in Myanmar. METHODS: Children with suspected subacute or acute encephalitis at Yangon Children's Hospital, Yangon, Myanmar, were prospectively recruited from 2016-2018. Cohort 1 (n = 65) had locally available diagnostic testing, whereas cohort 2 (n = 38) had advanced tests for autoantibodies (ie, cell-based assays, tissue immunostaining, studies with cultured neurons) and infections (ie, BioFire FilmArray multiplex Meningitis/Encephalitis multiplex PCR panel, metagenomic sequencing, and pan-viral serologic testing [VirScan] of cerebrospinal fluid). RESULTS: A total of 20 cases (13 in cohort 1 and 7 in cohort 2) were found to have illnesses other than encephalitis. Of the 52 remaining cases in cohort 1, 43 (83%) had presumed infectious encephalitis, of which 2 cases (4%) had a confirmed infectious etiology. Nine cases (17%) had presumed autoimmune encephalitis. Of the 31 cases in cohort 2, 23 (74%) had presumed infectious encephalitis, of which one (3%) had confirmed infectious etiology using local tests only, whereas 8 (26%) had presumed autoimmune encephalitis. Advanced tests confirmed an additional 10 (32%) infections, 4 (13%) possible infections, and 5 (16%) cases of N-methyl-D-aspartate receptor antibody encephalitis. INTERPRETATION: Pediatric encephalitis is prevalent in Myanmar, and advanced technologies increase identification of treatable infectious and autoimmune causes. Developing affordable advanced tests to use globally represents a high clinical and research priority to improve the diagnosis and prognosis of encephalitis. ANN NEUROL 2023;93:615-628.

Fecal microbiome and bile acid metabolome in adult short bowel syndrome.

Loss of functional small bowel surface area causes short bowel syndrome (SBS), intestinal failure, and parenteral nutrition (PN) dependence. The gut adaptive response following resection may be difficult to predict, and it may take up to 2 yr to determine which patients will wean from PN. Here, we examined features of gut microbiota and bile acid (BA) metabolism in determining adaptation and ability to wean from PN. Stool and sera were collected from healthy controls and from patients with SBS (n = 52) with ileostomy, jejunostomy, ileocolonic, and jejunocolonic anastomoses fed with PN plus enteral nutrition or who were exclusively enterally fed. We undertook 16S rRNA gene sequencing, BA profiling, and 7α-hydroxy-4-cholesten-3-one (C4) quantitation with LC-MS/MS and serum amino acid analyses. Patients with SBS exhibited altered gut microbiota with reduced gut microbial diversity compared with healthy controls. We observed differences in the microbiomes of patients with SBS with ileostomy versus jejunostomy, jejunocolonic versus ileocolonic anastomoses, and PN dependence compared with those who weaned from PN. Stool and serum BA composition and C4 concentrations were also altered in patients with SBS, reflecting adaptive changes in enterohepatic BA cycling. Stools from patients who were weaned from PN were enriched in secondary BAs including deoxycholic acid and lithocholic aicd. Shifts in gut microbiota and BA metabolites may generate a favorable luminal environment in select patients with SBS, promoting the ability to wean from PN. Proadaptive microbial species and select BA may provide novel targets for patient-specific therapies for SBS.NEW & NOTEWORTHY Loss of intestinal surface area causes short bowel syndrome, intestinal failure, and parenteral nutrition dependence. We analyzed the gut microbiota and bile acid metabolome of a large cohort of short bowel syndrome adult patients with different postsurgical anatomies. We report a novel analysis of the microbiome of patients with ileostomy and jejunostomy. Enrichment of specific microbial and bile acid species may be associated with the ability to wean from parenteral nutrition.

Methods and implementation of a pediatric asthma pharmacogenomic study in the emergency department setting.

OBJECTIVES: The emergency department (ED) is a challenging setting to conduct pharmacogenomic studies and integrate that data into fast-paced and potentially life-saving treatment decisions. Therefore, our objective is to present the methods and feasibility of a pilot pharmacogenomic study set in the ED that measured pediatric bronchodilator response (BDR) during acute asthma exacerbations. METHODS: This is an exploratory pilot study that collected buccal swabs for DNA and measured BDR during ED encounters for pediatric asthma exacerbations. We evaluated the study's feasibility with a qualitative analysis of ED provider surveys and quantitatively by the proportion of eligible patients enrolled. RESULTS: We enrolled 59 out of 90 patients (65%) that were identified and considered eligible during a 5-month period (target enrollment 60 patients over 12 months). The median patient age was 7 years (interquartile range 4-9 years), 61% (N = 36) were male, and 92% (N = 54) were African American. Quality DNA collection was successful for all 59 patients. The ED provider survey response rate was 100%. Most ED providers reported that the study did not impact their workflow (98% of physicians, 88% of nurses, and 90% of respiratory therapists). ED providers did report difficulties with spirometry in the younger age group. CONCLUSIONS: Pharmacogenomic studies can be conducted in the ED setting, and enroll a younger patient population with a high proportion of minority participants. By disseminating this study's methods and feasibility analysis, we aim to increase interest in pharmacogenomic studies set in the ED and aimed toward future ED-based pharmacogenomic decision-making.

Impaired Chylomicron Assembly Modifies Hepatic Metabolism Through Bile Acid-Dependent and Transmissible Microbial Adaptations.

The mechanisms by which alterations in intestinal bile acid (BA) metabolism improve systemic glucose tolerance and hepatic metabolic homeostasis are incompletely understood. We examined metabolic adaptations in mice with conditional intestinal deletion of the abetalipoproteinemia (ABL) gene microsomal triglyceride transfer protein (Mttp-IKO), which blocks chylomicron assembly and impairs intestinal lipid transport. Mttp-IKO mice exhibit improved hepatic glucose metabolism and augmented insulin signaling, without weight loss. These adaptations included decreased BA excretion, increased pool size, altered BA composition, and increased fibroblast growth factor 15 production. Mttp-IKO mice absorb fructose normally but are protected against dietary fructose-induced hepatic steatosis, without weight loss or changes in energy expenditure. In addition, Mttp-IKO mice exhibit altered cecal microbial communities, both at baseline and following fructose feeding, including increased abundance of Bacteroides and Lactobacillus genera. Transplantation of cecal microbiota from chow-fed Mttp-IKO mice into antibiotic-treated wild-type recipients conferred transmissible protection against fructose-induced hepatic steatosis in association with a bloom in Akkermansia and increased Clostridium XIVa genera, whose abundance was positively correlated with fecal coprostanol and total neutral sterol excretion in recipient mice. However, antibiotic-treated Mttp-IKO mice were still protected against fructose-induced hepatic steatosis, suggesting that changes in microbiota are not required for this phenotype. Nevertheless, we found increased abundance of fecal Akkermansia from two adult ABL subjects with MTTP mutations compared to their heterozygous parents and within the range noted in six healthy control subjects. Furthermore, Akkermansia abundance across all subjects was positively correlated with fecal coprostanol excretion. Conclusion: The findings collectively suggest multiple adaptive pathways of metabolic regulation following blocked chylomicron assembly, including shifts in BA signaling and altered microbial composition that confer a transmissible phenotype.

Host Gene Expression in Nose and Blood for the Diagnosis of Viral Respiratory Infection.

BACKGROUND: Recently there has been a growing interest in the potential for host transcriptomic analysis to augment the diagnosis of infectious diseases. METHODS: We compared nasal and blood samples for evaluation of the host transcriptomic response in children with acute respiratory syncytial virus (RSV) infection, symptomatic non-RSV respiratory virus infection, asymptomatic rhinovirus infection, and virus-negative asymptomatic controls. We used nested leave-one-pair-out cross-validation and supervised principal components analysis to define small sets of genes whose expression patterns accurately classified subjects. We validated gene classification scores using an external data set. RESULTS: Despite lower quality of nasal RNA, the number of genes detected by microarray in each sample type was equivalent. Nasal gene expression signal derived mainly from epithelial cells but also included a variable leukocyte contribution. The number of genes with increased expression in virus-infected children was comparable in nasal and blood samples, while nasal samples also had decreased expression of many genes associated with ciliary function and assembly. Nasal gene expression signatures were as good or better for discriminating between symptomatic, asymptomatic, and uninfected children. CONCLSUSIONS: Our results support the use of nasal samples to augment pathogen-based tests to diagnose viral respiratory infection.

Carriage of Cronobacter sakazakii in the Very Preterm Infant Gut.

Background: Cronobacter sakazakii causes severe neonatal infections, but we know little about gut carriage of this pathogen in very low birthweight infants. Methods: We sequenced 16S ribosomal RNA (rRNA) genes from 2304 stools from 121 children at St Louis Children's Hospital whose birthweight was ≤1500 g, attempted to isolate C. sakazakii from 157 of these stools, genome-sequenced the recovered isolates, and sought correlations between indices of Cronobacter excretion, host characteristics, and unit formula use. Results: Of these 2304 stools, 1271 (55.2%) contained Cronobacter rRNA gene sequences. The median (interquartile range) per-subject percentage of specimens with at least 1 Cronobacter sequence and the median per-subject read density were 57.1 (25.5-87.3) and 0.07 (0.01-0.67), respectively. There was no variation according to commercially prepared liquid vs powdered formula use in the neonatal intensive care unit, or the day of life that specimens were produced. However, the proportion of specimens containing >4.0% of reads mapping to Cronobacter fell from 4.3% to 0.9% after powdered infant formula was discontinued (P < .0001). We isolated sequence type 4 (ST4) C. sakazakii from multiple specimens from 2 subjects; 1 also harbored sequence type 233. The sequenced ST4 isolates from the 2 subjects had >99.9% sequence identity in the approximately 93% of best-match reference genome that they contained, and shared multiple virulence loci. Conclusions: Very low birthweight infants excrete putatively pathogenic Cronobacter. High-density Cronobacter sequence samples were more common during the use of powdered infant formula. Better understanding of the ecology of Cronobacter in infant guts will inform future prevention and control strategies.

Enhanced virome sequencing using targeted sequence capture.

Metagenomic shotgun sequencing (MSS) is an important tool for characterizing viral populations. It is culture independent, requires no a priori knowledge of the viruses in the sample, and may provide useful genomic information. However, MSS can lack sensitivity and may yield insufficient data for detailed analysis. We have created a targeted sequence capture panel, ViroCap, designed to enrich nucleic acid from DNA and RNA viruses from 34 families that infect vertebrate hosts. A computational approach condensed ∼1 billion bp of viral reference sequence into <200 million bp of unique, representative sequence suitable for targeted sequence capture. We compared the effectiveness of detecting viruses in standard MSS versus MSS following targeted sequence capture. First, we analyzed two sets of samples, one derived from samples submitted to a diagnostic virology laboratory and one derived from samples collected in a study of fever in children. We detected 14 and 18 viruses in the two sets, comprising 19 genera from 10 families, with dramatic enhancement of genome representation following capture enrichment. The median fold-increases in percentage viral reads post-capture were 674 and 296. Median breadth of coverage increased from 2.1% to 83.2% post-capture in the first set and from 2.0% to 75.6% in the second set. Next, we analyzed samples containing a set of diverse anellovirus sequences and demonstrated that ViroCap could be used to detect viral sequences with up to 58% variation from the references used to select capture probes. ViroCap substantially enhances MSS for a comprehensive set of viruses and has utility for research and clinical applications.

Detection of Viruses in Clinical Samples by Use of Metagenomic Sequencing and Targeted Sequence Capture.

Metagenomic shotgun sequencing (MSS) is a revolutionary approach to viral diagnostic testing that allows simultaneous detection of a broad range of viruses, detailed taxonomic assignment, and detection of mutations associated with antiviral drug resistance. To enhance sensitivity for virus detection, we previously developed ViroCap, a targeted sequence capture panel designed to enrich nucleic acid from a comprehensive set of eukaryotic viruses prior to sequencing. To demonstrate the utility of MSS with targeted sequence capture for detecting clinically important viruses and characterizing clinically important viral features, we used ViroCap to analyze clinical samples from a diagnostic virology laboratory containing a broad range of medically relevant viruses. From 26 samples, MSS with ViroCap detected all of the expected viruses and 30 additional viruses. Comparing sequencing after capture enrichment with standard MSS, we detected 13 viruses only with capture enrichment and observed a consistent increase in the number and percentage of viral sequence reads as well as the breadth and depth of coverage of the viral genomes. Compared with clinical testing, MSS enhanced taxonomic assignment for 15 viruses, and codons associated with antiviral drug resistance in influenza A virus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV) could be analyzed. Overall, in clinical samples, MSS with targeted sequence capture provides enhanced virus detection and information of clinical and epidemiologic relevance compared with clinical testing and MSS without targeted sequence capture.

The vaginal eukaryotic DNA virome and preterm birth.

BACKGROUND: Despite decades of attempts to link infectious agents to preterm birth, an exact causative microbe or community of microbes remains elusive. Culture-independent sequencing of vaginal bacterial communities demonstrates community characteristics are associated with preterm birth, although none are specific enough to apply clinically. Viruses are important components of the vaginal microbiome and have dynamic relationships with vaginal bacterial communities. We hypothesized that vaginal eukaryotic DNA viral communities (the "vaginal virome") either alone or in the context of bacterial communities are associated with preterm birth. OBJECTIVE: The objective of this study was to use high-throughput sequencing to examine the vaginal eukaryotic DNA virome in a cohort of pregnant women and examine associations between vaginal community characteristics and preterm birth. STUDY DESIGN: This is a nested case-control study within a prospective cohort study of women with singleton pregnancies, not on supplemental progesterone, and without cervical cerclage in situ. Serial midvaginal swabs were obtained at routine prenatal visits. DNA was extracted, bacterial communities were characterized by 16S ribosomal RNA gene sequencing, and eukaryotic viral communities were characterized by enrichment of viral nucleic acid with the ViroCap targeted sequence capture panel followed by nucleic acid sequencing. Viral communities were analyzed according to presence/absence of viruses, diversity, dynamics over time, and association with bacterial community data obtained from the same specimens. RESULTS: Sixty subjects contributed 128 vaginal swabs longitudinally across pregnancy. In all, 24 patients delivered preterm. Participants were predominantly African American (65%). Six families of eukaryotic DNA viruses were detected in the vaginal samples. At least 1 virus was detected in 80% of women. No specific virus or group of viruses was associated with preterm delivery. Higher viral richness was significantly associated with preterm delivery in the full group and in the African American subgroup (P = .0005 and P = .0003, respectively). Having both high bacterial diversity and high viral diversity in the first trimester was associated with the highest risk for preterm birth. CONCLUSION: Higher vaginal viral diversity is associated with preterm birth. Changes in vaginal virome diversity appear similar to changes in the vaginal bacterial microbiome over pregnancy, suggesting that underlying physiology of pregnancy may regulate both bacterial and viral communities.

Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting.

Cancer immunoediting, the process by which the immune system controls tumour outgrowth and shapes tumour immunogenicity, is comprised of three phases: elimination, equilibrium and escape. Although many immune components that participate in this process are known, its underlying mechanisms remain poorly defined. A central tenet of cancer immunoediting is that T-cell recognition of tumour antigens drives the immunological destruction or sculpting of a developing cancer. However, our current understanding of tumour antigens comes largely from analyses of cancers that develop in immunocompetent hosts and thus may have already been edited. Little is known about the antigens expressed in nascent tumour cells, whether they are sufficient to induce protective antitumour immune responses or whether their expression is modulated by the immune system. Here, using massively parallel sequencing, we characterize expressed mutations in highly immunogenic methylcholanthrene-induced sarcomas derived from immunodeficient Rag2(-/-) mice that phenotypically resemble nascent primary tumour cells. Using class I prediction algorithms, we identify mutant spectrin-ß2 as a potential rejection antigen of the d42m1 sarcoma and validate this prediction by conventional antigen expression cloning and detection. We also demonstrate that cancer immunoediting of d42m1 occurs via a T-cell-dependent immunoselection process that promotes outgrowth of pre-existing tumour cell clones lacking highly antigenic mutant spectrin-ß2 and other potential strong antigens. These results demonstrate that the strong immunogenicity of an unedited tumour can be ascribed to expression of highly antigenic mutant proteins and show that outgrowth of tumour cells that lack these strong antigens via a T-cell-dependent immunoselection process represents one mechanism of cancer immunoediting.

Acute Hypotonia in an Infant.

BACKGROUND: Acute flaccid myelitis (AFM) is increasing in incidence in the United States and presenting to emergency departments (EDs) across the country. This clinical entity presents as acute paralysis, with magnetic resonance imaging changes in the gray matter only in children younger than 21 years of age. The etiology is unknown, although preceding viral illnesses are common. There are no consensus guidelines regarding treatment. CASE REPORT: A 4-month-old girl presented with decreased bilateral arm movement. The history consisted of a recent upper respiratory illness and abrupt decline in movement. She was found to have truncal and peripheral hypotonia, while maintaining her airway. Magnetic resonance imaging found gray matter hyperintensity at C2-C6, with no white matter changes. The patient was positive for enterovirus. Intravenous steroids and intravenous immunoglobulin were given, with slight improvement prior to discharge to an inpatient rehabilitation center. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: AFM was largely nonexistent in the United States after implementation of the polio vaccine, but the incidence has recently increased. Pediatric patients are now presenting to EDs with acute hypotonia, and emergency physicians must recognize how to differentiate this emerging diagnosis from other causes of acute flaccid paralysis. The clinical course of AFM does not seem to change acutely, in stark contrast to disease entities like botulism, which can change in hours. Patients with AFM do not need aggressive ED diagnostic evaluation, but rather transfer to a pediatric hospital for further care. Therefore, discerning the etiology of pediatric hypotonia with history and physical examination alone is important.

Somatic neurofibromatosis type 1 (NF1) inactivation characterizes NF1-associated pilocytic astrocytoma.

Low-grade brain tumors (pilocytic astrocytomas) arising in the neurofibromatosis type 1 (NF1) inherited cancer predisposition syndrome are hypothesized to result from a combination of germline and acquired somatic NF1 tumor suppressor gene mutations. However, genetically engineered mice (GEM) in which mono-allelic germline Nf1 gene loss is coupled with bi-allelic somatic (glial progenitor cell) Nf1 gene inactivation develop brain tumors that do not fully recapitulate the neuropathological features of the human condition. These observations raise the intriguing possibility that, while loss of neurofibromin function is necessary for NF1-associated low-grade astrocytoma development, additional genetic changes may be required for full penetrance of the human brain tumor phenotype. To identify these potential cooperating genetic mutations, we performed whole-genome sequencing (WGS) analysis of three NF1-associated pilocytic astrocytoma (PA) tumors. We found that the mechanism of somatic NF1 loss was different in each tumor (frameshift mutation, loss of heterozygosity, and methylation). In addition, tumor purity analysis revealed that these tumors had a high proportion of stromal cells, such that only 50%-60% of cells in the tumor mass exhibited somatic NF1 loss. Importantly, we identified no additional recurrent pathogenic somatic mutations, supporting a model in which neuroglial progenitor cell NF1 loss is likely sufficient for PA formation in cooperation with a proper stromal environment.

Genome Modeling System: A Knowledge Management Platform for Genomics.

In this work, we present the Genome Modeling System (GMS), an analysis information management system capable of executing automated genome analysis pipelines at a massive scale. The GMS framework provides detailed tracking of samples and data coupled with reliable and repeatable analysis pipelines. The GMS also serves as a platform for bioinformatics development, allowing a large team to collaborate on data analysis, or an individual researcher to leverage the work of others effectively within its data management system. Rather than separating ad-hoc analysis from rigorous, reproducible pipelines, the GMS promotes systematic integration between the two. As a demonstration of the GMS, we performed an integrated analysis of whole genome, exome and transcriptome sequencing data from a breast cancer cell line (HCC1395) and matched lymphoblastoid line (HCC1395BL). These data are available for users to test the software, complete tutorials and develop novel GMS pipeline configurations. The GMS is available at https://github.com/genome/gms.

Impact of Video Discharge Instructions for Pediatric Fever and Closed Head Injury from the Emergency Department.

BACKGROUND: Lack of understanding of diagnosis and disease process remains a major complaint of caregivers who bring their children to the pediatric emergency department (PED). Misunderstanding of diagnosis and discharge instructions can lead to unnecessary return visits and health disparities. OBJECTIVE: We attempted to determine if video discharge instructions when added to standard of care written and verbal instruction improved caregivers' comprehension of their child's diagnosis, disease process, and discharge instructions. METHODS: Caregivers who presented to the PED with a child's chief complaint of fever or closed head injury (CHI) were included and randomized into a control or intervention group. Each group received standard discharge instructions, and the intervention group additionally viewed a video. Participants completed a post-test on knowledge and were followed 2 weeks post-visit to determine follow-up care. RESULTS: Sixty-three caregivers participated in the study. Eleven participants had less than a high school (HS) education and 52 had more than a HS education. Thirty-one children presented with fever and 32 with CHI. The intervention group had significantly higher percentage of correct answers on postintervention tests (median [Mdn] = 88.89) than the control (Mdn = 75.73; p < 0.0001). Participants in the intervention group with less than a HS education (Mdn = 89.47) and more than HS education (Mdn = 88.89) had similar test scores (p = 0.13), whereas those in the control group with less than a HS education (Mdn = 66.67) had significantly lower test scores than those with more than a HS education (Mdn = 77.78; p = 0.03). CONCLUSION: For caregivers with children who presented to the PED with fever and CHI, video discharge instructions improved caregiver comprehension of the child's diagnosis and disease process when added to verbal and written instructions.

RNA-sequencing reveals oligodendrocyte and neuronal transcripts in microglia relevant to central nervous system disease.

Expression profiling of distinct central nervous system (CNS) cell populations has been employed to facilitate disease classification and to provide insights into the molecular basis of brain pathology. One important cell type implicated in a wide variety of CNS disease states is the resident brain macrophage (microglia). In these studies, microglia are often isolated from dissociated brain tissue by flow sorting procedures [fluorescence-activated cell sorting (FACS)] or from postnatal glial cultures by mechanic isolation. Given the highly dynamic and state-dependent functions of these cells, the use of FACS or short-term culture methods may not accurately capture the biology of brain microglia. In the current study, we performed RNA-sequencing using Cx3cr1(+/GFP) labeled microglia isolated from the brainstem of 6-week-old mice to compare the transcriptomes of FACS-sorted versus laser capture microdissection (LCM). While both isolation techniques resulted in a large number of shared (common) transcripts, we identified transcripts unique to FACS-isolated and LCM-captured microglia. In particular, ∼50% of these LCM-isolated microglial transcripts represented genes typically associated with neurons and glia. While these transcripts clearly localized to microglia using complementary methods, they were not translated into protein. Following the induction of murine experimental autoimmune encephalomyelitis, increased oligodendrocyte and neuronal transcripts were detected in microglia, while only the myelin basic protein oligodendrocyte transcript was increased in microglia after traumatic brain injury. Collectively, these findings have implications for the design and interpretation of microglia transcriptome-based investigations.

The transcriptional profile of coronary arteritis in Kawasaki disease.

BACKGROUND: Kawasaki Disease (KD) can cause potentially life-threatening coronary arteritis in young children, and has a likely infectious etiology. Transcriptome profiling is a powerful approach to investigate gene expression in diseased tissues. RNA sequencing of KD coronary arteries could elucidate the etiology and the host response, with the potential to improve KD diagnosis and/or treatment. METHODS: Deep RNA sequencing was performed on KD (n = 8) and childhood control (n = 7) coronary artery tissues, revealing 1074 differentially expressed mRNAs. Non-human RNA sequences were subjected to a microbial discovery bioinformatics platform, and microbial sequences were analyzed by Metastats for association with KD. RESULTS: T lymphocyte activation, antigen presentation, immunoglobulin production, and type I interferon response were significantly upregulated in KD arteritis, while the tumor necrosis factor α pathway was not differentially expressed. Transcripts from known infectious agents were not specifically associated with KD coronary arteritis. CONCLUSIONS: The immune transcriptional profile in KD coronary artery tissues has features of an antiviral immune response such as activated cytotoxic T lymphocyte and type I interferon-induced gene upregulation. These results provide new insights into the pathogenesis of KD arteritis that can guide selection of new immunomodulatory therapies for high-risk KD patients, and provide direction for future etiologic studies.

Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay.

We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.

Genomic impact of transient low-dose decitabine treatment on primary AML cells.

Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized.

Sepsis from the gut: the enteric habitat of bacteria that cause late-onset neonatal bloodstream infections.

BACKGROUND: Late-onset sepsis is a major problem in neonatology, but the habitat of the pathogens before bloodstream invasion occurs is not well established. METHODS: We examined prospectively collected stools from premature infants with sepsis to find pathogens that subsequently invaded their bloodstreams, and sought the same organisms in stools of infants without sepsis. Culture-based techniques were used to isolate stool bacteria that provisionally matched the bloodstream organisms, which were then genome sequenced to confirm or refute commonality. RESULTS: Of 11 children with late-onset neonatal bloodstream infections, 7 produced at least 1 stool that contained group B Streptococcus (GBS), Serratia marcescens, or Escherichia coli before their sepsis episode with provisionally matching organisms. Of 96 overlap comparison subjects without sepsis temporally associated with these cases, 4 were colonized with provisionally matching GBS or S. marcescens. Of 175 comparisons of stools from randomly selected infants without sepsis, 1 contained a GBS (this infant had also served as an overlap comparison subject and both specimens contained provisionally matching GBS). Genome sequencing confirmed common origin of provisionally matching fecal and blood isolates. The invasive E. coli were present in all presepticemic stools since birth, but gut colonization with GBS and S. marcescens occurred closer to time of bloodstream infection. CONCLUSIONS: The neonatal gut harbors sepsis-causing pathogens, but such organisms are not inevitable members of the normal microbiota. Surveillance microbiology, decolonization, and augmented hygiene might prevent dissemination of invasive bacteria between and within premature infants.