Robust Transcriptional Profiling and Identification of Differentially Expressed Genes With Low Input RNA Sequencing of Adult Hippocampal Neural Stem and Progenitor Populations.

Multipotent neural stem cells (NSCs) are found in several isolated niches of the adult mammalian brain where they have unique potential to assist in tissue repair. Modern transcriptomics offer high-throughput methods for identifying disease or injury associated gene expression signatures in endogenous adult NSCs, but they require adaptation to accommodate the rarity of NSCs. Bulk RNA sequencing (RNAseq) of NSCs requires pooling several mice, which impedes application to labor-intensive injury models. Alternatively, single cell RNAseq can profile hundreds to thousands of cells from a single mouse and is increasingly used to study NSCs. The consequences of the low RNA input from a single NSC on downstream identification of differentially expressed genes (DEGs) remains insufficiently explored. Here, to clarify the role that low RNA input plays in NSC DEG identification, we directly compared DEGs in an oxidative stress model of cultured NSCs by bulk and single cell sequencing. While both methods yielded DEGs that were replicable, single cell sequencing using the 10X Chromium platform yielded DEGs derived from genes with higher relative transcript counts compared to non-DEGs and exhibited smaller fold changes than DEGs identified by bulk RNAseq. The loss of high fold-change DEGs in the single cell platform presents an important limitation for identifying disease-relevant genes. To facilitate identification of such genes, we determined an RNA-input threshold that enables transcriptional profiling of NSCs comparable to standard bulk sequencing and used it to establish a workflow for in vivo profiling of endogenous NSCs. We then applied this workflow to identify DEGs after lateral fluid percussion injury, a labor-intensive animal model of traumatic brain injury. Our work joins an emerging body of evidence suggesting that single cell RNA sequencing may underestimate the diversity of pathologic DEGs. However, our data also suggest that population level transcriptomic analysis can be adapted to capture more of these DEGs with similar efficacy and diversity as standard bulk sequencing. Together, our data and workflow will be useful for investigators interested in understanding and manipulating adult hippocampal NSC responses to various stimuli.

Liquid biopsies in pediatric oncology: opportunities and obstacles.

PURPOSE OF REVIEW: Liquid biopsies have emerged as a noninvasive alternative to tissue biopsy with potential applications during all stages of pediatric oncology care. The purpose of this review is to provide a survey of pediatric cell-free DNA (cfDNA) studies, illustrate their potential applications in pediatric oncology, and to discuss technological challenges and approaches to overcome these hurdles. RECENT FINDINGS: Recent literature has demonstrated liquid biopsies' ability to inform treatment selection at diagnosis, monitor clonal evolution during treatment, sensitively detect minimum residual disease following local control, and provide sensitive posttherapy surveillance. Advantages include reduced procedural anesthesia, molecular profiling unbiased by tissue heterogeneity, and ability to track clonal evolution. Challenges to wider implementation in pediatric oncology, however, include blood volume restrictions and relatively low mutational burden in childhood cancers. Multiomic approaches address challenges presented by low-mutational burden, and novel bioinformatic analyses allow a single assay to yield increasing amounts of information, reducing blood volume requirements. SUMMARY: Liquid biopsies hold tremendous promise in pediatric oncology, enabling noninvasive serial surveillance with adaptive care. Already integrated into adult care, recent advances in technologies and bioinformatics have improved applicability to the pediatric cancer landscape.

Mandatory Hepatology Education for Internal Medicine Residents: Long-Term Effects and Implications for Workforce Needs.

We previously created a mandatory, inpatient, hepatology resident curriculum that immediately improved comfort, knowledge, and career interest in chronic liver disease (CLD). The durability of these effects needs to be known to use this intervention to address the hepatologist shortage. Thus, we aimed to assess this curriculum's long-term outcomes on internal medicine (IM) residents' CLD comfort, knowledge, and career interest. From 2015 to 2019 at a single institution, one IM resident was always assigned to the rotation. Similar anonymous assessments were administered to incoming postgraduate year (PGY)-1 residents and graduating PGY-3 residents, including a historic control cohort that graduated in June 2015. At residency completion, the intervention cohort (n = 61) had significantly higher comfort (1, not at all comfortable/strongly disagree; 5, very comfortable/strongly agree) with both hepatology (e.g., hepatitis C, 2.5 vs. 3.3, P < 0.001) and common IM topics (e.g., heart failure, 3.6 vs. 4.8, P < 0.001) but not specialty topics lacking curricula (e.g., inflammatory bowel disease, 2.8 vs. 2.7, P = 0.54). Compared to the historic cohort (n = 27), the intervention cohort was more comfortable in several CLD topics (e.g., cirrhosis, 3.2 vs. 3.8; P = 0.005) and answered more questions correctly (65% vs. 55%; P = 0.04), but career interest was unchanged (1.9 vs. 1.8; P = 0.45). Many residents (33%) would consider a hepatology career if training were separated from gastroenterology. Conclusion: With the completion of a mandatory hepatology curriculum, residents' CLD comfort and knowledge durably improved and exceeded that of historic counterparts. Initial career interest was not sustained, perhaps due to prerequisite gastroenterology training. These findings suggest IM educational initiatives may better address hepatology workforce needs by generating comanagers than by recruiting trainees.

Outcomes of Open, Laparoscopic, and Percutaneous Drainage of Infected Walled-Off Pancreatic Necrosis: A Nationwide Inpatient Sample Study.

Background Walled-off pancreatic necrosis (WOPN) represents an encapsulated collection of necrotic pancreatic or peripancreatic tissue that tends to develop four weeks after the onset of acute necrotizing pancreatitis. When infected, it is managed initially by antibiotic therapy before drainage by endoscopic, percutaneous, or surgical means. This study aims to describe the morbidity, mortality, length of stay (LOS), and cost of care associated with open surgical, laparoscopic, and radiology-guided percutaneous drainage in adult patients with infected WOPN. Methods Using the Nationwide Inpatient Sample (NIS), patients aged 18 years and older discharged with the diagnosis of WOPN between January 1, 2016 and December 31, 2016 who underwent open, laparoscopic, or percutaneous drainage were included. Patients' characteristics including age, gender, and body mass index were reported. The primary endpoints were the mortality rate as well as length and cost of stay in each group. The secondary endpoint was the rate of procedural complications in each arm. Endpoints were reported and compared with studies assessing similar outcomes. Statistical Analysis System (SAS) statistical software (SAS Institute Inc., Cary, NC, USA) was used to perform the analysis. Results A total of 229 patients with the diagnosis of acute pancreatitis with infected necrosis were identified. Of these 229 patients, 27, 15, and 20 underwent open, laparoscopic, and percutaneous drainage, respectively. A total of eight studies were used for comparison of outcome variables. Mortality rate was found to be similar among comparison studies. LOS and costs varied widely among studies. There were significantly fewer pancreatic fistula and significantly more multi-organ failure complications as a result of open necrosectomy in the NIS study sample. Conclusion Overall, in analyzing the outcomes of patients undergoing intervention for infected WOPN through the 2016 NIS database, it appears that the database is representative of the majority of outcomes seen in similar clinical trials.

Atoh1 directs the formation of sensory mosaics and induces cell proliferation in the postnatal mammalian cochlea in vivo.

Hearing impairment due to the loss of sensory hair cells is permanent in humans. Considerable interest targets the hair cell differentiation factor Atoh1 as a potential tool with which to promote hair cell regeneration. We generated a novel mouse model to direct the expression of Atoh1 in a spatially and temporally specific manner in the postnatal mammalian cochlea to determine the competency of various types of cochlear epithelial cells for hair cell differentiation. Atoh1 can generate cells in young animals with morphological, molecular, and physiological properties reminiscent of hair cells. This competency is cell type specific and progressively restricted with age. Significantly, Atoh1 induces ectopic sensory patches through Notch signaling to form a cellular mosaic similar to the endogenous sensory epithelia and expansion of the sensory mosaic through the conversion of supporting cells and nonautonomous supporting cell production. Furthermore, Atoh1 also activates proliferation within the normally postmitotic cochlear epithelium. These results provide insight into the potential and limitations of Atoh1-mediated hair cell regeneration.