Development of an automated amplicon-based next-generation sequencing pipeline for rapid detection of bacteria and fungi directly from clinical specimens.

The timely identification of microbial pathogens is essential to guide targeted antimicrobial therapy and ultimately, successful treatment of an infection. However, the yield of standard microbiology testing (SMT) is directly related to the duration of antecedent antimicrobial therapy as SMT culture methods are dependent on the recovery of viable organisms, the fastidious nature of certain pathogens, and other pre-analytic factors. In the last decade, metagenomic next-generation sequencing (mNGS) has been successfully utilized as a diagnostic tool for various applications within the clinical laboratory. However, mNGS is resource, time, and labor-intensive-requiring extensive laborious preliminary benchwork, followed by complex bioinformatic analysis. We aimed to address these shortcomings by developing a largely Automated targeted Metagenomic next-generation sequencing (tmNGS) PipeLine for rapId inFectIous disEase Diagnosis (AMPLIFIED) to detect bacteria and fungi directly from clinical specimens. Therefore, AMPLIFIED may serve as an adjunctive approach to complement SMT. This tmNGS pipeline requires less than 1 hour of hands-on time before sequencing and less than 2 hours of total processing time, including bioinformatic analysis. We performed tmNGS on 50 clinical specimens with concomitant cultures to assess feasibility and performance in the hospital laboratory. Of the 50 specimens, 34 (68%) were from true clinical infections. Specimens from cases of true infection were more often tmNGS positive compared to those from the non-infected group (82.4% vs 43.8%, respectively, P = 0.0087). Overall, the clinical sensitivity of AMPLIFIED was 54.6% with 85.7% specificity, equating to 70.6% and 75% negative and positive predictive values, respectively. AMPLIFIED represents a rapid supplementary approach to SMT; the typical time from specimen receipt to identification of potential pathogens by AMPLIFIED is roughly 24 hours which is markedly faster than the days, weeks, and months required to recover bacterial, fungal, and mycobacterial pathogens by culture, respectively. IMPORTANCE: To our knowledge, this represents the first application of an automated sequencing and bioinformatics pipeline in an exclusively pediatric population. Next-generation sequencing is time-consuming, labor-intensive, and requires experienced personnel; perhaps contributing to hesitancy among clinical laboratories to adopt such a test. Here, we report a strong case for use by removing these barriers through near-total automation of our sequencing pipeline.

Reverse Plasticity Underlies Rapid Evolution by Clonal Selection within Populations of Fibroblasts Propagated on a Novel Soft Substrate.

Mechanical properties such as substrate stiffness are a ubiquitous feature of a cell's environment. Many types of animal cells exhibit canonical phenotypic plasticity when grown on substrates of differing stiffness, in vitro and in vivo. Whether such plasticity is a multivariate optimum due to hundreds of millions of years of animal evolution, or instead is a compromise between conflicting selective demands, is unknown. We addressed these questions by means of experimental evolution of populations of mouse fibroblasts propagated for approximately 90 cell generations on soft or stiff substrates. The ancestral cells grow twice as fast on stiff substrate as on soft substrate and exhibit the canonical phenotypic plasticity. Soft-selected lines derived from a genetically diverse ancestral population increased growth rate on soft substrate to the ancestral level on stiff substrate and evolved the same multivariate phenotype. The pattern of plasticity in the soft-selected lines was opposite of the ancestral pattern, suggesting that reverse plasticity underlies the observed rapid evolution. Conversely, growth rate and phenotypes did not change in selected lines derived from clonal cells. Overall, our results suggest that the changes were the result of genetic evolution and not phenotypic plasticity per se. Whole-transcriptome analysis revealed consistent differentiation between ancestral and soft-selected populations, and that both emergent phenotypes and gene expression tended to revert in the soft-selected lines. However, the selected populations appear to have achieved the same phenotypic outcome by means of at least two distinct transcriptional architectures related to mechanotransduction and proliferation.

K⁺ and Rb⁺ Affinities of the Na,K-ATPase α1 and α2 Isozymes: An Application of ICP-MS for Quantification of Na⁺ Pump Kinetics in Myofibers.

The potassium affinities of Na,K-ATPase isozymes are important determinants of their physiological roles in skeletal muscle. This study measured the apparent K⁺ and Rb⁺ affinities of the Na,K-ATPase α1 and α2 isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (α1S/Sα2R/R and skα2-/-). It also validates a new method to quantify cations in intact, dissociated myofibers, using inductively coupled plasma mass spectrometry (ICP-MS). Our findings were that: (1) The extracellular substrate sites of Na,K-ATPase bind Rb⁺ and K⁺ with comparable apparent affinities; however; turnover rate is reduced when Rb⁺ is the transported ion; (2) The rate of Rb⁺ uptake by the Na,K-ATPase is not constant but declines with a half-time of approximately 1.5 min; (3) The apparent K⁺ affinity of the α2 isozymes for K⁺ is significantly lower than α1. When measured in intact fibers of WT and α1S/Sα2R/R mice in the presence of 10 µM ouabain; the K1/2,K of α1 and α2 isozymes are 1.3 and 4 mM, respectively. Collectively, these results validate the single fiber model for studies of Na,K-ATPase transport and kinetic constants, and they imply the existence of mechanisms that dynamically limit pump activity during periods of active transport.

A method for measuring mitochondrial DNA copy number in pediatric populations.

The mitochondrion is a multifunctional organelle that modulates multiple systems critical for homeostasis during pathophysiological stress. Variation in mitochondrial DNA (mtDNA) copy number (mtDNAcn), a key mitochondrial change associated with chronic stress, is an emerging biomarker for disease pathology and progression. mtDNAcn can be quantified from whole blood samples using qPCR to determine the ratio of mtDNA to nuclear DNA. However, the collection of blood samples in pediatric populations, particularly in infants and young children, can be technically challenging, yield much smaller volume samples, and can be distressing for the patients and their caregivers. Therefore, we have validated a mtDNAcn assay utilizing DNA from simple buccal swabs (Isohelix SK-2S) and report here it's performance in specimens from infants (age = <12 months). Utilizing qPCR to amplify ∼200 bp regions from two mitochondrial (ND1, ND6) and two nuclear (BECN1, NEB) genes, we demonstrated absolute (100%) concordance with results from low-pass whole genome sequencing (lpWGS). We believe that this method overcomes key obstacles to measuring mtDNAcn in pediatric populations and creates the possibility for development of clinical assays to measure mitochondrial change during pathophysiological stress.

Pediatric Chordoma: A Tale of Two Genomes.

Little is known regarding the genomic alterations in chordoma, with the exception of loss of SMARCB1, a core member of the SWI/SNF complex, in poorly differentiated chordomas. A TBXT duplication and rs2305089 polymorphism, located at 6q27, are known genetic susceptibility loci. A comprehensive genomic analysis of the nuclear and mitochondrial genomes in pediatric chordoma has not yet been reported. In this study, we performed whole exome and mitochondrial DNA (mtDNA) genome sequencing on 29 chordomas from 23 pediatric patients. Findings were compared with that from whole genome sequencing datasets of 80 adult skull base chordoma patients. In the pediatric chordoma cohort, 81% percent of the somatic mtDNA mutations were observed in NADH complex genes, which is significantly enriched compared to the rest of the mtDNA genes (p=0.001). In adult chordomas, mtDNA mutations were also enriched in the NADH complex genes (p<0.0001). Furthermore, a progressive increase in heteroplasmy of non-synonymous mtDNA mutations was noted in patients with multiple tumors (p=0.0007). In the nuclear genome, rare likely germline in-frame indels in ARID1B, a member of the SWI/SNF complex located at 6q25.3, were observed in five pediatric patients (22%) and four patients in the adult cohort (5%). The frequency of rare ARID1B indels in the pediatric cohort is significantly higher than that of the adult cohort (p=0.0236, Fisher's exact test), but they were both significantly higher than that in the ethnicity-matched populations (p<5.9e-07 and p<0.0001174, respectively). Implications: germline ARID1B indels and mtDNA aberrations appear important for chordoma genesis, especially in pediatric chordoma.

Trafficking of mitochondrial double-stranded RNA from mitochondria to the cytosol.

In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.

Maintaining informatics training learning outcomes with a COVID-19 era shift to a fully online flipped course.

The emergence of the coronavirus disease 2019 pandemic forced us to adapt our recently developed informatics training serving a variety of students as well as faculty and staff. The successful flipped classroom course series (a hybrid-format with both asynchronous online learning and in-person synchronous components) was shifted to a fully online format with the synchronous portion now held via web-based video conference. We repeated our participant survey at the end of each of the 3 one-credit courses to compare student satisfaction and learning outcomes achievement to the original offering. The responses were overall very positive again with a slight response distribution improvement in some measures of satisfaction. Likewise, students reported similar achievement of the learning outcomes across all courses with some Unix coourse objectives receiving higher competency agreement in the new, fully online version. Overall, the fully online version of the course series was equally successful, if not more so, than the original version with a physical classroom session each week. Given that participants also had strong agreement with a new question that they would prefer online class meetings instead of in a classroom, even if there wasn't a global pandemic (citing a variety of logistical reasons such as "convenience of screen sharing," parking issues, and job-related time constraints), the fully online version of the informatics training will be retained.

Transcriptional Profile of Human Pancreatic Acinar Ductal Metaplasia.

BACKGROUND AND AIMS: Aberrant acinar to ductal metaplasia (ADM), one of the earliest events involved in exocrine pancreatic cancer development, is typically studied using pancreata from genetically engineered mouse models. METHODS: We used primary, human pancreatic acinar cells from organ donors to evaluate the transcriptional and pathway profiles during the course of ADM. RESULTS: Following 6 days of three-dimensional culture on Matrigel, acinar cells underwent morphological and molecular changes indicative of ADM. mRNA from 14 donors' paired cells (day 0, acinar phenotype and day 6, ductal phenotype) was subjected to whole transcriptome sequencing. Acinar cell specific genes were significantly downregulated in the samples from the day 6 cultures while ductal cell-specific genes were upregulated. Several regulons of ADM were identified including transcription factors with reduced activity (PTF1A, RBPJL, and BHLHA15) and those ductal and progenitor transcription factors with increased activity (HNF1B, SOX11, and SOX4). Cells with the ductal phenotype contained higher expression of genes increased in pancreatic cancer while cells with an acinar phenotype had lower expression of cancer-associated genes. CONCLUSION: Our findings support the relevancy of human in vitro models to study pancreas cancer pathogenesis and exocrine cell plasticity.

Toward Building Surge Capacity: Potentially Effective Spatial Configurations in Emergency Departments.

BACKGROUND: Emergency departments (EDs) have been struggling with overcrowding issues for years. Some spatial configurations have been proposed to improve ED performance in facing overcrowding. Despite similarities with mass casualty incidents (MCIs), when demand for care exceeds the capacity, little is documented about the application of the proposed configurations during MCIs to improve surge capacity. OBJECTIVES: We aimed to explore the potential of spatial configurations that have been proposed to handle ED overcrowding in daily operations so as to improve surge capacity during MCIs. METHODS: Using an online Likert-scale survey, 11 spatial design strategies were rated by ED care teams in terms of their potential to improve surge capacity during MCIs. RESULTS: Responses from 72 participants revealed that establishing an in-house lab was perceived as the most potential strategy, followed by rapid care area, internal waiting rooms, and in-house imaging. In contrast, separate entrance and exit doors, as well as decentralized nurse stations, were perceived as the least potential strategies but also exhibited the most variance in response. Respondents' comments implied that their choice of in-house ancillary services was primarily to improve communication and to reduce turnaround time and risk of errors. Their choice of rapid care and internal waiting areas related to improved flexibility. CONCLUSIONS: Understanding clinicians' perspectives on potentially effective spatial configurations aids in implementing balanced strategies to better equip EDs to handle overcrowding in daily operations and manage surges during MCIs.

The vaginal microbiome is associated with endometrial cancer grade and histology.

The human microbiome has been strongly correlated with disease pathology and outcomes, yet remains relatively underexplored in patients with malignant endometrial disease. In this study, vaginal microbiome samples were prospectively collected at the time of hysterectomy from 61 racially and ethnically diverse patients from three disease conditions: 1) benign gynecologic disease (controls, n=11), 2) low-grade endometrial carcinoma (n=30), and 3) high-grade endometrial carcinoma (n=20). Extracted DNA underwent shotgun metagenomics sequencing, and microbial α and ß diversities were calculated. Hierarchical clustering was used to describe community state types (CST), which were then compared by microbial diversity and grade. Differential abundance was calculated, and machine learning utilized to assess the predictive value of bacterial abundance to distinguish grade and histology. Both α- and ß-diversity were associated with patient tumor grade. Four vaginal CST were identified that associated with grade of disease. Different histologies also demonstrated variation in CST within tumor grades. Using supervised clustering algorithms, critical microbiome markers at the species level were used to build models that predicted benign vs carcinoma, high-grade carcinoma versus benign, and high-grade versus low-grade carcinoma with high accuracy. These results confirm that the vaginal microbiome segregates not just benign disease from endometrial cancer, but is predictive of histology and grade. Further characterization of these findings in large, prospective studies is needed to elucidate their potential clinical applications.

Pharmacological inhibition and reversal of pancreatic acinar ductal metaplasia.

Pancreatic acinar cells display a remarkable degree of plasticity and can dedifferentiate into ductal-like progenitor cells by a process known as acinar ductal metaplasia (ADM). ADM is believed to be one of the earliest precursor lesions toward the development of pancreatic ductal adenocarcinoma and maintaining the pancreatic acinar cell phenotype suppresses tumor formation. The effects of a novel pStat3 inhibitor (LLL12B) and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) were investigated using 3-D cultures from p48Cre/+ and p48Cre/+LSL-KrasG12D/+ (KC) mice. LLL12B and TSA inhibited ADM in both KC and p48Cre/+ mouse pancreatic organoids. Furthermore, treatment with LLL12B or TSA on dedifferentiated acini from p48Cre/+ and KC mice that had undergone ADM produced morphologic and gene expression changes that suggest a reversal of ADM. Validation experiments using qRT-PCR (p48Cre/+ and KC) and RNA sequencing (KC) of the LLL12B and TSA treated cultures showed that the ADM reversal was more robust for the TSA treatments. Pathway analysis showed that TSA inhibited Spink1 and PI3K/AKT signaling during ADM reversal. The ability of TSA to reverse ADM was also observed in primary human acinar cultures. We report that pStat3 and HDAC inhibition can attenuate ADM in vitro and reverse ADM in the context of wild-type Kras. Our findings suggest that pharmacological inhibition or reversal of pancreatic ADM represents a potential therapeutic strategy for blocking aberrant ductal reprogramming of acinar cells.

Data-Driven Design Strategies to Address Crowding and Boarding in an Emergency Department: A Discrete-Event Simulation Study.

OBJECTIVE: To address prolonged lengths of stay (LOS) in a Level 1 trauma center, we examined the impact of implementing two data-driven strategies with a focus on the physical environment. BACKGROUND: Crowding in emergency departments (EDs) is a widely reported problem leading to increased service times and patients leaving without being seen. METHODS: Using ED historical data and expert estimates, we created a discrete-event simulation model. We analyzed the likely impact of initiating care and boarding patients in the hallway (hallway care) instead of the exam rooms and adding a dedicated triage space for patients who arrive by emergency medical services (EMS triage) to decrease hallway congestion. The scenarios were compared in terms of LOS, time spent in exam rooms and hallway spaces, service time, blocked time, and utilization rate. RESULTS: The hallway care scenario resulted in significantly lower LOS and exam room time only for EMS patients but when implemented along with the EMS triage scenario, a significantly lower LOS and exam room time was observed for all patients (EMS and walk-in). The combination of two simulated scenarios resulted in significant improvements in other flow metrics as well. CONCLUSIONS: Our findings discourage boarding of admitted patients in ED exam rooms. If space limitations require that admitted patients be placed in ED hallways, designers and planners should consider enabling hallway spaces with features recommended in this article. Alternative locations for boarding should be prioritized in or out of the ED. Our findings also encourage establishing a triage area dedicated to EMS patients in the ED.

Enrichment of the erythrocyte miR-451a in brain extracellular vesicles following impairment of the blood-brain barrier.

Extracellular RNAs (exRNAs) are present in all biofluids and incorporate many types of RNAs including miRNA. To enhance their stability outside of the cell, exRNAs are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (EVs). The blood-brain barrier (BBB) is a dynamic interface between the systemic circulation and the CNS and is responsible for maintaining a stable extracellular environment for CNS cells. The intent of this study was to determine if EVs and their contents are transferred from the peripheral circulation to the CNS under conditions of an impaired BBB. The BBB of mice was disrupted by unilateral intracarotid artery infusion with hyperosmolar mannitol solution. To validate barrier opening, the uptake clearance of [13C12]-sucrose in the left forebrain (i.e. the ipsilateral, mannitol injected hemisphere) was quantified and revealed a 14-fold increase in the mannitol perfused hemisphere compared to sham treated mice. EVs were isolated from the extracellular spaces of the left forebrain following gentle tissue lysis and differential ultracentrifugation. EVs were confirmed using nanotracking analysis, electron microscopy and western blotting. qRT-PCR showed that the erythrocyte-enriched miR-451a in brain tissue EVs increased with mannitol treatment by 24-fold. Small RNA sequencing performed on the EVs isolated from the sham and mannitol treated mice showed that miR-9-5p was the most abundant miRNA contained within the brain EVs. qRT-PCR analysis of plasma EVs did not produce a statistically significant difference in the expression of the CNS-enriched miR-9-5p or miR-9-3p, suggesting that transfer of CNS EVs to the peripheral circulation did not occur under the conditions of our experiment. We demonstrate that EVs containing miR-451a, a highly abundant miRNA present within erythrocytes and erythrocyte EVs, are enhanced in the CNS upon BBB disruption.

Design Strategies to Improve Emergency Departments' Performance During Mass Casualty Incidents: A Survey of Caregivers.

OBJECTIVES: To identify effective facility design strategies to improve the performance of healthcare providers and patient flow during mass casualty incidents (MCIs) in emergency departments. BACKGROUND: Emergency departments (EDs) are the first line of medical care in MCIs. While operational surge management plans are well described in literature, physical design strategies to improve performance and patient flow during disasters are discussed scarcely. METHOD: An online questionnaire was sent to EDs' caregivers nationwide asking them to rate the effectiveness of nine physical design strategies, discussed in the literature, to improve caregivers' performance and patient flow during MCIs. Assessed strategies were about providing expandable departments and care areas, alternate care facilities for the least sick to maximize care areas for critical patients, care areas from nonemergency units, increased number of decontamination units, dedicated isolation units, within-hospital and close emergency operation centers, and within-hospital media areas. RESULTS: All suggested strategies were rated as effective. The most effective and agreed-upon solution was identified as maximizing the care area for critical patients by establishing an alternate care facility with separate entrance and exit doors from the emergency department for the least critical patients. The least effective and agreed-upon strategy was identified as locating a media unit within the hospital outside of the ED. CONCLUSIONS: Caregivers who work in EDs consider design strategies to be effective in surge management during disasters. Designers can consider implementing identified strategies in designing new emergency departments or expansion and renovation projects.

Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+.

The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K+ concentration in the T-tubules, through its K+ substrate affinity. Apparent K+ affinity was determined from measurements of the K1/2 for K+ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K+ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (-90 to -30 mV) and increased with depolarization in the subthreshold range, -90 to -50 mV. The Q10 was 2.1 over the range of 22-37°C. The K1/2,K of Ip was 4.3±0.3 mM at -90 mV and was relatively voltage independent. This K+ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K+ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K+ increases in proportion to the frequency and duration of action potential firing. This K1/2,K predicts a low fractional occupancy of K+ substrate sites at the resting extracellular K+ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K+ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.