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A guide to teaching elementary science : ten easy steps
This book helps teachers develop curricula compatible with the Next Generation Science Standards and the Common Core Standards, provides easy-to-implement steps for setting up a science classroom, plus strategies for using all available resources to assemble needed teaching materials, offers detailed sample lesson plans in each STEM subject, adaptable to age and ability and designed to embrace the needs of all learners, and presents bonus information about organizing field trips and managing science fairs.
Book
Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma
TP53 mutations are ubiquitous in high‐grade serous ovarian carcinomas (HGSOC), and the presence of TP53 mutation discriminates between high and low‐grade serous carcinomas and is now an important biomarker for clinical trials targeting mutant p53. p53 immunohistochemistry (IHC) is widely used as a surrogate for TP53 mutation but its accuracy has not been established. The objective of this study was to test whether improved methods for p53 IHC could reliably predict TP53 mutations independently identified by next generation sequencing (NGS). Four clinical p53 IHC assays and tagged‐amplicon NGS for TP53 were performed on 171 HGSOC and 80 endometrioid carcinomas (EC). p53 expression was scored as overexpression (OE), complete absence (CA), cytoplasmic (CY) or wild type (WT). p53 IHC was evaluated as a binary classifier where any abnormal staining predicted deleterious TP53 mutation and as a ternary classifier where OE, CA or WT staining predicted gain‐of‐function (GOF or nonsynonymous), loss‐of‐function (LOF including stopgain, indel, splicing) or no detectable TP53 mutations (NDM), respectively. Deleterious TP53 mutations were detected in 169/171 (99%) HGSOC and 7/80 (8.8%) EC. The overall accuracy for the best performing IHC assay for binary and ternary prediction was 0.94 and 0.91 respectively, which improved to 0.97 (sensitivity 0.96, specificity 1.00) and 0.95 after secondary analysis of discordant cases. The sensitivity for predicting LOF mutations was lower at 0.76 because p53 IHC detected mutant p53 protein in 13 HGSOC with LOF mutations. CY staining associated with LOF was seen in 4 (2.3%) of HGSOC. Optimized p53 IHC can approach 100% specificity for the presence of TP53 mutation and its high negative predictive value is clinically useful as it can exclude the possibility of a low‐grade serous tumour. 4.1% of HGSOC cases have detectable WT staining while harboring a TP53 LOF mutation, which limits sensitivity for binary prediction of mutation to 96%.
Journal Article
What would Captain Picard do?
Fans of Captain Jean Luc Picard from Star Trek, the Next Generation explore a galaxy of advice in this fun pop philosophy book narrated by the character and filled with full-color photos from the television show. The universe is filled with a great many wonders-- filled with mysteries, uncharted worlds, bizarre life forms, dangers, and unique challenges only the most experienced leader can handle. Within all of us is the raw potential to rise above our meager beginnings and excel beyond the stars. Keep an open mind as you navigate through this collection of scenarios as Captain Picard teaches you how to handle even the most challenging situations. Remain determined against all odds and stay the course with wisdom from U.S.S. Enterprise captain Jean-Luc Picard.
Book
Clinical Evaluation of an Improved Metagenomic Next-Generation Sequencing Test for the Diagnosis of Bloodstream Infections
Abstract
Background
Metagenomic next-generation sequencing (mNGS) of plasma cell-free DNA has emerged as a promising diagnostic technology for bloodstream infections. However, a major limitation of current mNGS assays is the high rate of false-positive results due to contamination.
Methods
We made novel use of 3 control groups—external negative controls under long-term surveillance, blood samples with a negative result in conventional tests, and a group of healthy people—that were combined and dedicated to distinguishing contaminants arising from specimen collection, sample processing, and human normal flora. We also proposed novel markers to filter out false-positive interspecies calls. This workflow was applied retrospectively to 209 clinical plasma samples from patients with suspected bloodstream infections. Every pathogen identified by the mNGS test was reviewed to assess the diagnostic performance of the workflow.
Results
Our mNGS workflow showed clinical sensitivity of 87.1%, clinical specificity of 80.2%, positive predictive value of 77.9%, and negative predictive value of 88.6% compared with the composite reference standard. Notably, mNGS showed great improvement in clinical specificity compared with the current test while keeping clinical sensitivity at a high level.
Conclusion
The mNGS workflow with multiple control groups dedicated to distinguishing nonpathogen microbes from real causal pathogens has reducing false-positive results. This contribution, with its optimization of workflow and careful use of controls, can help mNGS become a powerful tool for identifying the pathogens responsible for bloodstream infections.
Journal Article
Integrating host response and unbiased microbe detection for lower respiratory tract infection diagnosis in critically ill adults
Lower respiratory tract infections (LRTIs) lead to more deaths each year than any other infectious disease category. Despite this, etiologic LRTI pathogens are infrequently identified due to limitations of existing microbiologic tests. In critically ill patients, noninfectious inflammatory syndromes resembling LRTIs further complicate diagnosis. To address the need for improved LRTI diagnostics, we performed metagenomic next-generation sequencing (mNGS) on tracheal aspirates from 92 adults with acute respiratory failure and simultaneously assessed pathogens, the airway microbiome, and the host transcriptome. To differentiate pathogens from respiratory commensals, we developed a rules-based model (RBM) and logistic regression model (LRM) in a derivation cohort of 20 patients with LRTIs or noninfectious acute respiratory illnesses. When tested in an independent validation cohort of 24 patients, both models achieved accuracies of 95.5%. We next developed pathogen, microbiome diversity, and host gene expression metrics to identify LRTI-positive patients and differentiate them from critically ill controls with noninfectious acute respiratory illnesses. When tested in the validation cohort, the pathogen metric performed with an area under the receiver-operating curve (AUC) of 0.96 (95% CI, 0.86–1.00), the diversity metric with an AUC of 0.80 (95% CI, 0.63–0.98), and the host transcriptional classifier with an AUC of 0.88 (95% CI, 0.75–1.00). Combining these achieved a negative predictive value of 100%. This study suggests that a single streamlined protocol offering an integrated genomic portrait of pathogen, microbiome, and host transcriptome may hold promise as a tool for LRTI diagnosis.
Journal Article
Comparative diagnostic performance of metagenomic and two targeted sequencing methods in lower respiratory infection
Lower respiratory tract infections are characterized by high morbidity and mortality, the latter associated with the low sensitivity and long turnaround time (TAT) of traditional diagnostic methods. Advances in next-generation sequencing (NGS) offers a promising solution, but in the face of so many different NGS products, how to use them appropriately remains a great challenge for clinicians. This study included 205 patients with suspected lower respiratory tract infections from the department of respiratory and critical care medicine, and collected their lower respiratory tract samples for metagenomic NGS (mNGS) and two different targeted NGS (tNGS), amplification-based tNGS and capture-based tNGS. We analyzed their microorganisms reported, and evaluated their detection performance based on the comprehensive clinical diagnosis. Compared to the two tNGS, mNGS showed significant higher cost ($840) and longer TAT (20 h). Conversely, it identified the highest number of species, totaling 80, compared to 71 species identified by capture-based tNGS and 65 species by amplification-based tNGS. When benchmarked against the comprehensive clinical diagnosis, the capture-based tNGS demonstrated significantly higher diagnostic performance than the other two NGS, with an accuracy of 93.17% and a sensitivity of 99.43%. However, it showed lower specificity compared to the amplification-based tNGS in identifying DNA virus (74.78% vs. 98.25%). The amplification-based tNGS exhibited a poor sensitivity for both gram-positive (40.23%) and gram-negative bacteria (71.74%). Moreover, tNGS was able to identify genotypes, antimicrobial resistance genes and virulence factors. In conclusion, mNGS is suited for the detection of rare pathogens; the capture-based tNGS is preferable for routine diagnostic testing; the amplification-based tNGS can be an alternative in situations requiring rapid results and constrained by limited resources.
Journal Article
DNA barcoding, an effective tool for species identification: a review
DNA barcoding is a powerful taxonomic tool to identify and discover species. DNA barcoding utilizes one or more standardized short DNA regions for taxon identification. With the emergence of new sequencing techniques, such as Next-generation sequencing (NGS), ONT MinION nanopore sequencing, and Pac Bio sequencing, DNA barcoding has become more accurate, fast, and reliable. Rapid species identification by DNA barcodes has been used in a variety of fields, including forensic science, control of the food supply chain, and disease understanding. The Consortium for Barcode of Life (CBOL) presents various working groups to identify the universal barcode gene, such as COI in metazoans; rbcL, matK, and ITS in plants; ITS in fungi; 16S rRNA gene in bacteria and archaea, and creating a reference DNA barcode library. In this article, an attempt has been made to analyze the various proposed DNA barcode for different organisms, strengths & limitations, recent advancements in DNA barcoding, and methods to speed up the DNA barcode reference library construction. This study concludes that constructing a reference library with high species coverage would be a major step toward identifying species by DNA barcodes. This can be achieved in a short period of time by using advanced sequencing and data analysis methods.
Journal Article
The classification of the Compositae
The classification of the family Compositae (Asteraceae) has been much improved in the last decades by the application of molecular methods culminating in the recompilation published in 2009, Systematics, evolution, and biogeography of Compositae. Additional evidence of relationships has come from the use of high-throughput sequencing methods. Our late colleague Vicki Ann Funk (1947–2019) was a pioneer in this line of research. Together with her team, she contributed to the achievement of a mature classification of the family, which she left outlined. In this paper, we contribute this classification including all of the recent advances at the subtribal level and review in depth all contributions to Compositae classification made since the 2009 compilation.
Journal Article
Application of metagenomic next-generation sequencing for bronchoalveolar lavage diagnostics in critically ill patients
The purpose of this study was to assess the value of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) for the diagnosis of severe respiratory diseases based on interpretation of sequencing results. BALF samples were harvested and used for mNGS as well as microbiological detection. Infectious bacteria or fungi were defined according to relative abundance and number of unique reads. We performed mNGS on 35 BALF samples from 32 patients. The positive rate reached 100% in the mNGS analysis of nine immunocompromised patients. Compared with the culture method, mNGS had a diagnostic sensitivity of 88.89% and a specificity of 74.07% with an agreement rate of 77.78% between these two methods. Compared with the smear method and PCR, mNGS had a diagnostic sensitivity of 77.78% and a specificity of 70.00%. In 13 cases, detection results were positive by mNGS but negative by culture/smear and PCR. The mNGS findings in 11/32 (34.4%) cases led to changes in treatment strategies. Linear regression analysis showed that diversity was significantly correlated with interval between disease onset and sampling. Dynamic changes in reads could indirectly reflect therapeutic effectiveness. BALF mNGS improves sensitivity of pathogen detection and provides guidance in clinical practice. Potential pathogens can be identified based on relative abundance and number of unique reads.
Journal Article
Clinical diagnostic value of targeted next‑generation sequencing for infectious diseases (Review)
As sequencing technology transitions from research to clinical settings, due to technological maturity and cost reductions, metagenomic next-generation sequencing (mNGS) is increasingly used. This shift underscores the growing need for more cost-effective and universally accessible sequencing assays to improve patient care and public health. Therefore, targeted NGS (tNGS) is gaining prominence. tNGS involves enrichment of target pathogens in patient samples based on multiplex PCR amplification or probe capture with excellent sensitivity. It is increasingly used in clinical diagnostics due to its practicality and efficiency. The present review compares the principles of different enrichment methods. The high positivity rate of tNGS in the detection of pathogens was found in respiratory samples with specific instances. tNGS maintains high sensitivity (70.8-95.0%) in samples with low pathogen loads, including blood and cerebrospinal fluid. Furthermore, tNGS is effective in detecting drug-resistant strains of Mycobacterium tuberculosis, allowing identification of resistance genes and guiding clinical treatment decisions, which is difficult to achieve with mNGS. In the present review, the application of tNGS in clinical settings and its current limitations are assessed. The continued development of tNGS has the potential to refine diagnostic accuracy and treatment efficacy and improving infectious disease management. However, further research to overcome technical challenges such as workflow time and cost is required.
Journal Article