Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy.

Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.

Correction: High-resolution DNA size enrichment using a magnetic nano-platform and application in non-invasive prenatal testing.

Correction for 'High-resolution DNA size enrichment using a magnetic nano-platform and application in non-invasive prenatal testing' by Bo Zhang et al., Analyst, 2020, 145, 5733-5739, DOI: 10.1039/D0AN00813C.

miR-34a directly targets tRNAiMet precursors and affects cellular proliferation, cell cycle, and apoptosis.

It remains unknown whether microRNA (miRNA/miR) can target transfer RNA (tRNA) molecules. Here we provide evidence that miR-34a physically interacts with and functionally targets tRNAiMet precursors in both in vitro pulldown and Argonaute 2 (AGO2) cleavage assays. We find that miR-34a suppresses breast carcinogenesis, at least in part by lowering the levels of tRNAiMet through AGO2-mediated repression, consequently inhibiting the proliferation of breast cancer cells and inducing cell cycle arrest and apoptosis. Moreover, miR-34a expression is negatively correlated with tRNAiMet levels in cancer cell lines. Furthermore, we find that tRNAiMet knockdown also reduces cell proliferation while inducing cell cycle arrest and apoptosis. Conversely, ectopic expression of tRNAiMet promotes cell proliferation, inhibits apoptosis, and accelerates the S/G2 transition. Moreover, the enforced expression of modified tRNAiMet completely restores the phenotypic changes induced by miR-34a. Our results demonstrate that miR-34a directly targets tRNAiMet precursors via AGO2-mediated cleavage, and that tRNAiMet functions as an oncogene, potentially representing a target molecule for therapeutic intervention.

High-resolution DNA size enrichment using a magnetic nano-platform and application in non-invasive prenatal testing.

Precise DNA sizing can boost sequencing efficiency, reduce cost, improve data quality, and even allow sequencing of low-input samples, while current pervasive DNA sizing approaches are incapable of differentiating DNA fragments under 200 bp with high resolution (<20 bp). In non-invasive prenatal testing (NIPT), the size distribution of cell-free fetal DNA in maternal plasma (main peak at 143 bp) is significantly different from that of maternal cell-free DNA (main peak at 166 bp). The current pervasive workflow of NIPT and DNA sizing is unable to take advantage of this 20 bp difference, resulting in sample rejection, test inaccuracy, and restricted clinical utility. Here we report a simple, automatable, high-resolution DNA size enrichment workflow, named MiniEnrich, on a magnetic nano-platform to exploit this 20 bp size difference and to enrich fetal DNA fragments from maternal blood. Two types of magnetic nanoparticles were developed, with one able to filter high-molecular-weight DNA with high resolution and the other able to recover the remaining DNA fragments under the size threshold of interest with >95% yield. Using this method, the average fetal fraction was increased from 13% to 20% after the enrichment, as measured by plasma DNA sequencing. This approach provides a new tool for high-resolution DNA size enrichment under 200 bp, which may improve NIPT accuracy by rescuing rejected non-reportable clinical samples, and enable NIPT earlier in pregnancy. It also has the potential to improve non-invasive screening for fetal monogenic disorders, differentiate tumor-related DNA in liquid biopsy and find more applications in autoimmune disease diagnosis.

Noninvasive diagnosis of urothelial cancer in urine using DNA hypermethylation signatures-Gender matters.

Urothelial cancer (UCa) is the most predominant cancer of the urinary tract and noninvasive diagnosis using hypermethylation signatures in urinary cells is promising. Here, we assess gender differences in a newly identified set of methylation biomarkers. UCa-associated hypermethylated sites were identified in urine of a male screening cohort (n = 24) applying Infinium-450K-methylation arrays and verified in two separate mixed-gender study groups (n = 617 in total) using mass spectrometry as an independent technique. Additionally, tissue samples (n = 56) of mixed-gender UCa and urological controls (UCt) were analyzed. The hypermethylation signature of UCa in urine was specific and sensitive across all stages and grades of UCa and independent on hematuria. Individual CpG sensitivities reached up to 81.3% at 95% specificity. Albeit similar methylation differences in tissue of both genders, differences were less pronounced in urine from women, most likely due to the frequent presence of squamous epithelial cells and leukocytes. Increased repression of methylation levels was observed at leukocyte counts ≥500/µl urine which was apparent in 30% of female and 7% of male UCa cases, further confirming the significance of the relative amounts of cancerous and noncancerous cells in urine. Our study shows that gender difference is a most relevant issue when evaluating the performance of urinary biomarkers in cancer diagnostics. In case of UCa, the clinical benefits of methylation signatures to male patients may outweigh those in females due to the general composition of women's urine. Accordingly, these markers offer a diagnostic option specifically in males to decrease the number of invasive cystoscopies.

Bacterial charity work leads to population-wide resistance.

Bacteria show remarkable adaptability in the face of antibiotic therapeutics. Resistance alleles in drug target-specific sites and general stress responses have been identified in individual end-point isolates. Less is known, however, about the population dynamics during the development of antibiotic-resistant strains. Here we follow a continuous culture of Escherichia coli facing increasing levels of antibiotic and show that the vast majority of isolates are less resistant than the population as a whole. We find that the few highly resistant mutants improve the survival of the population's less resistant constituents, in part by producing indole, a signalling molecule generated by actively growing, unstressed cells. We show, through transcriptional profiling, that indole serves to turn on drug efflux pumps and oxidative-stress protective mechanisms. The indole production comes at a fitness cost to the highly resistant isolates, and whole-genome sequencing reveals that this bacterial altruism is made possible by drug-resistance mutations unrelated to indole production. This work establishes a population-based resistance mechanism constituting a form of kin selection whereby a small number of resistant mutants can, at some cost to themselves, provide protection to other, more vulnerable, cells, enhancing the survival capacity of the overall population in stressful environments.

Human-specific endogenous retroviral insert serves as an enhancer for the schizophrenia-linked gene PRODH.

Using a systematic, whole-genome analysis of enhancer activity of human-specific endogenous retroviral inserts (hsERVs), we identified an element, hsERVPRODH, that acts as a tissue-specific enhancer for the PRODH gene, which is required for proper CNS functioning. PRODH is one of the candidate genes for susceptibility to schizophrenia and other neurological disorders. It codes for a proline dehydrogenase enzyme, which catalyses the first step of proline catabolism and most likely is involved in neuromediator synthesis in the CNS. We investigated the mechanisms that regulate hsERVPRODH enhancer activity. We showed that the hsERVPRODH enhancer and the internal CpG island of PRODH synergistically activate its promoter. The enhancer activity of hsERVPRODH is regulated by methylation, and in an undermethylated state it can up-regulate PRODH expression in the hippocampus. The mechanism of hsERVPRODH enhancer activity involves the binding of the transcription factor SOX2, whch is preferentially expressed in hippocampus. We propose that the interaction of hsERVPRODH and PRODH may have contributed to human CNS evolution.

Genetic switchboard for synthetic biology applications.

A key next step in synthetic biology is to combine simple circuits into higher-order systems. In this work, we expanded our synthetic riboregulation platform into a genetic switchboard that independently controls the expression of multiple genes in parallel. First, we designed and characterized riboregulator variants to complete the foundation of the genetic switchboard; then we constructed the switchboard sensor, a testing platform that reported on quorum-signaling molecules, DNA damage, iron starvation, and extracellular magnesium concentration in single cells. As a demonstration of the biotechnological potential of our synthetic device, we built a metabolism switchboard that regulated four metabolic genes, pgi, zwf, edd, and gnd, to control carbon flow through three Escherichia coli glucose-utilization pathways: the Embden-Meyerhof, Entner-Doudoroff, and pentose phosphate pathways. We provide direct evidence for switchboard-mediated shunting of metabolic flux by measuring mRNA levels of the riboregulated genes, shifts in the activities of the relevant enzymes and pathways, and targeted changes to the E. coli metabolome. The design, testing, and implementation of the genetic switchboard illustrate the successful construction of a higher-order system that can be used for a broad range of practical applications in synthetic biology and biotechnology.

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale.

We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.

Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection.

Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.

Detection of hepatitis C virus transmission by use of DNA mass spectrometry.

The molecular detection of transmission of rapidly mutating pathogens such as hepatitis C virus (HCV) is commonly achieved by assessing the genetic relatedness of strains among infected patients. We describe the development of a novel mass spectrometry (MS)-based approach to identify HCV transmission. MS was used to detect products of base-specific cleavage of RNA molecules obtained from HCV polymerase chain reaction fragments. The MS-peak profiles were found to reflect variation in the HCV genomic sequence and the intrahost composition of the HCV population. Serum specimens originating from 60 case patients from 14 epidemiologically confirmed outbreaks and 25 unrelated controls were tested. Neighbor-joining trees constructed using MS-peak profile-based Hamming distances showed 100% accuracy, and linkage networks constructed using a threshold established from the Hamming distances between epidemiologically unrelated cases showed 100% sensitivity and 99.93% specificity in transmission detection. This MS-based approach is rapid, robust, reproducible, cost-effective, and applicable to investigating transmissions of other pathogens.

Tracking, tuning, and terminating microbial physiology using synthetic riboregulators.

The development of biomolecular devices that interface with biological systems to reveal new insights and produce novel functions is one of the defining goals of synthetic biology. Our lab previously described a synthetic, riboregulator system that affords for modular, tunable, and tight control of gene expression in vivo. Here we highlight several experimental advantages unique to this RNA-based system, including physiologically relevant protein production, component modularity, leakage minimization, rapid response time, tunable gene expression, and independent regulation of multiple genes. We demonstrate this utility in four sets of in vivo experiments with various microbial systems. Specifically, we show that the synthetic riboregulator is well suited for GFP fusion protein tracking in wild-type cells, tight regulation of toxic protein expression, and sensitive perturbation of stress response networks. We also show that the system can be used for logic-based computing of multiple, orthogonal inputs, resulting in the development of a programmable kill switch for bacteria. This work establishes a broad, easy-to-use synthetic biology platform for microbiology experiments and biotechnology applications.

Intron retention facilitates splice variant diversity in calcium-activated big potassium channel populations.

We report that the stress axis-regulated exon (STREX)-containing calcium-activated big potassium (BKCa) channel splice variant expression and physiology are regulated in part by cytoplasmic splicing and intron retention. NextGen sequencing of the mRNA complement of pooled hippocampal dendrite samples found intron 17a (i17a), the intron immediately preceding STREX, in the BKCa mRNA. Further molecular analyses of i17a revealed that the majority of i17a-containing BKCa channel mRNAs associate with STREX. i17a siRNA treatment followed by STREX protein immunocytochemistry demonstrated both reduced levels and altered subcellular distribution of STREX-containing BKCa channel protein. Selective reduction of i17a-BKCa or STREX-BKCa mRNAs induced similar changes in the burst firing properties of hippocampal neurons. Collectively, these data show that STREX splice variant regulation via cytoplasmic splicing and intron retention helps generate STREX-dependent BKCa current diversity in hippocampal neurons.

Non-invasive prenatal diagnosis by single molecule counting technologies.

Non-invasive prenatal diagnosis of fetal chromosomal aneuploidies and monogenic diseases by analysing fetal DNA present in maternal plasma poses a challenging goal. In particular, the presence of background maternal DNA interferes with the analysis of fetal DNA. Using single molecule counting methods, including digital PCR and massively parallel sequencing, many of the former problems have been solved. Digital mutation dosage assessment can detect the number of mutant alleles a fetus has inherited from its parents for fetal monogenic disease diagnosis, and massively parallel plasma DNA sequencing enables the direct detection of fetal chromosomal aneuploidies from maternal plasma. The analytical power of these methods, namely sensitivity, specificity, accuracy and precision, should catalyse the eventual clinical use of non-invasive prenatal diagnosis.

A bottom-up approach to gene regulation.

The ability to construct synthetic gene networks enables experimental investigations of deliberately simplified systems that can be compared to qualitative and quantitative models. If simple, well-characterized modules can be coupled together into more complex networks with behaviour that can be predicted from that of the individual components, we may begin to build an understanding of cellular regulatory processes from the 'bottom up'. Here we have engineered a promoter to allow simultaneous repression and activation of gene expression in Escherichia coli. We studied its behaviour in synthetic gene networks under increasingly complex conditions: unregulated, repressed, activated, and simultaneously repressed and activated. We develop a stochastic model that quantitatively captures the means and distributions of the expression from the engineered promoter of this modular system, and show that the model can be extended and used to accurately predict the in vivo behaviour of the network when it is expanded to include positive feedback. The model also reveals the counterintuitive prediction that noise in protein expression levels can increase upon arrest of cell growth and division, which we confirm experimentally. This work shows that the properties of regulatory subsystems can be used to predict the behaviour of larger, more complex regulatory networks, and that this bottom-up approach can provide insights into gene regulation.

A network biology approach to aging in yeast.

In this study, a reverse-engineering strategy was used to infer and analyze the structure and function of an aging and glucose repressed gene regulatory network in the budding yeast Saccharomyces cerevisiae. The method uses transcriptional perturbations to model the functional interactions between genes as a system of first-order ordinary differential equations. The resulting network model correctly identified the known interactions of key regulators in a 10-gene network from the Snf1 signaling pathway, which is required for expression of glucose-repressed genes upon calorie restriction. The majority of interactions predicted by the network model were confirmed using promoter-reporter gene fusions in gene-deletion mutants and chromatin immunoprecipitation experiments, revealing a more complex network architecture than previously appreciated. The reverse-engineered network model also predicted an unexpected role for transcriptional regulation of the SNF1 gene by hexose kinase enzyme/transcriptional repressor Hxk2, Mediator subunit Med8, and transcriptional repressor Mig1. These interactions were validated experimentally and used to design new experiments demonstrating Snf1 and its transcriptional regulators Hxk2 and Mig1 as modulators of chronological lifespan. This work demonstrates the value of using network inference methods to identify and characterize the regulators of complex phenotypes, such as aging.

Spatiotemporal patterns and transcription kinetics of induced RNA in single bacterial cells.

Bacteria have a complex internal organization with specific localization of many proteins and DNA, which dynamically move during the cell cycle and in response to changing environmental stimuli. Much less is known, however, about the localization and movements of RNA molecules. By modifying our previous RNA labeling system, we monitor the expression and localization of a model RNA transcript in live Escherichia coli cells. Our results reveal that the target RNA is not evenly distributed within the cell and localizes laterally along the long cell axis, in a pattern suggesting the existence of ordered helical RNA structures reminiscent of known bacterial cytoskeletal cellular elements.

Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting.

OBJECTIVE: We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA. STUDY DESIGN: Sample multiplexing and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed. RESULTS: In all, 480 prospectively collected samples were obtained from our third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed prespecified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was misclassified as trisomy 21. The overall classification showed 100% sensitivity (95% confidence interval, 89-100%) and 99.7% specificity (95% confidence interval, 98.5-99.9%). CONCLUSION: Extending the scope of previous reports, this study demonstrates that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.

Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma.

Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and beta-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background maternal DNA, which interferes with the analysis of fetal DNA in maternal plasma, noninvasive prenatal diagnosis of maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for the CD41/42 (-CTTT) and hemoglobin E mutations on HBB, digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma. To achieve fetal genotype diagnosis at lower volumes of maternal plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality.

Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma.

Chromosomal aneuploidy is the major reason why couples opt for prenatal diagnosis. Current methods for definitive diagnosis rely on invasive procedures, such as chorionic villus sampling and amniocentesis, and are associated with a risk of fetal miscarriage. Fetal DNA has been found in maternal plasma but exists as a minor fraction among a high background of maternal DNA. Hence, quantitative perturbations caused by an aneuploid chromosome in the fetal genome to the overall representation of sequences from that chromosome in maternal plasma would be small. Even with highly precise single molecule counting methods such as digital PCR, a large number of DNA molecules and hence maternal plasma volume would need to be analyzed to achieve the necessary analytical precision. Here we reasoned that instead of using approaches that target specific gene loci, the use of a locus-independent method would greatly increase the number of target molecules from the aneuploid chromosome that could be analyzed within the same fixed volume of plasma. Hence, we used massively parallel genomic sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. Twenty-eight first and second trimester maternal plasma samples were tested. All 14 trisomy 21 fetuses and 14 euploid fetuses were correctly identified. Massively parallel plasma DNA sequencing represents a new approach that is potentially applicable to all pregnancies for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.