Multiplexing Strategy Research Articles

R-MetaboList 2: A Flexible Tool for Metabolite Annotation from High-Resolution Data-Independent Acquisition Mass Spectrometry Analysis.

Technological advancements have permitted the development of innovative multiplexing strategies for data independent acquisition (DIA) mass spectrometry (MS). Software solutions and extensive compound libraries facilitate the efficient analysis of MS1 data, regardless of the analytical platform. However, the development of comparable tools for DIA data analysis has significantly lagged. This research introduces an update to the former MetaboList R package and a workflow for full-scan MS1 and MS/MS DIA processing of metabolomic data from multiplexed liquid chromatography high-resolution mass spectrometry (LC-HRMS) experiments. When compared to the former version, new functions have been added to address isolated MS1 and MS/MS workflows, processing of MS/MS data from stepped collision energies, performance scoring of metabolite annotations, and batch job analysis were incorporated into the update. The flexibility and efficiency of this strategy were assessed through the study of the metabolite profiles of human urine, leukemia cell culture, and medium samples analyzed by either liquid chromatography quadrupole time-of-flight (q-TOF) or quadrupole orbital (q-Orbitrap) instruments. This open-source alternative was designed to promote global metabolomic strategies based on recursive retrospective research of multiplexed DIA analysis.

Joint Vehicular and Static Users Multiplexing Transmission With Hierarchical Modulation for Throughput Maximization in Vehicular Networks

With the rapid development of intelligent transportation system (ITS), increasing the throughput of all kinds of users in the mobile communication network is a pressing problem. We consider there are two types of users, i.e. vehicular users (VUs) and static users (SUs), in a vehicular communication network. Since the channel characteristics of VUs and SUs are different, designing a multi-user multiplexing transmission strategy to provide an efficient and reliable transmission for two types of users is quite challenging. We propose a dynamic cross-layer transmission strategy with joint admission control, user association and resource allocation to increase the total throughput of VUs and SUs while satisfying the different quality of service (QoS) requirements. First, we formulate a throughput utility maximization problem subject to the QoS and maximum transmit power constraints. Second, with the help of Lyapunov optimization theory, the problem can be decomposed into three subproblems including traffic admission control, user association and resource allocation. Third, a novel robust resource allocation scheme with discrete-rate hierarchical modulation (HM) is designed for the reliable multiplexing transmission of two types of users. In addition, an optimal algorithm and a low complexity suboptimal algorithm are presented to solve the user association subproblem. For two types of users, comparing with uniform modulation schemes, the proposed transmission strategy with HM scheme can improve the throughput by more than 15%.

Mapping histone modifications in low cell number and single cells using antibody-guided chromatin tagmentation (ACT-seq)

Modern next-generation sequencing-based methods have empowered researchers to assay the epigenetic states of individual cells. Existing techniques for profiling epigenetic marks in single cells often require the use and optimization of time-intensive procedures such as drop fluidics, chromatin fragmentation, and end repair. Here we describe ACT-seq, a streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. We conclude that ACT-seq present an attractive alternative to existing techniques for mapping epigenetic marks in single cells.

Branched hybridization chain reaction\u2014using highly dimensional DNA nanostructures for label-free, reagent-less, multiplexed molecular diagnostics

The specific and multiplexed detection of DNA underpins many analytical methods, including the detection of microorganisms that are important in the medical, veterinary, and environmental sciences. To achieve such measurements generally requires enzyme-mediated amplification of the low concentrations of the target nucleic acid sequences present, together with the precise control of temperature, as well as the use of enzyme-compatible reagents. This inevitably leads to compromises between analytical performance and the complexity of the assay. The hybridization chain reaction (HCR) provides an attractive alternative, as a route to enzyme-free DNA amplification. To date, the linear nucleic acid products, produced during amplification, have not enabled the development of efficient multiplexing strategies, nor the use of label-free analysis. Here, we show that by designing new DNA nanoconstructs, we are able, for the first time, to increase the molecular dimensionality of HCR products, creating highly branched amplification products, which can be readily detected on label-free sensors. To show that this new, branching HCR system offers a route for enzyme-free, label-free DNA detection, we demonstrate the multiplexed detection of a target sequence (as the initiator) in whole blood. In the future, this technology will enable rapid point-of-care multiplexed clinical analysis or in-the-field environmental monitoring.

Wireless Power and Data Transmission System of Submarine Cable-Inspecting Robot Fish and Its Time-Sharing Multiplexing Method

In this paper, a hybrid system topology with one-way wireless charging function and the function of the bi-directional data communication is proposed for the problem of electric energy replenishment and data transmission faced by robot fish in the implementation of autonomous submarine cable inspection. Three working modes of the system and the time-sharing multiplexing method are studied. In the power transmission mode, high-efficiency wireless charging is realized by utilizing the transmission characteristics of a series–series (SS)-type resonant network which involves series resonant networks in both the primary side and the secondary side. In the alignment detection and handshake communication mode, the charging platform distance recognition and the handshake signal transmission are implemented through a series–parallel (SP)-type resonant network based on the ASK (amplitude shift keying) modulation method. In the high-speed data transmission mode, the reverse (secondary to primary) high-speed transmission of the inspection data is achieved through a SP-type resonant network based on the OFDM (orthogonal frequency division multiplexing) modulation method. The three modes share the same coupled coils via a reconfigurable resonant network. The working principle of the system is expounded, the system characteristics under each working mode are analyzed, and the time-division multiplexing control strategy is given. The rationality and effectiveness of the scheme are verified by experiments.

Fundamental study of ion trapping and multiplexing using drift tube-ion mobility time-of-flight mass spectrometry for non-targeted metabolomics.

This study of ion accumulation/release behavior relevant to ion mobility-mass spectrometry (IM-MS) as employed for non-targeted metabolomics involves insight from theoretical studies, and controlled reference experiments involving measurement of low and high molecular mass metabolites in varying concentrations within a complex matrix (yeast extracts). Instrumental settings influencing ion trapping (accumulation time) and release conditions in standard and multiplexed operation have been examined, and translation of these insights to liquid chromatography (LC) in combination with drift tube IM-MS measurements has been made. The focus of the application is non-targeted metabolomics using carefully selected samples to allow quantitative interpretations to be made. Experimental investigation of the IM-MS ion utilization efficiency particularly focusing on the use of the Hadamard transform multiplexing with 4-bit pseudo-random pulsing sequence for assessment of low and high molecular mass metabolites is compared with theoretical modeling of gas-phase behavior of small and large molecules in the IM trapping funnel. Increasing the trapping time for small metabolites with standard IM-MS operation is demonstrated to have a deleterious effect on maintaining a quantitative representation of the metabolite abundance. The application of these insights to real-world non-targeted metabolomics assessment of intracellular extracts from biotechnologically relevant production processes is presented, and the results were compared to LC×IM-MS measurements of the same samples. Spiking of a uniformly 13C-labeled yeast extract (as a standard matrix) with varying amounts of natural metabolites is used to assess the linearity and sensitivity according to the instrument mode of operation (i.e., LC-MS, LC×IM-MS, and LC×[multiplexed]IM-MS). When comparing metabolite quantification using standard and multiplexed operation, sensitivity gain factors of 2-8 were obtained for metabolites with m/z below 250. Taken together, the simulation and experimental results of this study provide insight for optimizing measurement conditions for metabolomics and highlight the need for implementation of multiplexing strategies using short trapping times as relative quantification (e.g., in the context with non-targeted differential analysis) with sufficient sensitivity and working range is a requirement in this field of application.

SMONoC: Optical network-on-chip using a statistical multiplexing strategy

Optical interconnect can provide high bandwidth and low latency, which is considered as a good candidate for future multicore systems. To maintain the scalability of optical networks and simultaneously support high performance communication, we propose a Statistical Multiplexing Optical Network-on-Chip (SMONoC), a new architecture targeting dynamic channel allocation for high channel utilization, a modest amount of photonic devices for low integration cost, and statistical multiplexing for a simple arbitration scheme. The SMONoC consists of two layers, one optical layer for data transmission and one electrical layer for controlling and scheduling. With the introduction of the statistical multiplexing strategy, the SMONoC avoids the confliction between different cores with low complexity. The simulation results show that the SMONoC is a low-latency and high-throughput architecture with high channel utilization under both synthetic traffic patterns and real traffic patterns.

Differentially synchronized spiking enables multiplexed neural coding

Multiplexing refers to the simultaneous encoding of two or more signals. Neurons have been shown to multiplex, but different stimuli require different multiplexing strategies. Whereas the frequency and amplitude of periodic stimuli can be encoded by the timing and rate of the same spikes, natural scenes, which comprise areas over which intensity varies gradually and sparse edges where intensity changes abruptly, require a different multiplexing strategy. Recording in vivo from neurons in primary somatosensory cortex during tactile stimulation, we found that stimulus onset and offset (edges) evoked highly synchronized spiking, whereas other spikes in the same neurons occurred asynchronously. Stimulus intensity modulated the rate of asynchronous spiking, but did not affect the timing of synchronous spikes. From this, we hypothesized that spikes driven by high- and low-contrast stimulus features can be distinguished on the basis of their synchronization, and that differentially synchronized spiking can thus be used to form multiplexed representations. Applying a Bayesian decoding method, we verified that information about high- and low-contrast features can be recovered from an ensemble of model neurons receiving common input. Equally good decoding was achieved by distinguishing synchronous from asynchronous spikes and applying reverse correlation methods separately to each spike type. This result, which we verified with patch clamp recordings in vitro, demonstrates that neurons receiving common input can use the rate of asynchronous spiking to encode the intensity of low-contrast features while using the timing of synchronous spikes to encode the occurrence of high-contrast features. We refer to this strategy as synchrony-division multiplexing.

124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics.

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.

Exploiting Molecular Barcodes in High-Throughput Cellular Assays.

Multiplexing strategies, which greatly increase the number of simultaneously measured parameters in single experiments, are now being widely implemented by both the pharmaceutical industry and academic researchers. Color has long been used to identify biological signals and, when combined with molecular barcodes, has substantially enhanced the depth of multiplexed sample characterization. Moreover, the recent advent of DNA barcodes has led to an explosion of innovative cell sequencing approaches. Novel barcoding strategies also show great promise for encoding spatial information in transcriptomic studies, and for precise assessment of molecular abundance. Both color- and DNA-based barcodes can be conveniently analyzed with either a microscope or a cytometer, or via DNA sequencing. Here we review the basic principles of several technologies used to create barcodes and detail the type of samples that can be identified with such tags.

Super-Resolution Microscopy with DNA Molecules: Towards Localizomics

Super-resolution fluorescence microscopy is a powerful tool for biological research. We use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT) for simple and easy-to-implement multiplexed super-resolution imaging that technically achieves sub-5-nm spatial resolution. However, this does not translate to achievable image resolution in biological specimen, mainly due to the lack of readily available small and specific affinity reagents. We introduce Slow Off-rate Modified Aptamers (SOMAmers) for DNA-PAINT super-resolution microscopy as efficient and quantitative labeling reagents. We demonstrate the achievable image resolution and specificity by labeling and imaging of transmembrane as well as intracellular targets in fixed and live cell-specimen. Apart from ever increasing spatial resolution, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. We now introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.

C1 CAGE detects transcription start sites and enhancer activity at single-cell resolution

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3′-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5′-ends with an original sample multiplexing strategy in the C1TM microfluidic system. We first quantifiy the performance of C1 CAGE and find it as accurate and sensitive as other methods in the C1 system. We then use it to profile promoter and enhancer activities in the cellular response to TGF-β of lung cancer cells and discover subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand in a mutually exclusive manner, validated using single molecule fluorescence in situ hybridization.

Resolving CO (2−1) in z ∼ 1.6 Gas-rich Cluster Galaxies with ALMA: Rotating Molecular Gas Disks with Possible Signatures of Gas Stripping

Abstract We present the first spatially resolved observations of molecular gas in a sample of cluster galaxies beyond z > 0.1. Using ALMA, we detect CO (2–1) in eight z ∼ 1.6 cluster galaxies, all within a single 70″ primary beam. The cluster, SpARCS-J0225, was discovered by the Spitzer Adaptation of the Red-sequence Cluster Survey, and is replete with gas-rich galaxies in close proximity, thus affording an efficient multiplexing strategy to amass the first sample of resolved CO in distant clusters. Mapping out the kinematic structure and morphology of molecular gas on ∼3.5 kpc scales reveals rotating gas disks in the majority of the galaxies, and some kinematic peculiarities, including a central gas void, a merger, and one-sided gas tails. We find that the extent of the molecular gas is slightly smaller than that of the optical HST stellar component; this is even more pronounced in low-redshift Virgo cluster galaxies. However, limited by small sample sizes of spatially resolved CO, we are unable to differentiate the distribution of stellar-to-gas radii between cluster and field environments at high redshift. Thus, at first glance, while the cluster galaxies generally look like galaxies infalling from the field, with typical main-sequence star formation rates and massive molecular gas reservoirs situated in rotating disks, they have potentially remarkable attributes, including elevated gas fractions, slightly smaller CO disks, and asymmetric gas tails. Taken in tandem, these signatures are tentative evidence for gas stripping in the z ∼ 1.6 cluster, though verification of these trends will require larger samples.

Single-color multiplexing by the integration of high-resolution melting pattern recognition with loop-mediated isothermal amplification.

We develop a single-color multiplexing strategy by the integration of high-resolution melting pattern recognition with loop-mediated isothermal amplification (LAMP). This strategy can identify multiple amplicons with a small DNA melting temperature (Tm) difference (∼0.2 °C) without the involvement of either multicolor labels or parallelized multiplexing, and it can sensitively detect LAMP amplicons with the initial DNA concentrations ranging from 10 to 108 copies.

A mmWave Automotive Joint Radar-Communications System

We propose a millimeter-wave joint radar-communications (JRC) system comprising a bi-static automotive radar and vehicle-to-vehicle communications. We study the applicability of known phase-modulated-continuous-wave (PMCW) and orthogonal-frequency-division-multiple-access waveforms for bi-static-JRC. In both cases, we design multiplexing strategies to ensure the parameter identifiability, derive JRC statistical bounds and numerically demonstrate superior performance of proposed low-complexity JRC super-resolution algorithms over conventional two-dimensional fast Fourier transform/MUltiple SIgnal Classification.

Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility.

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.

Highly tunable multiple narrow emissions of dyed dielectric-metal core–shell resonators: towards efficient fluorescent labels

We report a potential efficient fluorescent label based on the dyed dielectric-metal core–shell resonators (DMCSRs). By utilizing the near-field coupling between the dyes and the multipolar sharp cavity plasmon resonances, the dyed DMCSRs with diameter of 1.02 μm are demonstrated to be capable of supporting multiple spontaneous emission peaks with the linewidths as narrow as ∼ 10 nm in visible range, and these reshaped fluorescent emissions are insensitive to the surrounding dielectric environment. Furthermore, these multiple narrow emission peaks show a precise tunability on the spectrum by simply separating a nanometric dielectric layer between the dielectric core and the metallic shell, which may provide an attractive spectral multiplexing strategy in the fields of cell biology and medical sciences.

Web-Based Search Tool for Visualizing Instrument Performance Using the Triple Knockout (TKO) Proteome Standard.

Multiplexing strategies are at the forefront of mass-spectrometry-based proteomics, with SPS-MS3 methods becoming increasingly commonplace. A known caveat of isobaric multiplexing is interference resulting from coisolated and cofragmented ions that do not originate from the selected precursor of interest. The triple knockout (TKO) standard was designed to benchmark data collection strategies to minimize interference. However, a limitation to its widespread use has been the lack of an automated analysis platform. We present a TKO Visualization Tool (TVT). The TVT viewer allows for automated, web-based, database searching of the TKO standard, returning traditional figures of merit, such as peptide and protein counts, scan-specific ion accumulation times, as well as the TKO-specific metric, the IFI (interference-free index). Moreover, the TVT viewer allows for plotting of two TKO standards to assess protocol optimizations, compare instruments, or measure degradation of instrument performance over time. We showcase the TVT viewer by probing the selection of (1) stationary phase resin, (2) MS2 isolation window width, and (3) number of synchronous precursor selection (SPS) ions for SPS-MS3 analysis. Using the TVT viewer will allow the proteomics community to search and compare TKO results to optimize user-specific data collection workflows.

Syndrome Evaluation System for Simultaneous Detection Pathogens Causing Acute Encephalitic Syndrome in India, Part-1: Development and Standardization of the Assay.

A large number of organisms are known to cause acute encephalitic syndrome (AES). A number of diagnostic tests have to be performed in order to arrive at a probable pathogen causing AES thus making it a very time consuming, laborious and expensive. The problem is further compounded by the lack of availability of sufficient volume of Cerebrospinal fluid (CSF). Thus, there is an urgent need of a diagnostic tool for the simultaneous detection of all probable pathogens responsible for causing AES. Here we report the development of a novel diagnostic method, Syndrome Evaluation System (SES) for the simultaneous detection of 22 pathogens including RNA and DNA Viruses, bacteria, fungi, and parasite all endemic to India and Southeast Asia in a single sample using a novel multiplexing strategy. Syndrome Evaluation System (SES) involves isolation of nucleic acid, multiplex amplification of the DNA, and cDNA followed by identification of the amplified product by sequence specific hybridization on SES platform with the final read out being a visually recordable colored signal. The total time required to carry out this diagnostic procedure is 7 h. The SES was standardized using the commercially available vaccines, panels and cell culture grown quantified viruses/bacteria/fungi. The limit of detection (LOD) of SES ranged between 0.1 and 50 viral particles per ml of CSF and 100 to 200 bacterial cells or 5 parasites per ml of CSF, along with 100% specificity. Precision studies carried out as per the Clinical Laboratory Improvement Amendments (CLIA) guidelines, using two concentrations of each pathogen one the LOD and the other double the LOD, clearly demonstrated, that inter/intra assay variability was within the limits prescribed by the guidelines. SES is a rapid molecular diagnostic tool for simultaneous identification of 22 etiological agents of AES encountered both in sporadic and outbreak settings.

Latency-Driven Transport for 5G

A transport network is a key component for enabling future fifth-generation (5G) network deployments. The transport network will be under demand to support current and future radio access technologies (RATs), along with the various schemes for splitting radio functions, that should be supported with statistical multiplexing strategies compatible with the tight requirements of latency and bandwidth expected in 5G. In this paper, a different perspective is considered with respect to the conventional method proposed in the literature, where transport design is largely based on some radio design parameters that are vendor specific. This paper, applied to a network architecture named eXHaul, proposes a novel strategy for consistent bandwidth savings in the transport segment considering both the limitations of the optical transmission technology and the radio design requirements in a set of scenarios that vary in terms of distance between the antenna site and the baseband site, rate of the transport channels, end-to-end latency imposed by radio needs, and radio time transmission interval. The deployment in a real scenario, provided by Telefonica and referring to the urban area of Tijuana (Mexico), has also been evaluated to provide an indication in a real case.