Validation of the Awareness Atlas-a new measure of the manifestation of consciousness.

Consciousness has intrigued philosophers and scholars for millennia and has been the topic of considerable scientific investigation in recent decades. Despite its importance, there is no unifying definition of the term, nor are there widely accepted measures of consciousness. Indeed, it is likely that consciousness-by its very nature-eludes measurement. It is, however, possible to measure how consciousness manifests as a lived experience. Yet here, too, holistic measures are lacking. This investigation describes the development and validation of the Awareness Atlas, a measure of the manifestation of consciousness. The scale was informed by heart-based contemplative practices and the resulting lived experience with a focus on the impacts of manifestation of consciousness on daily life. Four hundred forty-nine individuals from the USA, Canada, India, and Europe participated in psychometric testing of the scale. Exploratory and confirmatory factor analyses were used for validation, demonstrating excellent validity in measuring manifestation of consciousness. The final model fit exceeded all required thresholds, indicating an excellent fitted model with a single dimensionality to measure the manifestation of consciousness comprised of four subscales: Relationship to Others; Listening to the Heart; Connection with Higher Self; and Acceptance and Letting Go. Number of years meditating and practicing Heartfulness meditation were positively related to the total and subscale scores. Test-retest reliability was excellent for the total scale, and good to excellent for the four subscales. Findings demonstrate that the Awareness Atlas is a well-constructed tool that will be useful in examining changes in manifestation of consciousness with various experiences (e.g., meditation, life-altering conditions).

Effect of Heartfulness meditation based integrative therapy on infertility outcomes: A retrospective case series evaluation.

BACKGROUND: In Indian context, infertility is often a silent struggle. Despite the high prevalence of infertility in the country, the majority of couples do not share their struggles with family or friends due to social stigma, thus increasing their psychological vulnerability. Heartfulness meditation has shown to decrease stress, anxiety, loneliness and improve sleep along with quality of life. OBJECTIVES: The current retrospective series evaluated the effectiveness of Heartfulness-based integrative therapy on infertility outcomes. METHODS: The program consisted of a 5- day onsite lifestyle modification workshop and online follow up meditation sessions. RESULTS: 54 couples with infertility participated in the program with a mean age of 30.74 years (SD 5.04) for females and 34.03 years (SD 4.54) for males. 15 couples presented with male infertility, 16 couples presented with female infertility and in 5 couples both partners had infertility problems. Further, 18 couples had unexplained infertility. 24 couples conceived with 18 natural conceptions, five via assisted reproductive technology and one spontaneous abortion. CONCLUSION: The program was beneficial in the cohort who utilized it as prescribed resulting in conception of 24 out of 54 couples. Future research investigating the causal relationship of Heartfulness meditation on fertility outcomes in a randomized control study could solidify this treatment method to be used independently or as an adjuvant therapy with assisted reproductive technologies.

Heartfulness Cleaning and Meditation: Short-Term Effects on Attention and Affect.

Heartfulness meditation (HM) trains the practitioner's attention as they progress towards reaching a super-conscious state. The process is supported by guided "Heartfulness cleaning," which helps clear the mind. This study aimed to examine the short-term effects of HM on affect and cognition and determine whether performing Heartfulness cleaning beforehand influenced the meditation outcome. Forty-eight experienced meditators (age range: 19-71 years and a male-to-female ratio: 27:21) were randomly assigned to 3 sessions: (i) HM, (ii) Heartfulness meditation preceded by cleaning, and (iii) quiet rest as a control. Mood state and emotional well-being were assessed before and after each intervention using established scales such as the Brief Mood Introspection Scale, Global Vigor and Affect Scale, Spielberger's State-Trait Anxiety Inventory, and the Digit Letter Substitution Test. After engaging in both HM and Heartfulness cleaning meditation (HCM) practices, there was a noticeable increase in feelings of pleasantness (7.3%, 7.0%, respectively) and positivity (7.5%, 7.8%, respectively), accompanied by a decrease in negative affect (14.4%, 16.5%, respectively). Additionally, HM and HCM increased in the net and total scores on a substitution test designed to measure associative learning. In contrast, there were no changes observed after 30 minutes of non-meditation. In summary, the findings of this study provide support for the positive impact of Heartfulness meditation and Heartfulness cleaning meditation on emotions, as well as their ability to enhance performance in tasks involving complex attention and associative learning. It should be noted that preceding Heartfulness meditation with 5 minutes of Heartfulness cleaning did not significantly alter the overall outcome of the meditation practice.

Heartfulness Meditation: A Yogic and Neuroscientific Perspective.

Today, as research into the contemplative sciences is being widely referenced, the research community would benefit from an understanding of the Heartfulness method of meditation. Heartfulness offers an in-depth experiential practice focused on the evolution of human consciousness using the ancient technique of Pranahuti (yogic Transmission) during Meditation, in combination with the more active mental practice of "Cleaning." Both are enabled by initiation into the Heartfulness practices. These unique features distinguish Heartfulness from other paths that have been described in the scientific literature thus far. In this introductory paper, we present the Heartfulness practices, the philosophy upon which the practices are based, and we reflect on the putative mechanisms through which Heartfulness could exert its effects on the human body and mind in the light of scientific research that has been done in other meditation systems. We conclude with suggestions for future research on the Heartfulness way of meditation.

Publisher Correction: Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria.

The Oxford MinION, the first commercial nanopore sequencer, is also the first to implement molecule-by-molecule real-time selective sequencing or "Read Until". As DNA transits a MinION nanopore, real-time pore current data can be accessed and analyzed to provide active feedback to that pore. Fragments of interest are sequenced by default, while DNA deemed non-informative is rejected by reversing the pore bias to eject the strand, providing a novel means of background depletion and/or target enrichment. In contrast to the previously published pattern-matching Read Until approach, our RUBRIC method is the first example of real-time selective sequencing where on-line basecalling enables alignment against conventional nucleic acid references to provide the basis for sequence/reject decisions. We evaluate RUBRIC performance across a range of optimizable parameters, apply it to mixed human/bacteria and CRISPR/Cas9-cut samples, and present a generalized model for estimating real-time selection performance as a function of sample composition and computing configuration.

Systematic and stochastic influences on the performance of the MinION nanopore sequencer across a range of nucleotide bias.

Emerging sequencing technologies are allowing us to characterize environmental, clinical and laboratory samples with increasing speed and detail, including real-time analysis and interpretation of data. One example of this is being able to rapidly and accurately detect a wide range of pathogenic organisms, both in the clinic and the field. Genomes can have radically different GC content however, such that accurate sequence analysis can be challenging depending upon the technology used. Here, we have characterized the performance of the Oxford MinION nanopore sequencer for detection and evaluation of organisms with a range of genomic nucleotide bias. We have diagnosed the quality of base-calling across individual reads and discovered that the position within the read affects base-calling and quality scores. Finally, we have evaluated the performance of the current state-of-the-art neural network-based MinION basecaller, characterizing its behavior with respect to systemic errors as well as context- and sequence-specific errors. Overall, we present a detailed characterization the capabilities of the MinION in terms of generating high-accuracy sequence data from genomes with a wide range of nucleotide content. This study provides a framework for designing the appropriate experiments that are the likely to lead to accurate and rapid field-forward diagnostics.

The rotary zone thermal cycler: a low-power system enabling automated rapid PCR.

Advances in molecular biology, microfluidics, and laboratory automation continue to expand the accessibility and applicability of these methods beyond the confines of conventional, centralized laboratory facilities and into point of use roles in clinical, military, forensic, and field-deployed applications. As a result, there is a growing need to adapt the unit operations of molecular biology (e.g., aliquoting, centrifuging, mixing, and thermal cycling) to compact, portable, low-power, and automation-ready formats. Here we present one such adaptation, the rotary zone thermal cycler (RZTC), a novel wheel-based device capable of cycling up to four different fixed-temperature blocks into contact with a stationary 4-microliter capillary-bound sample to realize 1-3 second transitions with steady state heater power of less than 10 W. We demonstrate the utility of the RZTC for DNA amplification as part of a highly integrated rotary zone PCR (rzPCR) system that uses low-volume valves and syringe-based fluid handling to automate sample loading and unloading, thermal cycling, and between-run cleaning functionalities in a compact, modular form factor. In addition to characterizing the performance of the RZTC and the efficacy of different online cleaning protocols, we present preliminary results for rapid single-plex PCR, multiplex short tandem repeat (STR) amplification, and second strand cDNA synthesis.

World-to-digital-microfluidic interface enabling extraction and purification of RNA from human whole blood.

Digital microfluidics (DMF) is a powerful technique for simple and precise manipulation of microscale droplets of fluid. This technique enables processing and analysis of a wide variety of samples and reagents and has proven useful in a broad range of chemical, biological, and medical applications. Handling of "real-world" samples has been a challenge, however, because typically their volumes are greater than those easily accommodated by DMF devices and contain analytes of interest at low concentration. To address this challenge, we have developed a novel "world-to-DMF" interface in which an integrated companion module drives the large-volume sample through a 10 µL droplet region on the DMF device, enabling magnet-mediated recovery of bead-bound analytes onto the device as they pass through the region. To demonstrate its utility, we use this system for extraction of RNA from human whole blood lysates (110-380 µL) and further purification in microscale volumes (5-15 µL) on the DMF device itself. Processing by the system was >2-fold faster and consumed 12-fold less reagents, yet produced RNA yields and quality fully comparable to conventional preparations and supporting qRT-PCR and RNA-Seq analyses. The world-to-DMF system is designed for flexibility in accommodating different sample types and volumes, as well as for facile integration of additional modules to enable execution of more complex protocols for sample processing and analysis. As the first technology of its kind, this innovation represents an important step forward for DMF, further enhancing its utility for a wide range of applications.

A versatile automated platform for micro-scale cell stimulation experiments.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (≥ 10(5)) in milliliter-scale volumes (≥ 0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100-2,000 cells) in micro-scale volumes (1-20 µl). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

A microfluidic DNA library preparation platform for next-generation sequencing.

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

Enriching pathogen transcripts from infected samples: a capture-based approach to enhanced host-pathogen RNA sequencing.

To fully understand the interactions of a pathogen with its host, it is necessary to analyze the RNA transcripts of both the host and pathogen throughout the course of an infection. Although this can be accomplished relatively easily on the host side, the analysis of pathogen transcripts is complicated by the overwhelming amount of host RNA isolated from an infected sample. Even with the read depth provided by second-generation sequencing, it is extremely difficult to get enough pathogen reads for an effective gene-level analysis. In this study, we describe a novel capture-based technique and device that considerably enriches for pathogen transcripts from infected samples. This versatile method can, in principle, enrich for any pathogen in any infected sample. To test the technique's efficacy, we performed time course tissue culture infections using Rift Valley fever virus and Francisella tularensis. At each time point, RNA sequencing (RNA-Seq) was performed and the results of the treated samples were compared with untreated controls. The capture of pathogen transcripts, in all cases, led to more than an order of magnitude enrichment of pathogen reads, greatly increasing the number of genes hit, the coverage of those genes, and the depth at which each transcript was sequenced.

cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications.

Second-generation sequencing (SGS) has become the preferred method for RNA transcriptome profiling of organisms and single cells. However, SGS analysis of transcriptome diversity (including protein-coding transcripts and regulatory non-coding RNAs) is inefficient unless the sample of interest is first depleted of nucleic acids derived from ribosomal RNA (rRNA), which typically account for up to 95% of total intracellular RNA content. Here we describe a novel microscale hydroxyapatite chromatography (HAC) normalization method to remove eukaryotic and prokaryotic high abundant rRNA species, thereby increasing sequence coverage depth and transcript diversity across non-rRNA populations. RNA-seq analysis of Escherichia coli K-12 and human intracellular total RNA showed that HAC-based normalization enriched for all non-ribosomal RNA species regardless of RNA transcript abundance or length when compared with untreated controls. Microcolumn HAC normalization generated rRNA-depleted cDNA libraries comparable to the well-established duplex specific nuclease (DSN) normalization and Ribo-Zero rRNA-depletion methods, thus establishing microscale HAC as an effective, cost saving, and non-destructive alternative normalization technique.

Quality control of next-generation sequencing library through an integrative digital microfluidic platform.

We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/µL, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments.

Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.

We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-α cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways.

Digital microfluidics: a versatile tool for applications in chemistry, biology and medicine.

Digital microfluidics (DMF) has recently emerged as a popular technology for a wide range of applications. In DMF, nanoliter to microliter droplets containing samples and reagents can be manipulated to carry out a range of discrete fluidic operations simply by applying a series of electrical potentials to an array of patterned electrodes coated with a hydrophobic insulator. DMF is distinct from microchannel-based fluidics as it allows for precise control over multiple reagent phases (liquids and solids) in heterogeneous systems with no need for complex networks of connections, microvalves, or pumps. In this review, we discuss the most recent developments in this technology with particular attention to the potential benefits and outstanding challenges for applications in chemistry, biology, and medicine.

Automated digital microfluidic sample preparation for next-generation DNA sequencing.

Next-generation sequencing (NGS) technology is a promising tool for identifying and characterizing unknown pathogens, but its usefulness in time-critical biodefense and public health applications is currently limited by the lack of fast, efficient, and reliable automated DNA sample preparation methods. To address this limitation, we are developing a digital microfluidic (DMF) platform to function as a fluid distribution hub, enabling the integration of multiple subsystem modules into an automated NGS library sample preparation system. A novel capillary interface enables highly repeatable transfer of liquid between the DMF device and the external fluidic modules, allowing both continuous-flow and droplet-based sample manipulations to be performed in one integrated system. Here, we highlight the utility of the DMF hub platform and capillary interface for automating two key operations in the NGS sample preparation workflow. Using an in-line contactless conductivity detector in conjunction with the capillary interface, we demonstrate closed-loop automated fraction collection of target analytes from a continuous-flow sample stream into droplets on the DMF device. Buffer exchange and sample cleanup, the most repeated steps in NGS library preparation, are also demonstrated on the DMF platform using a magnetic bead assay and achieving an average DNA recovery efficiency of 80%±4.8%.

An ultra-high temperature flow-through capillary device for bacterial spore lysis.

Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (195 degrees C) an ethylene glycol lysis buffer to perform rapid flow-through rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow-through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices.

Oligonucleotide hybridization and free-solution electrokinetic separation in a nanofluidic device.

There is significant interest in developing on-chip DNA hybridization assays to leverage the advantages of lab-on-a-chip systems, which include smaller sample and reagent volumes, faster processing speeds, and greater opportunities for large-scale integration. While much research has explored ways to integrate DNA microarrays on-chip, little work has been done to incorporate hybridization with existing microscale separation platforms. We present the first separation of single-stranded and double-stranded oligonucleotides in a nanofluidic device. We couple this separation with free-solution hybridization to develop a simple, electrokinetic technique that detects DNA hybridization without sample labeling. The technique is used both to detect target DNA sequences and to quantitatively measure hybridization kinetics. To demonstrate the method, we measured the second order reaction coefficient of complementary 20-mer oligonucleotides as a function of sodium ion concentration, which ranged from 0.0048 mol(-1).sec(-1) at 5 mM sodium to 0.42 mol(-1).sec(-1) at 50 mM. We also distinguished between a pair of complementary oligonucleotides and a pair with a single nucleotide mismatch, observing a two-fold difference in hybridization rate. Additionally, we observed a relative change in the mobility of single-stranded and double-stranded DNA with increasing sodium concentration, suggesting that our device may provide a useful platform for studying biomolecule transport in nanochannels.

Biologically functional cationic phospholipid-gold nanoplasmonic carriers of RNA.

Biologically functional cationic phospholipid-gold nanoplasmonic carriers have been designed to simultaneously exhibit carrier capabilities, demonstrate improved colloidal stability, and show no cytotoxicity under physiological conditions. Cargo, such as RNA, DNA, proteins, or drugs, can be adsorbed onto or incorporated into the cationic phospholipid bilayer membrane. These carriers are able to retain their unique nanoscale optical properties under physiological conditions, making them particularly useful in a wide range of imaging, therapeutic, and gene delivery applications that utilize selective nanoplasmonic properties.