Deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome.

Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is poorly defined, the pathophysiology is unknown, and no disease-modifying treatments are available. We used rigorous criteria to recruit PI-ME/CFS participants with matched controls to conduct deep phenotyping. Among the many physical and cognitive complaints, one defining feature of PI-ME/CFS was an alteration of effort preference, rather than physical or central fatigue, due to dysfunction of integrative brain regions potentially associated with central catechol pathway dysregulation, with consequences on autonomic functioning and physical conditioning. Immune profiling suggested chronic antigenic stimulation with increase in naïve and decrease in switched memory B-cells. Alterations in gene expression profiles of peripheral blood mononuclear cells and metabolic pathways were consistent with cellular phenotypic studies and demonstrated differences according to sex. Together these clinical abnormalities and biomarker differences provide unique insight into the underlying pathophysiology of PI-ME/CFS, which may guide future intervention.

Integrating Clinical Decision Support Into Electronic Health Record Systems Using a Novel Platform (EvidencePoint): Developmental Study.

BACKGROUND: Through our work, we have demonstrated how clinical decision support (CDS) tools integrated into the electronic health record (EHR) assist providers in adopting evidence-based practices. This requires confronting technical challenges that result from relying on the EHR as the foundation for tool development; for example, the individual CDS tools need to be built independently for each different EHR. OBJECTIVE: The objective of our research was to build and implement an EHR-agnostic platform for integrating CDS tools, which would remove the technical constraints inherent in relying on the EHR as the foundation and enable a single set of CDS tools that can work with any EHR. METHODS: We developed EvidencePoint, a novel, cloud-based, EHR-agnostic CDS platform, and we will describe the development of EvidencePoint and the deployment of its initial CDS tools, which include EHR-integrated applications for clinical use cases such as prediction of hospitalization survival for patients with COVID-19, venous thromboembolism prophylaxis, and pulmonary embolism diagnosis. RESULTS: The results below highlight the adoption of the CDS tools, the International Medical Prevention Registry on Venous Thromboembolism-D-Dimer, the Wells' criteria, and the Northwell COVID-19 Survival (NOCOS), following development, usability testing, and implementation. The International Medical Prevention Registry on Venous Thromboembolism-D-Dimer CDS was used in 5249 patients at the 2 clinical intervention sites. The intervention group tool adoption was 77.8% (4083/5249 possible uses). For the NOCOS tool, which was designed to assist with triaging patients with COVID-19 for hospital admission in the event of constrained hospital resources, the worst-case resourcing scenario never materialized and triaging was never required. As a result, the NOCOS tool was not frequently used, though the EvidencePoint platform's flexibility and customizability enabled the tool to be developed and deployed rapidly under the emergency conditions of the pandemic. Adoption rates for the Wells' criteria tool will be reported in a future publication. CONCLUSIONS: The EvidencePoint system successfully demonstrated that a flexible, user-friendly platform for hosting CDS tools outside of a specific EHR is feasible. The forthcoming results of our outcomes analyses will demonstrate the adoption rate of EvidencePoint tools as well as the impact of behavioral economics "nudges" on the adoption rate. Due to the EHR-agnostic nature of EvidencePoint, the development process for additional forms of CDS will be simpler than traditional and cumbersome IT integration approaches and will benefit from the capabilities provided by the core system of EvidencePoint.

Persistence of Human Immunodeficiency Virus-Associated Cerebral Toxoplasmosis Lesions in Successfully Treated Patients Receiving Combination Antiretroviral Therapy.

Background: Toxoplasmic encephalitis (TE) is a life-threatening complication of people with human immunodeficiency virus (PWH) with severe immunodeficiency, especially those with a CD4+ T-cell count <100 cells/µL. Following a clinical response to anti-Toxoplasma therapy, and immune reconstitution after initiation of combination antiretroviral therapy (ART), anti-Toxoplasma therapy can be discontinued with a low risk of relapse. Methods: To better understand the evolution of magnetic resonance imaging (MRI)-defined TE lesions in PWH receiving ART, we undertook a retrospective study of PWH initially seen at the National Institutes of Health between 2001 and 2012, who had at least 2 serial MRI scans. Lesion size and change over time were calculated and correlated with clinical parameters. Results: Among 24 PWH with TE and serial MRI scans, only 4 had complete clearance of lesions at the last MRI (follow-up, 0.09-5.8 years). Of 10 PWH off all anti-Toxoplasma therapy (median, 3.2 years after TE diagnosis), 6 had persistent MRI enhancement. In contrast, all 5 PWH seen in a pre-ART era study who were followed for >6 months had complete clearance of lesions. TE lesion area at diagnosis was associated with the absolute change in area (P < .0001). Conclusions: Contrast enhancement can persist even when TE has been successfully treated and anti-Toxoplasma therapy has been stopped, highlighting the need to consider diagnostic alternatives in successfully treated patients with immune reconstitution presenting with new neurologic symptoms.

Encoding Coacervate Droplets with Paramagnetism for Dynamical Reconfigurability and Spatial Addressability.

It is an ongoing endeavor in chemistry and materials science to regulate coacervate droplets on a physiologically relevant spatiotemporal scale to ultimately match or even surpass living cells' precision, complexity, and functionality. Herein, we develop a magnetic strategy orthogonal to the thermal, pH, light, or chemical counterparts that are commonly employed by biotic or artificial systems; its successful implementation thus adds a missing piece to the current arsenal of manipulative methodologies. Specifically, we paramagnetize the otherwise diamagnetic coacervate droplets by cooperatively combining paramagnetic ingredients (including organic radicals, metal ions, and Fe3O4 nanoparticles) and coacervate ingredients to obtain "MagCoa" droplets. A simple model is derived theoretically to account for migration and division of MagCoa droplets in an uneven magnetic field. Experimentally, we produce an array of compartmentalized and monodispersed droplets using microfluidics and magnetically steer them with uniformity and synchronicity. We design and fabricate spatial magnetic modulators to engineer the landscape of a magnetic field that, in turn, directs the MagCoa droplets into predesigned patterns in a reconfigurable fashion. These programmable liquid patterns can be potentially extended to dynamic assembly and information encryption. We envision that the toolbox established here is of generality and multitudes to serve as a practical guide to control droplets magnetically.

Cocaine restricts nucleus accumbens feedforward drive through a monoamine-independent mechanism.

Parvalbumin-expressing fast-spiking interneurons (PV-INs) within feedforward microcircuits in the nucleus accumbens (NAc) coordinate goal-directed motivational behavior. Feedforward inhibition of medium spiny projection neurons (MSNs) is initiated by glutamatergic input from corticolimbic brain structures. While corticolimbic synapses onto MSNs are targeted by the psychostimulant, cocaine, it remains unknown whether cocaine also exerts acute neuromodulatory actions at collateralizing synapses onto PV-INs. Using whole-cell patch-clamp electrophysiology, optogenetics, and pharmacological tools in transgenic reporter mice, we found that cocaine decreases thalamocortical glutamatergic drive onto PV-INs by engaging a monoamine-independent mechanism. This mechanism relies on postsynaptic sigma-1 (σ1) activity, leading to the mobilization of intracellular Ca2+ stores that trigger retrograde endocannabinoid signaling at presynaptic type-1 cannabinoid receptors (CB1R). Cocaine-evoked CB1R activity occludes the expression of CB1R-dependent long-term depression (LTD) at this synaptic locus. These findings provide evidence that acute cocaine exposure targets feedforward microcircuits in the NAc and extend existing models of cocaine action on mesolimbic reward circuits.

Kappa opioid receptor modulation of excitatory drive onto nucleus accumbens fast-spiking interneurons.

The dynorphin/kappa opioid receptor (KOR) system within the nucleus accumbens (NAc) contributes to affective states. Parvalbumin fast-spiking interneurons (PV-FSIs), a key component of feedforward inhibition, participate in integration of excitatory inputs to the NAc by robustly inhibiting select populations of medium spiny output neurons, therefore greatly influencing NAc dependent behavior. How the dynorphin/KOR system regulates feedforward inhibition in the NAc remains unknown. Here, we elucidate the molecular mechanisms of KOR inhibition of excitatory transmission onto NAc PV-FSIs using a combination of whole-cell patch-clamp electrophysiology, optogenetics, pharmacology, and a parvalbumin reporter mouse. We find that postsynaptic KOR stimulation induces long-term depression (LTD) of excitatory synapses onto PV-FSI by stimulating the endocytosis of AMPARs via a PKA and calcineurin-dependent mechanism. Furthermore, KOR regulation of PV-FSI synapses are input specific, inhibiting thalamic but not cortical inputs. Finally, following acute stress, a protocol known to elevate dynorphin/KOR signaling in the NAc, KOR agonists no longer inhibit excitatory transmission onto PV-FSI. In conclusion, we delineate pathway-specific mechanisms mediating KOR control of feedforward inhibitory circuits in the NAc and provide evidence for the recruitment of this system in response to stress.

Development of a Point-of-Care Assay for HIV-1 Viral Load Using Higher Refractive Index Antibody-Coated Microbeads.

The detection of viruses using imaging techniques is challenging because of the weak scattering of light generated by the targets of sizes in the nanometer range. The system we have developed overcomes the light scattering problems by utilizing antibody-coated microbeads of higher index of refraction that can specifically bind with viruses and increase the acceptance angle. Using the new technology, we have developed a portable, cost-effective, and field-deployable platform for the rapid quantification of HIV-1 viral load for point-of-care (POC) settings. The system combines microfluidics with a wide field of view lensless imaging technology. Highly specific antibodies are functionalized to a glass slide inside a microchip to capture HIV-1 virions. The captured virions are then bound by antibody-conjugated microbeads, which have a higher refraction index. The microbeads-HIV-1 virions complexes generate diffraction patterns that are detected with a custom-built imaging setup and rapidly and accurately quantified by computational analysis. This platform technology enables fast nanoscale virus imaging and quantification from biological samples and thus can play a significant role in the detection and management of viral diseases.

Noradrenergic Signaling Disengages Feedforward Transmission in the Nucleus Accumbens Shell.

The nucleus accumbens shell (NAcSh) receives extensive monoaminergic input from multiple midbrain structures. However, little is known how norepinephrine (NE) modulates NAc circuit dynamics. Using a dynamic electrophysiological approach with optogenetics, pharmacology, and drugs acutely restricted by tethering (DART), we explored microcircuit-specific neuromodulatory mechanisms recruited by NE signaling in the NAcSh of parvalbumin (PV)-specific reporter mice. Surprisingly, NE had little direct effect on modulation of synaptic input at medium spiny projection neurons (MSNs). In contrast, we report that NE transmission selectively modulates glutamatergic synapses onto PV-expressing fast-spiking interneurons (PV-INs) by recruiting postsynaptically-localized α2-adrenergic receptors (ARs). The synaptic effects of α2-AR activity decrease PV-IN-dependent feedforward inhibition onto MSNs evoked via optogenetic stimulation of cortical afferents to the NAcSh. These findings provide insight into a new circuit motif in which NE has a privileged line of communication to tune feedforward inhibition in the NAcSh.SIGNIFICANCE STATEMENT The nucleus accumbens (NAc) directs reward-related motivational output by integrating glutamatergic input with diverse neuromodulatory input from monoamine centers. The present study reveals a synapse-specific regulatory mechanism recruited by norepinephrine (NE) signaling within parvalbumin-expressing interneuron (PV-IN) feedforward inhibitory microcircuits. PV-IN-mediated feedforward inhibition in the NAc is instrumental in coordinating NAc output by synchronizing the activity of medium spiny projection neurons (MSNs). By negatively regulating glutamatergic transmission onto PV-INs via α2-adrenergic receptors (ARs), NE diminishes feedforward inhibition onto MSNs to promote NAc output. These findings elucidate previously unknown microcircuit mechanisms recruited by the historically overlooked NE system in the NAc.

Spherical Nucleic Acids: Integrating Nanotechnology Concepts into General Chemistry Curricula.

Nanoscience and technology research offer exciting avenues to modernize undergraduate-level General Chemistry curricula. In particular, spherical nucleic acid (SNA) nanoconjugates, which behave as "programmable atom equivalents" (PAEs) in the context of colloidal crystals, are one system that one can use to reinforce foundational concepts in chemistry including matter and atoms, the Periodic Table, Lewis dot structures and the octet rule, valency and valence-shell electron-pair repulsion (VSEPR) theory, and Pauling's rules, ultimately leading to enriching discussions centered on materials chemistry and biochemistry with key implications in medicine, optics, catalysis, and other areas. These lessons connect historical and modern concepts in chemistry, relate course content to current professional and popular science topics, inspire critical and creative thinking, and spur some students to continue their science, technology, engineering, and mathematics (STEM) education and attain careers in STEM fields. Ultimately, and perhaps most importantly, these lessons may expand the pool of young students interested in chemistry by making connections to a broader group of contemporary concepts and technologies that impact their lives and enhance their view of the field. Herein, a way of teaching aspects of General Chemistry in the context of modern nanoscience concepts is introduced to instructors and curricula developers at research institutions, primarily undergraduate institutions, and community colleges worldwide.

To Petabytes and beyond: recent advances in probabilistic and signal processing algorithms and their application to metagenomics.

As computational biologists continue to be inundated by ever increasing amounts of metagenomic data, the need for data analysis approaches that keep up with the pace of sequence archives has remained a challenge. In recent years, the accelerated pace of genomic data availability has been accompanied by the application of a wide array of highly efficient approaches from other fields to the field of metagenomics. For instance, sketching algorithms such as MinHash have seen a rapid and widespread adoption. These techniques handle increasingly large datasets with minimal sacrifices in quality for tasks such as sequence similarity calculations. Here, we briefly review the fundamentals of the most impactful probabilistic and signal processing algorithms. We also highlight more recent advances to augment previous reviews in these areas that have taken a broader approach. We then explore the application of these techniques to metagenomics, discuss their pros and cons, and speculate on their future directions.

Development of a Flow-Free Magnetic Actuation Platform for an Automated Microfluidic ELISA.

There is a need to create an easily deployable and point-of-care (POC) diagnostic platform for disease outbreaks and for monitoring and maintenance of chronic illnesses. Such platforms are useful in regions where access to clinical laboratories may be limited or constrained using cost-effective solutions to quickly process high numbers of samples. Using oil and water liquid-liquid interphase separation, immunoassays developed for microfluidic chips can potentially meet this need when leveraged with electromagnetic actuation and antibody-coated superparamagnetic beads. We have developed a microfluidic immunoassay detection platform, which enables assay automation and maintains successful liquid containment for future use in the field. The assay was studied through a series of magnetic and fluid simulations to demonstrate these optimizations, and an optimized chip was tested using a target model for HIV-1, the p24 capsid antigen. The use of minimal reagents further lowers the cost of each assay and lowers the required sample volume for testing (<50 µL), that can offer easy turnaround for sample collection and assay results. The developed microfluidic immunoassay platform can be easily scaled for multiplex or multi-panel specific testing at the POC.

Virtual Reality Assessment of Classroom - Related Attention: An Ecologically Relevant Approach to Evaluating the Effectiveness of Working Memory Training.

Computerized cognitive interventions to improve working memory also purport to improve ADHD-related inattention and off task behavior. Such interventions have been shown to improve working memory, executive functioning, and fluid reasoning on standardized neuropsychological measures. However, debate continues as to whether such programs lead to improvement on ecologically relevant outcomes, such as classroom behavior. This study sought to propose a novel, ecologically relevant approach to evaluate the effectiveness of working memory training on real-world attention performance. Participants included 15 children, aged 6-15, identified as having attention problems were assessed via the virtual classroom continuous performance task (VCCPT) before and after completing 5 weeks of Cogmed working memory training. The VCCPT is a validated measure of sustained and selective attention set within a virtual reality (VR) environment. Several key areas of attention performance were observed to improve, including omission errors, reaction time, reaction time variability, and hit variability. Results suggest that working memory training led to substantial improvements in sustained attention in a real-life scenario of classroom learning. Moreover, the use of psychometrically validated VR measurement provides incremental validity beyond that of teacher or parent report of behavior. Observing such improvements on ecologically relevant measures of attention adds to the discussion around how to evaluate the effectiveness of working memory training as it pertains to real-life improvements and serves to inform consumer awareness of such products and their claims.

Point-of-care Colorimetric Analysis through Smartphone Video.

Point-of-care (POC) tests often rely on smartphone image methods for colorimetric analysis, but the results of such methods are frequently difficult to reproduce or standardize. The problem is aggravated by unpredictable image capture conditions, which pose a significant challenge when low limits of detection (LOD) are needed. Application-specific smartphone attachments are often used to standardize imaging conditions, but there has recently been an interest in equipment-free POC colorimetric analysis. Improved output metrics and preprocessing methods have been developed, but equipment-free imaging often has a high LOD and is inappropriate for quantitative tasks. Additional work is necessary to replace external smartphone attachments with algorithms. Towards this end, we have developed a video processing method that synthesizes many images into a single output metric. We use image features to select the best inputs from a large set of video frames and demonstrate that the resulting output values have a stronger correlation with laboratory methods and a lower standard error. The developed algorithm only requires 20 seconds of video and can easily be integrated with existing processing methods. We apply our algorithm to the NS1-based sandwich ELISA for Zika detection and show that the LOD is two times lower when our video-based method is used.

Cell phone based colorimetric analysis for point-of-care settings.

Cell phones show considerable promise for point-of-care (POC) diagnostic procedures because they are accessible, connected, and computationally powerful. Cell phone image processing methods are being developed for the detection and quantification of a wide range of targets, employing methods from microscopy to fluorescence techniques. However, most of the lab-based biological and biochemical assays still lack a robust and repeatable cell phone analogue. Existing solutions require external smartphone hardware to obtain quantitative results, imposing a design tradeoff between accessibility and accuracy. Here, we develop a cell phone imaging algorithm that enables analysis of assays that would typically be evaluated via spectroscopy. The developed technique uses the saturation parameter of hue-saturation-value color space to enable POC diagnosis. Through the analysis of over 10 000 images, we show that the saturation method consistently outperforms existing algorithms under a wide range of operating field conditions. The performance improvement is also proven analytically via the mathematic relationship between the saturation method and existing techniques. The method presented here is a step forward towards the development of POC diagnostics by reducing the required equipment, improving the limit of detection (LOD), and increasing the precision of quantitative results.

Histone H3 lysine K4 methylation and its role in learning and memory.

Epigenetic modifications such as histone methylation permit change in chromatin structure without accompanying change in the underlying genomic sequence. A number of studies in animal models have shown that dysregulation of various components of the epigenetic machinery causes cognitive deficits at the behavioral level, suggesting that proper epigenetic control is necessary for the fundamental processes of learning and memory. Histone H3 lysine K4 (H3K4) methylation comprises one component of such epigenetic control, and global levels of this mark are increased in the hippocampus during memory formation. Modifiers of H3K4 methylation are needed for memory formation, shown through animal studies, and many of the same modifiers are mutated in human cognitive diseases. Indeed, all of the known H3K4 methyltransferases and four of the known six H3K4 demethylases have been associated with impaired cognition in a neurologic or psychiatric disorder. Cognitive impairment in such patients often manifests as intellectual disability, consistent with a role for H3K4 methylation in learning and memory. As a modification quintessentially, but not exclusively, associated with transcriptional activity, H3K4 methylation provides unique insights into the regulatory complexity of writing, reading, and erasing chromatin marks within an activated neuron. The following review will discuss H3K4 methylation and connect it to transcriptional events required for learning and memory within the developed nervous system. This will include an initial discussion of the most recent advances in the developing methodology to analyze H3K4 methylation, namely mass spectrometry and deep sequencing, as well as how these methods can be applied to more deeply understand the biology of this mark in the brain. We will then introduce the core enzymatic machinery mediating addition and removal of H3K4 methylation marks and the resulting epigenetic signatures of these marks throughout the neuronal genome. We next foray into the brain, discussing changes in H3K4 methylation marks within the hippocampus during memory formation and retrieval, as well as the behavioral correlates of H3K4 methyltransferase deficiency in this region. Finally, we discuss the human cognitive diseases connected to each H3K4 methylation modulator and summarize advances in developing drugs to target them.

SARNAclust: Semi-automatic detection of RNA protein binding motifs from immunoprecipitation data.

RNA-protein binding is critical to gene regulation, controlling fundamental processes including splicing, translation, localization and stability, and aberrant RNA-protein interactions are known to play a role in a wide variety of diseases. However, molecular understanding of RNA-protein interactions remains limited; in particular, identification of RNA motifs that bind proteins has long been challenging, especially when such motifs depend on both sequence and structure. Moreover, although RNA binding proteins (RBPs) often contain more than one binding domain, algorithms capable of identifying more than one binding motif simultaneously have not been developed. In this paper we present a novel pipeline to determine binding peaks in crosslinking immunoprecipitation (CLIP) data, to discover multiple possible RNA sequence/structure motifs among them, and to experimentally validate such motifs. At the core is a new semi-automatic algorithm SARNAclust, the first unsupervised method to identify and deconvolve multiple sequence/structure motifs simultaneously. SARNAclust computes similarity between sequence/structure objects using a graph kernel, providing the ability to isolate the impact of specific features through the bulge graph formalism. Application of SARNAclust to synthetic data shows its capability of clustering 5 motifs at once with a V-measure value of over 0.95, while GraphClust achieves only a V-measure of 0.083 and RNAcontext cannot detect any of the motifs. When applied to existing eCLIP sets, SARNAclust finds known motifs for SLBP and HNRNPC and novel motifs for several other RBPs such as AGGF1, AKAP8L and ILF3. We demonstrate an experimental validation protocol, a targeted Bind-n-Seq-like high-throughput sequencing approach that relies on RNA inverse folding for oligo pool design, that can validate the components within the SLBP motif. Finally, we use this protocol to experimentally interrogate the SARNAclust motif predictions for protein ILF3. Our results support a newly identified partially double-stranded UUUUUGAGA motif similar to that known for the splicing factor HNRNPC.

The contribution of active and passive leisure to children's well-being.

The relation between leisure and well-being, including happiness and self-concept, was examined in 375 children aged 8-12 years. Active leisure (e.g. physical activity) was positively correlated with well-being. Passive leisure (e.g. television and video games) was negatively correlated with well-being. Aspects of active leisure (e.g. the importance of sport to the child and how sports made the child feel) as judged by both parents and children accounted for unique variance in children's wellbeing; passive leisure did not. Similar to previous research on adolescents and adults, active leisure activities were related to children's well-being.

Loss of heterozygosity identifies genetic changes in chronic myeloid disorders, including myeloproliferative disorders, myelodysplastic syndromes and chronic myelomonocytic leukemia.

This study evaluates changes in genetic loci of chronic myeloid disorders using loss of heterozygosity (LOH) techniques. We present the combined results of three experiments. First, examination of a panel of genetic loci in groups of myeloproliferative disorders was evaluated. The second experiment involved microdissection of megakaryocytes from myeloproliferative disorders and comparison of their genetic changes to surrounding neoplastic marrow elements. Finally, we compared results of LOH studies of myeloproliferative disorders to those of myelodysplastic syndromes and chronic myelomonocytic leukemia. A total of 41 bone marrow biopsies were evaluated. Twenty-seven were myeloproliferative disorders (11 chronic idiopathic myelofibrosis, 11 essential thrombocythemia, 5 polycythemia vera). The remaining cases consisted of myelodysplastic syndromes (total=5; RAEB-1=2; RAEB-2=2; MDS, not otherwise specified=1) and chronic myelomonocytic leukemia (n=8). The abnormalities in myeloproliferative disorders were distributed as follows: D7S2554-4/14 (5/14); D8S263-4/15 (5/15); D9S157-5/15 (5/15); D9S161-7/17 (6/17); D13S319-5/14 (4/14); TP53-5/16 (5/16); D20S108-4/15 (4/15). In 75% cases diagnosed as essential thrombocythemia (6/8), both cases of polycythemia vera (2/2), and 29% cases of chronic idiopathic myelofibrosis (2/7), there were genetic differences between the megakaryocytes and the surrounding marrow. These results suggest that in some cases, megakaryocytes have different clonal abnormalities than surrounding hematopoietic tissue. The genetic profiles of myeloproliferative disorders had several differences from those of myelodysplastic syndromes. Although different from both, chronic myelomonocytic leukemia appeared more similar to myeloproliferative disorders using these techniques.