Spatial Variation of False Map Turtle (Graptemys pseudogeographica) Bacterial Microbiota in the Lower Missouri River, United States

Turtle populations around the world are continually confronted with changing environments that affect their ecology and conservation status. Among freshwater turtles, population dynamics are thought to be mediated by complex yet often cryptic causes. One recent direction of focus in addressing these causes is the turtle-associated microbiota. In turtles, the gut- associated microbiota is of exceptional interest due to its continual association with host species under changing conditions. Diet-based fluctuations and changes in microbial diversity may correspond to varying external environments at both the individual and population level. Environmental responses are of particular interest due to the anthropogenic changes that may underlie them. Pollutants, disruption of climatic patterns, and habitat fragmentation all have the potential to affect turtle-associated microbiota and subsequent population and species conservation. To better understand potential human-induced changes, the diversity of turtle-associated microbiota over local spatial gradients must be better understood. We examined microbial community alpha- and beta-diversity among 30 adult False Map Turtles (Graptemys pseudogeographica) at three sites within the lower Missouri River, United States. Our results indicate significant microbial community centroid differences among sites (beta-diversity), which are likely mediated by various local environmental factors. Such factors will have to be carefully considered in any future attribution of anthropogenic determinants on turtle-associated microbiota as it relates to turtle population dynamics

Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels

Multiplexed, real-time fluorescence detection at the single-molecule level is highly desirable to reveal the stoichiometry, dynamics, and interactions of individual molecular species within complex systems. However, traditionally fluorescence sensing is limited to 3-4 concurrently detected labels, due to low signal-to-noise, high spectral overlap between labels, and the need to avoid dissimilar dye chemistries. We have engineered a palette of several dozen fluorescent labels, called FRETfluors, for spectroscopic multiplexing at the single-molecule level. Each FRETfluor is a compact nanostructure formed from the same three chemical building blocks (DNA, Cy3, and Cy5). The composition and dye-dye geometries create a characteristic F\"orster Resonance Energy Transfer (FRET) efficiency for each construct. In addition, we varied the local DNA sequence and attachment chemistry to alter the Cy3 and Cy5 emission properties and thereby shift the emission signatures of an entire series of FRET constructs to new sectors of the multi-parameter detection space. Unique spectroscopic emission of each FRETfluor is therefore conferred by a combination of FRET and this site-specific tuning of individual fluorophore photophysics. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs statistically selected to minimize classification errors, measured using an Anti-Brownian ELectrokinetic (ABEL) trap which provides precise multi-parameter spectroscopic measurements. The ABEL trap also enables discrimination between FRETfluors attached to a target (here: mRNA) and unbound FRETfluors, eliminating the need for washes or removal of excess label by purification. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs selected to minimize classification errors.Comment: 43 pages, 6 figures, 13 Supplementary figures, 3 Supplementary tables, 5 Supplementary note

Fluorogenic Atom Transfer Radical Polymerization in Aqueous Media as a Strategy for Detection

The development of novel approaches to signal amplification in aqueous media could enable new diagnostic platforms for the detection of water-soluble analytes, including biomolecules. This paper describes a fluorogenic polymerization approach to amplify initiator signal by the detection of visible fluorescence upon polymerization in real-time. Fluorogenic monomers were synthesized and co-polymerized by atom transfer radical polymerization (ATRP) in water to reveal increasing polymer fluorescence as a function of both reaction time and initiator concentration. Optimization of the fluorogenic ATRP reaction conditions allowed for the quantitative detection of a small-molecule initiator as a model analyte over a broad linear concentration range (pM to mM). Raising the reaction temperature from 30 C to 60 C facilitated sensitive initiator detection at sub-picomolar concentrations in as little as 1 h of polymerization. This method was then applied to the detection of streptavidin as a model biological analyte by fluorogenic polymerization from a designed biotinylated ATRP initiator. Taken together, these studies represent the first example of a fluorogenic ATRP reaction and establish fluorogenic polymerization as a promising approach for the direct detection of aqueous analytes and biomolecular recognition events

Unbiased Metagenomic Sequencing for Pediatric Meningitis in Bangladesh Reveals Neuroinvasive Chikungunya Virus Outbreak and Other Unrealized Pathogens.

The burden of meningitis in low-and-middle-income countries remains significant, but the infectious causes remain largely unknown, impeding institution of evidence-based treatment and prevention decisions. We conducted a validation and application study of unbiased metagenomic next-generation sequencing (mNGS) to elucidate etiologies of meningitis in Bangladesh. This RNA mNGS study was performed on cerebrospinal fluid (CSF) specimens from patients admitted in the largest pediatric hospital, a World Health Organization sentinel site, with known neurologic infections (n = 36), with idiopathic meningitis (n = 25), and with no infection (n = 30), and six environmental samples, collected between 2012 and 2018. We used the IDseq bioinformatics pipeline and machine learning to identify potentially pathogenic microbes, which we then confirmed orthogonally and followed up through phone/home visits. In samples with known etiology and without infections, there was 83% concordance between mNGS and conventional testing. In idiopathic cases, mNGS identified a potential bacterial or viral etiology in 40%. There were three instances of neuroinvasive Chikungunya virus (CHIKV), whose genomes were >99% identical to each other and to a Bangladeshi strain only previously recognized to cause febrile illness in 2017. CHIKV-specific qPCR of all remaining stored CSF samples from children who presented with idiopathic meningitis in 2017 (n = 472) revealed 17 additional CHIKV meningitis cases, exposing an unrecognized meningitis outbreak. Orthogonal molecular confirmation, case-based clinical data, and patient follow-up substantiated the findings. Case-control CSF mNGS surveys can complement conventional diagnostic methods to identify etiologies of meningitis, conduct surveillance, and predict outbreaks. The improved patient- and population-level data can inform evidence-based policy decisions.IMPORTANCE Globally, there are an estimated 10.6 million cases of meningitis and 288,000 deaths every year, with the vast majority occurring in low- and middle-income countries. In addition, many survivors suffer from long-term neurological sequelae. Most laboratories assay only for common bacterial etiologies using culture and directed PCR, and the majority of meningitis cases lack microbiological diagnoses, impeding institution of evidence-based treatment and prevention strategies. We report here the results of a validation and application study of using unbiased metagenomic sequencing to determine etiologies of idiopathic (of unknown cause) cases. This included CSF from patients with known neurologic infections, with idiopathic meningitis, and without infection admitted in the largest children's hospital of Bangladesh and environmental samples. Using mNGS and machine learning, we identified and confirmed an etiology (viral or bacterial) in 40% of idiopathic cases. We detected three instances of Chikungunya virus (CHIKV) that were >99% identical to each other and to a strain previously recognized to cause systemic illness only in 2017. CHIKV qPCR of all remaining stored 472 CSF samples from children who presented with idiopathic meningitis in 2017 at the same hospital uncovered an unrecognized CHIKV meningitis outbreak. CSF mNGS can complement conventional diagnostic methods to identify etiologies of meningitis, and the improved patient- and population-level data can inform better policy decisions

Adaptation of a mobile interactive obesity treatment approach for early severe mental illness: Protocol for a mixed methods implementation and pilot randomized controlled trial

BACKGROUND: Obesity is common in individuals with severe mental illness (SMI), contributing to a significantly shortened lifespan when compared to the general population. Available weight loss treatments have attenuated efficacy in this population, underscoring the importance of prevention and early intervention. OBJECTIVE: Here, we describe a type 1 hybrid study design for adapting and pilot-testing an existing mobile health intervention for obesity prevention in individuals with early SMI and Class I or early-stage obesity, defined as a BMI of 30-35. METHODS: An existing, evidence-based interactive obesity treatment approach using low-cost, semiautomated SMS text messaging was selected for adaptation. Community mental health clinics and Clubhouse settings in Eastern Missouri and South Florida were identified to participate. This study has the following 3 aims. First, using the Enhanced Framework for Reporting Adaptations and Modifications to Evidence-based interventions, contextual aspects of the clinical and digital treatment environments are identified for adaptation, considering 5 main stakeholder groups (clinical administrators, prescribing clinicians, case managers, nurses, and patients). Following a 2-week trial of unadapted SMS text messaging, Innovation Corps methods are used to discover needed intervention adaptations by stakeholder group and clinical setting. Second, adaptations to digital functionality and intervention content will be made based on themes identified in aim 1, followed by rapid usability testing with key stakeholders. A process for iterative treatment adaptation will be developed for making unplanned modifications during the aim 3 implementation pilot study. Individuals working in partner community mental health clinics and Clubhouse settings will be trained in intervention delivery. Third, in a randomized pilot and feasibility trial, adults with 5 years or less of treatment for an SMI diagnosis will be randomized 2:1 to 6 months of an adapted interactive obesity treatment approach or to an attentional control condition, followed by a 3-month extension phase of SMS text messages only. Changes in weight, BMI, and behavioral outcomes, as well as implementation challenges, will be evaluated at 6 and 9 months. RESULTS: Institutional review board approval for aims 1 and 2 was granted on August 12, 2018, with 72 focus group participants enrolled; institutional review board approval for aim 3 was granted on May 6, 2020. To date, 52 participants have been enrolled in the study protocol. CONCLUSIONS: In this type 1 hybrid study design, we apply an evidence-based treatment adaptation framework to plan, adapt, and feasibility test a mobile health intervention in real-world treatment settings. Resting at the intersection of community mental health treatment and physical health promotion, this study aims to advance the use of simple technology for obesity prevention in individuals with early-stage mental illness. TRIAL REGISTRATION: ClinicalTrials.gov NCT03980743; https://clinicaltrials.gov/ct2/show/NCT03980743. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/42114

GDE6 promotes progenitor identity in the vertebrate neural tube

The generation of neurons in the central nervous system is a complex, stepwise process necessitating the coordinated activity of mitotic progenitors known as radial glia. Following neural tube closure, radial glia undergo a period of active proliferation to rapidly expand their population, creating a densely packed neurepithelium. Simultaneously, radial glia positioned across the neural tube are uniquely specified to produce diverse neuronal sub-types. Although these cellular dynamics are well studied, the molecular mechanisms governing them are poorly understood. The six-transmembrane Glycerophosphodiester Phosphodiesterase proteins (GDE2, GDE3, and GDE6) comprise a family of cell-surface enzymes expressed in the embryonic nervous system. GDE proteins can release Glycosylphosphatidylinositol-anchored proteins from the cell surface via cleavage of their lipid anchor. GDE2 has established roles in motor neuron differentiation and oligodendrocyte maturation, and GDE3 regulates oligodendrocyte precursor cell proliferation. Here, we describe a role for GDE6 in early neural tube development. Using RNAscope, we show that Gde6 mRNA is expressed by ventricular zone progenitors in the caudal neural tube. Utilizing in-ovo electroporation, we show that GDE6 overexpression promotes neural tube hyperplasia and ectopic growths of the neurepithelium. At later stages, electroporated embryos exhibit an expansion of the ventral patterning domains accompanied by reduced cross-repression. Ultimately, electroporated embryos fail to produce the full complement of post-mitotic motor neurons. Our findings indicate that GDE6 overexpression significantly affects radial glia function and positions GDE6 as a complementary factor to GDE2 during neurogenesis

Pregnancy-Associated Hypertension in Glucose-Intolerant Pregnancy and Subsequent Metabolic Syndrome

To evaluate whether pregnancy-associated hypertension (preeclampsia or gestational hypertension), among women with varying degrees of glucose intolerance during pregnancy is associated with maternal metabolic syndrome 5-10 years later

Amplification dynamics of platy-1 retrotransposons in the cebidae platyrrhine lineage

© 2019 The Author(s). Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. Platy-1 elements are Platyrrhine-specific, short interspersed elements originally discovered in the Callithrix jacchus (common marmoset) genome. To date,only themarmoset genomehas been analyzed for Platy-1 repeat content.Here,we report full-length Platy-1 insertions in other NewWorld monkey (NWM) genomes (Saimiri boliviensis, squirrel monkey; Cebus imitator, capuchin monkey; and Aotus nancymaae, owl monkey) and analyze the amplification dynamics of lineage-specific Platy-1 insertions. A relatively small number of full-length and lineage-specific Platy-1 elements were found in the squirrel, capuchin, and owl monkey genomes compared with the marmoset genome. In addition, only a few older Platy-1 subfamilies were recovered in this study, with no Platy-1 subfamilies younger than Platy-1-6. By contrast, 62 Platy-1 subfamilieswere discovered in themarmoset genome.All of the lineagespecific insertions found in the squirrel and capuchin monkeys were fixed present. However, 15%of the lineage-specific Platy-1 loci in Aotus were polymorphic for insertion presence/absence. In addition, two new Platy-1 subfamilies were identified in the owl monkey genome with low nucleotide divergences compared with their respective consensus sequences, suggesting minimal ongoing retrotransposition in the Aotus genus and no current activity in the Saimiri, Cebus, and Sapajus genera. These comparative analyses highlight the finding that the high number of Platy-1 elements discovered in themarmoset genome is an exception among NWManalyzed thus far, rather than the rule. Future studies are needed to expand upon our knowledge of Platy-1 amplification in other NWM genomes

Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children

This article is made available for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing.BACKGROUND: Despite improved diagnostics, pulmonary pathogens in immunocompromised children frequently evade detection, leading to significant mortality. Therefore, we aimed to develop a highly sensitive metagenomic next-generation sequencing (mNGS) assay capable of evaluating the pulmonary microbiome and identifying diverse pathogens in the lungs of immunocompromised children. METHODS: We collected 41 lower respiratory specimens from 34 immunocompromised children undergoing evaluation for pulmonary disease at 3 children's hospitals from 2014-2016. Samples underwent mechanical homogenization, parallel RNA/DNA extraction, and metagenomic sequencing. Sequencing reads were aligned to the National Center for Biotechnology Information nucleotide reference database to determine taxonomic identities. Statistical outliers were determined based on abundance within each sample and relative to other samples in the cohort. RESULTS: We identified a rich cross-domain pulmonary microbiome that contained bacteria, fungi, RNA viruses, and DNA viruses in each patient. Potentially pathogenic bacteria were ubiquitous among samples but could be distinguished as possible causes of disease by parsing for outlier organisms. Samples with bacterial outliers had significantly depressed alpha-diversity (median, 0.61; interquartile range [IQR], 0.33-0.72 vs median, 0.96; IQR, 0.94-0.96; P < .001). Potential pathogens were detected in half of samples previously negative by clinical diagnostics, demonstrating increased sensitivity for missed pulmonary pathogens (P < .001). CONCLUSIONS: An optimized mNGS assay for pulmonary microbes demonstrates significant inoculation of the lower airways of immunocompromised children with diverse bacteria, fungi, and viruses. Potential pathogens can be identified based on absolute and relative abundance. Ongoing investigation is needed to determine the pathogenic significance of outlier microbes in the lungs of immunocompromised children with pulmonary disease

Host Plants and Climate Structure Habitat Associations of the Western Monarch Butterfly

The monarch butterfly is one of the most easily recognized and frequently studied insects in the world, and has recently come into the spotlight of public attention and conservation concern because of declining numbers of individuals associated with both the eastern and western migrations. Historically, the larger eastern migration has received the most scientific attention, but this has been changing in recent years, and here we report the largest-ever attempt to map and characterize non-overwintering habitat for the western monarch butterfly. Across the environmentally and topographically complex western landscape, we include 8,427 observations of adults and juvenile monarchs, as well as 20,696 records from 13 milkweed host plant species. We find high heterogeneity of suitable habitats across the geographic range, with extensive concentrations in the California floristic province in particular. We also find habitat suitability for the butterfly to be structured primarily by host plant habitat associations, which are in turn structured by a diverse suite of climatic variables. These results add to our knowledge of range and occupancy determinants for migratory species and provide a tool that can be used by conservation biologists and researchers interested in interactions among climate, hosts and host-specific animals, and by managers for prioritizing future conservation actions at regional to watershed scales