Plasma microRNA Interindividual Variability in Healthy Individuals, Pregnant Women, and an Individual with a Stably Altered Neuroendocrine Phenotype.

BACKGROUND: Extracellular RNAs (exRNAs) in biofluids are amenable to quantitative analysis and proposed as noninvasive biomarkers for monitoring organ function. Cell-lineage-specific microRNAs (miRNAs) are present in plasma as soluble ribonucleoproteins or enclosed in exRNA carriers and transported through the vasculature. However, more extensive studies of healthy individuals are needed to gain insights into the variability of plasma miRNA abundance and composition. METHODS: The exRNA composition of platelet-depleted plasma collected twice from 236 healthy individuals was characterized by small RNA sequencing. Plasma of pregnant women featuring dramatically increased placental miRNAs and samples from subject P12 with noticeably increased epithelial- and neuroendocrine-origin miRNAs were included for comparison. The miRNA content of 10 000g and 100 000g pellet fractions of plasma generated by ultracentrifugation was also determined. Data analysis methods included Pearson correlation, differential gene expression, and unsupervised clustering. RESULTS: The abundance changes for more variable miRNAs in plasma of normal individuals correlated between coexpressed cell-lineage-specific miRNAs of the liver, neuroendocrine organs, epithelial cells, and muscle. ExRNA of pellet fractions contained <2% of total plasma miRNA with modest enrichment of lineage-specific and variable miRNAs compared to supernatant. The abundance fold changes of miRNAs observed in pregnancy and P12 compared to normal exceeded interquartile variability of healthy individuals. The neuroendocrine miRNA signature of P12 persisted for more than 4 years and was absent in other individuals. CONCLUSIONS: This study defines the framework and effect size for screening of extensive plasma collections for miRNA phenotypes and biomarker discovery.

Mechanisms of lactic acid gustatory attraction in Drosophila.

Sour has been studied almost exclusively as an aversive taste modality. Yet recent work in Drosophila demonstrates that specific carboxylic acids are attractive at ecologically relevant concentrations. Here, we demonstrate that lactic acid is an appetitive and energetic tastant, which stimulates feeding through activation of sweet gustatory receptor neurons (GRNs). This activation displays distinct, mechanistically separable stimulus onset and removal phases. Ionotropic receptor 25a (IR25a) primarily mediates the onset response, which shows specificity for the lactate anion and drives feeding initiation through proboscis extension. Conversely, sweet gustatory receptors (Gr64a-f) mediate a non-specific removal response to low pH that primarily impacts ingestion. While mutations in either receptor family have marginal impacts on feeding, lactic acid attraction is completely abolished in combined mutants. Thus, specific components of lactic acid are detected through two classes of receptors to activate a single set of sensory neurons in physiologically distinct ways, ultimately leading to robust behavioral attraction.

Morphological characterization of Etv2 vascular explants using fractal analysis and atomic force microscopy.

The rapid engraftment of vascular networks is critical for functional incorporation of tissue explants. However, existing methods for inducing angiogenesis utilize approaches that yield vasculature with poor temporal stability or inadequate mechanical integrity, which reduce their robustness in vivo. The transcription factor Ets variant 2 (Etv2) specifies embryonic hematopoietic and vascular endothelial cell (EC) development, and is transiently reactivated during postnatal vascular regeneration and tumor angiogenesis. This study investigates the role for Etv2 upregulation in forming stable vascular beds both in vitro and in vivo. Control and Etv2+ prototypical fetal-derived human umbilical vein ECs (HUVECs) and adult ECs were angiogenically grown into vascular beds. These vessel beds were characterized using fractal dimension and lacunarity, to quantify their branching complexity and space-filling homogeneity, respectively. Atomic force microscopy (AFM) was used to explore whether greater complexity and homogeneity lead to more mechanically stable vessels. Additionally, markers of EC integrity were used to probe for mechanistic clues. Etv2+ HUVECs exhibit greater branching, vessel density, and structural homogeneity, and decreased stiffness in vitro and in vivo, indicating a greater propensity for stable vessel formation. When co-cultured with colon tumor organoid tissue, Etv2+ HUVECs had decreased fractal dimension and lacunarity compared to Etv2+ HUVECs cultured alone, indicating that vessel density and homogeneity of vessel spacing increased due to the presence of Etv2. This study sets forth the novel concept that fractal dimension, lacunarity, and AFM are as informative as conventional angiogenic measurements, including vessel branching and density, to assess vascular perfusion and stability.

Illumination pattern design with deep learning for single-shot Fourier ptychographic microscopy.

Fourier ptychographic microscopy allows for the collection of images with a high space-bandwidth product at the cost of temporal resolution. In Fourier ptychographic microscopy, the light source of a conventional widefield microscope is replaced with a light-emitting diode (LED) matrix, and multiple images are collected with different LED illumination patterns. From these images, a higher-resolution image can be computationally reconstructed without sacrificing field-of-view. We use deep learning to achieve single-shot imaging without sacrificing the space-bandwidth product, reducing the acquisition time in Fourier ptychographic microscopy by a factor of 69. In our deep learning approach, a training dataset of high-resolution images is used to jointly optimize a single LED illumination pattern with the parameters of a reconstruction algorithm. Our work paves the way for high-throughput imaging in biological studies.

A complex peripheral code for salt taste in Drosophila.

Each taste modality is generally encoded by a single, molecularly defined, population of sensory cells. However, salt stimulates multiple taste pathways in mammals and insects, suggesting a more complex code for salt taste. Here, we examine salt coding in Drosophila. After creating a comprehensive molecular map comprised of five discrete sensory neuron classes across the fly labellum, we find that four are activated by salt: two exhibiting characteristics of 'low salt' cells, and two 'high salt' classes. Behaviorally, low salt attraction depends primarily on 'sweet' neurons, with additional input from neurons expressing the ionotropic receptor IR94e. High salt avoidance is mediated by 'bitter' neurons and a population of glutamatergic neurons expressing Ppk23. Interestingly, the impact of these glutamatergic neurons depends on prior salt consumption. These results support a complex model for salt coding in flies that combinatorially integrates inputs from across cell types to afford robust and flexible salt behaviors.

Rapid preimplantation genetic screening using a handheld, nanopore-based DNA sequencer.

OBJECTIVE: To determine if a handheld, nanopore-based DNA sequencer can be used for rapid preimplantation genetic screening (PGS). DESIGN: Laboratory study. SETTING: Academic medical center. PATIENT(S): Amplified genomic DNA from euploid and aneuploid trophectoderm biopsy samples (n=9) that was also tested using traditional next generation sequencing (NGS). INTERVENTION(S): Short-read DNA library preparation and nanopore-based sequencing using a hand-held MinION sequencer. MAIN OUTCOME MEASURE(S): Comparison of cytogenetic testing result from NGS and nanopore-based sequencing and the time required for library preparation and sequencing. RESULT(S): Multiplexed short-read DNA library preparation was completed in 45 minutes. Sequencing on a single sample was completed within 20 minutes and 5 samples were simultaneously sequenced in under 2 hours. Whole-chromosome aneuploidy screening results obtained from nanopore-based sequencing were identical to those obtained using NGS. CONCLUSION(S): Here we report the first application of nanopore-based sequencing for PGS on trophectoderm biopsy samples using a novel rapid multiplxed short-read nanopore sequencing library preparation protocol. Sequencing for aneuploidy screening could be performed on a single sample in 20 minutes and on 5 samples, simultaneously, within 2 hours. Overall, nanopore sequencing is a promising tool to perform rapid PGS onsite, enabling same day testing and embryo transfer, thus obviating the need for complex, large and expensive DNA sequencers or embryo freezing.

Human plasma and serum extracellular small RNA reference profiles and their clinical utility.

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell-specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell-specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies.

Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform.

Real-time sequencing of short DNA reads has a wide variety of clinical and research applications including screening for mutations, target sequences and aneuploidy. We recently demonstrated that MinION, a nanopore-based DNA sequencing device the size of a USB drive, could be used for short-read DNA sequencing. In this study, an ultra-rapid multiplex library preparation and sequencing method for the MinION is presented and applied to accurately test normal diploid and aneuploidy samples' genomic DNA in under three hours, including library preparation and sequencing. This novel method shows great promise as a clinical diagnostic test for applications requiring rapid short-read DNA sequencing.

Recurrent idiopathic painless shoulder weakness in a Navy F/A-18 aviator.

Brachial plexopathies of various etiologies are commonly discussed in the literature; however, recurrent painless shoulder weakness is an uncommon event, especially in an otherwise healthy adult man. A designated Naval F/A-18 E/F aviator presented with acute right-sided, painless shoulder girdle weakness that initially presented 2 yr earlier in a similar fashion. Extensive medical workups during both episodes did not reveal any identifiable cause. This case report discusses the most common etiologies of shoulder weakness. Additionally, we discuss the aviator's most recent presentation and evaluation for acute shoulder weakness. Lastly, we propose a hypothesis as to the cause of the patient's symptoms based on a review of the literature.