Merged magnetic resonance and light sheet microscopy of the whole mouse brain.

We have developed workflows to align 3D magnetic resonance histology (MRH) of the mouse brain with light sheet microscopy (LSM) and 3D delineations of the same specimen. We start with MRH of the brain in the skull with gradient echo and diffusion tensor imaging (DTI) at 15 µm isotropic resolution which is ~ 1,000 times higher than that of most preclinical MRI. Connectomes are generated with superresolution tract density images of ~5 µm. Brains are cleared, stained for selected proteins, and imaged by LSM at 1.8 µm/pixel. LSM data are registered into the reference MRH space with labels derived from the ABA common coordinate framework. The result is a high-dimensional integrated volume with registration (HiDiver) with alignment precision better than 50 µm. Throughput is sufficiently high that HiDiver is being used in quantitative studies of the impact of gene variants and aging on mouse brain cytoarchitecture and connectomics.

Targeted Analysis of the Size Distribution of Heavy Chain-Modified Hyaluronan with Solid-State Nanopores.

The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HC•HA) complexes. While limited assessments of HC•HA have been performed previously, determining the size distribution of its HA component remains a challenge. Here, we describe a selective method for extracting HC•HA from mixtures that yields material amenable to MW analysis with a solid-state nanopore sensor. After demonstrating the approach in vitro, we validate extraction of HC•HA from osteoarthritic human synovial fluid as a model complex biological matrix. Finally, we apply our technique to pathophysiology by measuring the size distributions of HC•HA and total HA in an equine model of synovitis.

A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury. METHODS: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. RESULTS: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. CONCLUSION: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD. IMPACT AND IMPLICATIONS: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

High Sensitivity Extended Nano-Coulter Counter for Detection of Viral Particles and Extracellular Vesicles.

We present a chip-based extended nano-Coulter counter (XnCC) that can detect nanoparticles affinity-selected from biological samples with low concentration limit-of-detection that surpasses existing resistive pulse sensors by 2-3 orders of magnitude. The XnCC was engineered to contain 5 in-plane pores each with an effective diameter of 350 nm placed in parallel and can provide high detection efficiency for single particles translocating both hydrodynamically and electrokinetically through these pores. The XnCC was fabricated in cyclic olefin polymer (COP) via nanoinjection molding to allow for high-scale production. The concentration limit-of-detection of the XnCC was 5.5 × 103 particles/mL, which was a 1,100-fold improvement compared to a single in-plane pore device. The application examples of the XnCC included counting affinity selected SARS-CoV-2 viral particles from saliva samples using an aptamer and pillared microchip; the selection/XnCC assay could distinguish the COVID-19(+) saliva samples from those that were COVID-19(-). In the second example, ovarian cancer extracellular vesicles (EVs) were affinity selected using a pillared chip modified with a MUC16 monoclonal antibody. The affinity selection chip coupled with the XnCC was successful in discriminating between patients with high grade serous ovarian cancer and healthy donors using blood plasma as the input sample.

Methods for isolating and analyzing physiological hyaluronan: a review.

The carbohydrate hyaluronan (or hyaluronic acid, HA) is found in all human tissues and biofluids where it has wide-ranging functions in health and disease that are dictated by both its abundance and size. Consequently, hyaluronan evaluation in physiological samples has significant translational potential. Although the analytical tools and techniques for probing other biomolecules such as proteins and nucleic acids have become standard approaches in biochemistry, those available for investigating hyaluronan are less well established. In this review, we survey methods related to the assessment of native hyaluronan in biological specimens, including protocols for separating it from biological matrices and technologies for determining its concentration and molecular weight.

Mechanistic insights into non-coding Y RNA processing.

Y RNAs (84-112 nt) are non-coding RNAs transcribed by RNA polymerase III and are characterized by a distinctive secondary structure. Human Y RNAs interact with the autoimmune proteins SSB and RO60 that together form a ribonucleoprotein (RNP) complex termed RoRNP and Y RNAs also perform regulatory roles in DNA and RNA replication and stability, which has major implications for diseases including cancer. During cellular stress and apoptosis, Y RNAs are cleaved into 3' and 5' end fragments termed Y RNA-derived small RNAs (ysRNAs). Although some ysRNA functions in stress, apoptosis and cancer have been reported, their fundamental biogenesis has not been described. Here we report that 3' end RNY5 cleavage is structure dependent. In high throughput mutagenesis experiments, cleavage occurred between the 2nd and 3rd nt above a double stranded stem comprising high GC content. We demonstrate that an internal loop above stem S3 is critical for producing 3' end ysRNAs (31 nt) with mutants resulting in longer or no ysRNAs. We show a UGGGU sequence motif at position 22 of RNY5 is critical for producing 5' end ysRNAs (22-25 nt). We show that intact RO60 is critical for ysRNA biogenesis. We conclude that ribonuclease L (RNASEL) contributes to Y RNA cleavage in mouse embryonic fibroblasts but is not the only endoribonuclease important in human cells.

Association Between Afterhours Discharge From the Intensive Care Unit and Hospital Mortality: A Multi-Center Retrospective Cohort Study.

BACKGROUND: There is conflicting evidence on the association between afterhours discharge from the intensive care unit (ICU) and hospital mortality. We examined the effects of afterhours discharge, including the potential effect of residual organ dysfunction, on hospital mortality in a large integrated health region. METHODS: We performed a multi-center retrospective cohort study of 10,463 adults discharged from 9 mixed medical/surgical ICUs in Alberta from June 2012 to December 2014. We applied a 2-stage modeling strategy to investigate the association between afterhours discharge (19:00h to 07:59h) and post-ICU hospital mortality. We applied mixed-effect multi-variable linear regression to assess the relationship between discharge organ dysfunction and afterhours discharge. We then applied mixed-effect multi-variable logistic regression to evaluate the direct, indirect and integrated associations of afterhours discharge on hospital mortality and hospitalization duration. RESULTS: Of 10,463 patients, 23.7% (n = 2,480) were discharged afterhours, of which 27.4% occurred on a holiday or weekend. This varied significantly by ICU size, type, and site. Patients discharged afterhours were more likely medical admissions, had greater multi-morbidity and illness acuity. A greater average SOFA score in the 72 hours prior to ICU discharge was not associated with afterhours discharge. However, a greater average SOFA score was associated with hospital mortality (adjusted-odds ratio [OR], 1.23; 95% CI, 1.18-1.28). Afterhours discharge was associated with higher hospital mortality (adjusted-OR, 1.19; 95% CI, 1.01-1.39), increased hospital stay (adjusted-risk ratio [RR], 1.10; 95% CI, 1.09-1.11) and increased post-ICU stay (adjusted-RR, 1.16; 95% CI, 1.14-1.17) when compared with workhours discharge. CONCLUSIONS: Afterhours discharge is common, occurring in 1 in 4 discharges, and is widely variable across ICUs. Patients discharged afterhours have greater risk of hospital mortality and prolonged hospitalization.

Structure and function analysis of the C. elegans aminophospholipid translocase TAT-1.

The Caenorhabditis elegans aminophospholipid translocase TAT-1 maintains phosphatidylserine (PS) asymmetry in the plasma membrane and regulates endocytic transport. Despite these important functions, the structure-function relationship of this protein is poorly understood. Taking advantage of the tat-1 mutations identified by the C. elegans million mutation project, we investigated the effects of 16 single amino acid substitutions on the two functions of the TAT-1 protein. Two substitutions that alter a highly conserved PISL motif in the fourth transmembrane domain and a highly conserved DKTGT phosphorylation motif, respectively, disrupt both functions of TAT-1, leading to a vesicular gut defect and ectopic PS exposure on the cell surface, whereas most other substitutions across the TAT-1 protein, often predicted to be deleterious by bioinformatics programs, do not affect the functions of TAT-1. These results provide in vivo evidence for the importance of the PISL and DKTGT motifs in P4-type ATPases and improve our understanding of the structure-function relationship of TAT-1. Our study also provides an example of how the C. elegans million mutation project helps decipher the structure, functions, and mechanisms of action of important genes.

Solid-state nanopore analysis of human genomic DNA shows unaltered global 5-hydroxymethylcytosine content associated with early-stage breast cancer.

5-Hydroxymethylcytosine (5hmC), the first oxidized form of the well-known epigenetic modification 5-methylcytosine, is an independent regulator of gene expression and therefore a potential marker for disease. Here, we report on methods developed for a selective solid-state nanopore assay that enable direct analysis of global 5hmC content in human tissue. We first describe protocols for preparing genomic DNA derived from both healthy breast tissue and stage 1 breast tumor tissue and then use our approach to probe the net abundance of the modified base in each cohort. Then, we employ empirical data to adjust for the impact of nanopore diameter on the quantification. Correcting for variations in nanopore diameter among the devices used for analysis reveals no detectable difference in global 5hmC content between healthy and tumor tissue. These results suggest that 5hmC changes may not be associated with early-stage breast cancer and instead are a downstream consequence of the disease.

Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection.

The biotin-streptavidin bond is the strongest noncovalent bond in nature and is thus used extensively in biotechnology applications. However, the difficulty of releasing the bond without high temperatures or corrosive solutions can be a barrier to applications involving nucleic acids and other delicate substrates. Here, room-temperature phenol is employed to release biotin-tagged DNA constructs from streptavidin rapidly and efficiently. It is demonstrated that synthetic biotinylated DNA can be recovered at yields approaching 100% from both solution-phase and bead-bound streptavidin with as little as 12% (v/v) phenol, leaving the biotin tag active and reusable after extraction. As an application of this recovery method, biotinylated DNA fragments are isolated from a mixed solution to provide selectivity for solid-state nanopore detection.

Direct analysis in real time-Mass spectrometry (DART-MS) in forensic and security applications.

Over the last decade, direct analysis in real time (DART) has emerged as a viable method for fast, easy, and reliable "ambient ionization" for forensic analysis. The ability of DART to generate ions from chemicals that might be present at the scene of a criminal activity, whether they are in the gas, liquid, or solid phase, with limited sample preparation has made the technology a useful analytical tool in numerous forensic applications. This review paper summarizes many of those applications, ranging from the analysis of trace evidence to security applications, with a focus on providing the forensic scientist with a resource for developing their own applications. The most common uses for DART in forensics are in studying seized drugs, drugs of abuse and their metabolites, bulk and detonated explosives, toxic chemicals, chemical warfare agents, inks and dyes, and commercial plant and animal products that have been adulterated for economic gain. This review is meant to complement recent reviews that have described the fundamentals of the ionization mechanism and the general use of DART. We describe a wide range of forensic applications beyond the field of analyzing drugs of abuse, which dominates the literature, including common experimental and data analysis methods. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:171-187, 2018.

Wafer-scale fabrication of fused silica chips for low-noise recording of resistive pulses through nanopores.

This paper presents a maskless method to manufacture fused silica chips for low-noise resistive-pulse sensing. The fabrication includes wafer-scale density modification of fused silica with a femtosecond-pulsed laser, low-pressure chemical vapor deposition (LPVCD) of silicon nitride (SiN x ) and accelerated chemical wet etching of the laser-exposed regions. This procedure leads to a freestanding SiN x window, which is permanently attached to a fused silica support chip and the resulting chips are robust towards Piranha cleaning at ∼80 °C. After parallel chip manufacturing, we created a single nanopore in each chip by focused helium-ion beam or by controlled breakdown. Compared to silicon chips, the resulting fused silica nanopore chips resulted in a four-fold improvement of both the signal-to-noise ratio and the capture rate for signals from the translocation of IgG1 proteins at a recording bandwidth of 50 kHz. At a bandwidth of ∼1 MHz, the noise from the fused silica nanopore chips was three- to six-fold reduced compared to silicon chips. In contrast to silicon chips, fused silica chips showed no laser-induced current noise-a significant benefit for experiments that strive to combine nanopore-based electrical and optical measurements.

Fluid surface coatings for solid-state nanopores: comparison of phospholipid bilayers and archaea-inspired lipid monolayers.

In the context of sensing and characterizing single proteins with synthetic nanopores, lipid bilayer coatings provide at least four benefits: first, they minimize unwanted protein adhesion to the pore walls by exposing a zwitterionic, fluid surface. Second, they can slow down protein translocation and rotation by the opportunity to tether proteins with a lipid anchor to the fluid bilayer coating. Third, they provide the possibility to impart analyte specificity by including lipid anchors with a specific receptor or ligand in the coating. Fourth, they offer a method for tuning nanopore diameters by choice of the length of the lipid's acyl chains. The work presented here compares four properties of various lipid compositions with regard to their suitability as nanopore coatings for protein sensing experiments: (1) electrical noise during current recordings through solid-state nanopores before and after lipid coating, (2) long-term stability of the recorded current baseline and, by inference, of the coating, (3) viscosity of the coating as quantified by the lateral diffusion coefficient of lipids in the coating, and (4) the success rate of generating a suitable coating for quantitative nanopore-based resistive pulse recordings. We surveyed lipid coatings prepared from bolaamphiphilic, monolayer-forming lipids inspired by extremophile archaea and compared them to typical bilayer-forming phosphatidylcholine lipids containing various fractions of curvature-inducing lipids or cholesterol. We found that coatings from archaea-inspired lipids provide several advantages compared to conventional phospholipids; the stable, low noise baseline qualities and high viscosity make these membranes especially suitable for analysis that estimates physical protein parameters such as the net charge of proteins as they enable translocation events with sufficiently long duration to time-resolve dwell time distributions completely. The work presented here reveals that the ease or difficulty of coating a nanopore with lipid membranes did not depend significantly on the composition of the lipid mixture, but rather on the geometry and surface chemistry of the nanopore in the solid state substrate. In particular, annealing substrates containing the nanopore increased the success rate of generating stable lipid coatings.

Association between afterhours admission to the intensive care unit, strained capacity, and mortality: a retrospective cohort study.

BACKGROUND: Admission to the intensive care unit (ICU) outside daytime hours has been shown to be variably associated with increased morbidity and mortality. We aimed to describe the characteristics and outcomes of patients admitted to the ICU afterhours (22:00-06:59 h) in a large Canadian health region. We further hypothesized that the association between afterhours admission and mortality would be modified by indicators of strained ICU capacity. METHODS: This is a population-based cohort study of 12,265 adults admitted to nine ICUs in Alberta from June 2012 to December 2014. We used a path-analysis modeling strategy and mixed-effects multivariate regression analysis to evaluate direct and integrated associations (mediated through Acute Physiology and Chronic Health Evaluation (APACHE) II score) between afterhours admission (22:00-06:59 h) and ICU mortality. Further analysis examined the effects of strained ICU capacity and varied definitions of afterhours and weekend admissions. ICU occupancy ≥ 90% or clustering of admissions (≥ 0.15, defined as number of admissions 2 h before or after the index admission, divided by the number of ICU beds) were used as indicators of strained capacity. RESULTS: Of 12,265 admissions, 34.7% (n = 4251) occurred afterhours. The proportion of afterhours admissions varied amongst ICUs (range 26.7-37.8%). Patients admitted afterhours were younger (median (IQR) 58 (44-70) vs 60 (47-70) years, p < 0.0001), more likely to have a medical diagnosis (75.9% vs 72.1%, p < 0.0001), and had higher APACHE II scores (20.9 (8.6) vs 19.9 (8.3), p < 0.0001). Crude ICU mortality was greater for those admitted afterhours (15.9% vs 14.1%, p = 0.007), but following multivariate adjustment there was no direct or integrated effect on ICU mortality (odds ratio (OR) 1.024; 95% confidence interval (CI) 0.923-1.135, p = 0.658). Furthermore, direct and integrated analysis showed no association of afterhours admission and hospital mortality (p = 0.90) or hospital length of stay (LOS) (p = 0.27), although ICU LOS was shorter (p = 0.049). Early-morning admission (00:00-06:59 h) with ICU occupancy ≥ 90% was associated with short-term (≤ 7 days) and all-cause ICU mortality. CONCLUSIONS: One-third of critically ill patients are admitted to the ICU afterhours. Afterhours ICU admission was not associated with greater mortality risk in most circumstances but was sensitive to strained ICU capacity.

Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle.

The candidate phylum TM7 is globally distributed and often associated with human inflammatory mucosal diseases. Despite its prevalence, the TM7 phylum remains recalcitrant to cultivation, making it one of the most enigmatic phyla known. In this study, we cultivated a TM7 phylotype (TM7x) from the human oral cavity. This extremely small coccus (200-300 nm) has a distinctive lifestyle not previously observed in human-associated microbes. It is an obligate epibiont of an Actinomyces odontolyticus strain (XH001) yet also has a parasitic phase, thereby killing its host. This first completed genome (705 kb) for a human-associated TM7 phylotype revealed a complete lack of amino acid biosynthetic capacity. Comparative genomics analyses with uncultivated environmental TM7 assemblies show remarkable conserved gene synteny and only minimal gene loss/gain that may have occurred as TM7x adapted to conditions within the human host. Transcriptomic and metabolomic profiles provided the first indications, to our knowledge, that there is signaling interaction between TM7x and XH001. Furthermore, the induction of TNF-α production in macrophages by XH001 was repressed in the presence of TM7x, suggesting its potential immune suppression ability. Overall, our data provide intriguing insights into the uncultivability, pathogenicity, and unique lifestyle of this previously uncharacterized oral TM7 phylotype.

Interobserver and intraobserver reliability of radiographic classification of acromioclavicular joint dislocations.

HYPOTHESIS AND BACKGROUND: The classification and treatment of acromioclavicular (AC) joint dislocations remain controversial. The purpose of this study was to determine the interobserver and intraobserver reliability of the Rockwood classification system. We hypothesized poor interobserver and intraobserver reliability, limiting the role of the Rockwood classification system in determining severity of AC joint dislocations and accurately guiding treatment decisions. METHODS: We identified 200 patients with AC joint injuries using the International Classification of Diseases, Ninth Revision code 831.04. Fifty patients met inclusion criteria. Deidentified radiographs were compiled and presented to 6 fellowship-trained upper extremity orthopedic surgeons. The surgeons classified each patient into 1 of the 6 classification types described by Rockwood. A second review was performed several months later by 2 surgeons. A κ value was calculated to determine the interobserver and intraobserver reliability. RESULTS: The interobserver and intraobserver κ values were fair (κ = 0.278) and moderate (κ = 0.468), respectively. Interobserver results showed that 4 of the 50 radiographic images had a unanimous classification. Intraobserver results for the 2 surgeons showed that 18 of the 50 images were rated the same on second review by the first surgeon and 38 of the 50 images were rated the same on second review by the second surgeon. CONCLUSION: We found that the Rockwood classification system has limited interobserver and intraobserver reliability. We believe that unreliable classification may account for some of the inconsistent treatment outcomes among patients with similarly classified injuries. We suggest that a better classification system is needed to use radiographic imaging for diagnosis and treatment of AC joint dislocations.

Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process.

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.

Solid-state nanopore analysis of alcohol-soluble molecules.

We report on a modified solid-state nanopore measurement scheme to probe alcohol-soluble proteins. Taking advantage of the intrinsic alcohol solubility of LiCl as an electrolyte, we show that the devices can be operated in azeotropic mixtures of ethanol and water. We first characterize nanopore conductivity across a range of ethanol content as a function of both nanopore diameter and salt concentration, showing ionic response that can be understood through established models. Then, as a demonstration of resistive-pulse sensing, we measure and interpret electrical translocations of zeins, a class of alcohol-soluble maize protein.

Solid-state nanopore localization by controlled breakdown of selectively thinned membranes.

We demonstrate precise positioning of nanopores fabricated by controlled breakdown (CBD) on solid-state membranes by spatially varying the electric field strength with localized membrane thinning. We show 100 × 100 nm2 precision in standard SiN x membranes (30-100 nm thick) after selective thinning by as little as 25% with a helium ion beam. Control over nanopore position is achieved through the strong dependence of the electric field-driven CBD mechanism on membrane thickness. Confinement of pore formation to the thinned region of the membrane is confirmed by TEM imaging and by analysis of DNA translocations. These results enhance the functionality of CBD as a fabrication approach and enable the production of advanced nanopore devices for single-molecule sensing applications.