Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection.

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

Ligament Tension and Balance before and after Robotic-Assisted Total Knee Arthroplasty - Dynamic Changes with Increasing Applied Force.

The optimal force applied during ligament balancing in total knee arthroplasty (TKA) is not well understood. We quantified the effect of increasing distraction force on medial and lateral gaps throughout the range of knee motion, both prior to and after femoral resections in tibial-first gap-balancing TKA. Twenty-five consecutive knees in 21 patients underwent robotic-assisted TKA. The posterior cruciate ligament was resected, and the tibia was cut neutral to the mechanical axis. A digital ligament tensioning tool recorded gaps and applied equal mediolateral loads of 70 N (baseline), 90 N, and 110 N from 90 degrees to full extension. A gap-balancing algorithm planned the femoral implant position to achieve a balanced knee throughout flexion. After femoral resections, gap measurements were repeated under the same conditions. Paired t-tests identified gap differences between load levels, medial/lateral compartments, and flexion angle. Gaps increased from 0 to 20 degrees in flexion, then remain consistent through 90 degrees of flexion. Baseline medial gap was significantly smaller than lateral gap throughout flexion (p <0.05). Increasing load had a larger effect on the lateral versus medial gaps (p <0.05) and on flexion versus extension gaps. Increasing distraction force resulted in non-linear and asymmetric gap changes mediolaterally and from flexion to extension. Digital ligament tensioning devices can give better understanding of the relationship between joint distraction, ligament tension, and knee stiffness throughout the range of flexion. This can aid in informed surgical decision making and optimal soft tissue tensioning during TKA.

In vivo noninvasive systemic myography of acute systemic vasoactivity in female pregnant mice.

Altered vasoactivity is a major characteristic of cardiovascular and oncological diseases, and many therapies are therefore targeted to the vasculature. Therapeutics which are selective for the diseased vasculature are ideal, but whole-body selectivity of a therapeutic is challenging to assess in practice. Vessel myography is used to determine the functional mechanisms and evaluate pharmacological responses of vascularly-targeted therapeutics. However, myography can only be performed on ex vivo sections of individual arteries. We have developed methods for implementation of spherical-view photoacoustic tomography for non-invasive and in vivo myography. Using photoacoustic tomography, we demonstrate the measurement of acute vascular reactivity in the systemic vasculature and the placenta of female pregnant mice in response to two vasodilators. Photoacoustic tomography simultaneously captures the significant acute vasodilation of major arteries and detects selective vasoactivity of the maternal-fetal vasculature. Photoacoustic tomography has the potential to provide invaluable preclinical information on vascular response that cannot be obtained by other established methods.

Characterizing a photoacoustic and fluorescence imaging platform for preclinical murine longitudinal studies.

Significance: To effectively study preclinical animal models, medical imaging technology must be developed with a high enough resolution and sensitivity to perform anatomical, functional, and molecular assessments. Photoacoustic (PA) tomography provides high resolution and specificity, and fluorescence (FL) molecular tomography provides high sensitivity; the combination of these imaging modes will enable a wide range of research applications to be studied in small animals. Aim: We introduce and characterize a dual-modality PA and FL imaging platform using in vivo and phantom experiments. Approach: The imaging platform's detection limits were characterized through phantom studies that determined the PA spatial resolution, PA sensitivity, optical spatial resolution, and FL sensitivity. Results: The system characterization yielded a PA spatial resolution of 173 ± 17 µ m in the transverse plane and 640 ± 120 µ m in the longitudinal axis, a PA sensitivity detection limit not less than that of a sample with absorption coefficient µ a = 0.258 cm - 1 , an optical spatial resolution of 70 µ m in the vertical axis and 112 µ m in the horizontal axis, and a FL sensitivity detection limit not < 0.9 µ M concentration of IR-800. The scanned animals displayed in three-dimensional renders showed high-resolution anatomical detail of organs. Conclusions: The combined PA and FL imaging system has been characterized and has demonstrated its ability to image mice in vivo, proving its suitability for biomedical imaging research applications.

Size-tunable ICG-based contrast agent platform for targeted near-infrared photoacoustic imaging.

Near-infrared photoacoustic imaging (NIR-PAI) combines the advantages of optical and ultrasound imaging to provide anatomical and functional information of tissues with high resolution. Although NIR-PAI is promising, its widespread use is hindered by the limited availability of NIR contrast agents. J-aggregates (JA) made of indocyanine green dye (ICG) represents an attractive class of biocompatible contrast agents for PAI. Here, we present a facile synthesis method that combines ICG and ICG-azide dyes for producing contrast agents with tunable size down to 230 nm and direct functionalization with targeting moieties. The ICG-JA platform has a detectable PA signal in vitro that is two times stronger than whole blood and high photostability. The targeting ability of ICG-JA was measured in vitro using HeLa cells. The ICG-JA platform was then injected into mice and in vivo NIR-PAI showed enhanced visualization of liver and spleen for 90 min post-injection with a contrast-to-noise ratio of 2.42.

Photoacoustic imaging provides an in vivo assessment of the preeclamptic placenta remodeling and function in response to therapy.

INTRODUCTION: There is a lack of effective therapeutic interventions for preeclampsia. A central factor in the etiology of the disease is the development of placental hypoxia due to abnormal vascular remodeling. However, methods to assess the impact of potential therapies on placental growth and remodeling are currently lacking. Here, we develop and validate ultrasound-guided photoacoustic imaging methods to monitor the placental response to therapeutic intervention. Establishing non-invasive tools to image placental function opens up previously unachievable understandings of placental therapeutic response. METHODS: Studies were performed in the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia. Preclinical research has identified tempol, a superoxide dismutase mimetic, and the phosphodiesterase inhibitor sildenafil as potential therapeutics for preeclampsia, as both improve in vivo maternal outcomes. PA images of the placental environment were acquired in RUPP rats receiving tempol (n = 8) or sildenafil (n = 8) to assess the longitudinal effects of treatment on placental oxygenation and vascular remodeling. Imaging measurements were validated with ex vivo histological analysis. RESULTS: Spectral photoacoustic imaging non-invasively measured placental hypoxia and impaired vascular growth two days after the RUPP procedure was implemented. Sildenafil significantly improved (p < 0.05) placental oxygenation and promoted vascular remodeling in RUPP animals, while RUPP animals treated with tempol had a diminished placental therapeutic response. DISCUSSION: We demonstrate that photoacoustic imaging provides in vivo measures of placental oxygenation and vascular remodeling, a previously unobtainable assessment of preeclamptic therapeutic response. These imaging tools have tremendous potential to accelerate the search for effective therapies for preeclampsia.

Single-cell genomics for resolution of conserved bacterial genes and mobile genetic elements of the human intestinal microbiota using flow cytometry.

As our understanding of the importance of the human microbiota in health and disease grows, so does our need to carefully resolve and delineate its genomic content. 16S rRNA gene-based analyses yield important insights into taxonomic composition, and metagenomics-based approaches reveal the functional potential of microbial communities. However, these methods generally fail to directly link genetic features, including bacterial genes and mobile genetic elements, to each other and to their source bacterial genomes. Further, they are inadequate to capture the microdiversity present within a genus, species, or strain of bacteria within these complex communities. Here, we present a method utilizing fluorescence-activated cell sorting for isolation of single bacterial cells, amplifying their genomes, screening them by 16S rRNA gene analysis, and selecting cells for genomic sequencing. We apply this method to both a cultured laboratory strain of Escherichia coli and human stool samples. Our analyses reveal the capacity of this method to provide nearly complete coverage of bacterial genomes when applied to isolates and partial genomes of bacterial species recovered from complex communities. Additionally, this method permits exploration and comparison of conserved and variable genomic features between individual cells. We generate assemblies of novel genomes within the Ruminococcaceae family and the Holdemanella genus by combining several 16S rRNA gene-matched single cells, and report novel prophages and conjugative transposons for both Bifidobacterium and Ruminococcaceae. Thus, we demonstrate an approach for flow cytometric separation and sequencing of single bacterial cells from the human microbiota, which yields a variety of critical insights into both the functional potential of individual microbes and the variation among those microbes. This method definitively links a variety of conserved and mobile genomic features, and can be extended to further resolve diverse elements present in the human microbiota.

Intestinal antiviral signaling is controlled by autophagy gene Epg5 independent of the microbiota.

Mutations in the macroautophagy/autophagy gene EPG5 are responsible for Vici syndrome, a human genetic disease characterized by combined immunodeficiency. Previously, we found that epg5-/- mice exhibit hyperinflammation in the lungs mediated by IL1B/IL-1ß and TNF/TNFα, resulting in resistance to influenza. Here, we find that disruption of Epg5 results in protection against multiple enteric viruses including norovirus and rotavirus. Gene expression analysis reveals IFNL/IFN-λ responsive genes as a key alteration. Further, mice lacking Epg5 exhibit substantial alterations of the intestinal microbiota. Surprisingly, germ-free mouse studies indicate Epg5-associated inflammation of both the intestine and lung is microbiota-independent. Genetic studies support IFNL signaling as the primary mediator of resistance to enteric viruses, but not of microbial dysbiosis, in epg5-/- mice. This study unveils an important role, unexpectedly independent of the microbiota, for autophagy gene Epg5 in host organism protection by modulating intestinal IFNL responses.Abbreviations: CTNNB1: catenin (cadherin associated protein), beta 1; DAPI: 4',6-diamidino-2-phenylindole; EPG5: ectopic P-granules autophagy protein 5 homolog (C. elegans); FT: fecal transplant; IFI44: interferon-induced protein 44; IFIT1: interferon-induced protein with tetratricopeptide repeats 1; IFNG/IFN-γ: interferon gamma; IFNL/IFN-λ: interferon lambda; IFNLR1: interferon lambda receptor 1; IL1B/IL-1ß: interleukin 1 beta; ISG: interferon stimulated gene; GF: germ-free; LEfSe: linear discriminant analysis effect size; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MNoV: murine norovirus; MX2: MX dynamin-like GTPase 2; OAS1A: 2'-5' oligoadenylate synthetase 1A; RV: rotavirus; SPF: specific-pathogen free; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1; TNF/TNFα: tumor necrosis factor.

Sex and the G Protein-Coupled Estrogen Receptor Impact Vascular Stiffness.

[Figure: see text].

Transferrable protection by gut microbes against STING-associated lung disease.

STING modulates immunity by responding to bacterial and endogenous cyclic dinucleotides (CDNs). Humans and mice with STING gain-of-function mutations develop a syndrome known as STING-associated vasculopathy with onset in infancy (SAVI), which is characterized by inflammatory or fibrosing lung disease. We hypothesized that hyperresponsiveness of gain-of-function STING to bacterial CDNs might explain autoinflammatory lung disease in SAVI mice. We report that depletion of gut microbes with oral antibiotics (vancomycin, neomycin, and ampicillin [VNA]) nearly eliminates lung disease in SAVI mice, implying that gut microbes might promote STING-associated autoinflammation. However, we show that germ-free SAVI mice still develop severe autoinflammatory disease and that transferring gut microbiota from antibiotics-treated mice to germ-free animals eliminates lung inflammation. Depletion of anaerobes with metronidazole abolishes the protective effect of the VNA antibiotics cocktail, and recolonization with the metronidazole-sensitive anaerobe Bacteroides thetaiotaomicron prevents disease, confirming a protective role of a metronidazole-sensitive microbe in a model of SAVI.

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid.

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.

Using Apache Spark on genome assembly for scalable overlap-graph reduction.

BACKGROUND: De novo genome assembly is a technique that builds the genome of a specimen using overlaps of genomic fragments without additional work with reference sequence. Sequence fragments (called reads) are assembled as contigs and scaffolds by the overlaps. The quality of the de novo assembly depends on the length and continuity of the assembly. To enable faster and more accurate assembly of species, existing sequencing techniques have been proposed, for example, high-throughput next-generation sequencing and long-reads-producing third-generation sequencing. However, these techniques require a large amounts of computer memory when very huge-size overlap graphs are resolved. Also, it is challenging for parallel computation. RESULTS: To address the limitations, we propose an innovative algorithmic approach, called Scalable Overlap-graph Reduction Algorithms (SORA). SORA is an algorithm package that performs string graph reduction algorithms by Apache Spark. The SORA's implementations are designed to execute de novo genome assembly on either a single machine or a distributed computing platform. SORA efficiently compacts the number of edges on enormous graphing paths by adapting scalable features of graph processing libraries provided by Apache Spark, GraphX and GraphFrames. CONCLUSIONS: We shared the algorithms and the experimental results at our project website, https://github.com/BioHPC/SORA . We evaluated SORA with the human genome samples. First, it processed a nearly one billion edge graph on a distributed cloud cluster. Second, it processed mid-to-small size graphs on a single workstation within a short time frame. Overall, SORA achieved the linear-scaling simulations for the increased computing instances.

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph.

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). Smooth muscle cells exhibit a contractile function within the vaginal and cervical walls. Depending on the biochemical environment and the mechanical distension of the organ walls, the smooth muscle cells alter the contractile conditions. The contribution of the smooth muscle cells under baseline physiological conditions is classified as a basal tone. More specifically, a basal tone is the baseline partial constriction of smooth muscle cells in the absence of hormonal and neural stimulation. Furthermore, the ECM provides structural support for the organ walls and functions as a reservoir for biochemical cues. These biochemical cues are vital to various organ functions, such as inciting growth and maintaining homeostasis. The ECM of each organ is composed primarily of collagen fibers (mostly collagen types I, III, and V), elastic fibers, and glycosaminoglycans/proteoglycans. The composition and organization of the ECM dictate the mechanical properties of each organ. A change in ECM composition may lead to the development of reproductive pathologies, such as pelvic organ prolapse or premature cervical remodeling. Furthermore, changes in ECM microstructure and stiffness may alter smooth muscle cell activity and phenotype, thus resulting in the loss of the contractile force. In this work, the reported protocols are used to assess the basal tone and passive mechanical properties of the nonpregnant murine vagina and cervix at 4-6 months of age in estrus. The organs were mounted in a commercially available pressure myograph and both pressure-diameter and force-length tests were performed. Sample data and data analysis techniques for the mechanical characterization of the reproductive organs are included. Such information may be useful for constructing mathematical models and rationally designing therapeutic interventions for women's health pathologies.

Longitudinal characterization of local perfusion of the rat placenta using contrast-enhanced ultrasound imaging.

The placenta performs many physiological functions critical for development. Insufficient placental perfusion, due to improper vascular remodelling, has been linked to many pregnancy-related diseases. To study longitudinal in vivo placental perfusion, we have implemented a pixel-wise time-intensity curve (TIC) analysis of contrast-enhanced ultrasound (CEUS) images. CEUS images were acquired of pregnant Sprague Dawley rats after bolus injections of gas-filled microbubble contrast agents. Conventionally, perfusion can be quantified using a TIC of contrast enhancement in an averaged region of interest. However, the placenta has a complex structure and flow profile, which is insufficiently described using the conventional technique. In this work, we apply curve fitting in each pixel of the CEUS image series in order to quantify haemodynamic parameters in the placenta and surrounding tissue. The methods quantified an increase in mean placental blood volume and relative blood flow from gestational day (GD) 14 to GD18, while the mean transit time of the microbubbles decreased, demonstrating an overall rise in placental perfusion during gestation. The variance of all three parameters increased during gestation, showing that regional differences in perfusion are observable using the pixel-wise TIC approach. Additionally, the high-resolution parametric images show distinct regions of high blood flow developing during late gestation. The developed methods could be applied to assess placental vascular remodelling during the treatment of the pathologies of pregnancy.

Smooth muscle regional contribution to vaginal wall function.

Pelvic organ prolapse is characterized as the descent of the pelvic organs into the vaginal canal. In the USA, there is a 12% lifetime risk for requiring surgical intervention. Although vaginal childbirth is a well-established risk factor for prolapse, the underlying mechanisms are not fully understood. Decreased smooth muscle organization, composition and maximum muscle tone are characteristics of prolapsed vaginal tissue. Maximum muscle tone of the vaginal wall was previously investigated in the circumferential or axial direction under uniaxial loading; however, the vaginal wall is subjected to multiaxial loads. Further, the contribution of vaginal smooth muscle basal (resting) tone to mechanical function remains undetermined. The objectives of this study were to determine the contribution of smooth muscle basal and maximum tone to the regional biaxial mechanical behaviour of the murine vagina. Vaginal tissue from C57BL/6 mice was subjected to extension-inflation protocols (n = 10) with and without basal smooth muscle tone. Maximum tone was induced with KCl under various circumferential (n = 5) and axial (n = 5) loading conditions. The microstructure was visualized with multiphoton microscopy (n = 1), multiaxial histology (n = 4) and multiaxial immunohistochemistry (n = 4). Smooth muscle basal tone decreased material stiffness and increased anisotropy. In addition, maximum vaginal tone was decreased with increasing intraluminal pressures. This study demonstrated that vaginal muscle tone contributed to the biaxial mechanical response of murine vaginal tissue. This may be important in further elucidating the underlying mechanisms of prolapse, in order to improve current preventative and treatment strategies.

Phages and Human Health: More Than Idle Hitchhikers.

Bacteriophages, or phages, are viruses that infect bacteria and archaea. Phages have diverse morphologies and can be coded in DNA or RNA and as single or double strands with a large range of genome sizes. With the increasing use of metagenomic sequencing approaches to analyze complex samples, many studies generate massive amounts of "viral dark matter", or sequences of viral origin unable to be classified either functionally or taxonomically. Metagenomic analysis of phages is still in its infancy, and uncovering novel phages continues to be a challenge. Work over the past two decades has begun to uncover key roles for phages in different environments, including the human gut. Recent studies in humans have identified expanded phage populations in both healthy infants and in inflammatory bowel disease patients, suggesting distinct phage activity during development and in specific disease states. In this review, we examine our current knowledge of phage biology and discuss recent efforts to improve the analysis and discovery of novel phages. We explore the roles phages may play in human health and disease and discuss the future of phage research.

Spectral photoacoustic imaging to estimate in vivo placental oxygenation during preeclampsia.

Preeclampsia is a pregnancy-related hypertensive disorder accounting for 14% of global maternal deaths annually. Preeclampsia - maternal hypertension and proteinuria - is promoted by placental ischemia resulting from reduced uteroplacental perfusion. Here, we assess longitudinal changes in placental oxygenation during preeclampsia using spectral photoacoustic imaging. Spectral photoacoustic images were acquired of the placenta of normal pregnant (NP) and preeclamptic reduced uterine perfusion pressure (RUPP) Sprague Dawley rats on gestational days (GD) 14, 16, and 18, corresponding to mid- to late gestation (n = 10 per cohort). Two days after implementation of the RUPP surgical model, placental oxygen saturation decreased 12% in comparison with NP. Proteinuria was determined from a 24-hour urine collection prior to imaging on GD18. Blood pressure measurements were obtained on GD18 after imaging. Placental hypoxia in the RUPP was confirmed with histological staining for hypoxia-inducible factor (HIF)-1α, a cellular transcription regulator which responds to local oxygen levels. Using in vivo, longitudinal imaging methods we determined that the placenta in the reduced uterine perfusion pressure rat model of preeclampsia is hypoxic, and that this hypoxia is maintained through late gestation. Future work will utilize these methods to assess the impact of novel therapeutics on placental ischemia and the progression of preeclampsia.