Donor-Derived Engineered Microvessels for Cardiovascular Risk Stratification of Patients with Kidney Failure.

Cardiovascular disease is the cause of death in ≈50% of hemodialysis patients. Accumulation of uremic solutes in systemic circulation is thought to be a key driver of the endothelial dysfunction that underlies elevated cardiovascular events. A challenge in understanding the mechanisms relating chronic kidney disease to cardiovascular disease is the lack of in vitro models that allow screening of the effects of the uremic environment on the endothelium. Here, a method is described for microfabrication of human blood vessels from donor cells and perfused with donor serum. The resulting donor-derived microvessels are used to quantify vascular permeability, a hallmark of endothelial dysfunction, in response to serum spiked with pathophysiological levels of indoxyl sulfate, and in response to serum from patients with chronic kidney disease and from uremic pigs. The uremic environment has pronounced effects on microvascular integrity as demonstrated by irregular cell-cell junctions and increased permeability in comparison to cell culture media and healthy serum. Moreover, the engineered microvessels demonstrate an increase in sensitivity compared to traditional 2D assays. Thus, the devices and the methods presented here have the potential to be utilized to risk stratify and to direct personalized treatments for patients with chronic kidney disease.

The Expanding Role of Extracellular Traps in Inflammation and Autoimmunity: The New Players in Casting Dark Webs.

The first description of a new form of neutrophil cell death distinct from that of apoptosis or necrosis was discovered in 2004 and coined neutrophil extracellular traps "(NETs)" or "NETosis". Different stimuli for NET formation, and pathways that drive neutrophils to commit to NETosis have been elucidated in the years that followed. Critical enzymes required for NET formation have been discovered and targeted therapeutically. NET formation is no longer restricted to neutrophils but has been discovered in other innate cells: macrophages/monocytes, mast Cells, basophils, dendritic cells, and eosinophils. Furthermore, extracellular DNA can also be extruded from both B and T cells. It has become clear that although this mechanism is thought to enhance host defense by ensnaring bacteria within large webs of DNA to increase bactericidal killing capacity, it is also injurious to innocent bystander tissue. Proteases and enzymes released from extracellular traps (ETs), injure epithelial and endothelial cells perpetuating inflammation. In the context of autoimmunity, ETs release over 70 well-known autoantigens. ETs are associated with pathology in multiple diseases: lung diseases, vasculitis, autoimmune kidney diseases, atherosclerosis, rheumatoid arthritis, cancer, and psoriasis. Defining these pathways that drive ET release will provide insight into mechanisms of pathological insult and provide potential therapeutic targets.

Microfluidics for the study of mechanotransduction.

Mechanical forces regulate a diverse set of biological processes at cellular, tissue, and organismal length scales. Investigating the cellular and molecular mechanisms that underlie the conversion of mechanical forces to biological responses is challenged by limitations of traditional animal models and in vitro cell culture, including poor control over applied force and highly artificial cell culture environments. Recent advances in fabrication methods and material processing have enabled the development of microfluidic platforms that provide precise control over the mechanical microenvironment of cultured cells. These devices and systems have proven to be powerful for uncovering and defining mechanisms of mechanotransduction. In this review, we first give an overview of the main mechanotransduction pathways that function at sites of cell adhesion, many of which have been investigated with microfluidics. We then discuss how distinct microfluidic fabrication methods can be harnessed to gain biological insight, with description of both monolithic and replica molding approaches. Finally, we present examples of how microfluidics can be used to apply both solid forces (substrate mechanics, strain, and compression) and fluid forces (luminal, interstitial) to cells. Throughout the review, we emphasize the advantages and disadvantages of different fabrication methods and applications of force in order to provide perspective to investigators looking to apply forces to cells in their own research.

An objective methodology capturing online commodity marketing and other harms.

Increasingly life is lived online, yet little is known about the actual nature and extent of online content that people view due to the difficulty of recording real time exposure. This includes people's exposure to harmful commodity marketing. This study aimed to develop a methodology to assess the nature and extent of exposure to, and engagement with, unhealthy commodity marketing and other public health harms online, particularly children's exposure. A convenience sample of 16 young adult participants (aged 21-29) recorded their device usage for 2 days using Zoom software. Data were coded and analysed to assess the nature and extent of marketing for alcohol, gambling, junk food and smoking products. Four focus groups were conducted with participants to explore their data collection and coding experiences, and results assessed using thematic analysis. The study found that, with some modifications, this method was feasible for gathering real-time objective data from the online world that can be analysed for a range of public health harms, including marketing of unhealthy commodities. Larger studies are recommended to build global evidence for public health action in the online world.

Physical activity and risk of testicular cancer: a systematic review.

BACKGROUND: Physical activity has been implicated as a risk factor in the development of testicular cancer (TC), but the relationship remains controversial. This systematic review pooled available evidence regarding this association. METHODS: Using Boolean search terms and following PRISMA guidelines, we examined the risk of TC across three categories of exposure: intensity (i.e. comparison of risk between those previously exposed to high, moderate and low levels of physical activity); dose-response (i.e. whether risk of TC increases or decreases with increasing exposure to physical activity); and the role of timing of physical activity (i.e. during early childhood or adolescence). RESULTS: Thirteen studies (11 case-control studies, 2 cohort studies) were included in the review. While some studies have reported a strong protective effect of high levels of physical activity on risk of TC, others have reported either no relationship or a weak direct association; and while a dose-response relationship has been identified across several studies, this relationship has been observed in both directions. Similarly conflicting results exist in terms of individual types of activity and the lifecourse timing of the physical activity. Reasons for this inconsistency may include the absence of any association, heterogeneous assessment of physical activity, misclassification bias and difference in sample sizes. CONCLUSIONS: On balance, there is presently no strong evidence of an association between physical activity and risk of subsequent TC. This review highlights key areas for future investigation that may clarify any association between physical activity and risk of testicular cancer.

Dairy Consumption and Risk of Testicular Cancer: A Systematic Review.

Dairy consumption has been studied extensively in terms of its relationship with testicular cancer (TC), yet this relationship remains unclear. In this systematic review, we aimed to answer whether TC development is associated with (a) high amounts of dairy product consumption, (b) the type of dairy product consumed, (c) increasing levels of dairy product consumption, and (d) dairy consumption during certain periods during the lifecourse. Following a systematic review of the literature, eight studies (all case-control studies) were included in our review. The included studies varied in terms of the dairy product(s) investigated (milk, cheese, cream, butter, and yoghurt) as well as the type of exposure to dairy consumption (e.g., high vs. low exposure, dose-response, and timing during lifecourse). We found that there was no strong evidence that high levels of dairy consumption are associated with risk of TC, conflicting evidence of a dose-response relationship, inconsistent evidence on whether certain types of dairy are more strongly associated with TC than others, and conflicting evidence that exposure during certain life-course periods affects TC risk more than other periods. There is no consistent evidence to support the premise that dairy product consumption is associated with the risk of TC development.

Postconcussive Symptoms Over the First 14 Days After Mild Traumatic Brain Injury: An Experience Sampling Study.

OBJECTIVE: This study examined changes in postconcussive symptoms (PCS) over the acute postinjury recovery period, focusing on how daily PCSs differ between mild traumatic brain injury (mTBI) and other injury types. SETTING: An urban emergency department (ED) in Western Pennsylvania. SUBJECTS: A total of 108 adult patients with trauma being discharged from the ED were recruited and grouped by injury type: mild TBI (mTBI; n = 39), head injury without mTBI (HI: n = 16), and non-head-injured trauma controls (TCs: n = 53). MAIN MEASURES: Subjects completed a baseline assessment and an experience sampling method (ESM) protocol for 14 consecutive days postinjury: outcomes were daily reports of headaches, anxiety, and concentration difficulties. RESULTS: Controlling for confounders, multilevel modeling revealed greater odds of headache and concentration difficulties on day 1 postinjury among the HI and mTBI groups (vs TCs). These odds decreased over time, with greater reductions for the HI and mTBI groups compared with TCs. By day 14, there were no group differences in PCS. In addition, only the HI group reported higher initial levels of anxiety and a steeper slope relative to TCs. CONCLUSION: Patients with HI, regardless of whether they meet the American Congress of Rehabilitation Medicines definition of mTBI, have higher odds of typical PCS immediately postinjury, but faster rates of recovery than TCs. ESM can improve understanding the dynamic nature of postinjury PCS.

Identifying Robertsonian Translocation Carriers by Microarray-Based DNA Analysis.

OBJECTIVE: To develop a noninvasive prenatal testing improvement that allows identification of Robertsonian translocation carriers. METHODS: Blood samples from 191 subjects, including 7 pregnant and 9 non-pregnant Robertsonian translocation carriers, were analyzed for fetal trisomy and Robertsonian translocation status. Digital Analysis of Selected Regions (DANSR™) assays targeting sequences common to the p arms of 5 acrocentric chromosomes were developed and added to existing DANSR assays. DANSR products were hybridized onto a custom DNA microarray for DNA analysis. The Fetal-Fraction Optimized Risk of Trisomy Evaluation (FORTE™) algorithm measures the fraction of fetal DNA and accounts for both the fetal and maternal fractions in the cell-free DNA sample to determine Robertsonian risk. The expectation in a Robertsonian translocation carrier is that DANSR assays on acrocentric p arms should have a concentration 20% less than that of controls. RESULTS: The FORTE algorithm correctly classified the fetal trisomy status and maternal Robertsonian translocation status in all 191 samples. Sixteen samples had a Robertsonian risk score above 99%, while 175 samples had a Robertsonian risk score below 0.01%. CONCLUSIONS: Robertsonian translocations are the most common chromosomal translocations and can have significant reproductive consequences. A maternal screen for Robertsonian translocation carriers would provide women valuable information regarding the risk of fetal trisomy.

Making magnetic yeast.

This study demonstrates that normal yeast cells can be magnetized, and identifies local redox control via carbon metabolism and iron supply as key factors involved in magnetization.

Sphere-forming cells from peripheral cornea demonstrate a wound-healing response to injury.

The cornea is the initial refractive interface of the eye. Its transparency is critical for clear vision and is maintained by stem cells which also act to repair injury inflicted by external insults, such as chemical and thermal burns. Damage to the epithelium compromises its clarity and can reduce or eliminate the stem cell population, diminishing the ability for self-repair. This condition has been termed "limbal stem cell deficiency"; severe cases can lead to corneal blindness. Sphere-forming cells isolated from peripheral cornea are a potential source of stem and progenitor cells for corneal repair. When provided with appropriate substrate, these spheres have the ability to adhere and for cells to migrate outwards akin to that of their natural environment. Direct compression injury and remote scratch injury experiments were conducted on the sphere cells to gauge their wound healing capacity. Measures of proliferation, differentiation, and migration were assessed by immunohistochemical detection of EdU incorporation, α-smooth muscle actin expression and confocal image analysis, respectively. Both modes of injury were observed to draw responses from the spheres indicating wound healing processes. Direct wounding induced a rapid, but transient increase in expression of α-SMA, a marker of corneal myofibroblasts, followed by a proliferative and increasing migratory response. The spheres were observed to respond to remote injury as entire units, with no directional response seen for targeted repair over the scratch injury area. These results give strength to the future use of these peripheral corneal spheres as transplantable units for the regeneration of corneal tissue.

The QTc effect of low-dose methadone for chronic pain: a prospective pilot study.

OBJECTIVE: Methadone is associated with QT prolongation and serious cardiac complications, but this has been primarily demonstrated in opioid dependent patients receiving moderate to high doses. This study investigates the effect of low-dose methadone on the QTc interval in a chronic pain population. DESIGN AND SUBJECTS: We conducted a prospective cohort study in a chronic pain clinic including 82 patients receiving methadone and 102 patients receiving non-methadone opioid therapy. METHODS: We analyzed automated QTc calculations from 12-lead electrocardiograms at baseline and during the subsequent 6 months. The primary outcome of interest was the incidence of QTc greater than 470 milliseconds or an increase from baseline of greater than 60 milliseconds. RESULTS: The methadone group did not manifest an overall higher frequency of QTc > 470 milliseconds (6% for the methadone group vs 5% for controls, P = 0.722) or an increase in the QTc of > 60 milliseconds (4% for the methadone group vs 4% for controls, P = 0.94). In the first month after initiating methadone, patients demonstrated an increase in QTc compared to controls (5% for the methadone group vs 0% for the controls, P = 0.073) but the difference disappeared in the third and sixth months. CONCLUSION: Data from our chronic pain clinic support a potential association of QTc prolongation during the initiation of methadone, but this effect is small and short lived. We believe larger scale studies to further characterize the safety profile of low-dose methadone are warranted.

CDX2 is an amplified lineage-survival oncogene in colorectal cancer.

The mutational activation of oncogenes drives cancer development and progression. Classic oncogenes, such as MYC and RAS, are active across many different cancer types. In contrast, "lineage-survival" oncogenes represent a distinct and emerging class typically comprising transcriptional regulators of a specific cell lineage that, when deregulated, support the proliferation and survival of cancers derived from that lineage. Here, in a large collection of colorectal cancer cell lines and tumors, we identify recurrent amplification of chromosome 13, an alteration highly restricted to colorectal-derived cancers. A minimal region of amplification on 13q12.2 pinpoints caudal type homeobox transcription factor 2 (CDX2), a regulator of normal intestinal lineage development and differentiation, as a target of the amplification. In contrast to its described role as a colorectal tumor suppressor, CDX2 when amplified is required for the proliferation and survival of colorectal cancer cells. Further, transcriptional profiling, binding-site analysis, and functional studies link CDX2 to Wnt/ß-catenin signaling, itself a key oncogenic pathway in colorectal cancer. These data characterize CDX2 as a lineage-survival oncogene deregulated in colorectal cancer. Our findings challenge a prevailing view that CDX2 is a tumor suppressor in colorectal cancer and uncover an additional piece in the multistep model of colorectal tumorigenesis.

Microarray-based cell-free DNA analysis improves noninvasive prenatal testing.

OBJECTIVE: To develop a microarray-based method for noninvasive prenatal testing (NIPT) and compare it with next-generation sequencing. METHODS: Maternal plasma from 878 pregnant women, including 187 trisomy cases (18 trisomy 13, 37 trisomy 18, 132 trisomy 21), was evaluated for trisomy risk. Targeted chromosomes were analyzed using Digital Analysis of Selected Regions (DANSR™) assays. DANSR products were subsequently divided between two DNA quantification methods: microarrays and next-generation sequencing. For both microarray and sequencing methodologies, the Fetal-Fraction Optimized Risk of Trisomy Evaluation (FORTE™) algorithm was used to determine trisomy risk, assay variability across samples, and compute fetal fraction variability within samples. RESULTS: NIPT using microarrays provided faster and more accurate cell-free DNA (cfDNA) measurements than sequencing. The assay variability, a measure of variance of chromosomal cfDNA counts, was lower for microarrays than for sequencing, 0.051 versus 0.099 (p < 0.0001). Analysis time using microarrays was faster, 7.5 versus 56 h for sequencing. Additionally, fetal fraction precision was improved 1.6-fold by assaying more polymorphic sites with microarrays (p < 0.0001). Microarrays correctly classified all trisomy and nontrisomy cases. CONCLUSIONS: NIPT using microarrays delivers more accurate cfDNA analysis than next-generation sequencing and can be performed in less time.

Motor neurons are dispensable for the assembly of a sensorimotor circuit for gaze stabilization.

Sensorimotor reflex circuits engage distinct neuronal subtypes, defined by precise connectivity, to transform sensation into compensatory behavior. Whether and how motor neuron populations specify the subtype fate and/or sensory connectivity of their pre-motor partners remains controversial. Here, we discovered that motor neurons are dispensable for proper connectivity in the vestibular reflex circuit that stabilizes gaze. We first measured activity following vestibular sensation in pre-motor projection neurons after constitutive loss of their extraocular motor neuron partners. We observed normal responses and topography indicative of unchanged functional connectivity between sensory neurons and projection neurons. Next, we show that projection neurons remain anatomically and molecularly poised to connect appropriately with their downstream partners. Lastly, we show that the transcriptional signatures that typify projection neurons develop independently of motor partners. Our findings comprehensively overturn a long-standing model: that connectivity in the circuit for gaze stabilization is retrogradely determined by motor partner-derived signals. By defining the contribution of motor neurons to specification of an archetypal sensorimotor circuit, our work speaks to comparable processes in the spinal cord and advances our understanding of general principles of neural development.

Deciphering the Genetic Code of Autoimmune Kidney Diseases.

Autoimmune kidney diseases occur due to the loss of tolerance to self-antigens, resulting in inflammation and pathological damage to the kidneys. This review focuses on the known genetic associations of the major autoimmune kidney diseases that result in the development of glomerulonephritis: lupus nephritis (LN), anti-neutrophil cytoplasmic associated vasculitis (AAV), anti-glomerular basement disease (also known as Goodpasture's disease), IgA nephropathy (IgAN), and membranous nephritis (MN). Genetic associations with an increased risk of disease are not only associated with polymorphisms in the human leukocyte antigen (HLA) II region, which governs underlying processes in the development of autoimmunity, but are also associated with genes regulating inflammation, such as NFkB, IRF4, and FC γ receptors (FCGR). Critical genome-wide association studies are discussed both to reveal similarities in gene polymorphisms between autoimmune kidney diseases and to explicate differential risks in different ethnicities. Lastly, we review the role of neutrophil extracellular traps, critical inducers of inflammation in LN, AAV, and anti-GBM disease, where inefficient clearance due to polymorphisms in DNase I and genes that regulate neutrophil extracellular trap production are associated with autoimmune kidney diseases.

A facile fluid pressure system reveals differential cellular response to interstitial pressure gradients and flow.

Interstitial fluid pressure gradients and interstitial flow have been shown to drive morphogenic processes that shape tissues and influence progression of diseases including cancer. The advent of porous media microfluidic approaches has enabled investigation of the cellular response to interstitial flow, but questions remain as to the critical biophysical and biochemical signals imparted by interstitial fluid pressure gradients and resulting flow on resident cells and extracellular matrix (ECM). Here, we introduce a low-cost method to maintain physiological interstitial fluid pressures that is built from commonly accessible laboratory equipment, including a laser pointer, camera, Arduino board, and a commercially available linear actuator. We demonstrate that when the system is connected to a microfluidic device containing a 3D porous hydrogel, physiologic pressure is maintained with sub-Pascal resolution and when basic feedback control is directed using an Arduino, constant pressure and pressure gradient can be maintained even as cells remodel and degrade the ECM hydrogel over time. Using this model, we characterized breast cancer cell growth and ECM changes to ECM fibril structure and porosity in response to constant interstitial fluid pressure or constant interstitial flow. We observe increased collagen fibril bundling and the formation of porous structures in the vicinity of cancer cells in response to constant interstitial fluid pressure as compared to constant interstitial flow. Collectively, these results further define interstitial fluid pressure as a driver of key pathogenic responses in cells, and the systems and methods developed here will allow for future mechanistic work investigating mechanotransduction of interstitial fluid pressures and flows.

Neuronal birthdate reveals topography in a vestibular brainstem circuit for gaze stabilization.

Across the nervous system, neurons with similar attributes are topographically organized. This topography reflects developmental pressures. Oddly, vestibular (balance) nuclei are thought to be disorganized. By measuring activity in birthdated neurons, we revealed a functional map within the central vestibular projection nucleus that stabilizes gaze in the larval zebrafish. We first discovered that both somatic position and stimulus selectivity follow projection neuron birthdate. Next, with electron microscopy and loss-of-function assays, we found that patterns of peripheral innervation to projection neurons were similarly organized by birthdate. Finally, birthdate revealed spatial patterns of axonal arborization and synapse formation to projection neuron outputs. Collectively, we find that development reveals previously hidden organization to the input, processing, and output layers of a highly conserved vertebrate sensorimotor circuit. The spatial and temporal attributes we uncover constrain the developmental mechanisms that may specify the fate, function, and organization of vestibulo-ocular reflex neurons. More broadly, our data suggest that, like invertebrates, temporal mechanisms may assemble vertebrate sensorimotor architecture.

Microphysiological model of PIK3CA-driven vascular malformations reveals a role of dysregulated Rac1 and mTORC1/2 in lesion formation.

Somatic activating mutations of PIK3CA are associated with development of vascular malformations (VMs). Here, we describe a microfluidic model of PIK3CA-driven VMs consisting of human umbilical vein endothelial cells expressing PIK3CA activating mutations embedded in three-dimensional hydrogels. We observed enlarged, irregular vessel phenotypes and the formation of cyst-like structures consistent with clinical signatures and not previously observed in cell culture models. Pathologic morphologies occurred concomitant with up-regulation of Rac1/p21-activated kinase (PAK), mitogen-activated protein kinase cascades (MEK/ERK), and mammalian target of rapamycin (mTORC1/2) signaling networks. We observed differential effects between alpelisib, a PIK3CA inhibitor, and rapamycin, an mTORC1 inhibitor, in mitigating matrix degradation and network topology. While both were effective in preventing vessel enlargement, rapamycin failed to reduce MEK/ERK and mTORC2 activity and resulted in hyperbranching, while inhibiting PAK, MEK1/2, and mTORC1/2 mitigates abnormal growth and vascular dilation. Collectively, these findings demonstrate an in vitro platform for VMs and establish a role of dysregulated Rac1/PAK and mTORC1/2 signaling in PIK3CA-driven VMs.

COSMOS: a platform for real-time morphology-based, label-free cell sorting using deep learning.

Cells are the singular building blocks of life, and a comprehensive understanding of morphology, among other properties, is crucial to the assessment of underlying heterogeneity. We developed Computational Sorting and Mapping of Single Cells (COSMOS), a platform based on Artificial Intelligence (AI) and microfluidics to characterize and sort single cells based on real-time deep learning interpretation of high-resolution brightfield images. Supervised deep learning models were applied to characterize and sort cell lines and dissociated primary tissue based on high-dimensional embedding vectors of morphology without the need for biomarker labels and stains/dyes. We demonstrate COSMOS capabilities with multiple human cell lines and tissue samples. These early results suggest that our neural networks embedding space can capture and recapitulate deep visual characteristics and can be used to efficiently purify unlabeled viable cells with desired morphological traits. Our approach resolves a technical gap in the ability to perform real-time deep learning assessment and sorting of cells based on high-resolution brightfield images.

The regulatory effect of miRNAs is a heritable genetic trait in humans.

BACKGROUND: microRNAs (miRNAs) have been shown to regulate the expression of a large number of genes and play key roles in many biological processes. Several previous studies have quantified the inhibitory effect of a miRNA indirectly by considering the expression levels of genes that are predicted to be targeted by the miRNA and this approach has been shown to be robust to the choice of prediction algorithm. Given a gene expression dataset, Cheng et al. defined the regulatory effect score (RE-score) of a miRNA as the difference in the gene expression rank of targets of the miRNA compared to non-targeted genes. RESULTS: Using microarray data from parent-offspring trios from the International HapMap project, we show that the RE-score of most miRNAs is correlated between parents and offspring and, thus, inter-individual variation in RE-score has a genetic component in humans. Indeed, the mean RE-score across miRNAs is correlated between parents and offspring, suggesting genetic differences in the overall efficiency of the miRNA biogenesis pathway between individuals. To explore the genetics of this quantitative trait further, we carried out a genome-wide association study of the mean RE-score separately in two HapMap populations (CEU and YRI). No genome-wide significant associations were discovered; however, a SNP rs17409624, in an intron of DROSHA, was significantly associated with mean RE-score in the CEU population following permutation-based control for multiple testing based on all SNPs mapped to the canonical miRNA biogenesis pathway; of 244 individual miRNA RE-scores assessed in the CEU, 214 were associated (p < 0.05) with rs17409624. The SNP was also nominally significantly associated (p = 0.04) with mean RE-score in the YRI population. Interestingly, the same SNP was associated with 17 (8.5% of all expressed) miRNA expression levels in the CEU. We also show here that the expression of the targets of most miRNAs is more highly correlated with global changes in miRNA regulatory effect than with the expression of the miRNA itself. CONCLUSIONS: We present evidence that miRNA regulatory effect is a heritable trait in humans and that a polymorphism of the DROSHA gene contributes to the observed inter-individual differences.