A Strategic Blend of Stabilizing Polymers to Control Particle Surface Charge for Enhanced Mucus Transport and Cell Binding.

Mucus layers, viscoelastic gels abundant in anionic mucin glycoproteins, obstruct therapeutic delivery across all mucosal surfaces. We found that strongly positively charged nanoparticles (NPs) rapidly adsorb a mucin protein corona in mucus, impeding cell binding and uptake. To overcome this, we developed mucus-evading, cell-adhesive (MECS) NPs with variable surface charge using Flash NanoPrecipitation, by blending a neutral poly(ethylene glycol) (PEG) corona for mucus transport with a small amount, 5 wt%, of polycationic dimethylaminoethyl methacrylate (PDMAEMA) for increased cell targeting. In vitro experiments confirmed rapid mucus penetration and binding to epithelial cells by MECS NPs, suggesting a breakthrough in mucosal drug delivery.

Human Mesofluidic Intestinal Model for Studying Transport of Drug Carriers and Bacteria Through a Live Mucosal Barrier.

The intestinal mucosal barrier forms a critical interface between lumen contents such as bacteria, drugs, and drug carriers and the underlying tissue. Current in vitro intestinal models, while recapitulating certain aspects of this barrier, generally present challenges with respect to imaging transport across mucus and uptake into enterocytes. A human mesofluidic small intestinal chip was designed to enable facile visualization of a mucosal interface created by growing primary human intestinal cells on a vertical hydrogel wall separating channels representing the intestinal lumen and circulatory flow. Type I collagen, fortified via cross-linking to prevent deformation and leaking during culture, was identified as a suitable gel wall material for supporting primary organoid-derived human duodenal epithelial cell attachment and monolayer formation. Addition of DAPT and PGE2 to culture medium paired with air-liquid interface culture increased the thickness of the mucus layer on epithelium grown within the device for 5 days from approximately 5 mm to 50 µm, making the model suitable for revealing intriguing features of interactions between luminal contents and the mucus barrier using live cell imaging. Time-lapse imaging of nanoparticle diffusion within mucus revealed a zone adjacent to the epithelium largely devoid of nanoparticles up to 4.5 hr after introduction to the lumen channel, as well as pockets of dimly lectin-stained mucus within which particles freely diffused, and apparent clumping of particles by mucus components. Multiple particle tracking conducted on the intact mucus layer in the chip revealed significant size-dependent differences in measured diffusion coefficients. E. coli introduced to the lumen channel were freely mobile within the mucus layer and appeared to intermittently contact the epithelial surface over 30 minute periods of culture. Mucus shedding into the lumen and turnover of mucus components within cells were visualized. Taken together, this system represents a powerful tool for visualization of interactions between luminal contents and an intact live mucosal barrier.

Using deep learning for dermatologist-level detection of suspicious pigmented skin lesions from wide-field images.

A reported 96,480 people were diagnosed with melanoma in the United States in 2019, leading to 7230 reported deaths. Early-stage identification of suspicious pigmented lesions (SPLs) in primary care settings can lead to improved melanoma prognosis and a possible 20-fold reduction in treatment cost. Despite this clinical and economic value, efficient tools for SPL detection are mostly absent. To bridge this gap, we developed an SPL analysis system for wide-field images using deep convolutional neural networks (DCNNs) and applied it to a 38,283 dermatological dataset collected from 133 patients and publicly available images. These images were obtained from a variety of consumer-grade cameras (15,244 nondermoscopy) and classified by three board-certified dermatologists. Our system achieved more than 90.3% sensitivity (95% confidence interval, 90 to 90.6) and 89.9% specificity (89.6 to 90.2%) in distinguishing SPLs from nonsuspicious lesions, skin, and complex backgrounds, avoiding the need for cumbersome individual lesion imaging. We also present a new method to extract intrapatient lesion saliency (ugly duckling criteria) on the basis of DCNN features from detected lesions. This saliency ranking was validated against three board-certified dermatologists using a set of 135 individual wide-field images from 68 dermatological patients not included in the DCNN training set, exhibiting 82.96% (67.88 to 88.26%) agreement with at least one of the top three lesions in the dermatological consensus ranking. This method could allow for rapid and accurate assessments of pigmented lesion suspiciousness within a primary care visit and could enable improved patient triaging, utilization of resources, and earlier treatment of melanoma.

Fully synthetic matrices for in vitro culture of primary human intestinal enteroids and endometrial organoids.

Epithelial organoids derived from human donor tissues are important tools in fields ranging from regenerative medicine to drug discovery. Organoid culture requires expansion of stem/progenitor cells in Matrigel, a tumor-derived extracellular matrix (ECM). An alternative completely synthetic ECM could improve reproducibility, clarify mechanistic phenomena, and enable human implantation of organoids. We designed synthetic ECMs with tunable biomolecular and biophysical properties to identify gel compositions supporting human tissue-derived stem/progenitor epithelial cells as enteroids and organoids starting with single cells rather than tissue fragments. The synthetic ECMs consist of 8-arm PEG-macromers modified with ECM-binding peptides and different combinations of integrin-binding peptides, crosslinked with peptides susceptible to matrix metalloprotease (MMP) degradation, and tuned to exhibit a range of biophysical properties. A gel containing an α2ß1 integrin-binding peptide (GFOGER) and matrix binder peptides grafted to a 20 kDa 8-arm PEG macromer showed the most robust support of human duodenal and colon enteroids and endometrial organoids. In this synthetic ECM, human intestinal enteroids and endometrial organoids emerge from single cells and show cell-specific and apicobasal polarity markers upon differentiation. Intestinal enteroids, in addition, retain their proliferative capacity, are functionally responsive to basolateral stimulation, express canonical markers of intestinal crypt cells including Paneth cells, and can be serially passaged. The success of this synthetic ECM in supporting human postnatal organoid culture from multiple different donors and from both the intestine and endometrium suggests it may be broadly useful for other epithelial organoid culture.

OrgaQuant: Human Intestinal Organoid Localization and Quantification Using Deep Convolutional Neural Networks.

Organoid cultures are proving to be powerful in vitro models that closely mimic the cellular constituents of their native tissue. Organoids are typically expanded and cultured in a 3D environment using either naturally derived or synthetic extracellular matrices. Assessing the morphology and growth characteristics of these cultures has been difficult due to the many imaging artifacts that accompany the corresponding images. Unlike single cell cultures, there are no reliable automated segmentation techniques that allow for the localization and quantification of organoids in their 3D culture environment. Here we describe OrgaQuant, a deep convolutional neural network implementation that can locate and quantify the size distribution of human intestinal organoids in brightfield images. OrgaQuant is an end-to-end trained neural network that requires no parameter tweaking; thus, it can be fully automated to analyze thousands of images with no user intervention. To develop OrgaQuant, we created a unique dataset of manually annotated human intestinal organoid images with bounding boxes and trained an object detection pipeline using TensorFlow. We have made the dataset, trained model and inference scripts publicly available along with detailed usage instructions.

Interconnected Microphysiological Systems for Quantitative Biology and Pharmacology Studies.

Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs - "4-way", "7-way", and "10-way" - each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS "physiome-on-a-chip" approaches in drug discovery.

How do research faculty in the biosciences evaluate paper authorship criteria?

Authorship of peer-reviewed journal articles and abstracts has become the primary currency and reward unit in academia. Such a reward is crucial for students and postdocs who are often under-compensated and thus highly value authorship as an incentive. While numerous scientific and publishing organizations have written guidelines for determining author qualifications and author order, there remains much ambiguity when it comes to how these criteria are weighed by research faculty. Here, we sought to provide some initial insight on how faculty view the relative importance of 11 criteria for scientific authorship. We distributed an online survey to 564 biomedical engineering, biology, and bioengineering faculty members at 10 research institutions across the United States. The response rate was approximately 18%, resulting in a final sample of 102 respondents. Results revealed an agreement on some criteria, such as time spent conducting experiments, but there was a lack of agreement regarding the role of funding procurement. This study provides quantitative assessments of how faculty members in the biosciences evaluate authorship criteria. We discuss the implications of these findings for researchers, especially new graduate students, to help navigate the discrepancy between official policies for authorship and the contributions that faculty truly value.

Postprandial lymphatic pump function after a high-fat meal: a characterization of contractility, flow, and viscosity.

Dietary lipids are transported from the intestine through contractile lymphatics. Chronic lipid loads can adversely affect lymphatic function. However, the acute lymphatic pump response in the mesentery to a postprandial lipid meal has gone unexplored. In this study, we used the rat mesenteric collecting vessel as an in vivo model to quantify the effect of lipoproteins on vessel function. Lipid load was continuously monitored by using the intensity of a fluorescent fatty-acid analog, which we infused along with a fat emulsion through a duodenal cannula. The vessel contractility was simultaneously quantified. We demonstrated for the first time that collecting lymphatic vessels respond to an acute lipid load by reducing pump function. High lipid levels decreased contraction frequency and amplitude. We also showed a strong tonic response through a reduction in the end-diastolic and systolic diameters. We further characterized the changes in flow rate and viscosity and showed that both increase postprandially. In addition, shear-mediated Ca(2+) signaling in lymphatic endothelial cells differed when cultured with lipoproteins. Together these results show that the in vivo response could be both shear and lipid mediated and provide the first evidence that high postprandial lipid has an immediate negative effect on lymphatic function even in the acute setting.

S-Nitrosated Polypropylene Sulfide Nanoparticles for Thiol-Dependent Transnitrosation and Toxicity Against Adult Female Filarial Worms.

A synthetic polymer nanoparticle formulation utilizing the physiological nitrosothiol chemistry for nitric oxide delivery is shown. Toxicity of S-nitroso nanoparticles against adult female Brugia malayi worms, which are responsible for lymphatic filariasis, is dependent on nitric oxide release through transnitrosation as S-nitrosocysteine, a potent endogenous nitric oxide donor.

An integrated in vitro imaging platform for characterizing filarial parasite behavior within a multicellular microenvironment.

Lymphatic Filariasis, a Neglected Tropical Disease, is caused by thread-like parasitic worms, including B. malayi, which migrate to the human lymphatic system following transmission. The parasites reside in collecting lymphatic vessels and lymph nodes for years, often resulting in lymphedema, elephantiasis or hydrocele. The mechanisms driving worm migration and retention within the lymphatics are currently unknown. We have developed an integrated in vitro imaging platform capable of quantifying B. malayi migration and behavior in a multicellular microenvironment relevant to the initial site of worm injection by incorporating the worm in a Polydimethylsiloxane (PDMS) microchannel in the presence of human dermal lymphatic endothelial cells (LECs) and human dermal fibroblasts (HDFs). The platform utilizes a motorized controllable microscope with CO2 and temperature regulation to allow for worm tracking experiments with high resolution over large length and time scales. Using post-acquisition algorithms, we quantified four parameters: 1) speed, 2) thrashing intensity, 3) percentage of time spent in a given cell region and 4) persistence ratio. We demonstrated the utility of our system by quantifying these parameters for L3 B. malayi in the presence of LECs and HDFs. Speed and thrashing increased in the presence of both cell types and were altered within minutes upon exposure to the anthelmintic drug, tetramisole. The worms displayed no targeted migration towards either cell type for the time course of this study (3 hours). When cells were not present in the chamber, worm thrashing correlated directly with worm speed. However, this correlation was lost in the presence of cells. The described platform provides the ability to further study B. malayi migration and behavior.

Minimally invasive method for determining the effective lymphatic pumping pressure in rats using near-infrared imaging.

The ability to quantify collecting vessel function in a minimally invasive fashion is crucial to the study of lymphatic physiology and the role of lymphatic pump function in disease progression. Therefore, we developed a highly sensitive, minimally invasive research platform for quantifying the pumping capacity of collecting lymphatic vessels in the rodent tail and forelimb. To achieve this, we have integrated a near-infrared lymphatic imaging system with a feedback-controlled pressure cuff to modulate lymph flow. After occluding lymphatic flow by inflating a pressure cuff on the limb or tail, we gradually deflate the cuff while imaging flow restoration proximal to the cuff. Using prescribed pressure applications and automated image processing of fluorescence intensity levels in the vessels, we were able to noninvasively quantify the effective pumping pressure (P(eff), pressure at which flow is restored after occlusion) and vessel emptying rate (rate of fluorescence clearance during flow occlusion) of lymphatics in the rat. To demonstrate the sensitivity of this system to changes in lymphatic function, a nitric oxide (NO) donor cream, glyceryl trinitrate ointment (GTNO), was applied to the tails. GTNO decreased P(eff) of the vessels by nearly 50% and the average emptying rate by more than 60%. We also demonstrate the suitability of this approach for acquiring measurements on the rat forelimb. Thus, this novel research platform provides the first minimally invasive measurements of P(eff) and emptying rate in rodents. This experimental platform holds strong potential for future in vivo studies that seek to evaluate changes in lymphatic health and disease.

Dual-channel in-situ optical imaging system for quantifying lipid uptake and lymphatic pump function.

Nearly all dietary lipids are transported from the intestine to venous circulation through the lymphatic system, yet the mechanisms that regulate this process remain unclear. Elucidating the mechanisms involved in the functional response of lymphatics to changes in lipid load would provide valuable insight into recent implications of lymphatic dysfunction in lipid related diseases. Therefore, we sought to develop an in situ imaging system to quantify and correlate lymphatic function as it relates to lipid transport. The imaging platform provides the capability of dual-channel imaging of both high-speed bright-field video and fluorescence simultaneously. Utilizing post-acquisition image processing algorithms, we can quantify correlations between vessel pump function, lymph flow, and lipid concentration of mesenteric lymphatic vessels in situ. All image analysis is automated with customized LabVIEW virtual instruments; local flow is measured through lymphocyte velocity tracking, vessel contraction through measurements of the vessel wall displacement, and lipid uptake through fluorescence intensity tracking of an orally administered fluorescently labelled fatty acid analogue, BODIPY FL C16. This system will prove to be an invaluable tool for scientists studying intestinal lymphatic function in health and disease, and those investigating strategies for targeting the lymphatics with orally delivered drugs to avoid first pass metabolism.

Sensitivity analysis of near-infrared functional lymphatic imaging.

Near-infrared imaging of lymphatic drainage of injected indocyanine green (ICG) has emerged as a new technology for clinical imaging of lymphatic architecture and quantification of vessel function, yet the imaging capabilities of this approach have yet to be quantitatively characterized. We seek to quantify its capabilities as a diagnostic tool for lymphatic disease. Imaging is performed in a tissue phantom for sensitivity analysis and in hairless rats for in vivo testing. To demonstrate the efficacy of this imaging approach to quantifying immediate functional changes in lymphatics, we investigate the effects of a topically applied nitric oxide (NO) donor glyceryl trinitrate ointment. Premixing ICG with albumin induces greater fluorescence intensity, with the ideal concentration being 150 µg/mL ICG and 60 g/L albumin. ICG fluorescence can be detected at a concentration of 150 µg/mL as deep as 6 mm with our system, but spatial resolution deteriorates below 3 mm, skewing measurements of vessel geometry. NO treatment slows lymphatic transport, which is reflected in increased transport time, reduced packet frequency, reduced packet velocity, and reduced effective contraction length. NIR imaging may be an alternative to invasive procedures measuring lymphatic function in vivo in real time.