AR loss in prostate cancer stroma mediated by NF-κB and p38-MAPK signaling disrupts stromal morphogen production.

Androgen Receptor (AR) activity in prostate stroma is required to maintain prostate homeostasis. This is mediated through androgen-dependent induction and secretion of morphogenic factors that drive epithelial cell differentiation. However, stromal AR expression is lost in aggressive prostate cancer. The mechanisms leading to stromal AR loss and morphogen production are unknown. We identified TGFß1 and TNFα as tumor-secreted factors capable of suppressing AR mRNA and protein expression in prostate stromal fibroblasts. Pharmacological and RNAi approaches identified NF-κB as the major signaling pathway involved in suppressing AR expression by TNFα. In addition, p38α- and p38δ-MAPK were identified as suppressors of AR expression independent of TNFα. Two regions of the AR promoter were responsible for AR suppression through TNFα. FGF10 and Wnt16 were identified as androgen-induced morphogens, whose expression was lost upon TNFα treatment and enhanced upon p38-MAPK inhibition. Wnt16, through non-canonical Jnk signaling, was required for prostate basal epithelial cell survival. These findings indicate that stromal AR loss is mediated by secreted factors within the TME. We identified TNFα/TGFß as two possible factors, with TNFα mediating its effects through NF-κB or p38-MAPK to suppress AR mRNA transcription. This leads to loss of androgen-regulated stromal morphogens necessary to maintain normal epithelial homeostasis.

Microfluidic-based human prostate-cancer-on-chip.

Lack of adequate models significantly hinders advances in prostate cancer treatment, where resistance to androgen-deprivation therapies and bone metastasis remain as major challenges. Current in vitro models fail to faithfully mimic the complex prostate physiology. In vivo animal models can shed light on the oncogenes involved in prostate cancer development and progression; however, the animal prostate gland is fundamentally different from that of human, and the underlying genetic mechanisms are different. To address this problem, we developed the first in vitro microfluidic human Prostate-Cancer-on-Chip (PCoC) model, where human prostate cancer and stromal fibroblast cells were co-cultivated in two channels separated by a porous membrane under culture medium flow. The established microenvironment enables soluble signaling factors secreted by each culture to locally diffuse through the membrane pores affecting the neighboring culture. We particularly explored the conversion of the stromal fibroblasts into cancer-associated fibroblasts (CAFs) due to the interaction between the 2 cell types. Immunofluorescence microscopy revealed that tumor cells induced CAF biomarkers, αSMA and COL1A1, in stromal fibroblasts. The stromal CAF conversion level was observed to increase along the flow direction in response to diffusion agents, consistent with simulations of solute concentration gradients. The tumor cells also downregulated androgen receptor (AR) expression in stromal fibroblasts, while an adequate level of stromal AR expression is maintained in normal prostate homeostasis. We further investigated tumor invasion into the stroma, an early step in the metastatic cascade, in devices featuring a serpentine channel with orthogonal channel segments overlaying a straight channel and separated by an 8 µm-pore membrane. Both tumor cells and stromal CAFs were observed to cross over into their neighboring channel, and the stroma's role seemed to be proactive in promoting cell invasion. As control, normal epithelial cells neither induced CAF conversion nor promoted cell invasion. In summary, the developed PCoC model allows spatiotemporal analysis of the tumor-stroma dynamic interactions, due to bi-directional signaling and physical contact, recapitulating tissue-level multicellular responses associated with prostate cancer in vivo. Hence, it can serve as an in vitro model to dissect mechanisms in human prostate cancer development and seek advanced therapeutic strategies.

Polarization properties and Umov effect of human hair.

This study delves into the polarization properties of various hair colors using several techniques, including polarization ray tracing, full Stokes, and Mueller matrix imaging. Our analysis involved studying hair in both indoor and outdoor settings under varying lighting conditions. Our results demonstrate a strong correlation between hair color and the degree of linear polarization. Specifically, light-colored hair, such as white and blond, exhibits high albedo and low DoLP. In contrast, dark hair, like black and brown hair, has low albedo and high DoLP. Our research also revealed that a single hair strand displays high diattenuation near specular reflections but high depolarization in areas with diffuse reflections. Additionally, we investigated the wavelength dependency of the polarization properties by comparing the Mueller matrix under illumination at 450 nm and 589 nm. Our investigation demonstrates the impact of hair shade and color on polarization properties and the Umov effect.

Drosophila CASK regulates brain size and neuronal morphogenesis, providing a genetic model of postnatal microcephaly suitable for drug discovery.

BACKGROUND: CASK-related neurodevelopmental disorders are untreatable. Affected children show variable severity, with microcephaly, intellectual disability (ID), and short stature as common features. X-linked human CASK shows dosage sensitivity with haploinsufficiency in females. CASK protein has multiple domains, binding partners, and proposed functions at synapses and in the nucleus. Human and Drosophila CASK show high amino-acid-sequence similarity in all functional domains. Flies homozygous for a hypomorphic CASK mutation (∆18) have motor and cognitive deficits. A Drosophila genetic model of CASK-related disorders could have great scientific and translational value. METHODS: We assessed the effects of CASK loss of function on morphological phenotypes in Drosophila using established genetic, histological, and primary neuronal culture approaches. NeuronMetrics software was used to quantify neurite-arbor morphology. Standard nonparametric statistics methods were supplemented by linear mixed effects modeling in some cases. Microfluidic devices of varied dimensions were fabricated and numerous fluid-flow parameters were used to induce oscillatory stress fields on CNS tissue. Dissociation into viable neurons and neurite outgrowth in vitro were assessed. RESULTS: We demonstrated that ∆18 homozygous flies have small brains, small heads, and short bodies. When neurons from developing CASK-mutant CNS were cultured in vitro, they grew small neurite arbors with a distinctive, quantifiable "bushy" morphology that was significantly rescued by transgenic CASK+. As in humans, the bushy phenotype showed dosage-sensitive severity. To overcome the limitations of manual tissue trituration for neuronal culture, we optimized the design and operation of a microfluidic system for standardized, automated dissociation of CNS tissue into individual viable neurons. Neurons from CASK-mutant CNS dissociated in the microfluidic system recapitulate the bushy morphology. Moreover, for any given genotype, device-dissociated neurons grew larger arbors than did manually dissociated neurons. This automated dissociation method is also effective for rodent CNS. CONCLUSIONS: These biological and engineering advances set the stage for drug discovery using the Drosophila model of CASK-related disorders. The bushy phenotype provides a cell-based assay for compound screening. Nearly a dozen genes encoding CASK-binding proteins or transcriptional targets also have brain-development mutant phenotypes, including ID. Hence, drugs that improve CASK phenotypes might also benefit children with disorders due to mutant CASK partners.

Birefringent coating to remove polarization aberrations.

Polarization aberrations are found in most optical components due to a materials-differing response to s- and p-polarizations. This differing response can manifest either as diattenuation, retardance, or both. Correction of polarization aberrations, such as these, are critical in many applications such as interferometry, polarimetry, display, and high contrast imaging, including astronomy. In this work, compensators based on liquid crystal polymer and anti-reflection thin-films are presented to correct polarization aberrations in both transmission and reflection configurations. Our method is versatile, allowing for good correction in transmission and reflection due to optical components possessing differing diattenuation and retardance dispersions. Through simulation and experimental validation we show two designs, one correcting the polarization aberrations of a dichroic spectral filter over a 170nm wavelength band, and the other correcting the polarization aberration of an aluminum-coated mirror over a 400nm wavelength band and a 55-degree cone of angles. The measured performance of the polarization aberration compensators shows good agreement with theory.

Generalized elliptical retarder design and construction using nematic and cholesteric phase liquid crystal polymers.

Elliptical retarders have important applications in interferometry and polarimetry, as well as imaging and display technologies. In this work, we discuss the traditional elliptical retarder decomposition using Pauli matrices as basis sets and then introduce a solution to the inverse problem: how an arbitrary elliptical retarder with desired eigenpolarizations and retardance can be constructed using a combination of linear and circular retarders. We present a simple design process, based on eigen-decomposition, with a solution determined by the intrinsic properties of each individual retarder layer. Additionally, a novel use of cholesteric liquid crystal polymer as a circular retarder is presented. Through simulation and experimental validation, we show cholesteric phase liquid crystal has an achromatic region of circular retardance at shorter wavelengths, outside of the Bragg regime. Finally, we verify our design process by fabricating and testing an elliptical retarder using both nematic and cholesteric phase liquid crystal polymers. The performance of the elliptical retarders shows excellent agreement with theory.

Extracellular Fluid Flow Induces Shallow Quiescence Through Physical and Biochemical Cues.

The balance between cell quiescence and proliferation is fundamental to tissue physiology and homeostasis. Recent studies have shown that quiescence is not a passive and homogeneous state but actively maintained and heterogeneous. These cellular characteristics associated with quiescence were observed primarily in cultured cells under a static medium. However, cells in vivo face different microenvironmental conditions, particularly, under interstitial fluid flows distributed through extracellular matrices. Interstitial fluid flow exerts shear stress on cells and matrix strain, and results in continuous replacement of extracellular factors. In this study, we analyzed individual cells under varying fluid flow rates in microfluidic devices. We found quiescence characteristics previously identified under conventional static medium, including serum signal-dependant quiescence entry and exit and time-dependant quiescence deepening, are also present under continuous fluid flow. Furthermore, increasing the flow rate drives cells to shallower quiescence and become more likely to reenter the cell cycle upon growth stimulation. This effect is due to flow-induced physical and biochemical cues. Specifically, increasing shear stress or extracellular factor replacement individually, without altering other parameters, results in shallow quiescence. We show our experimental results can be quantitatively explained by a mathematical model connecting extracellular fluid flow to an Rb-E2f bistable switch that regulates the quiescence-to-proliferation transition. Our findings uncover a previously unappreciated mechanism that likely underlies the heterogeneous responses of quiescent cells for tissue repair and regeneration in different physiological tissue microenvironments.

Patterned achromatic elliptical polarizer for short-wave infrared imaging polarimetry.

Short-wave infrared (SWIR) imaging polarimetry has widespread applications in telecommunication, medical imaging, surveillance, remote-sensing, and industrial metrology. In this work, we design, fabricate, and test an achromatic SWIR elliptical polarizer, which is a key component of SWIR imaging polarimetry. The elliptical polarizer is made of a patterned linear polarizer and a patterned optical elliptical retarder. The linear polarizer is a wire grid polarizer. The elliptical retarder is constructed with three layers of nematic phase A-plate liquid crystal polymer (LCP) films with different fast axis orientations and physical film thicknesses. For each LCP layer, four arrays of hexagonal patterns with individual fast-axis orientations are realized utilizing selective linearly polarized ultraviolet (UV) irradiation on a photo-alignment polymer film. The Mueller matrices of the optical filters were measured in the wavelength range 1000 nm to 1600 nm and compared with theory. Our results demonstrate the functionality and quality of the patterned retarders with normalized analyzer vector parameter deviation below 7% over this wavelength range. To the best of our knowledge, this work represents the first polymer-based patterned elliptical polarizer for SWIR polarimetry imaging applications.

Application of Microfluidic Systems for Breast Cancer Research.

Cancer is a disease in which cells in the body grow out of control; breast cancer is the most common cancer in women in the United States. Due to early screening and advancements in therapeutic interventions, deaths from breast cancer have declined over time, although breast cancer remains the second leading cause of cancer death among women. Most deaths are due to metastasis, as cancer cells from the primary tumor in the breast form secondary tumors in remote sites in distant organs. Over many years, the basic biological mechanisms of breast cancer initiation and progression, as well as the subsequent metastatic cascade, have been studied using cell cultures and animal models. These models, although extremely useful for delineating cellular mechanisms, are poor predictors of physiological responses, primarily due to lack of proper microenvironments. In the last decade, microfluidics has emerged as a technology that could lead to a paradigm shift in breast cancer research. With the introduction of the organ-on-a-chip concept, microfluidic-based systems have been developed to reconstitute the dominant functions of several organs. These systems enable the construction of 3D cellular co-cultures mimicking in vivo tissue-level microenvironments, including that of breast cancer. Several reviews have been presented focusing on breast cancer formation, growth and metastasis, including invasion, intravasation, and extravasation. In this review, realizing that breast cancer can recur decades following post-treatment disease-free survival, we expand the discussion to account for microfluidic applications in the important areas of breast cancer detection, dormancy, and therapeutic development. It appears that, in the future, the role of microfluidics will only increase in the effort to eradicate breast cancer.

Patterned liquid crystal polymer C-plate retarder and color polarizer.

The patternability and guest-host interaction with dichroic dye and C-plate liquid crystal polymer (LCP) materials are investigated, and the optical properties of a thin film C-plate retarder and polarizer are studied and compared with theory. The C-plate retarder is a waveplate made of a uniaxial LCP where the optical axis of the LCP is parallel to the surface normal of the optic. No retardance is observed at a normal angle of incidence and retardance grows as the angle of incidence increases. The C-plate polarizer is a C-plate retarder with LCP as the host and a dichroic dye as the guest. The linear diattenuation (LD) of the linear polarizer is zero at a normal angle of incidence and grows with an increasing angle of incidence. Both the C-plate retarder and polarizer can be patterned with minimum feature size down to 2 µm by using ultraviolet photolithography and plasma etching. A planarization process is also developed to deposit a cover layer on top of the pattern to reduce optical loss and to serve as a barrier for subsequent layers.

Division of focal plane red-green-blue full-Stokes imaging polarimeter.

We calibrate and test a division-of-focal-plane red-green-blue (RGB) full-Stokes imaging polarimeter in a variety of indoor and outdoor environments. The polarimeter, acting as a polarization camera, utilizes a low dispersion microretarder array on top of a sensor with Bayer filters and wire-grid linear polarizers. We also present the design and fabrication of the microretarder array and the assembly of the camera and validate the performance of the camera by taking multiple RGB full-Stokes images and videos. Our camera has a small form factor due to its single-sensor design and the unique capability to measure the intensity, color, and polarization of an optical field in a single shot.

Microfabrication of a color filter array utilizing colored SU-8 photoresists.

Patterned color filter arrays are important components in digital cameras, camcorders, scanners, and multispectral detection and imaging instruments. In addition to the rapid and continuous progress to improve camera resolution and the efficiency of imaging sensors, research into the design of color filter arrays is important to extend the imaging capability beyond conventional applications. This paper reports the use of colored SU-8 photoresists as a material to fabricate color filter arrays. Optical properties, fabrication parameters, and pattern spatial resolution are systematically studied for five color photoresists: violet, blue, green, yellow, and red. An end-to-end fabrication process is developed to realize a five-color filter array designed for a wide angle multiband artificial compound eye camera system for pentachromatic and polarization imaging. Colored SU-8 photoresists present notable advantages, including patternability, color tunability, low-temperature compatibility, and process simplicity. The results regarding the optical properties and the fabrication process for a colored SU-8 photoresist provide significant insight into its usage as an optical material to investigate nonconventional color filter designs.

Pharmacokinetic Analysis of Epithelial/Endothelial Cell Barriers in Microfluidic Bilayer Devices with an Air-Liquid Interface.

As the range of applications of organs-on-chips is broadening, the evaluation of aerosol-based therapies using a lung-on-a-chip model has become an attractive approach. Inhalation therapies are not only minimally invasive but also provide optimal pharmacokinetic conditions for drug absorption. As drug development evolves, it is likely that better screening through use of organs-on-chips can significantly save time and cost. In this work, bio-aerosols of various compounds including insulin were generated using a jet nebulizer. The aerosol flows were driven through microfluidic bilayer devices establishing an air-liquid interface to mimic the blood-air barrier in human small airways. The aerosol flow in the microfluidic devices has been characterized and adjusted to closely match physiological values. The permeability of several compounds, including paracellular and transcellular biomarkers, across epithelial/endothelial cell barriers was measured. Concentration-time plots were established in microfluidic devices with and without cells; the curves were then utilized to extract standard pharmacokinetic parameters such as the area under the curve, maximum concentration, and time to maximum concentration. The cell barrier significantly affected the measured pharmacokinetic parameters, as compound absorption through the barrier decreases with its increasing molecular size. Aerosolizing insulin can lead to the formation of fibrils, prior to its entry to the microfluidic device, with a substantially larger apparent molecular size effectively blocking its paracellular transport. The results demonstrate the advantage of using lung-on-a-chip for drug discovery with applications such as development of novel inhaled therapies.

Observation of elliptically polarized light from total internal reflection in bubbles.

Bubbles are ubiquitous in the natural environment, where different substances and phases of the same substance forms globules due to differences in pressure and surface tension. Total internal reflection occurs at the interface of a bubble, where light travels from the higher refractive index material outside a bubble to the lower index material inside a bubble at appropriate angles of incidence, which can lead to a phase shift in the reflected light. Linearly polarized skylight can be converted to elliptically polarized light with efficiency up to 53% by single scattering from the water-air interface. Total internal reflection from air bubble in water is one of the few sources of elliptical polarization in the natural world. Stationary and dynamic scenes of air bubbles in water in both indoor and outdoor settings are studied using an imaging polarimeter. Our results are important for studies in fluid dynamics, remote sensing, and polarimetry.

Permeability of Epithelial/Endothelial Barriers in Transwells and Microfluidic Bilayer Devices.

Lung-on-a-chip (LoC) models hold the potential to rapidly change the landscape for pulmonary drug screening and therapy, giving patients more advanced and less invasive treatment options. Understanding the drug absorption in these microphysiological systems, modeling the lung-blood barrier is essential for increasing the role of the organ-on-a-chip technology in drug development. In this work, epithelial/endothelial barrier tissue interfaces were established in microfluidic bilayer devices and transwells, with porous membranes, for permeability characterization. The effect of shear stress on the molecular transport was assessed using known paracellular and transcellular biomarkers. The permeability of porous membranes without cells, in both models, is inversely proportional to the molecular size due to its diffusivity. Paracellular transport, between epithelial/endothelial cell junctions, of large molecules such as transferrin, as well as transcellular transport, through cell lacking required active transporters, of molecules such as dextrans, is negligible. When subjected to shear stress, paracellular transport of intermediate-size molecules such as dextran was enhanced in microfluidic devices when compared to transwells. Similarly, shear stress enhances paracellular transport of small molecules such as Lucifer yellow, but its effect on transcellular transport is not clear. The results highlight the important role that LoC can play in drug absorption studies to accelerate pulmonary drug development.

Polarizing beam splitter cube for circularly and elliptically polarized light.

A method of designing an arbitrary polarizing beam splitter (PBS) cube using multiple layers of thin-film liquid crystal polymer is demonstrated. This methodology utilizes cholesteric phase liquid crystal polymer (Ch-LCP) to transmit one handedness of elliptically polarized light and reflect the orthogonal state when unpolarized light is incident. Using additional nematic liquid crystal polymer layers, the polarization state for the transmitted and reflected light can be controlled and output to any two orthogonal states represented on the Poincaré sphere. Two cubes are designed, fabricated, tested, and compared with theory. One cube is constructed with a single layer of Ch-LCP, and another cube is constructed with a layer of Ch-LCP and an additional nematic liquid crystal polymer layer.

De novo transcriptome analysis and differentially expressed genes in the ovary and testis of the Japanese mantis shrimp Oratosquilla oratoria by RNA-Seq.

The mantis shrimp Oratosquilla oratoria is a widely distributed, commercially important crustacean species. Although its conservation and the development of successful artificial breeding technologies have recently received considerable attention, there are currently no available data regarding the molecular mechanisms in controlling reproduction. In this study, we performed transcriptome sequencing of the testis, ovary, female and male eyestalks and the androgenic gland of O. oratoria, and compared the expression pattern of transcripts from the testis and ovary libraries to identify genes involved in gonadal development. A total of 147,130,937 clean reads were retrieved after removing the adapters in reads and filtering out low-quality data. All the reads were assembled into 94,990 unigenes (23,133 in testis and ovary) with an average length of 783 base pairs (bp) and N50 of 1502 bp. A search of all-unigenes against COG, GO, KEGG, KOG, Pfam, Swiss-Prot and Nr databases resulted in a total of 19,404 annotated unigenes. Comparison of the sequences in the ovary and testis libraries revealed that 1188 unigenes were up-regulated in the ovary and 2732 were up-regulated in the testis. Twenty ovary-up-regulated and 21 testis-up-regulated unigenes were confirmed by quantitative real-time PCR. Additionally, 13,437 simple sequence repeats (SSRs) and 275,799 putative single nucleotide polymorphisms (SNPs) were identified. The important functional genes and pathways identified here provide a valuable dataset for understanding the molecular mechanisms controlling gonad development in O. oratoria, and the numerous (13,437 SSRs and 275,799 SNPs) molecular markers obtained here will provide fundamental basis for functional genomic and population genetic studies of O. oratoria.

Design, fabrication and testing of achromatic elliptical polarizer.

A method of designing achromatic elliptical polarizers using a combination of multiple birefringent waveplates is demonstrated. This approach has a simple geometric interpretation and simplifies the problem of designing an achromatic elliptical polarizer to find overlapping arcs on the Poincaré sphere. The technique is applied to the design of achromatic elliptical polarizers for a broadband division-of-focal-plane full-Stokes imaging polarimeter for visible wavelength band (λ = 450nm to 650nm). An achromatic elliptical polarizer sample with a two-layer retarder is fabricated using liquid crystal polymer. The performance of the polarizer sample is measured and compared with the theoretical calculation. For comparison, a superachromatic polarizer design (λ = 400nm to 1µm) is also presented by using three-layer and four-layer retarder configurations.

Specific interactions between functionalised particles and circulating tumour cells.

Receptor-ligand binding has been one of the more popular approaches to specifically targeting tumour cells. In this work, targeting efficiency was quantitatively characterized using silica particles functionalized with EpCAM antibodies and EpCAM-expressing BT-20 breast cancer cells. The effects of incubation time and particle concentration on the number of functionalised particles bound to target cells were experimentally investigated. The number of bound particles was found to increase with particle concentration, but not necessarily with incubation time. Binding affinity loss because of cell-particle-cell interaction was identified as a limiting mechanism for the number of particles bound to target cells. While cell-surface coverage because of bound particles rises exponentially under low particle concentration, it features a peak value at high particle concentration. The current findings suggest that separation of a bound particle from a cell may be detrimental to cellular binding affinity.

Isolation of viable cancer cells in antibody-functionalized microfluidic devices.

Microfluidic devices functionalized with EpCAM antibodies were utilized for the capture of target cancer cells representing circulating tumor cells (CTCs). The fraction of cancer cells captured from homogeneous suspensions is mainly a function of flow shear rate, and can be described by an exponential function. A characteristic shear rate emerges as the most dominant parameter affecting the cell attachment ratio. Utilizing this characteristic shear rate as a scaling factor, all attachment ratio results for various combinations of receptor and ligand densities collapsed onto a single curve described by the empirical formula. The characteristic shear rate increases with both cell-receptor and surface-ligand densities, and empirical formulae featuring a product of two independent cumulative distributions described well these relationships. The minimum detection limit in isolation of target cancer cells from binary mixtures was experimentally explored utilizing microchannel arrays that allow high-throughput processing of suspensions about 0.5 ml in volume, which are clinically relevant, within a short time. Under a two-step attachment/detachment flow rate, both high sensitivity (almost 1.0) and high specificity (about 0.985) can be achieved in isolating target cancer cells from binary mixtures even for the lowest target/non-target cell concentration ratio of 1:100 000; this is a realistic ratio between CTCs and white blood cells in blood of cancer patients. Detection of CTCs from blood samples was also demonstrated using whole blood from healthy donors spiked with cancer cells. Finally, the viability of target cancer cells released after capture was confirmed by observing continuous cell growth in culture.