Ryan Green

Intracellular processing of disease-associated α-synuclein in the human brain suggests prion-like cell-to-cell spread.

Dementia with Lewy bodies (DLB), Parkinsons disease (PD) and multiple system atrophy are characterized by the deposition of disease-associated α-synuclein. In the present study we 1) examined the molecular specificity of the novel anti-α-synuclein 5G4 antibody; 2) evaluated immunoreactivity patterns and their correlation in human brain tissue with micro- and astrogliosis in 57 cases with PD or DLB; and 3) performed a systematic immunoelectron microscopical mapping of subcellular localizations. 5G4 strongly binds to the high molecular weight fraction of β-sheet rich oligomers, while no binding to primarily disordered oligomers or monomers was observed. We show novel localizations of disease-associated α-synuclein including perivascular macrophages, ependyma and cranial nerves. α-Synuclein immunoreactive neuropil dots and thin threads associate more with glial reaction than Lewy bodies alone. Astrocytic α-synuclein is an important component of the pathology. Furthermore, we document ultrastructurally the pathway of processing of disease-associated α-synuclein within neurons and astroglial cells. Interaction of mitochondria and disease-associated α-synuclein plays a key role in the molecular-structural cytopathogenesis of disorders with Lewy bodies. We conclude that 1) the 5G4 antibody has strong selectivity for β-sheet rich α-synuclein oligomers; 2) Lewy bodies themselves are not the most relevant morphological substrate that evokes tissue lesioning; 3) both neurons and astrocytes internalize disease-associated α-synuclein in the human brain, suggesting prion-like cell-to-cell spread of α-synuclein by uptake from surrounding structures, as shown previously in experimental observations.

A 3D Fibrous Scaffold Inducing Tumoroids: A Platform for Anticancer Drug Development

The development of a suitable three dimensional (3D) culture system for anticancer drug development remains an unmet need. Despite progress, a simple, rapid, scalable and inexpensive 3D-tumor model that recapitulates in vivo tumorigenesis is lacking. Herein, we report on the development and characterization of a 3D nanofibrous scaffold produced by electrospinning a mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of polylactic acid (PLA) and mono-methoxypolyethylene glycol (mPEG) designated as 3P. Cancer cells cultured on the 3P scaffold formed tight irregular aggregates similar to in vivo tumors, referred to as tumoroids that depended on the topography and net charge of the scaffold. 3P scaffolds induced tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) as demonstrated by up-regulation of vimentin and loss of E-cadherin expression. 3P tumoroids showed higher resistance to anticancer drugs than the same tumor cells grown as monolayers. Inhibition of ERK and PI3K signal pathways prevented EMT and reduced tumoroid formation, diameter and number. Fine needle aspirates, collected from tumor cells implanted in mice when cultured on 3P scaffolds formed tumoroids, but showed decreased sensitivity to anticancer drugs, compared to tumoroids formed by direct seeding. These results show that 3P scaffolds provide an excellent platform for producing tumoroids from tumor cell lines and from biopsies and that the platform can be used to culture patient biopsies, test for anticancer compounds and tailor a personalized cancer treatment.

NOT THAT RIGID MIDGETS AND NOT SO FLEXIBLE GIANTS: ON THE ABUNDANCE AND ROLES OF INTRINSIC DISORDER IN SHORT AND LONG PROTEINS

Intrinsically disordered proteins or proteins with disordered regions are very common in nature. These proteins have numerous biological functions which are complementary to the biological activities of traditional ordered proteins. A noticeable difference in the amino acid sequences encoding long and short disordered regions was found and this difference was used in the development of length-dependent predictors of intrinsic disorder. In this study, we analyze the scaling of intrinsic disorder in eukaryotic proteins and investigate the presence of length-dependent functions attributed to proteins containing long disordered regions.

Surface Acoustic Waves (SAW)-Based Biosensing for Quantification of Cell Growth in 2D and 3D Cultures

Detection and quantification of cell viability and growth in two-dimensional (2D) and three-dimensional (3D) cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose–response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs) permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW) device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS) well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control) were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids) and the results showed the devices ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in 3D cell culture models, which may have potential applications in both longitudinal 3D cell cultures in cancer biology and in regenerative medicine.

Respiratory Syncytial Virus-Infected Mesenchymal Stem Cells Regulate Immunity via Interferon Beta and Indoleamine-2,3-Dioxygenase

Respiratory syncytial virus (RSV) has been reported to infect human mesenchymal stem cells (MSCs) but the consequences are poorly understood. MSCs are present in nearly every organ including the nasal mucosa and the lung and play a role in regulating immune responses and mediating tissue repair. We sought to determine whether RSV infection of MSCs enhances their immune regulatory functions and contributes to RSV-associated lung disease. RSV was shown to replicate in human MSCs by fluorescence microscopy, plaque assay, and expression of RSV transcripts. RSV-infected MSCs showed differentially altered expression of cytokines and chemokines such as IL-1β, IL6, IL-8 and SDF-1 compared to epithelial cells. Notably, RSV-infected MSCs exhibited significantly increased expression of IFN-β (~100-fold) and indoleamine-2,3-dioxygenase (IDO) (~70-fold) than in mock-infected MSCs. IDO was identified in cytosolic protein of infected cells by Western blots and enzymatic activity was detected by tryptophan catabolism assay. Treatment of PBMCs with culture supernatants from RSV-infected MSCs reduced their proliferation in a dose dependent manner. This effect on PBMC activation was reversed by treatment of MSCs with the IDO inhibitors 1-methyltryptophan and vitamin K3 during RSV infection, a result we confirmed by CRISPR/Cas9-mediated knockout of IDO in MSCs. Neutralizing IFN-β prevented IDO expression and activity. Treatment of MSCs with an endosomal TLR inhibitor, as well as a specific inhibitor of the TLR3/dsRNA complex, prevented IFN-β and IDO expression. Together, these results suggest that RSV infection of MSCs alters their immune regulatory function by upregulating IFN-β and IDO, affecting immune cell proliferation, which may account for the lack of protective RSV immunity and for chronicity of RSV-associated lung diseases such as asthma and COPD.

Three- and Four-Dimensional Spheroid and FiSS Tumoroid Cultures: Platforms for Drug Discovery and Development and Translational Research

There have been remarkable improvements in our understanding of cancer biology. However, therapeutic improvements, with a few exceptions, have been minimal. Also, significant challenges remain in translating fundamental discoveries in cancer biology and genetics into effective drugs and cures. Traditional two-dimensional monolayer cell cultures lack predictive value, resulting in a >90% failure rate of compounds in clinical trials. A developing cancer is a symbiotic tissue consisting of cancer cells, including cancer stem cells (CSCs), and cohabitating with the components of its environment to form a tumor microenvironment (TME) niche. Throughout the process of tumorigenesis, ubiquitous autocrine and paracrine signaling between the cellular and noncellular components of the TME dictates the milieu and structure of this niche. Arising out of such interactions are the cancer cells phenotypic characteristics, such as stemness, epithelial mesenchymal transformation (EMT), and drug resistance which in turn greatly affect the response of these cells to drug therapy. For these reasons, in order to delineate the mechanism of tumorigenesis and in the process discover drugs that will have greatest impact on tumor growth, it becomes imperative to study the cancer cell in context of its microenvironment. In the present review, we enumerate the advantages of three- and four-dimensional (3D and 4D) cell cultures and describe the various cell culture platforms that are being used to study tumorigenesis in vitro. These culture systems will not only aid in the study of tumor progression complexities in a cost-effective and rapid manner; they also are expected to facilitate the discovery and delivery of therapeutic regimens that will have more success making it to the clinic.

Osteopontin plays a pivotal role in increasing severity of respiratory syncytial virus infection

The molecular mechanisms underlying susceptibility to severe respiratory syncytial virus (RSV) infection remain poorly understood. Herein, we report on the role of osteopontin (OPN) in regulation of RSV infection in human epithelial cells and how interleukin-1 beta (IL-1β), a cytokine secreted soon after RSV infection, when persistently expressed can induce OPN expression leading to increased viral infection. We first compared OPN expression in two human epithelial cell lines: HEK-293 and HEp-2. In contrast to HEp-2, HEK-293 expresses low levels of pro-caspase-1 resulting in decreased IL-1β expression in response to RSV infection. We found a correlation between low IL-1β levels and a delay in induction of OPN expression in RSV-infected HEK-293 cells compared to HEp-2. This phenomenon could partially explain the high susceptibility of HEp-2 cells to RSV infection versus the moderate susceptibility of HEK-293 cells. Also, HEK-293 cells expressing low levels of pro-caspase-1 exhibit decreased IL-1β expression and delayed OPN expression in response to RSV infection. HEK-293 cells incubated with human rIL-1β showed a dose-dependent increase in OPN expression upon RSV infection. Also, incubation with rOPN increased RSV viral load. Moreover, HEp-2 cells or mice infected with a mucogenic RSV strain RSV-L19F showed elevated levels of OPN in contrast to mice infected with the laboratory RSV strain rA2. This correlated with elevated levels of OPN following infection with RSV-L19F compared to rA2. Together, these results demonstrate that increased OPN expression is regulated in part by IL-1β, and the interplay between IL-1β and OPN signaling may play a pivotal role in the spread of RSV infection.

Multiple-Layer Guided Surface Acoustic Wave (SAW)-Based pH Sensing in Longitudinal FiSS-Tumoroid Cultures

A constitutively increased intracellular pH that is higher than the extracellular pH is emerging as a hallmark of cancer and determining pH could play a significant role in the measurement of drug responsiveness of tumor cells. However, a non-invasive, touch-free and real-time pH sensing as a research tool is lacking and remains a major unmet need. The purpose of the current study is to investigate a microfluidic surface acoustic wave (SAW) sensor platform capable of monitoring pH in cell and tumoroid cultures. A novel multi-layer guided SAW sensor integrated into a microfluidic channel was investigated theoretically and experimentally in detail for pH bio-sensing. Sensitivity and capability of the layer guided Love wave device was modeled using the finite element simulation. The model was verified experimentally, and a study monitoring pH of cell growth media is presented. This novel pH sensor is based on a 13.91 MHz center frequency SAW device coated with ZnO (500 nm) and IrO2 (30 nm) layers to increase the sensitivity. A change in mechanical and electrical properties of the conductive IrO2 layer was observed resulting from electrical corrosion induced by pH solutions affecting the charge distribution, SAW phase velocity and attenuation. By measuring the frequency shift induced by the change in SAW phase velocity between the test group and control group, the pH value of cell culture media from H460 cancer cell culture plates from day 0 to day 5 can easily be determined. To improve the sensitivity and stability of the sensor, a finite element method was used to optimize the layer thicknesses. Taken together, the results of experiments show the potential application of this device to be integrated with microfluidic channels and used in determining pH changes in longitudinal tumor cell cultures.