Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on‐chip EV isolation is showing promise with respect to improved ...recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity‐based microfluidic EV isolation using anti‐CD63 is widely used; however, anti‐CD63 is not specific to cancer‐EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer‐associated EVs. A new exosome isolation microfluidic device (newExoChip), conjugated with a PS‐specific protein, to isolate cancer‐associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549‐derived exosomes than an anti‐CD63‐conjugated device. Immobilized exosomes are then easily released using Ca2+ chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western‐blot analysis, revealing exosomal shapes and exosomal protein expressions. The newExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.
On‐chip exosome isolation using immunoaffinity capture improves recovery from small sample volumes compared to ultracentrifugation. However, tetraspanins are not specific to cancer exosomes and some cancers secrete exosomes with low expression of tetraspanins. Here, a microfluidic‐device conjugated with proteins against phosphatidylserine (PS), shown to be expressed on the outer surface of cancer‐associated exosome, is used to isolate cancer‐associated exosomes.
•PI switches at the cell surface prime endocytic membranes for endosomal fusion.•Endosomal PI switches determine the fate of internalized cargo.•Lysosomal PI switches couple degradation to nutrient ...signals.
Phosphoinositides (PIs) and their spatiotemporally controlled production, turnover, and interconversion is catalyzed by specific PI phosphatases and kinases and their regulators to allow rapid switching of subcompartmental PI identity. Recent studies have begun to decipher such PI switches at the molecular level and to unravel their physiological functions in endocytosis and endolysosomal membrane dynamics as well as in the control of autophagy and nutrient signaling at late endosomes/lysosomes. In this review, we summarize recent conceptual progress in the field and outline remaining perspectives and challenges for future research. As dysfunctional PI switches underlie a growing number of developmental disturbances and diseases, understanding how PI switches control endocytosis and endolysosomal function may serve to delineate new avenues for potential drug-based therapies to combat these disorders.
Nutrient sensing by mechanistic target of rapamycin complex 1 (mTORC1) on lysosomes and late endosomes (LyLEs) regulates cell growth. Many factors stimulate mTORC1 activity, including the production ...of phosphatidylinositol 3,4,5-trisphosphate PI(3,4,5)P3 by class I phosphatidylinositol 3-kinases (PI3Ks) at the plasma membrane. We investigated mechanisms that repress mTORC1 under conditions of growth factor deprivation. We identified phosphatidylinositol 3,4-bisphosphate PI(3,4)P₂, synthesized by class II PI3K β (PI3KC2β) at LyLEs, as a negative regulator of mTORC1, whereas loss of PI3KC2β hyperactivated mTORC1. Growth factor deprivation induced the association of PI3KC2β with the Raptor subunit of mTORC1. Local PI(3,4)P₂ synthesis triggered repression of mTORC1 activity through association of Raptor with inhibitory 14-3-3 proteins. These results unravel an unexpected function for local PI(3,4)P₂ production in shutting off mTORC1.
Immunoaffinity based EV isolation technologies use antibodies targeting surface markers on EVs to provide higher isolation specificity and purity compared to existing approaches. One standing ...challenge for researchers is how to release captured EVs from the substrate to increase downstream and biological studies. The strong binding between the antibody and antigen or the antibody and substrate is commonly unbreakable without operating at conditions outside of the critical physiological range, making the release of EVs problematic. Additionally, immuno-affinity approaches are usually low-throughput due to their low flow velocity to ensure adequate time for antibody-antigen binding. To overcome these limitations, we modified the OncoBean chip, a previously reported circulating tumor cell isolation microfluidic device. The OncoBean chip is a radial flow microfluidic device with bean-shape microposts functionalized with biotin-conjugated EPCAM antibody through biotin-avidin link chemistry. It was demonstrated that the high surface area and varying shear rate provided by the bean-shaped posts and the radial flow design in the chip, enabled efficient capture of CTCs at high flow rate. We replace the anti-EPCAM with antibodies that recognize common EV surface markers to achieve high-throughput EV isolation. Moreover, by incorporating desthiobiotin-conjugated antibodies, EVs can be released from the device after capture, which offers a significant improvement over the existing isolation. The released EVs were found to be functional by confirming their uptake by cells using flow cytometry and fluorescent microscopy. We believe the proposed technology can facilitate both the study of EVs as cell-to-cell communicators and the further identification of EV markers.
Immunoaffinity based EV isolation technologies use antibodies targeting surface markers on EVs to provide higher isolation specificity and purity compared to existing approaches.
As the recognition between natural killer (NK) cells and cancer cells does not require antigen presentation, NK cells are being actively studied for use in adoptive cell therapies in the rapidly ...evolving armamentarium of cancer immunotherapy. In addition to utilizing NK cells, recent studies have shown that exosomes derived from NK cells also exhibit antitumor properties. Furthermore, these NK cell‐derived exosomes exhibit higher stability, greater modification potentials and less immunogenicity compared to NK cells. Therefore, technologies that allow highly sensitive and specific isolation of NK cells and NK cell‐derived exosomes can enable personalized NK‐mediated cancer therapeutics in the future. Here, a novel microfluidic system to collect patient‐specific NK cells and on‐chip biogenesis of NK‐exosomes is proposed. In a small cohort of non‐small cell lung cancer (NSCLC) patients, both NK cells and circulating tumor cells (CTCs) were isolated, and it is found NSCLC patients have high numbers of NK and NK‐exosomes compared with healthy donors, and these concentrations show a trend of positive and negative correlations with bloodborne CTC numbers, respectively. It is further demonstrated that the NK‐exosomes harvested from NK‐graphene oxide chip exhibit cytotoxic effect on CTCs. This versatile system is expected to be used for patient‐specific NK‐based immunotherapies along with CTCs for potential prognostic/diagnostic applications.
Natural killer (NK) cells are lymphocytes that have the ability to destroy cancer cells without antigen presentation. The NK cell‐derived exosomes exhibit higher stability, greater modification potentials, and less immunogenicity compared to NK cells. Here, a novel microfluidic system to collect patient‐specific NK cells and on‐chip biogenesis of NK‐exosomes for diagnostic and therapeutic use of NK‐exosomes is proposed.
This study shifts the focus away from demonstrating the existence of the effect toward understanding the mechanism by which the effect of AR operates in museum learning. By uncovering and describing ...the contingencies of AR from the perspectives of learner control, this study investigates how and when AR affects museum learning experiences, and to give insights into curation with AR. A between-subjects experiment was conducted with 48 college students divided into three groups. This study considered both qualitative and quantitative features of learner control and designed the AR control tools and experiment accordingly, and the findings supported the success of integrating the immersive technology of AR and the theoretical framework of learner control to construct museum exhibits. The results showed that visitors are willing to use the provided tools in museum AR and perform steadily in knowledge acquisition. In addition to offering more learner control in museums, AR promotes positive behaviors and attitudes. This study contributes to the field studies of learner control by linking learner control with the critical dimensions of AR-enhanced museum learning to provide more guidance in exhibit design. Based on the findings, practical suggestions on incorporating learner control in AR-based interactive exhibits are provided.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Clathrin-mediated endocytosis (CME) involves membrane-associated scaffolds of the bin-amphiphysin-rvs (BAR) domain protein family as well as the GTPase dynamin, and is accompanied and perhaps ...triggered by changes in local lipid composition. How protein recruitment, scaffold assembly and membrane deformation is spatiotemporally controlled and coupled to fission is poorly understood. We show by computational modelling and super-resolution imaging that phosphatidylinositol 3,4-bisphosphate PI(3,4)P
synthesis within the clathrin-coated area of endocytic intermediates triggers selective recruitment of the PX-BAR domain protein SNX9, as a result of complex interactions of endocytic proteins competing for phospholipids. The specific architecture induces positioning of SNX9 at the invagination neck where its self-assembly regulates membrane constriction, thereby providing a template for dynamin fission. These data explain how lipid conversion at endocytic pits couples local membrane constriction to fission. Our work demonstrates how computational modelling and super-resolution imaging can be combined to unravel function and mechanisms of complex cellular processes.
Melanoma is among the most aggressive cancers, and its rate of incidence continues to grow. Early detection of melanoma has been hampered due to the lack of promising markers for testing. Recent ...advances in liquid biopsy have proposed noninvasive alternatives for cancer diagnosis and monitoring. Circulating tumor cells (CTCs) and cancer‐exosomes are gaining influence as promising biomarkers because of their cancer‐associated molecular markers and signatures. However, technologies that offer the dual‐isolation of CTCs and exosomes using a single sample have not been thoroughly developed. The dual‐utilization OncoBean (DUO) device is conjugated with melanoma specific antibodies, MCAM and MCSP, enabling simultaneous CTC and exosome isolations. Using blood samples from patients, CTCs and exosomes are specifically isolated from a single sample and then undergo molecular profiling for comprehensive study. Melanoma patients have 0–17CTCs mL−1 and 299 µg exosomal protein mL−1 while healthy donors display fewer than 2CTCs and 75.6 µg of exosomes mL−1, respectively. It is also demonstrated that both markers express melanoma‐associated genes using multiplex qRT‐PCR to test for expression pattern of a 96 gene panel. The dual isolation and molecular characterization will allow for further research into melanoma to identify viable markers for disease progression and treatment efficacy.
Circulating tumor cells (CTCs) and cancer exosomes are gaining influence as promising biomarkers in liquid biopsy, however, technologies that offer the dual‐isolation of CTCs and exosomes have not been developed. Here, a microfluidic device is devised conjugated with melanoma cell adhesion molecule (MCAM) and melanoma‐associated chondroitin sulfate proteoglycan (MCSP) for isolation and molecular profiling of both melanoma CTCs and exosomes.
The site selectivity for lysine conjugation on a native protein is difficult to control and characterize. Here, we applied mass spectrometry to examine the conjugation kinetics of Trastuzumab-IgG ...(Her-IgG) and α-lactalbumin under excess linker concentration (L
) based on the modified Michaelis-Menten equation, in which the initial rate constant per amine (k
= V
/K
) was determined by the maximum reaction rate (V
) under saturated accessible sites and initial amine-linker affinity (1/K
). Reductive amination (RA) displayed 3-4 times greater V
and a different panel of conjugation sites than that observed for N-hydroxysuccinimide ester (NHS) chemistry using the same length of polyethylene glycol (PEG) linkers. Moreover, faster conversion power rendered RA site selectivity among accessible amine groups and a greater tunable range of linker/protein ratio for aldehyde-linkers compared to those of the same length of NHS-linkers. Single conjugation with high yield or poly-conjugations with site homogeneity was demonstrated by controlling L
or gradual addition to minimize the L
/K
ratio. Formaldehyde, the shortest aldehyde-linker with the greatest 1/K
, exhibited the highest selectivity and was shown to be a suitable probe to predict conjugation profile of aldehyde-linkers. Four linkers on the few probe-predicted hot spots were elucidated by kinetically controlled RA with conserved drug efficacy when conjugated with the payload. This study provides insights into controlling factors for homogenous and predictable amine bioconjugation.
This study shifts the focus away from demonstrating the existence of the effect toward understanding the mechanism by which the effect of AR operates in museum learning. By uncovering and describing ...the contingencies of AR from the perspectives of learner control, this study investigates how and when AR affects museum learning experiences, and to give insights into curation with AR. A between-subjects experiment was conducted with 48 college students divided into three groups. This study considered both qualitative and quantitative features of learner control and designed the AR control tools and experiment accordingly, and the findings supported the success of integrating the immersive technology of AR and the theoretical framework of learner control to construct museum exhibits. The results showed that visitors are willing to use the provided tools in museum AR and perform steadily in knowledge acquisition. In addition to offering more learner control in museums, AR promotes positive behaviors and attitudes. This study contributes to the field studies of learner control by linking learner control with the critical dimensions of AR-enhanced museum learning to provide more guidance in exhibit design. Based on the findings, practical suggestions on incorporating learner control in AR-based interactive exhibits are provided.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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