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The treatment of bladder cancers has improved with new immunotherapy such as PD-L1. However, not all bladder cancers respond to PD-L1 immunotherapy which is why new targets are being assessed. Melanoma-associated antigen gene A (MAGE-A) family proteins are expressed in a variety of tumors, with each MAGE-A protein having unique roles in cancer pathogenesis. One advantage of targeting MAGE-A family members is the lack of expression in normal tissues which makes them well-suited for targeted cancer immunotherapy. The challenge with screening the MAGE-A family is to find a specific antibody since the 12-member family has over 60% sequence in homology. In this study, we evaluate multiple MAGE-A3 and MAGE-A4 antibodies using CytoSections, a reproducible alternative to control tissue with an expression of target biomarkers. A series of antibodies specific to each MAGE-A3 and MAGE-A4 protein were discovered and used to screen twenty-five bladder cancer tissues. The results showed that MAGE-A3 was present in 9 of the 25 bladder cancers, while MAGE-A4 was present in 10 of the 25 bladder cancers evaluated; in the meantime, 7 bladder cancers were positive for both targets after double immunofluorescence staining with MAGE-A3 or A4 specific antibodies. The data presented here could be an important development in treatment protocols for pre-screening patients who are at risk. Our findings show that MAGE-A3 and MAGE-A4 can be both present in bladder cancer, suggesting that targeting both genes may be necessary for the success of immunotherapy treatment. Citation Format: Bailey Gilmore, Rachel Gonzalez, Aubrey Su, Dehe Kong, Eden Zewdu, Jina Yom, Tianli Qu, Zhaoying Guo, Xiaomin Hu, Qi Ren, Ranran Zhang, Eric Christenson, Yan Ma, Dezhong Yin, Patrick Yin, Wei Fu, Xuan Liu, Krishnan Allampallam. Colocalization of MAGE-A3 and MAGE-A4 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 999.

Tet-On is a powerful inducible system and a classical tool to regulate gene expression in mammalian cells. It has also been applied to regulate Pol III-driven transcription, such as shRNA or gRNA driven by a U6 or H1 promoter. However, all of the current versions of Tet-On shRNA vectors are based on H1-2O2 or U6-2O2 promoters, which are only compatible with first-generation tetracycline repressor TetR. In addition, these promoters have the problem of driving downstream transcription without the binding of the Tet regulatory protein. Here, we developed a new system that is built upon tetracycline activator protein, Tet-On 3G, combined with a new structure of Tet responsive promoter H1-4O4, which tightly regulates the downstream transcription of gRNA or shRNA. The responsiveness of our system to Doxycycline regulation is dramatically improved compared with the current versions. The new Tet-On system is further optimized into a compact structure to be compatible with the lentivirus package (All-In-One Lenti-Tet-On system), which still keeps the leaky expression at an undetectable level. Combined with all these features, the new generation of the Tet-On system offers broad applications in gene knocking down and genomic editing. Citation Format: Lipeng Wu, Hua Su, Justin Fellows, Mao Fu, Julian Heller, Brian Park, Dezhong Yin. Development of a new All-In-One inducible lentiviral shRNA/gRNA Vector. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3727.

In this paper, BiOBr/BiOCl heterostructure microspheres are prepared successfully via a simple solvothermal way. Research shows that BiOBr/BiOCl microspheres exhibit significantly much higher photocatalytic performance than pure BiOBr or BiOCl. The optimum photodegradation rate of BiOBr/BiOCl for rhodamine B (RhB) is 0.0290 min−1, which is approximately 4.5 times as high as pure BiOBr (k = 0.0065 min−1) and 8.8 times as high as pure BiOCl (k = 0.0033 min−1) under visible light. For this new photocatalyst, the enhanced photoactivity is presumably attributed to the formation of heterojunction which improves the separation of photoinduced electrons and holes to a great extent. Mechanism investigation reveals that O.− plays significant roles during the photocatalysis process.

Neurological diseases and disorders are leading causes of death and disability worldwide. Many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. As the global burden of these pathologies continues to rise there is a significant need for the development of novel therapeutics. Due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. It is now believed that paracrine factors, such as extracellular vesicles (EVs), play a critical role in the functionality associated with stem cells. The diverse biological cargo contained within EVs are proposed to mediate these effects and, to date, the reparative and regenerative effects of stem cell EVs have been demonstrated in a wide range of cell types. While a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the CNS. Here, we report the isolation, characterization, and functional assessment of EVs from two sources of human stem cells, mesenchymal stem cells and induced pluripotent stem cells. We demonstrate the ability of these EVs to enhance the processes of cellular migration and angiogenesis, which are critical for both normal cellular development as well as cellular repair. Furthermore, we investigate their reparative effects on damaged cells, specifically those with relevance to the central nervous system. Collectively, our data highlight the similarities and differences among these EV populations and support the view that stem cells EV can be used to repair or partially reverse cellular damage. Graphical Abstract Stem cell-derived Extracellular Vesicles (EVs) for repair of damaged cells. EVs isolated from human induced pluripotent stem cells and mesenchymal stem cells contribute to the partial reversal of phenotypes induced by different sources of cellular damage.

Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide. Hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13), which was newly identified as a liver lipid droplet associated protein, was recently found to be down-regulated in HCC. However, the effects of HSD17B13 on the underlying mechanisms of HCC have not yet been fully explored. For this purpose, HSD17B13 gene was knockdown with CRISPER/Cas9 technique in several HCC cell lines. HSD17B13 expression was detected by Flow cytometry and Western blotting with recombinant rabbit monoclonal antibodies (clone OTIR3G2 and OTIR5C10) against HSD17B13 which were screened for use in immunohistochemistry, western blot, and ELISA application. Overexpressed HSD17B13 induces hepatic cell line G1 capture and increases several G1 arrest-related protein expression, including p21, p27, MMP2 and MMP3. Then the proliferation and cell cycle were suppressed. HSD17B13 gene knockdown will reverse the suppression. These data indicated that HSD17B13 play important roles in the progression of HCC lesions. HSD17B13 might be a potential immune-oncology marker for HCC. Citation Format: Tianli Qu, Lipeng Wu, Jina Yom, Zhaoying Guo, Xiaomin Hu, Qi Ren, Qian Niu, Ruei-Shiuan Lin, Dezhong Yin, Xuan Liu, Fu Wei. Overexpression of HSD17B13 induced G1 arrest in hepatic cell lines detected by anti-HSD17B13 recombinant rabbit monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3695.

Melanoma-associated antigen gene A (MAGEA) family proteins are expressed in a variety of tumors with each MAGEA protein having unique roles in cancer pathogenesis. The MAGEA members lack of expression in normal tissue and their role in cancer make them well suited for targeted cancer immunotherapy. Thus, knowing which MAGEA protein expression exists in a tumor is important. However, the MAGEA family shares sequence similarity that makes it difficult to find antibodies that are specific to just one family member. It is also challenging to source tissue generally with HIPAA regulation and even more so to get tissue representation of all 12 MAGEA family members. Here we present CytoSections as an alternative to patient control tissues. CytoSections are formalin fixed, paraffin embedded (FFPE) section of over-expression cell pellets. These cells were transfected with expression plasmids coding for genes of individual MAGES family. In this study, CytoSections are used to screen antibodies to all 12-member MAGEA family of proteins. The MAGEA family members were initially shown positive by targeted protein expression in western and immunohistochemistry using DDK tag antibody. Then multiple MAGEA3, MAGEA4, MAGEA9 antibodies were assessed by IHC to determine their specificity to their intended target. Using MAGEA family as a pilot project, we show that CytoSections is a verified, reproducible, and renewable alternative to human control tissues and serves as an ideal tool for antibody specificity assessment. Citation Format: Eden B. Zewdu, Rachel M. Gonzalez, Yan Ma, Derek Ling, Xiaomin Hu, Qi Ren, Ranran Zhang, Li-Hui Lei, Krishnan Allampallam, Dezhong Yin, Xuan Liu, Wei Fu. A renewable IHC control tool CytoSections࣪ for defining MAGEA3, MAGEA4, and MAGEA9 antibody specificity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2803.

Neurodegeneration is a hallmark of many diseases and disorders of the central nervous system (CNS). High levels of neuroinflammation are often associated with irreparable damage to CNS cells due to the dysregulation of signaling cascades that are unable to restore a homeostatic balance. Due to the inherent complexity of the CNS, development of CNS-related therapeutics has met limited success. While stem cell therapy has been evaluated in the context of CNS repair, the mechanisms responsible for their functional properties have not been clearly defined. In recent years, there has been growing interest in the use of stem cell extracellular vesicles (EVs) for the treatment of various CNS pathologies as these vesicles are believed to mediate many of the functional effects associated with their donor stem cells. The potency of stem cell EVs is believed to be largely driven by their biological cargo which includes various types of RNAs, proteins, and cytokines. In this review, we describe the characteristic properties of stem cell EVs and summarize their reported neuroprotective and immunomodulatory functions. A special emphasis is placed on the identification of specific biological cargo, including proteins and non-coding RNA molecules, that have been found to be associated with stem cell EVs. Collectively, this review highlights the potential of stem cell EVs as an alternative to traditional stem cell therapy for the repair of cellular damage associated with diverse CNS pathologies.

Microencapsulated phase change materials (MePCMs) based on swelling polymerization in Pickering emulsion were demonstrated. Monodisperse poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) P(GMA-co-HEMA) particles were prepared by dispersion polymerization. The introduction of hydrophilic monomer HEMA endowed the obtained particles with suitable surface hydrophilicity for stabilizing Pickering emulsion. MePCMs with PGMA–polystyrene interpenetrating composite shell were formed when non-crosslinked particles were employed as stabilizer, while MePCMs with particles-embedded shell were prepared with crosslinked particles as Pickering stabilizer. Phase change property, thermal stability and thermal reliability of two kinds of MePCMs were investigated by TGA, FT-IR, DSC and cycling test. The temperature of 10% weight loss of MePCMs with interpenetrating shell was 11 °C higher than that of MePCMs with particles-embedded shell. After cycling test, the percentage of leached core materials from MePCMs with interpenetrating shell was approximately one-third of that from MePCMs with particles-embedded shell. The results showed that MePCMs with an integral shell presented better thermal stability, tightness and thermal reliability than MePCMs with particles-embedded shell. The research developed a simple process for MePCMs with excellent performance.

A polymerized high internal phase emulsion (polyHIPE) was prepared using styrene, divinylbenzene and butyl acrylate as raw material and employed as stationary phase of thin layer chromatography (TLC). SEM, mercury intrusion porosimetry and FT-IR spectrum were employed to characterize the pore structure and chemical composition. To verify the feasibility as stationary phase of TLC, identification of plant extracts, Chinese herbs and traditional Chinese medicine were practiced on the prepared polyHIPE. Relative retardation factor (α), theoretical plate number (Nplate) and resolution (R) were used to evaluate the performance of TLC. Large difference in α value make it is feasible to optimize the chromatographic condition. The resolution R and Nplate indicates that the introduction of BA in polyHIPE facilitates the preparation and improves the performance for TLC separation. Our results show that polyHIPE monolith is a reusable stationary phase of TLC with high performance.

In this paper, BiOBr/BiOCl/CQDs heterostructure microspheres were successfully synthesized via a facile solvothermal method. Then, the structures, morphologies, optical properties and photocatalytic performances were investigated. This research showed that the BiOBr/BiOCl/CQDs microspheres exhibited significantly enhanced photocatalytic performance compared with BiOBr/BiOCl. In the photocatalysis process of rhodamine B (RhB) under visible light irradiation, the highest photodegradation rate (0.0609 min−1) would be obtained when the weight percentage of CQDs was appropriate, which was about 2.8 times higher than that of BiOBr/BiOCl (0.0217 min−1). In this photocatalytic system, the enhanced photoactivity was mainly attributed to the heterojunction interface among CQDs, BiOBr and BiOCl, and enhanced light harvesting for the appropriate CQD introduction. The radical trapping experiments revealed that O2˙−, e− and h+ were the main active species during the photocatalysis process.

Surface-initiated activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) was applied to graft poly(glycidyl methacrylate) (PGMA) brushes from macroporous hydrogel templating from oil-in-water high internal phase emulsion (O/W HIPE), and the grafted hydrogel was further heparinized as stationary phase of affinity chromatography for Enterovirus 71 (EV71) purification. Acrylate functional monomer containing isobutyl bromide (HEMA-BiBB) was synthesized and introduced into oil phase for macroporous hydrogel with ATRP initiator-anchored surface. With the increase of HEMA-BIBB dosage, the average pore size of hydrogel increased and the specific surface area decreased. Bromine atomic percent of hydrogel reached the maximum of 1.56 % when the HEMA-BiBB dosage was 1.0 g. The corresponding heparinized hydrogel possessed the highest amount of carboxyl group (56.8 μmol per column). Interconnected macropore (1.71 μm) and high porosity (84.2 %) of heparinized hydrogel ensured an excellent permeability (2.1 × 10−13 m2). Meanwhile, grafted PGMA brushes afforded abundant epoxy groups for immobilizing high density of heparin, which endowed hydrogel with outstanding adsorption capability for EV71. Immobilized heparin ligands were competent in specific capture of EV71 by receptor-ligand interaction. Besides, enhanced hydrophilicity of heparinized hydrogel avoided the irreversible and nonspecific adsorption of proteins. As a result, heparinized hydrogel demonstrated superior adsorption selectivity and capability for EV71 (dynamic adsorption capacity: 1275 ng per column, recovery: 86.2 %). The developed heparinized PGMA-grafted macroporous hydrogel was promising to be applied in the large-scale production of EV71 for vaccine industry.

Monolithic macroporous hydrogels was prepared from oil-in-water high internal phase emulsions (HIPEs) and heparinized using polydopamine (PDA) and polyethyleneimine (PEI) as mediators, and then used as a support of affinity chromatography to purify Enterovirus 71 (EV71) from culture supernatant. Our innovative strategy was characterized by two remits. (1) Interconnected macropores (1–5 μm) and high porosity (76–93%) of hydrophilic hydrogels ensured an excellent permeability (6.95 × 10−13 m2); (2) PDA/PEI composite provided abundant amino groups to immobilize heparin on macroporous hydrogels and increased the amount of carboxyl group to 46.5 μmol per column. A selective recognition of EV71 was achieved by receptor-ligand interaction between heparin and EV71. The adsorption capacity for EV71 was 986.0 ng per column and the recovery for EV71 reached a maximum of 79.9%. It was verified that these high performances were ascribed to the high density of heparin immobilized via PDA and PEI as mediators. Our innovative strategy provided a useful paradigm as a preparation method for heparin-affinity chromatographic medium for virus purification.

Herein we present a systematic study of the antagonistic effect of nanoparticles and nonionic surfactant on pore structure of polymerized high internal phase emulsion (polyHIPE). Interconnected polyHIPE with small voids and large RSD in void size was obtained by surfactant-stabilized emulsion, while a close-cellar polyHIPE with large voids and low RSD in void size was prepared by particles-stabilized emulsion. This antagonistic effect was originated from the different stabilizing mechanism of two different stabilizers. Particles improve the stability of HIPE, and resultantly, endow the polyHIPE with excellent homogeneity; On contrary, surfactant lead to the formation of interconnected void. To obtain polyHIPE with interconnected homogeneous voids, an optimal ratio of surfactant/particle is necessary. The interconnectivity of polyHIPE was first investigated by the loading and releasing property of polyHIPE.

We propose a novel route for the stabilization of oil-in-water Pickering emulsions using inherently hydrophilic nanoparticles. In the case of dialkyl adipate oils, in situ hydrophobisation of the particles by dissolved oil molecules in the aqueous phase enables stable emulsions to be formed. Emulsion stability is enhanced upon decreasing the chain length of the oil due to its increased solubility in the precursor aqueous phase. The oil thus acts like a surfactant in this respect in which hydrogen bonds form between the carbonyl group of the ester oil and the hydroxyl group on particle surfaces. The particles chosen include both fumed and precipitated anionic silica and cationic zirconia. Complementary experiments including relevant oil-water-solid contact angles and infra-red analysis of dried particles after contact with oil support the proposed mechanism.

In this paper, poly(n-butyl acrylate)-g-poly(methyl methacrylate) multigraft copolymers were synthesized by macromonomer technique and miniemulsion copolymerization. The PMMA macromonomers were obtained by an activator generated by electron transfer atom transfer radical polymerization (AGET ATRP) in emulsion system and subsequent allylation. Then the copolymerization of different macromonomers with nBA was carried out in miniemulsion system, obtaining multigraft copolymers with high molecular weight. The latex particles and distribution of emulsion AGET ATRP and miniemulsion copolymerization were characterized using laser light scattering. The molecular weight and polydispersity indices of macromonomers and multigraft copolymers were analyzed by gel permeation chromatography, and the number-average molecular weight range is 187,600–554,800 g/mol for PnBA-g-PMMA copolymers. In addition, the structural characteristics of macromonomer and brush-like copolymers were determined by infrared spectra and 1H nuclear magnetic resonance spectroscopy. The thermal performance of brush-like copolymers were characterized by differential scanning calorimetry and thermogravimetric analysis. Atomic force microscopy results showed that the degree of microphase separation was varying with increasing PMMA content in PnBA-g-PMMA. The dynamic rheometer analysis revealed that multigraft copolymer with PMMA content of 31.4% exhibited good elastomeric properties to function as a TPE. These multigraft copolymers show a promising low cost and environmental friendly thermoplastic elastomer.

Core–shell polymeric microspheres of tens microns in size with perfect spherical virus-like surface morphology were prepared through Pickering emulsion polymerization. Pickering stabilizer, monodisperse Poly(Glycidyl methacrylate) (PGMA) microsphere were obtained by two-stage dispersion polymerization and acid catalyzed hydrolysis was employed to transform the epoxy groups into glycol groups, rendering the P(GMA) microspheres moderately hydrophilic to stabilize Pickering emulsion. The P(GMA) microspheres and core–shell microspheres were characterized by scanning electronic microscope(SEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering(DLS). The results show that monodisperse P(GMA) microspheres were arrayed in a compact hexagonal arrangement on the surface of core–shell microspheres, forming a perfect spherical virus-like surface morphology.

Three-dimensional “organoid” growth of tumors may represent a more physiologically relevant in vitro model system than traditional two-dimensional monolayer cultures of cancer cell lines. With the increased availability of cryopreserved human cancer organoids generated by academic laboratories, large-scale biobanking initiatives, and commercial sources, there is an unmet need for simplified, standardized, and cost-effective methods for preparation of the complex growth media required by these models. Human organoid culture media contains a variety of recombinant proteins, small molecules, and other growth factors that are costly to purchase in small-scale, time consuming to reconstitute and aliquot, and have varying stability and shelf life once prepared. Organoid culture media often also utilizes undefined conditioned media (CM) from one or more engineered cell lines that must be cultured separately, requiring additional time and resources to maintain. These lines secrete critical growth factors and the CM generated must be carefully prepared, collected, and stored. CM is subject to variability in activity levels due to batch-to-batch and protocol-to-protocol differences that can affect subsequent organoid culture performance. To address these challenges and to facilitate the wide-spread adoption of human organoid culture we are developing reagent kits containing essential growth medium components in an individually lyophilized format for long-term storage and easy single-use preparation of organoid growth media for a variety of tissue types including human colon, mammary, and lung. We compared growth medium formulated with our components with small-scale “homebrew” preparations and found equivalent or better culture performance (as calculated by doubling times) and similar morphology. Both approaches were able to generate media capable of supporting growth for at least three passages (~30 days) in 15 different organoid models. Additionally, we compared protocols and cell lines for CM generation to support organoid culture and found that an optimized, scaled-up production method could generate Wnt-3a and R-spondin1 CM with higher activity levels (up to 52%) and reduced lot-lot variability in comparison with commonly used small-scale protocols. We found that the optimized protocols produced CM that was stable for at least 2 months at 4°C and at least 6 months at -20°C, and was resilient against freezing and thawing. Previously frozen CM was also able to support organoid growth in culture. These results show that our tissue-specific kits and ready-to-use CM can provide a simplified, cost effective method to support the long-term propagation of human cancer organoids from multiple tissues and cancer types. Citation Format: James Clinton, Allison Ruchinskas, Himanshi Desai, Nanda Mahashetty, Dezhong Yin. Propagation of human colon, mammary, and lung cancer organoids in growth medium utilizing tissue-specific reagent kits and ready-to-use Wnt-3a and R-Spondin1 conditioned media [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 51.

Exosomes are subcellular particles 50-200 nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. Exosomes are currently being evaluated as potential diagnostic tools in a number of diseases, including cancer. Exosomes are stable carriers of cell-free cargo in the form of DNA, RNA, and protein, thereby making them an attractive candidate for early detection of cancer via liquid biopsy. Tumor-derived exosomes have been linked to stimulation of tumor cell growth, angiogenesis, metastasis, and suppression of the immune system. However, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards for research and diagnostic applications. We have developed a novel method employing tangential flow filtration for the isolation of large quantities of highly purified, ready to use exosomes from cell culture supernatant. Exosomes from a variety of cancer cell lines representing the most prevalent cancer types, including prostate (LNCaP), colorectal (HCT116), breast (MDA-MB-231, MCF-7), lung (A549), cervical (HeLa), and glioblastoma (U87-MG) were isolated and characterized in this study. We employed a stringent quality control approach in order to define and characterize these exosomes. Our method generated a high yield (>1×1010 exosomes/mL) and average protein equivalent of 2 mg/mL. Our data demonstrated expression of a number of different characteristic exosomal proteins including tetraspanin, which was confirmed through western blot and PCR. Isolated exosomes had a median size of 102 nm through nanoparticle tracking analysis. Western blot data revealed cell line specific protein marker expression of CD63, CD81, flotillin 1, Alix and TSG101 protein. We assessed the functionality of these purified exosomes through cellular uptake, migration assay, and anchorage independent growth assay in this study. We also evaluated exosomal RNA from these purified exosomes for the presence of common mutations associated with cancer through PCR. Here we report that exosomes isolated by our method not only had high exosome yield and quality, but also retained relevant functionality and thereby make ideal reference standards/controls in the field of cancer research and diagnostics development. Citation Format: Siddhartha Paul, Nanda Mahashetty, Joy Wells, Dezhong Yin, Fang Tian. Development and characterization of cancer cell line exosomes as reference standards in cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-038.

In this work, Fe3O4/P(acrylic acid-methyl methacrylate-N-isopropylacrylamide) (Fe3O4/P(AA-MMA-NIPAm)) thermoresponsive magnetic composite microspheres have been prepared by controlled radical polymerization in the presence of 1,1-diphenylethene (DPE). The shell thickness of thermosensitive polymer (PNIPAm), which was on the surface of the microspheres, can be controlled by using DPE method. The morphology and thermosensitive properties of the composite microspheres, polymerization mechanism of the shell were characterized by TEM, FTIR, VSM, Laser Particle Sizer, TGA, NMR, and GPC. The microspheres with narrow particle size distribution show high saturation magnetization and superparamagnetism. The thermosensitive properties of the composite microspheres can be adjusted indirectly via controlling the addition amount of monomer (NIPAm) in the second step during controlled radical polymerization. Phenolphthalein was chosen as a model drug to investigate drug release behavior of the thermoresponsive magnetic composite microspheres with different shell thickness. Controlled drug release testing reveals that the release behavior depends on the thickness of polymer on the surface of the microspheres.

The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

Facile one-step Pickering emulsion polymerization was employed to prepare magnetic polymer microspheres with high magnetism and armored inorganic particles. Partially hydrophilic CTAB-modified Fe 3 O 4 particles were employed as stabilizer of Pickering emulsion and were armored on the as-prepared microspheres, while totally hydrophobic oleic acid-modified Fe 3 O 4 particles were encapsulated in the obtained microspheres. The microspheres were characterized by scanning electron microscopy (SEM), energy dispersive X-ray microanalyses (EDX) and magnetic measurements. Total Fe 3 O 4 , encapsulated Fe 3 O 4 and armored Fe 3 O 4 were detected and catalytic activity of microspheres for Fenton reaction was evaluated. The results showed that steady barrier formed by CTAB-modified Fe 3 O 4 on droplet surface can prevent oleic acid-modified Fe 3 O 4 from escaping away the polymerization vessel, which lead to an efficient encapsulation to oleic acid-modified Fe 3 O 4 . The encapsulated Fe 3 O 4 enables the microspheres to be separated by external magnetic field and the armored Fe 3 O 4 endows the composites with special catalytic property.

One-dimensional (1D) magnetic Fe(3)O(4)/P(GMA-DVB) peapod-like nanochains have been successfully synthesized by magnetic-field-induced precipitation polymerization using Fe(3)O(4) as building blocks and P(GMA-DVB) as linker. The Fe(3)O(4) microspheres without surface modification can be arranged with the direction of the external magnetic field in a line via the dipolar interaction between Fe(3)O(4) microspheres and linked permanently via P(GMA-DVB) coating during precipitation polymerization. The length of peapod-like nanochains can be controlled by magnetic field intensity, and the thickness of polymer shell can be tuned by the amount of monomers. Magnetic measurement revealed that these 1D peapod-like nanochains showed highly magnetic sensitivity. In the presence of magnetic field, 1D magnetic Fe(3)O(4)/P(GMA-DVB) peapod-like nanochains can be oriented and aligned along the direction of external magnetic field.

A magnetically separable Fe 3 O 4 –NH 2 –Pd (0) catalyst was easily synthesized by immobilizing Pd nanoparticles on the surface of magnetic Fe 3 O 4 –NH 2 microspheres. It was found that the combination of Fe 3 O 4 and triethylene tetramine (TETA) could give rise to structurally stable catalytic sites. Furthermore, the high-magnetization Fe 3 O 4 –NH 2 –Pd(0) catalyst can be recovered by magnet and reused for six runs for Heck reaction without significant loss in catalytic activity.

Pickering emulsion polymerization is of particular interest as a simple one-step method to prepare inorganic-covered microspheres. In this article, droplets with different particle coverages of

To meet the need for improved approaches to study cancer in vitro, there has been a surge in the development of novel research models utilizing advanced culture methods. These methods permit in vitro growth of cancer types previously not possible, and/or models with enhanced in vivo relevancy compared to traditional continuous cell lines. However, availability of these early-stage research models is currently limited and there is a lack of data on the ability to scale up production of these models to support the needs of the global research community. We sought to investigate the protocols, expansion capacity, cryopreservation ability, genetic stability, and feasibility of larger-scale bioproduction of a subset of the models generated by the Human Cancer Models Initiative (HCMI), an international collaborative effort between the National Cancer Institute, the foundation Hubrecht Organoid Technology, Cancer Research UK, and the Wellcome Trust Sanger Institute. The HCMI's initial goal is the development of 1,000 novel human cancer models, paired with bioinformatics and patient clinical data, particularly from rare or underrepresented cancer types. One advanced culture method being utilized, three-dimensional organoid “microtissue” culture, potentially poses challenges for traditional large-scale bioproduction processes. It requires growth embedded within an undefined extracellular matrix and complex media formulations containing multiple small molecules and recombinant proteins with unknown stability and shelf-life. Additionally, organoid growth media typically includes multiple sources of undefined conditioned media containing critical growth factors. We cultured organoid models derived from human colon, pancreas, esophagus, and mammary tissues developed by laboratories contributing to the HCMI. Multiple unique donors were available for all tissues and both cancer and non-cancer models were available for two tissue types. Most models were maintained in culture continuously for at least 60 days (7-27 population doublings, > 10 passages). Tissue and donor variability was evident in model characteristics, including morphology (assessed by microscopy and immunocytochemistry), growth rate, and genetic stability (measured by short tandem repeats analysis). All models were amenable to scale up beyond multiwell plates, and all models could recover from cryopreservation. While organoid culture represents a significant divergence from typical two-dimensional monolayer culture of continuous cell lines, our results show that these next-generation in vitro models are suitable for larger-scale bioproduction. This is vital to ensure the widespread availability of these models within the research community to facilitate applications like pre-clinical drug discovery and basic cancer research. Citation Format: James Clinton, Penney McWilliams-Koeppen, Siddhartha Paul, Allison Ruchinskas, Dezhong Yin, Robert Newman. Feasibility of manufacturing-scale bioproduction of novel next-generation 3D organoid cancer models in support of the Human Cancer Models Initiative [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5012.

Bone marrow transplantation can provide an effective cell-based strategy to enhance bone repair. However, the fate of implanted cells and the extent of their contribution to bone osteoinduction remain uncertain. To define the fate of bone marrow–derived cells and their contribution in vivo, we used a bone-specific collagen I promoter (2.3Col) driving green fluorescent protein (GFP) (2.3ColGFP) within a lentiviral vector. Prior to in vivo cell fate determination, we verified a high efficiency of lentiviral transduction in human bone marrow stromal cells (hBMSCs), without altering the proliferation or differentiation potential of these cells. We showed that the 2.3ColGFP marker responded to endogenous transcriptional regulation signals. In a mouse ossicle model, we demonstrated that the 2.3ColGFP marker is able to specifically define human bone marrow–derived stem cells that enter the osteoblast lineage in vivo. In addition, cells labeled with 2.3ColGFP with the donor origin, directly make a major contribution to bone formation. Furthermore, we also demonstrated in a calvarial defect model that a mixture of human bone marrow–derived populations, have stronger bone regenerative potential than that of hBMSCs, and an optimal dose is required for bone regeneration by the mixed populations.

Insulin counterregulates catecholamine action in part by inducing the sequestration of beta2-adrenergic receptors. Although similar to agonist-induced sequestration, insulin-induced internalization of beta2-adrenergic receptors operates through a distinct and better-understood cellular pathway. The effects of insulin treatment on the function and trafficking of both beta1- and beta2-adrenergic receptors were tested. The beta2-adrenergic receptors were counterregulated and internalized in response to insulin. The beta1-adrenergic receptors, in sharp contrast, are shown to be resistant to the ability of insulin to counterregulate function and induce receptor internalization. Using chimeric receptors composed of beta1-/beta2-adrenergic receptors in tandem with mutagenesis, we explored the role of the C-terminal cytoplasmic tail of the beta2-adrenergic receptors for insulin-induced counterregulation. Substitution of the C-terminal cytoplasmic tail of the beta2-adrenergic receptor on the beta1-adrenergic receptor enabled the chimeric G protein-coupled receptor to be functionally and spatially regulated by insulin. Truncation of the beta2-adrenergic receptor C-terminal cytoplasmic tail to a 15-amino acid motif harboring a potential Src homology 2-binding domain at Y350 and an Akt phosphorylation site at S345,346 was sufficient to enable receptor regulation by insulin.

Validation of antibiotic mode of action in whole bacterial cells is a key step for antibiotic drug discovery. In this study, one potential drug target, enoyl-acyl carrier protein reductase (FabI), an essential enzyme in the fatty acid biosynthesis pathway, was used to evaluate the feasibility of using a regulated antisense RNA interference approach to determine antibiotic mode of action. Antisense isogenic strains expressing antisense RNA to fabI were created using a tetracycline-regulated vector in Staphylococcus aureus. We demonstrated that down-regulation of FabI expression by induction of fabI antisense RNA induces a conditional lethal phenotype. In contrast, partial down-regulation gives a viable cell with a significant increase in sensitivity to FabI-specific inhibitors (i.e., a sensitized phenotype). More importantly, the mode of action for novel FabI inhibitors has been confirmed using this genetic approach in whole cell assay. These results indicate that controlled antisense technology provides a robust tool for defining and tracking the mode of action of novel antibacterial agents.

International audience; The present study was conducted to determine whether porcine somatotropin (pST) differentially regulates expression of the GLUT4 and fatty acid synthase (FAS) genes in pig adipose tissue. Three different experiments were conducted in which pigs were treated daily with different doses of pST for different time periods (7 or 14 d and from 60 to 90 kg of body wt). In these experiments, pST significantly and consistently decreased FAS mRNA levels (80%, 66% and 85%, respectively); however, GLUT4 mRNA was not affected by pST in two of the three experiments, and in the one showing an effect (Experiment 2), the decrease was less than observed for FAS (44%). Because of these results, we conducted subsequent experiments to see if the effects of pST on glucose metabolism in cultured pig adipose tissue (48 h) differed when glucose concentrations were changed from 1 to 5 mmol/L. These studies revealed that the antagonistic effect of pST on insulin action was more potent when glucose transport was saturated (5 mmol/L) than when glucose concentration limited glucose entry into the cell (1 mmol/L). In summary, these results suggest that the effects of pST on glucose transport in pig adipocytes are secondary to changes elicited by the hormone on intracellular glucose use for lipogenesis. When considered in the context of the decrease previously observed in glucose transport in pig adipocytes, the findings reported herein suggest that pST acts to decrease GLUT4 protein activity and/or distribution between the plasma membrane and the intracellular pool with little alteration in GLUT4 gene expression or total cell GLUT4 protein.

Hormones that activate receptor tyrosine kinases have been shown to regulate G protein-coupled receptors, and herein we investigate the ability of IGF-I to regulate the beta(1)-adrenergic receptor. Treating Chinese hamster ovary cells in culture with IGF-I is shown to functionally antagonize the ability of expressed beta(1)-adrenergic receptors to accumulate intracellular cAMP in response to stimulation by the beta-adrenergic agonist Iso. The attenuation of beta(1)-adrenergic action was accompanied by internalization of beta(1)-adrenergic receptors in response to IGF-I. Inhibiting either phosphatidylinositol 3-kinase or the serine/threonine protein kinase Akt blocks the ability of IGF-I to antagonize and to internalize beta(1)-adrenergic receptors. Mutation of one potential Akt substrate site Ser412Ala, but not another Ser312Ala, of the beta(1)-adrenergic receptor abolishes the ability of IGF-I to functionally antagonize and to sequester the beta(1)-adrenergic receptor. We also tested the ability of IGF-I to regulate beta(1)-adrenergic receptors and their signaling in adult canine cardiac myocytes. IGF-I attenuates the ability of beta(1)-adrenergic receptors to accumulate intracellular cAMP in response to Iso and promotes internalization of beta(1)-adrenergic receptors in these cardiac myocytes.

Background: Mammalian receptors that couple to effectors via heterotrimeric G proteins (e.g., beta 2 -adrenergic receptors) and receptors with intrinsic tyrosine kinase activity ( e.g ., insulin and IGF-I receptors) constitute the proximal points of two dominant cell signaling pathways. Receptors coupled to G proteins can be substrates for tyrosine kinases, integrating signals from both pathways. Yeast cells, in contrast, display G protein-coupled receptors ( e.g ., alpha-factor pheromone receptor Ste2) that have evolved in the absence of receptor tyrosine kinases, such as those found in higher organisms. We sought to understand the motifs in G protein-coupled receptors that act as substrates for receptor tyrosine kinases and the functional consequence of such phosphorylation on receptor biology. We expressed in human HEK 293 cells yeast wild-type Ste2 as well as a Ste2 chimera engineered with cytoplasmic domains of the beta 2 -adrenergic receptor and tested receptor sequestration in response to activation of the insulin receptor tyrosine kinase. Results: The yeast Ste2 was successfully expressed in HEK 293 cells. In response to alpha-factor, Ste2 signals to the mitogen-activated protein kinase pathway and internalizes. Wash out of agonist and addition of antagonist does not lead to Ste2 recycling to the cell membrane. Internalized Ste2 is not significantly degraded. Beta 2 -adrenergic receptors display internalization in response to agonist (isoproterenol), but rapidly recycle to the cell membrane following wash out of agonist and addition of antagonist. Beta 2 -adrenergic receptors display internalization in response to activation of insulin receptors ( i.e ., cross-regulation), whereas Ste2 does not. Substitution of the cytoplasmic domains of the β2-adrenergic receptor for those of Ste2 creates a Ste2/beta2-adrenergic receptor chimera displaying insulin-stimulated internalization. Conclusion: Chimera composed of yeast Ste2 into which domains of mammalian G proteincoupled receptors have been substituted, when expressed in animal cells, provide a unique tool for study of the regulation of G protein-coupled receptor trafficking by mammalian receptor tyrosine kinases and adaptor proteins.

The objective of this study was to investigate the effect of individual PUFAs on LDL receptor (LDLr) expression in human fibroblasts and HepG2 cells, and to evaluate whether acyl CoA:cholesterol acyltransferase (ACAT) and sterol regulatory element-binding protein 1 (SREBP-1) were involved in the regulation of LDLr expression by fatty acids. When fibroblasts and HepG2 cells were cultured with serum-free defined medium for 48 h, there was a 3- to 5-fold (P 0.05) increase in LDLr protein and mRNA levels. Incubation of fibroblasts and HepG2 cells in serum-free medium supplemented with 25-hydroxycholesterol (25OH-cholesterol, 5 mg/L) for 24 h decreased LDLr protein and mRNA levels by 50 -90% (P 0.05). Arachidonic acid (AA, 20:4(n-6)), EPA (20:5(n-3)), and DHA (22:6(n-3)) antagonized the depression of LDLr gene expression by 25OH-cholesterol and increased LDLr protein abundance 1- to 3-fold (P 0.05), but had no significant effects on LDLr mRNA levels. Oleic (18:1), linoleic (18:2), and -linolenic acids (18:3(n-3)) did not significantly affect LDLr expression. ACAT inhibitor (58 - 035, 1 mg/L) attenuated the regulatory effect of AA on LDLr protein abundance by 40% (P 0.05), but did not modify the regulatory effects of other unsaturated fatty acids in HepG2 cells. The present results suggest that AA, EPA, and DHA increase LDLr protein levels, and that ACAT plays a role in modulating the effects of AA on LDLr protein levels. Furthermore, the effects of the fatty acids appeared to be independent of any change in SREBP-1 protein. J. Nutr. 135: 2541-2545, 2005.

Conjugated linoleic acid (CLA) exerts anticarcinogenic and antiatherosclerotic effects in animals. The present study was conducted to examine the effects of CLA on LDL receptor (LDLr) expression in HepG2 cells, and to evaluate whether the sterol response element binding protein 1 (SREBP-1) and acyl CoA:cholesterol acyltransferase (ACAT) were involved in the regulation of LDLr expression by CLA. When HepG2 cells were cultured with serum-free DMEM for 48 h, there was a three- to fivefold (P

Human gamma-glutamyl hydrolase (hGH) plays an important role in the metabolism of folic acid and the pharmacology of antifolates such as methotrexate. We have previously cloned and characterized hGH cDNA and its gene. We report here that the levels of hGH gene expression are high in tissues such as liver, kidney, and placenta as determined by Northern blot and RT-PCR analyses. In contrast, hGH expression is relatively low in spleen, lung, small intestine, and peripheral blood leukocytes. In addition, high levels of hGH mRNA were detected in most cancer cell lines examined. There was no significant difference in hGH mRNA levels between breast cancer tissues and their normal counterparts, although breast cancer tissues generally appeared to have heterogeneous expression of hGH mRNA. Deletion analysis and transient transfection assays were performed. A positive regulatory element located from -52 to +4 relative to the transcriptional start site was found to be required for basal promoter activity in both HepG2 and MCF-7 cells. Also, transcriptional inhibitory elements were found at -96 to-52 and +88 to+104 in MCF-7 cells, but not in HepG2 cells. These data provide novel insights into the regulation of hGH gene transcription in HepG2 and MCF-7 cells.

How to balance high efficiency of transgene expression with low cytotoxicity is a central concern for cell biology. Developing lipid-based transfection reagents that have those attributes and can be conveniently used for multiple cell types is critical for studies in cancer biology and cell biology. To address these concerns, ATCC has developed two lipid-based transfection reagents, GeneXPlus and TransfeX™ that provide a powerful tool for efficiently transfecting a broad spectrum of cell types including continuous cancer cell lines, stem cells, immortalized cells, and primary cells with little or no cytotoxicity. For recognized hard-to-transfect cancer cell lines, like THP-1, SH-SY5Y, and Raw 264.7 cells, we have achieved over 30% GFP expression without obvious cytotoxicity by using the GeneXPlus and an EF1α-GFP vector. TransfeX™ has been found to be highly suitable for the transfection of stem cells, immortalized cell lines, and many types of primary cells such as mammary, renal and bronchial epithelial cells, dermal microvascular endothelial cells, and human dermal fibroblasts. We have developed optimized transfection protocols for the TransfeX™ transfection reagent for different cell types and achieved over 50% transfection efficiency and low cytotoxicity. ATCC will provide data showing the broad spectrum of cell types successfully transfected with either GeneXPlus or TransfeX™. Citation Format: Dezhong Yin, Joy Wells, James Clinton, Lysa-Anne Volpe, Kevin Grady, Jeanmarie Curley. Development of lipid-based transfection reagents for efficient expression of transgenes in hard-to-transfect cell types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-248. doi:10.1158/1538-7445.AM2014-LB-248

γ-Glutamyl hydrolase (GH) plays an important role in the metabolism of folic acid and the pharmacology of antifolates such as methotrexate. We have previously cloned and characterized the human GH cDNA. In this report, the complete organization and structure of the human GH gene was determined. The human GH gene spans 24 kb in the human genome, with nine exons sized from 51 to 371 bp. All of exon–intron splice junctions follow the GT–AG rule. The sequence upstream of exon 1 consists of a promoter-like, GC-rich region and a number of putative cis active elements including Sp1, AP1, and MZF1 sites. A TATA sequence in the 5′ region of human GH gene was not observed, similar to housekeeping genes known to be tissue-specific and differentially expressed. S1 nuclease protection analysis with human liver, prostate, brain, and mammary gland revealed a major transcription start point at nucleotide −125 relative to the ATG start codon and several minor transcription start points. Analysis of GH cDNA isolated from human liver indicated a nucleotide change, T→C, in the leader sequence of GH, which suggested a polymorphism. Studies of cDNA from different human tissue sources provided evidence that there is a single spliced cDNA species in human.

Somatotropin (ST) markedly decreases lipogenesis, fatty acid synthase (FAS) enzyme activity and mRNA abundance in pig adipocytes. The present study was conducted to determine whether the decrease in FAS mRNA in 3T3-F442A adipocytes was the result of a decrease in transcription of the FAS gene and/or a change in FAS mRNA stability. Insulin increased the abundance of FAS mRNA 2–13-fold and fatty acid synthesis 3–7-fold. Somatotropin decreased the stimulatory effect of insulin on the abundance of FAS mRNA and lipogenesis by 40–70% and 20–60% respectively. Subsequent run-on analyses demonstrated that the decrease observed in FAS mRNA in response to ST was associated with an 82% decrease in transcription; ST significantly shortened the half-life of FAS mRNA from 35 to 11 h. To corroborate the run-on analyses, cells were stably transfected with a pFAS–CAT5 (in which CAT stands for chloramphenicol acetyltransferase) reporter construct that contained 2195 bp of the 5´ flanking region of the rat FAS gene. Insulin treatment increased FAS–CAT activity 4.7-fold. When ST was added to the insulin-containing medium there was an approx. 60% reduction in FAS–CAT activity. In summary, our results indicate that ST decreases FAS mRNA levels and that this is the result of a marked decrease in both transcription of the FAS gene and stability of the FAS mRNA.

Background: Wnt3a regulates a canonical signaling pathway in early development that controls the nuclear accumulation of β-catenin and its activation of Lef/Tcf-sensitive transcription of developmentally important genes. Results: Using totipotent mouse F9 teratocarcinoma cells expressing Frizzled-1 and biochemical analyses, we detail the influence of Wnt3a stimulation on the expression, complexation, and subcellular trafficking of key signaling elements of the canonical pathway, i.e., Dishevelled-2, Axin, glycogen synthase kinase-3β, and β-catenin. Cellular content of β-catenin and Axin, and phosphoglycogen synthase kinase-3β, but not Dishevelled-2, increases in response to Wnt3a. Subcellular localization of Axin in the absence of Wnt3a is symmetric, found evenly distributed among plasma membrane-, cytosol-, and nuclear-enriched fractions. Dishevelled-2, in contrast, is found predominately in the cytosol, whereas β-catenin is localized to the plasma membrane-enriched fraction. Wnt3a stimulates trafficking of Dishevelled-2, Axin, and glycogen synthase kinase-3β initially to the plasma membrane, later to the nucleus. Bioluminescence resonance energy transfer measurements reveal that complexes of Axin with Dishevelled-2, with glycogen synthase kinase- 3β, and with β-catenin are demonstrable and they remain relatively stable in response to Wnt3a stimulation, although trafficking has occurred. Mammalian Dishevelled-1 and Dishevelled-2 display similar patterns of trafficking in response to Wnt3a, whereas that of Dishevelled-3 differs from the other two. Conclusion: This study provides a detailed biochemical analysis of signaling elements key to Wnt3a regulation of the canonical pathway. We quantify, for the first time, the Wnt-dependent regulation of cellular abundance and intracellular trafficking of these signaling molecules. In contrast, we observe little effect of Wnt3a stimulation on the level of protein-protein interactions among these constituents of Axin-based complexes themselves.