Secondary Sphere Research Articles

Abstract 1907: Claudin 6 is a carcinoembryonic antigen with cancer stem cell marker features

Abstract Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers, such as ovarian cancer (OC) and testicular cancer (TC). A monoclonal antibody against CLDN6, IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. In this series of nonclinical studies, we investigated CLDN6 expression in normal and cancer tissues, as well as the localization and possible function of CLDN6 in cancer cells. Methods Expression of CLDN6 was assessed in a wide range of human tissues (eg, lung, colon, skin, ovary) and cultured cells by quantitative RT-PCR, immunohistochemistry (IHC), flow cytometry, and western blotting. To investigate the effect of dedifferentiation on CLDN6 expression, human-induced pluripotent cells were generated by transfecting foreskin fibroblasts with a reprogramming cocktail, and then CLDN6 expression was evaluated. To characterize CLDN6 as a potential novel marker to identify cancer stem cells (CSCs) in OC, coexpression of CLDN6 with known CSC surface markers were analyzed by flow cytometry, and CLDN6+ and CLDN6-negative cells were tested in colony formation and sphere formation assay. Human OC cell lines were transplanted intraperitoneally into nude mice and assessed for metastasis to investigate tumorigenecity of CLDN6+ cells. Results Except for low mRNA levels measured in placenta, testis, umbilical cord, cerebellum, and lung samples, no CLDN6 (mRNA or protein) was detected in the vast majority of normal tissues. Additionally, there was also a lack of CLDN6 protein expression in tissue zones where stem cells for tissue homeostasis would normally be found as determined by IHC with an anti-CLDN6 antibody. CLDN6 was expressed on the cell surface of several solid tumors, including ovarian, testicular, uterine, and lung cancer tissues; OC and TC samples had high level expression. CLDN6 expression was strongly activated in human-induced pluripotent stem cells generated from fibroblasts. CLDN6 showed selective coexpression with known CSC markers such as CD44, CD24, and CD90 in OC and TC cell lines. In addition, some CLDN6+ OC cells exhibited CSC-like behavior in vitro: CLDN6+ populations were clonogenic and formed well-defined spheres in low attachment conditions; these spheres had the ability to self-renew into secondary spheres. Analysis of OC metastases in mouse xenografts showed when xenografts were generated by OC cells that had <10% of CLDN6+ cells, the metastases were enriched in CLDN6+ cells, suggesting CLDN6+ cells had selective growth advantage. Conclusions CLDN6 is a cancer cell-specific surface molecule aberrantly expressed in several cancers, and its expression may be an identifier for cells with CSC-like traits. These characteristics make CLDN6 an attractive target candidate for tumor-specific therapeutic antibodies. Citation Format: Özlem Türeci, Meike Wagner, Claudia Paret, Maria M. Kreuzberg, Stefan Wöll, Korden Walter, Sabine C. Häcker, Ikumi Nakajo, Tomohiro Yamada, Ugur Sahin. Claudin 6 is a carcinoembryonic antigen with cancer stem cell marker features [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 1907.

Abstract 896: Adjutant therapeutic strategy for sorafenib-resistant hepatocellular carcinoma

Abstract Purpose: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is the leading cause of cancer mortality worldwide. To date, Sorafenib remains the standard of care for advanced HCCs not amenable to curative therapies such as surgery or local ablation. Unfortunately, clinical efficacy of Sorafenib remains marginal as most HCC patients receiving Sorafenib treatment develop Sorafenib resistance. The recently approved regorafenib is the only rescue therapy for patients who fail Sorafenib treatment. However, the effect of regorafenib is also far from satisfactory. Therefore, new strategies to overcome sorefenib resistance are critically warranted. Recently, one FDA-approved drug, here we tentatively named as “FA”, was shown to suppress different cancer malignancy by various mechanisms including inhibiting the development of drug resistance in cancer. These findings provide a rationale for combination treatment of this FA drug with Sorafenib in Sorafenib-resistant HCC. Results: Two Sorafenib-resistant HCC cell lines of Hep3B and HepG2215 were established. The Sorafenib resistance characteristics of the HCC cells were further verified by western blotting assay detecting protein tyrosine phosphorylation. We also demonstarted the resistant HCC cells expressed stemness-related genes and showed high ability of cell migration and metastasis. Combination use of FA drug and Sorafenib significantly suppressed the cell viability as well as the stemness-related properties of Sorafenib-resistant HCC cells, including stemness-related gene/protein expression, secondary sphere formation, and the cell migration. Conclusion: Combination use of FA drug and Sorafenib would be a potential therapeutic strategy for Sorafenib-resistant HCC treatment. Citation Format: Syue-Wei Peng, Yi-Heng Lin, Ming-Hao Teng, Hung-Cheng Lai, Te-Sheng Chang, Yen-Hua Huang. Adjutant therapeutic strategy for sorafenib-resistant hepatocellular carcinoma [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 896.

Carbon Footprint Measurement and Management: Case Study of the School Forest Enterprise

This paper applies a corporate accounting standard approach for measuring greenhouse gas emissions for a particular entity, the GHG Protocol, for a specific type of company in the primary sector. The main goal was to measure the total carbon footprint and carbon balance of the School Forest Enterprise of the Czech University of Life Sciences Prague. The total carbon footprint for Scope 1, Scope 2, and Scope 3 of the forestland managed by the SFE in 2017 was 686 t CO2-eq or 3.5 t/employee, and 3.8 CZK/1000 CZK of turnover or 99 kg CO2-eq/ha. These findings suggested a specific role of forest management in terms of climate change, where, in contrast with other companies in the secondary and tertiary spheres, the sinks outweigh the greenhouse gas production.

Hydrogen Transfer Catalysis beyond the Primary Coordination Sphere

In this Perspective, we outline recent examples of homogeneous transition-metal hydrogen transfer catalysts for which functionality within the complex’s outer coordination sphere influences the outcome of a reaction. Secondary-sphere groups are often applied to hydrogen transfer reactions, but their specific role during catalysis is not always well-understood. New experimental and theoretical work details the complexity associated with predicting secondary-sphere interactions and therefore designing improved catalysts. This Perspective highlights examples of catalysts containing secondary-sphere groups that (1) accelerate a key turnover-limiting step such as H2 heterolysis or hydride transfer, (2) limit competing catalytic cycles, (3) prevent catalyst decomposition, and/or (4) provide access to new catalysts through postmetalation modifications. The examples described herein emphasize numerous roles of the secondary sphere in hydrogen transfer catalysis and illustrate how the optimal use of these interact...

Artificial Metalloproteins Containing Co4O4 Cubane Active Sites.

Artificial metalloproteins (ArMs) containing Co4O4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial CoIII-OH2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e-/1H+ chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co4O4 active site provided a single H-bond to one of a set of cofacial CoIII-OH2 groups. With this variant, multi-e-/multi-H+ chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. With structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co4O4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e-/multi-H+ reactivity.

A photoactive semisynthetic metalloenzyme exhibits complete selectivity for CO2 reduction in water.

A series of artificial metalloenzymes containing a ruthenium chromophore and [NiII(cyclam)]2+, both incorporated site-selectively, have been constructed within an azurin protein scaffold. These light-driven, semisynthetic enzymes do not evolve hydrogen, thus displaying complete selectivity for CO2 reduction to CO. Electrostatic effects rather than direct excited-state electron transfer dominate the ruthenium photophysics, suggesting that intramolecular electron transfer from photogenerated RuI to [NiII(cyclam)]2+ represents the first step in catalysis. Stern-Volmer analyses rationalize the observation that ascorbate is the only sacrificial electron donor that supports turnover. Collectively, these results highlight the important interplay of elements that must be considered when developing and characterizing molecular catalysts.

Interplay between CN- Ligands and the Secondary Coordination Sphere of the H-Cluster in [FeFe]-Hydrogenases.

The catalytic cofactor of [FeFe]-hydrogenses (H-cluster) is composed of a generic cubane [4Fe-4S]-cluster (4FeH) linked to a binuclear iron-sulfur cluster (2FeH) that has an open coordination site at which the reversible conversion of protons to molecular hydrogen occurs. The (2FeH) subsite features a diatomic coordination sphere composed of three CO and two CN- ligands affecting its redox properties and providing excellent probes for FTIR spectroscopy. The CO stretch vibrations are very sensitive to the redox changes within the H-cluster occurring during the catalytic cycle, whereas the CN- signals seem to be relatively inert to these effects. This could be due to the more structural role of the CN- ligands tightly anchoring the (2FeH) unit to the protein environment through hydrogen bonding. In this work we explore the effects of structural changes within the secondary ligand sphere affecting the CN- ligands on FTIR spectroscopy and catalysis. By comparing the FTIR spectra of wild-type enzyme and two mutagenesis variants, we are able to assign the IR signals of the individual CN- ligands of the (2FeH) site for different redox states of the H-cluster. Moreover, protein film electrochemistry reveals that targeted manipulation of the secondary coordination sphere of the proximal CN- ligand (i.e., closest to the (4FeH) site) can affect the catalytic bias. These findings highlight the importance of the protein environment for re-adjusting the catalytic features of the H-cluster in individual enzymes and provide valuable information for the design of artificial hydrogenase mimics.

Modulation of fatty acid metabolism and immune suppression are features of in vitro tumour sphere formation in ontogenetically distinct dog cancers

Non-adherent, 3-dimensional sphere formation is used as an in vitro surrogate to evaluate cellular potential for tumour initiation and self-renewal. To determine if a shared molecular program underlies the capacity for sphere formation by cells originating from diverse tumour types, we characterized molecular and functional properties of 10 independent cell lines derived from 3 ontogenetically distinct dog cancers: hemangiosarcoma, osteosarcoma and glial brain tumours. Genome-wide gene expression profiling identified tumour-of-origin-dependent patterns of adjustment to sphere formation in a uniform culture condition. However, expression of the stem/progenitor markers CD34 and CD117, resistance to cytotoxic drugs and dye efflux (side population assays) showed no association with these gene expression profiles. Instead, primary sphere-forming capacity was inversely correlated with the ability to reform secondary spheres, regardless of tumour ontogeny. Primary sphere formation seemed to be proportional to the number of pre-existing cells with sphere-forming capacity in the cell lines. Cell lines where secondary sphere formation was more proficient than primary sphere formation showed enrichment of genes involved in fatty acid synthesis and immunosuppressive cytokines. In contrast, cell lines where secondary sphere formation was approximately equivalent to or less proficient than primary sphere formation showed upregulation of CD40 and enrichment of genes involved in fatty acid oxidation. Our data suggest that in vitro sphere formation is associated with upregulation of gene clusters involved in metabolic and immunosuppressive functions, which might be necessary for self-renewal and for tumour initiation and/or tumour propagation in vivo.

Effect of leucine-rich repeat-containing G-protein coupled receptor 5 gene silencing on biological behaviors of colon cancer stem cells

Objective To investigate the effect of leucine-rich repeat-containing G-protein coupled receptor 5 gene (LGR5) silencing on biological behaviors of colon cancer spheroid cells with stemness. Methods RNA interfering methods were used to knock down the LGR5 expression. Small interfering RNA (siRNA) targeting LGR5 and control siRNA were transfected into HT29 colon cancer spheroid cells by Lipofectamine RNAi MAX. Cells transfected with control siRNA or untransfected were used as negative control and blank control respectively. The expression of LGR5 mRNA and protein was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. The proliferation of spheroid cells was detected by cell counting kit-8 (CCK-8) assay at 1st, 2nd, 3rd, and 4th day after transfection respectively. The self-renewal, invasion and cell cycle of spheroid cells were determined by sphere formation assay, Transwell assay and flow cytometry respectively at 48th h after transfection. Results At 48 h after transfection, the expression of LGR5 mRNA in experimental group (0.32±0.02) was remarkably lower than that in blank control group (1.00±0.16) and negative control group (0.99±0.12) (P=0.000). The expression of LGR5 protein in experimental group was also significantly down-regulated (P=0.000). CCK-8 assay showed that the absorbance (A) value in blank control group, negative control group and experimental group at 4th day was 0.540±0.010, 0.528±0.016 and 0.390±0.022, respectively (P=0.000). In addition, the experimental group (21.33±2.08) formed fewer secondary spheres than blank control group (72.33±3.06) and negative control group (70.67±2.52, P=0.000). Flow cytometry revealed that the cells in the experimental group were significantly arrested in G0/G1 phase (P=0.000). Furthermore, the number of cells passing through the Transwell membrane in experimental group (35.60±6.74) was remarkably decreased as compared with that in the blank control group (86.47±7.85) and negative control group (83.53±8.58, P=0.000). Conclusion LGR5 gene silencing markly inhibited the proliferation, self-renewal and invasion of HT29 spheroid cells, indicating that LGR5 may play an important role in manipulating the biological behaviors of colon cancer stem cells. Key words: Colon cancer; Cancer stem cells; Leucine-rich repeat-containing G-protein coupled receptor 5; Proliferation; Invasion

Molybdenum Substituted Vanadyl Phosphate ε-VOPO4 for Lithium-Ion Batteries

Maintaining the structural reversibility of multiple electron transfer cathodes is a challenge one has to address in order to increase the energy density of current lithium-ion batteries. ε-VOPO4 is a promising cathode material with two redox transitions of V3+/V4+ and V4+/V5+ at about 2.5 and 4 V, respectively. However, the reversibility and cycling stability is compromised with more than one lithium cycling. The goal of this work is to investigate if the substitution of molybdenum can enhance the electrochemical performance of ε-VOPO4. We have focused on molybdenum because it is predicted by the first-principle calculations to display multiple redox potentials in useful voltage window and to form relatively stable substituted ε-VOPO4 phases. We have refined the synthesis method, where by changing a solvent we can control morphology and obtain either loose uniform 100 nm particles, or make those particles form secondary micron-sized spheres. Combing the analysis of X-ray diffraction, and inductively coupled plasma mass spectrometry, we have determined the limits of molybdenum substitution in the structure. The effects of the substitution and morphology on the electrochemical performance will be discussed. This research is supported as part of the NorthEast Center for Chemical Energy Storage (NECCES), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0012583. CS gratefully acknowledges the financial support from Graduate Assistance in Areas of National Need (GAANN) Fellowship and Binghamton University Provost's Doctoral Summer Fellowship.

Hinokitiol suppresses cancer stemness and oncogenicity in glioma stem cells by Nrf2 regulation.

Glioma is one of the lethal malignancies with poor prognosis. In addition, glioma stem cells (GSCs) have been considered as the crucial player that attributed to the tumorigenesis and drug resistance. In the current study, we investigated the therapeutic effect of hinokitiol, a natural bioactive compound of aromatic tropolone, on the characteristics of GSCs and the possible mechanism. U87MG and T98G glioma cells were used to isolate GSCs. CD133 positivity and ALDH1 activity of GSCs following hinokitiol treatment were assessed by flow cytometry analysis. Secondary sphere formation, migration, invasion, and colony-forming assays were performed to examine the self-renewal capacity and oncogenicity in GCS after hinokitiol administration. The expression of Nrf2 was evaluated by RT-PCR and western blot analyses. We demonstrated that hinokitiol effectively inhibited the CD133 positivity and ALDH1 activity along with the reduced self-renewal, migration, invasion, and colony formation properties of GSCs. In addition, hinokitiol repressed the gene and protein expression of Nrf2, which has been shown to be critical for those GSCs features. Furthermore, we showed that administration of exogenous Nrf2 counteracted the inhibitory effect of hinokitiol on self-renewal and invasiveness of GSCs. These evidences suggest that treatment of hinokitiol significantly attenuates the hallmarks of GSCs due to downregulation of Nrf2 expression. Hence, hinokitiol may serve as a promising agent for the therapy of glioma.

Exploring secondary-sphere interactions in Fe-N x H y complexes relevant to N2 fixation.

Hydrogen bonding and other types of secondary-sphere interactions are ubiquitous in metalloenzyme active sites and are critical to the transformations they mediate. Exploiting secondary sphere interactions in synthetic catalysts to study the role(s) they might play in biological systems, and to develop increasingly efficient catalysts, is an important challenge. Whereas model studies in this broad context are increasingly abundant, as yet there has been relatively little progress in the area of synthetic catalysts for nitrogen fixation that incorporate secondary sphere design elements. Herein we present our first study of Fe-N x H y complexes supported by new tris(phosphine)silyl ligands, abbreviated as [SiPNMe3] and [SiPiPr2PNMe], that incorporate remote tertiary amine hydrogen-bond acceptors within a tertiary phosphine/amine 6-membered ring. These remote amine sites facilitate hydrogen-bonding interactions via a boat conformation of the 6-membered ring when certain nitrogenous substrates (e.g., NH3 and N2H4) are coordinated to the apical site of a trigonal bipyramidal iron complex, and adopt a chair conformation when no H-bonding is possible (e.g., N2). Countercation binding at the cyclic amine is also observed for anionic {Fe-N2}- complexes. Reactivity studies in the presence of proton/electron sources show that the incorporated amine functionality leads to rapid generation of catalytically inactive Fe-H species, thereby substantiating a hydride termination pathway that we have previously proposed deactivates catalysts of the type [EPR3]FeN2 (E = Si, C).

Sport Helps to Answer Fundamental Questions: Interview with Robert Edelman

The interview with Robert Edelmann, the renown American scholar of Soviet sport, Professor of Russian history and history of sport at California University in San Diego, is dedicated to the heuristic questions of study of contemporary bodily practices. In the beginning, the scientist tells about his way into the historical science, on the emergence of his interest to Russian history, in general, and to contemporary forms of work with the body, in particular. R. Edelmann’s reminiscences regarding the situation with American academic community in 1960-1970s, especially the influence of the leading theoreticians of the Frankfurt School such as T. Adorno and H. Marcuse, are of special value from the standpoint of social-theoretical interest. The common thread of the whole interview is the claim on the inadequacy of many, especially the left-wing, intellectuals’ approach towards sport as some secondary sphere, at best distracting the people from real problems. It is further said about the heuristics of social-theoretical, cultural and historical studies of sport related to the names of leading representatives of Birmingham, Lester schools, and other branches of the humanities. Several investigations putting sport in the wide sociocultural context, are specified. Then Edelmann explicates his methodological approaches to the phenomenon of Soviet sport, which allowed him to analyze the mass bodily practices from the perspective of the participants themselves, i.e. “from below”, instead of the widespread standpoint “from above”. In conclusion, Edelmann remarks on the problem of academic acceptance of the disciplines explicitly investigating sport and other cultural practices, which were traditionally evaluated as “low”.

HER2 and EGFR Overexpression Support Metastatic Progression of Prostate Cancer to Bone.

Activation of the EGF receptors EGFR (ErbB1) and HER2 (ErbB2) drives the progression of multiple cancer types through complex mechanisms that are still not fully understood. In this study, we report that HER2 expression is elevated in bone metastases of prostate cancer independently of gene amplification. An examination of HER2 and NF-κB receptor (RANK) coexpression revealed increased levels of both proteins in aggressive prostate tumors and metastatic deposits. Inhibiting HER2 expression in bone tumor xenografts reduced proliferation and RANK expression while maintaining EGFR expression. In examining the role of EGFR in tumor-initiating cells (TIC), we found that EGFR expression was required for primary and secondary sphere formation of prostate cancer cells. EGFR expression was also observed in circulating tumor cells (CTC) during prostate cancer metastasis. Dual inhibition of HER2 and EGFR resulted in significant inhibition of tumor xenograft growth, further supporting the significance of these receptors in prostate cancer progression. Overall, our results indicate that EGFR promotes survival of prostate TIC and CTC that metastasize to bone, whereas HER2 supports the growth of prostate cancer cells once they are established at metastatic sites. Cancer Res; 77(1); 74-85. ©2016 AACR.

Biosynthetic approach to modeling and understanding metalloproteins using unnatural amino acids

Metalloproteins have inspired chemists for many years to synthesize artificial catalysts that mimic native enzymes. As a complementary approach to studying native enzymes or making synthetic models, biosynthetic approach using small and stable proteins to model native enzymes has offered advantages of incorporating non-covalent secondary sphere interactions under physiological conditions. However, most biosynthetic models are restricted to natural amino acids. To overcome this limitation, incorporating unnatural amino acids into the biosynthetic models has shown promises. In this review, we summarize first synthetic, semisynthetic and biological methods of incorporates unnatural amino acids (UAAs) into proteins, followed by progress made in incorporating UAAs into both native metalloproteins and their biosynthetic models to fine-tune functional properties beyond native enzymes or their variants containing natural amino acids, such as reduction potentials of azurin, O2 reduction rates and percentages of product formation of HCO models in Mb, the rate of radical transport in ribonucleotide reductase (RNR) and the proton and electron transfer pathways in photosystem II (PS II). We also discuss how this endeavour has allowed systematic investigations of precise roles of conserved residues in metalloproteins, such as Met121 in azurin, Tyr244 that is cross-linked to one of the three His ligands to CuB in HCO, Tyr122, 356, 730 and 731 in RNR and TyrZ in PS II. These examples have demonstrated that incorporating UAAs has provided a new dimension in our efforts to mimic native enzymes and in providing deeper insights into structural features responsible high enzymatic activity and reaction mechanisms, making it possible to design highly efficient artificial catalysts with similar or even higher activity than native enzymes.

193 ROBUST PROPAGATION OF SELF-RENEWING PORCINE NEURAL STEM CELLS ISOLATED FROM TRANSGENIC PIGS WITH A GFAP-CreERT2 SYSTEM CAPABLE OF CONTROLLING THE EXPRESSION OF EGFP GENES

Ample evidence has demonstrated the important roles of pigs because their anatomical, immunologic, and physiological characteristics are fairly similar to humans. In particular, their gyrencephalic brain are more comparable to humans than rodents with similar grey and white matter composition and size. In this study, we isolated and propagated the neural stem cells (GFAP-CreERT2-NSCs) from the transgenic piglet with expression of CreERT2, a fusion protein of the DNA recombinase Cre and mutated ligand-binding domain of the human oestrogen receptor, under the control of the GFAP promoter. The primary culture from tissue of porcine CreERT2 brain led to floating spherical masses of cells that revealed similar morphology and size distribution to neurospheres reported by previous studies. Quantitative analysis indicated a yield of 2.50 ± 0.44 primary spheres per 1,000 viable cells from the neocortex, versus 12.92 ± 1.67 primary spheres per 1,000 viable cells from the periventricular region (PVR) including subventricular zone. Secondary spheres (6.67 ± 1.10 spheres from neocortex versus 23.08 ± 1.96 spheres from PVR cells) were formed from primary spheres at 10 days after passage. Tertiary spheres (8.42 ± 0.99 spheres from neocortex versus 23.08 ± 1.91 spheres from PVR cells) could also be obtained after a second passage, indicating that they were proliferating in vitro. The CreERT2-NSCs showed normal 36+XY karyotype and representative NSC markers, such as NESTIN, SOX2, and VIMENTIN. After differentiation, we were able to obtain populations of astrocytes and neurons expressing GFAP and TUJ1, respectively. In summary, we verified and propagated the isolated GFAP promoter-driven CreERT2-NSCs, which would be considered a promising source of cells for treatment of central nervous system diseases.

Stabilization of cytochrome b5 by a conserved tyrosine in the secondary sphere of heme active site: A spectroscopic and computational study

Heme proteins perform a large array of biological functions, with the heme group bound non-covalently or covalently. To probe the stabilization role of conserved tyrosine residue in the secondary sphere of heme site in heme proteins, we herein used cytochrome b5 (Cyt b5) as a model protein, and mutated Tyr30 to Phe or His by removal of Tyr30 associated H-bond network and hydrophobic interaction. We performed thermal-induced unfolding studies for the two mutants, Y30F Cyt b5 and Y30H Cyt b5, as monitored by both UV–Vis and CD spectroscopy, as well as heme transfer studies from these proteins to apo-myoglobin, with wild-type Cyt b5 under the same conditions for comparison. The reduced stability of both mutants indicates that both the H-bonding and hydrophobic interactions associated with Tyr30 contribute to the protein stability. Moreover, we performed molecular modeling studies, which revealed that the hydrophobic interaction in the local region of Y30F Cyt b5 was well-remained, whereas Y30H Cyt b5 formed an H-bond network. These observations suggest that the conserved Tyr30 in Cyt b5 is not replaceable due to the presence of both the H-bond network and hydrophobic interaction in the secondary sphere of the heme active site. As demonstrated here for Cyt b5, it may be of practical importance for design of artificial heme proteins by engineering a Tyr in the secondary sphere with improved properties and functions.

Tailored Transition Metal Oxide Spheres Using Versatile Polymer-Template for Sodium-Ion Battery Cathodes

Transition metal oxides have been studied as positive- and negative-electrode materials for sodium-ion batteries (SIBs). In this study, we report the strategic synthesis methods of tailored cathode materials for high performance SIBs using external-stimuli responsive polymer particles and water-in-oil system. The primary particles consisting of the polymer and metal precursors are fabricated by introduction of various metal ions and polar molecules into the three-dimensional cross-linked polymer spheres in aqueous solution. The assembled secondary metal oxide spheres with tailored structures were prepared by water-in-oil system and heat treatment in high purity oxygen. When tested in an electrochemical Na-half cell, the tailored transition metal oxide spheres exhibit good Na storage capabilities.

Primary Salivary Human Stem/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture.

Radiotherapy for head and neck cancer often has undesirable effects on salivary glands that lead to xerostomia or severe dry mouth, which can increase oral infections. Our goal is to engineer functional, three‐dimensional (3D) salivary gland neotissue for autologous implantation to provide permanent relief. An immediate need exists to obtain autologous adult progenitor cells as the use of embryonic and induced pluripotent stem cells potentially pose serious risks such as teratogenicity and immunogenic rejection. Here, we report an expandable population of primary salivary human stem/progenitor cells (hS/PCs) that can be reproducibly and scalably isolated and propagated from tissue biopsies. These cells have increased expression of progenitor markers (K5, K14, MYC, ETV4, ETV5) compared with differentiation markers of the parotid gland (acinar: MIST1/BHLHA15 and AMY1A; ductal: K19 and TFCP2L1). Isolated hS/PCs grown in suspension formed primary and secondary spheres and could be maintained in long‐term 3D hydrogel culture. When grown in a customized 3D modular hyaluronate‐based hydrogel system modified with bioactive basement membrane‐derived peptides, levels of progenitor markers, indices of proliferation, and viability of hS/PCs were enhanced. When appropriate microenvironmental cues were provided in a controlled manner in 3D, such as stimulation with β‐adrenergic and cholinergic agonists, hS/PCs differentiated into an acinar‐like lineage, needed for saliva production. We conclude that the stem/progenitor potential of adult hS/PCs isolated without antigenic sorting or clonal expansion in suspension, combined with their ability to differentiate into specialized salivary cell lineages in a human‐compatible culture system, makes them ideal for use in 3D bioengineered salivary gland applications. Stem Cells Translational Medicine 2017;6:110–120

Ultrathin and Uniform Carbon Coating on Active Materials Using Liquid CO2 As a Coating Solvent

Some of cathode and anode active materials (e.g., LiFePO4, Li4Ti5O12, Li2FeSiO4, TiO2, MoO2) exhibit inherently low electronic conductivities and low ionic conductivities; thus, these materials typically suffer from poor electrochemical performance. The reduction of particle size to a nanoscale dimension to enhance lithium ion diffusivity and carbon coating to increase electronic conductivity are the most widely used strategies to improve the performance of the potential active materials. Incorporation of too much carbon content, thick carbon layer coating, and non-uniform carbon coating on individual particles can also deteriorate energy density and battery performance. The complete, uniform, and thin carbon layer coverage ensures that the particles receive electrons from all directions and the electrons penetrate easily through the carbon layer, resulting in low polarization and high-rate performance. Herein, the use of liquid carbon dioxide (l-CO2) for uniform and ultrathin carbon layer coverage on highly porous LiFePO4 and MoO2 particles is discussed. The extremely low surface tension and the low viscosity associated with l-CO2 make it an excellent coating solvent for uniform and ultrathin carbon layer formation on the highly porous active materials. In addition, the carbon content in the range of 1-10 wt% can be easily controlled by adjusting process parameters such as solution concentration or evaporation velocity. The hierarchically porous MoO2 microspheres are synthesized in supercritical methanol without using any surfactants or structure-directing chemicals. The highly porous LiFePO4 particles are synthesized using a co-precipitation method. The MoO2 and LiFePO4 particles exhibits hierarchically porous nano/micro structure, in which primary particles with 30-65 mn size are loosely aggregated and form secondary spheres with 0.5-10 mm size. Owing to the low viscosity and low surface tension of l-CO2, the coating solution is penetrated into the pores of the particles and uniform and ultrathin carbon layer is covered on the primary particles. The thickness of carbon layers were in range of 0.8-2.0 nm by adjusting the carbon precursor concentration in l-CO2. The TEM analysis on the cross-sectional area of the particles shows that the uniform incorporation of the carbon layer on the whole surface of the micron-sized secondary particles. The carbon-coated LiFePO4 exhibits ~108.6 mAh g-1 at a high charge-discharge rate of 30 C. In addition, after 1000 cycles of 10 C, ~ 85% of an initial capacity is maintained. The carbon-coated MoO2 exhibits enhanced high-rate performance and cell stability. Figure 1