Sensitive Detection of Tumor Cells Using Protein Nanoparticles with Multiple Displays of DNA Aptamers and Bioluminescent Reporters.

Simple and effective detection methods for circulating tumor cells are essential for early detection and progression monitoring of tumors. The use of DNA aptamer and bioluminescence is expected to be a key tool for the simple, effective, and sensitive detection of tumor cells. Herein, we designed multifunctional protein nanoparticles for the detection of tumor cells using DNA aptamer and bioluminescence. Fusion proteins (ELP-poly(d)-POIs), composed of elastin-like polypeptide (ELP) fused with protein of interests (POIs) via poly(aspartic acid) (poly(d)), formed the protein nanoparticles based on the temperature responsivity of ELP sequences, leading to multiply displayed POIs on the protein nanoparticles. In the present study, we focused on porcine circovirus type 2 replication initiation protein (Rep), which covalently conjugated with DNA aptamers, and NanoLuc luciferase (Nluc), which emitted a strong bioluminescence, as POIs. ELP-poly(d)-Rep and ELP-poly(d)-Nluc were constructed and formed the protein nanoparticles with multiply displayed Nluc and Rep (DNA aptamer) that amplified the bioluminescence signal and tumor recognition ability. Mucin-1 (MUC1)-overexpressing human breast tumor MCF7 cells and MUC1-recognizing aptamer (MUC1 aptamer) were selected as models. The MUC1 aptamer-conjugated protein nanoparticles exhibited a 13.7-fold higher bioluminescence signal to MCF-7 cells than to human embryonic kidney 293 (HEK293) cells, which express low levels of MUC1. Furthermore, the protein nanoparticles could detect up to 70.7 cells/mL of MCF-7 cells from a cell suspension containing HEK-293. The protein nanoparticles with multiple Rep and Nluc show a great potential as a material for detecting CTCs.

Differences in the Intracellular Localization of Methylated ß-Cyclodextrins-Threaded Polyrotaxanes Lead to Different Cellular States.

Activation of autophagy represents a potential therapeutic strategy for the treatment of diseases that are caused by the accumulation of defective proteins and the formation of abnormal organelles. Methylated ß-cyclodextrins-threaded polyrotaxane (Me-PRX), a supramolecular structured polymer, induces autophagy by interacting with the endoplasmic reticulum. We previously reported on the successful activation of mitochondria-targeted autophagy by delivering Me-RRX to mitochondria using a MITO-Porter, a mitochondria-targeted nanocarrier. The same level of autophagy induction was achieved at one-twentieth the dosage for the MITO-Porter (Me-PRX) compared to the naked Me-PRX. We report herein on the quantitative evaluation of the intracellular organelle localization of both naked Me-PRX and the MITO-Porter (Me-PRX). Mitochondria, endoplasmic reticulum and lysosomes were selected as target organelles because they would be involved in autophagy induction. In addition, organelle injury and cell viability assays were performed. The results showed that the naked Me-PRX and the MITO-Porter (Me-PRX) were localized in different intracellular organelles, and organelle injury was different, depending on the route of administration, indicating that different organelles contribute to autophagy induction. These findings indicate that the organelle to which the autophagy-inducing molecules are delivered plays an important role in the level of induction of autophagy.

Cografting of Zwitterionic Sulfobetaines and Cationic Amines on ß-Cyclodextrin-Threaded Polyrotaxanes Facilitates Cellular Association and Tissue Accumulation with High Biocompatibility.

ß-Cyclodextrins (ß-CDs) and ß-CD-containing polymers have attracted considerable attention as potential candidates for the treatment of cholesterol-related metabolic and intractable diseases. We have advocated the use of ß-CD-threaded acid-degradable polyrotaxanes (PRXs) as intracellular delivery carriers for ß-CDs. As unmodified PRXs are insoluble in aqueous solutions, chemical modification of PRXs is an essential process to improve their solubility and impart novel functionalities. In this study, we investigated the effect of the modification of zwitterionic sulfobetaines on PRXs due to their excellent solubility, biocompatibility, and bioinert properties. Sulfobetaine-modified PRXs were synthesized by converting the tertiary amino groups of precursor 2-(N,N-dimethylamino)ethyl carbamate-modified PRXs (DMAE-PRXs) using 1,3-propanesultone. The resulting sulfobetaine-modified PRXs showed high solubility in aqueous solutions and no cytotoxicity, while their intracellular uptake levels were low. To further improve this system, we designed PRXs cografted with zwitterionic sulfobetaine and cationic DMAE groups via partial betainization of the DMAE groups. Consequently, the interaction with proteins, intracellular uptake levels, and liver accumulation of partly betainized PRXs were found to be higher than those of completely betainized PRXs. Additionally, partly betainized PRXs showed no toxicity in vitro or in vivo despite the presence of residual cationic DMAE groups. Furthermore, partly betainized PRXs ameliorated the abnormal free cholesterol accumulation in Niemann-Pick type C disease patient-derived cells at lower concentrations than ß-CD derivatives and previously designed PRXs. Overall, the cografting of sulfobetaines and amines on PRXs is a promising chemical modification for therapeutic applications due to the high cholesterol-reducing ability and biocompatibility of such modified PRXs. In addition, modification with both zwitterionic and cationic groups can be used for the design of various polymeric materials exhibiting both bioinert and bioactive characteristics.

Nanoscopic analyses of cell-adhesive protein adsorption on poly(2-methoxyethyl acrylate) surfaces.

Regulation of protein adsorption on the surface of biomaterials is important for modulating cell adhesion. Two important proteins in this regard are fibrinogen and fibronectin. Poly(2-methoxyethyl acrylate) (PMEA) and its derivatives have been developed as promising coating materials for biomaterial surfaces. Previous studies have highlighted that PMEA-coated substrates suppress thrombogenicity but promote cell adhesiveness. However, it was unclear what was responsible for these differences in adhesion. In this study, we focused on the correlation between protein adsorption and the nanometer-scale structures on the surfaces of the PMEA substrates. An atomic force microscope using protein- or antibody-conjugated cantilevers was used to perform nanoscopic analyses of the adsorption forces and conformational changes in fibrinogen and fibronectin adsorbed on the nanometer-scale PMEA structures. The adsorption force of fibronectin in the polymer-poor region was higher than that of fibrinogen, whereas the polymer-rich region showed a negligible difference in adsorption force between the two proteins. Interestingly, a greater conformational change in the adsorbed fibronectin was induced in the polymer-poor region than that in fibronectin in the polymer-rich region or fibrinogen in either regions, resulting in the induction of cell adhesion. Nanoscopic analyses of protein adsorption on biomaterial surfaces provide promising insights into the design of novel biomaterials that control protein adsorption and cell adhesion.

Roles of interfacial water states on advanced biomedical material design.

When biomedical materials come into contact with body fluids, the first reaction that occurs on the material surface is hydration; proteins are then adsorbed and denatured on the hydrated material surface. The amount and degree of denaturation of adsorbed proteins affect subsequent cell behavior, including cell adhesion, migration, proliferation, and differentiation. Biomolecules are important for understanding the interactions and biological reactions of biomedical materials to elucidate the role of hydration in biomedical materials and their interaction partners. Analysis of the water states of hydrated materials is complicated and remains controversial; however, knowledge about interfacial water is useful for the design and development of advanced biomaterials. Herein, we summarize recent findings on the hydration of synthetic polymers, supramolecular materials, inorganic materials, proteins, and lipid membranes. Furthermore, we present recent advances in our understanding of the classification of interfacial water and advanced polymer biomaterials, based on the intermediate water concept.

Selective Accumulation to Tumor Cells with Coacervate Droplets Formed from a Water-Insoluble Acrylate Polymer.

Selective targeting of specific cells without the use of biological ligands has not been achieved. In the present study, we revealed that the coacervate droplets formed from poly(2-methoxyethyl acrylate) (PMEA) and its derivatives selectively accumulated to tumor cells. PMEA derivatives, which are insoluble acrylate polymers, induced coacervation in water to form polymer-dense droplets via hydrophobic interaction. Interestingly, the accumulation of coacervate droplets to tumor cells was involved in the bound water content of PMEA derivatives. Coacervate droplets with a high bound water content accumulated and internalized up to 36.6-fold higher in HeLa cervical tumor cells than in normal human fibroblasts (NHDF). Moreover, the interactions between coacervate droplets and plasma membrane components such as CD44 played a key role in this accumulation process. Therefore, coacervate droplets formed from PMEA derivatives have great clinical potential in tumor cell detection, development of alternative tumor-targeting ligands, and optimization of drug delivery carriers.

Modulation of Biological Responses of Tumor Cells Adhered to Poly(2-methoxyethyl acrylate) with Increasing Cell Viability under Serum-Free Conditions.

Circulating tumor cells in body fluids are important biomarkers in cancer diagnosis. The culture of tumor cells isolated from body fluids can provide intrinsic information about tumors and can be used to screen for the best anticancer drugs. However, the culture of primary tumor cells has been hindered by their low viability and difficulties in recapitulating the phenotype of primary tumors in in vitro culture. The culture of tumor cells under serum-free conditions is one of the methodologies to maintain the phenotype and genotype of primary tumors. Poly(2-methoxyethyl acrylate) (PMEA)-coated substrates have been investigated to prolong the proliferation of tumor cells under serum-free conditions. In this study, we investigated the detailed behavior and the mechanism of the increase in tumor cell viability after adherence to PMEA substrates. The blebbing formation of tumor cells on PMEA was attributed not to apoptosis but to the low adhesion strength of cells on PMEA. Moreover, blebbing tumor cells showed amoeboid movement and formed clusters with other cells via N-cadherin, leading to an increase in tumor cell viability. Furthermore, the behaviors of tumor cells adhered to PMEA under serum-free conditions were involved in the activation of the PI3K and Rho-associated protein kinase pathways. Thus, we propose that PMEA would be suitable for the development of devices to cultivate primary tumor cells under serum-free conditions for the label-free diagnosis of cancer.

Relationship between diarrhoea risk and the combinations of drinking water sources in the Kathmandu Valley, Nepal.

In Nepal, the number of diarrhoea hospitalizations in all ages is seriously high. According to the World Health Organization, diarrheal diseases can be substantially prevented through safe drinking water sources. In the Kathmandu Valley, because of the shortage of piped water, local residents use alternative water sources, such as groundwater, jars and tanker water. However, these alternative water sources can be contaminated. This study aimed to clarify the relationship between diarrhoea risk and the combinations of drinking water sources. A survey using multiple questionnaires on diarrhoea occurrence, water sources and water treatment was conducted three time between 2015 and 2016. The odds ratios (ORs) of developing diarrhoea were significantly high for drinking jar (OR 6.1) and tanker water (OR 8.4) compared with not drinking. The combined drinking of jar and tanker water obtained the 1 log higher OR compared with drinking only piped water. Conversely, drinking groundwater had a low OR, implying that the residents refrained from drinking polluted groundwater. In conclusion, diarrhoea occurrence was related not only to the level of water contamination, but also to a behavioural factor, i.e. people's careful management of the choice of multiple water sources.

A ß-hairpin peptide with pH-controlled affinity for tumor cells.

Considering that the pH in the tumor microenvironment is dysregulated, we designed a ß-hairpin peptide (SSRFEWEFESSDPRGDPSSRFEWEFESS). The configuration of the peptide switched from a flexible linear to a rigid loop structure under weakly acidic conditions. The peptide internalized by tumor cells increased significantly under weakly acidic conditions.

Carborane as an Alternative Efficient Hydrophobic Tag for Protein Degradation.

Carboranes 1 and 2 were designed and synthesized for hydrophobic tag (HyT)-induced degradation of HaloTag fusion proteins. The levels of the hemagglutinin (HA)-HaloTag2-green fluorescent protein (EGFP) stably expressed in Flp-In 293 cells were significantly reduced by HyT13, HyT55, and carboranes 1 and 2, with expression levels of 49, 79, 43, and 65%, respectively, indicating that carborane is an alternative novel hydrophobic tag (HyT) for protein degradation under an intracellular environment. To clarify the mechanism of HyT-induced proteolysis, bovine serum albumin (BSA) was chosen as an extracellular protein and modified with maleimide-conjugated m-carborane (MIC). The measurement of the ζ-potentials and the lysine residue modification with fluorescein isothiocyanate (FITC) of BSA-MIC conjugates suggested that the conjugation of carborane induced the exposure of lysine residues on BSA, resulting in the degradation via ubiquitin E3 ligase-related proteasome pathways in the cell.

Using insights from water isotopes to improve simulation of surface water-groundwater interactions.

Understanding interactions between surface water (SW) and groundwater (GW) is challenging because of the lack of information available and the complexity of the processes involved. In this paper, SW-GW interactions are simulated using numerical modelling integrated with insights from water isotopes. Isotope analysis can be used to (i) determine the relative contribution from different sources of water into aquifer recharge and (ii) identify areas with high SW-GW interactions. The results, for the Shiraz catchment in Iran, show that (i) SWAT-MODFLOW has better performance than stand-alone SWAT (R2 improvement from 0.50 to 0.54) and (ii) SWAT-MODFLOW calibrated with insights from isotope data (SWAT-MODFLOW-ISO) has significantly better performance than SWAT-MODFLOW (R2 improvement from 0.54 to 0.68 and RMSE reduction from 1.67 to 1.33). This demonstrates that insights into SW-GW interactions that are revealed by isotopes can be used to improve hydrological modelling performance.

A protocol for nitrogen isotopic measurement of dissolved organic nitrogen with a combination of oxidation-denitrification and gas phase diffusion methods.

Isotopic tracing technique is one of the most effective methods to identify nitrogen source and fate in aquatic environments. Although dissolved organic nitrogen (DON) is a key component in nitrogen cycles, information on nitrogen stable isotope ratios in DON (δ15N-DON) is limitedly available for its low recovery through a direct measurement. Indirect measurement is based on mass balance calculations and easy to use with high recovery of DON. However, in theory, the result from mass balance calculation is sensitive to the level of DON content, and its applicability remains to be examined for waters containing a variety of DON content in total dissolved nitrogen (TDN). In this study, we established a protocol for indirect measurement of δ15N-DON values based on the combination of multiple analytical methods. Precision and accuracy in the measurement were assessed by varying the composition of DON and dissolved inorganic nitrogen, and quantitation thresholds were presented at different acceptable levels. The results illustrated an advantage of the developed protocol possibly applicable to water samples particularly with low DON content that is commonly detected in freshwater. This method is expected to expand the use of isotope tracing techniques for understanding the nitrogen cycle in water environments.

Effect of bound water content on cell adhesion strength to water-insoluble polymers.

Adhesion of cells on biomaterials plays an essential role in modulating cellular functions. Although hydration of biomaterials occurs under biological conditions, it is challenging to systematically evaluate the correlation of hydrated water content in biomaterials with the cell adhesion strength. In this report, we investigated the effect of bound water content on the adhesion strength of cells on poly(2-methoxyethyl acrylate) (PMEA) analogue substrates. Water-insoluble PMEA analogues were synthesized to fabricate substrates with a systemically controlled bound water content. To assess the surface properties of their substrates, contact angle measurement, atomic force microscopy (AFM), and fluorescence measurement were conducted. To reflect the effect of bound water of PMEA analogues, the relationship between the bound water content and cell adhesion behavior was evaluated under serum-free condition. From the single cell force spectrometry (SCFS) and microscopic analysis, it revealed that the increment of bound water content on the substrates decreased cell adhesion strength and cell spreading on the substrates. The bound water content exhibited a good correlation with adhesion strength, spreading area, circularity, and aspect ratio of cells. Our findings indicate that the bound water content could contribute to the development of a novel biomaterial and evaluation of cell behaviors on biomaterials. STATEMENT OF SIGNIFICANCE: For coordinating cell functions, such as growth, mobility, and differentiation, modulating the adhesion strength between cells and their environments is important. Although the hydration to biomaterials has been reported to be closely related to a antifouling property, the effect of hydration water on the cell adhesion behavior is not well understood. We present the first demonstration of essential relationship between cell adhesion strength and hydrated water on a biomaterials surface using the water-insoluble polymers with different hydrated water content. The results reveal that the hydrated water content of polymer substrates strong correlation with adhesion strength of cells. Collectively, the hydrated water content of the biomaterials will be a predominant factor affecting the cell adhesion strength and behavior.

Expression Patterns and Levels of All Tubulin Isotypes Analyzed in GFP Knock-In C. elegans Strains.

Most organisms have multiple α- and ß-tubulin isotypes that likely contribute to the diversity of microtubule (MT) functions. To understand the functional differences of tubulin isotypes in Caenorhabditis elegans, which has nine α-tubulin isotypes and six ß-tubulin isotypes, we systematically constructed null mutants and GFP-fusion strains for all tubulin isotypes with the CRISPR/Cas9 system and analyzed their expression patterns and levels in adult hermaphrodites. Four isotypes-α-tubulins TBA-1 and TBA-2 and ß-tubulins TBB-1 and TBB-2-were expressed in virtually all tissues, with a distinct tissue-specific spectrum. Other isotypes were expressed in specific tissues or cell types at significantly lower levels than the broadly expressed isotypes. Four isotypes (TBA-5, TBA-6, TBA-9, and TBB-4) were expressed in different subsets of ciliated sensory neurons, and TBB-4 was inefficiently incorporated into mitotic spindle MTs. Taken together, we propose that MTs in C. elegans are mainly composed of four broadly expressed tubulin isotypes and that incorporation of a small amount of tissue-specific isotypes may contribute to tissue-specific MT properties. These newly constructed strains will be useful for further elucidating the distinct roles of tubulin isotypes.Key words: tubulin isotypes, microtubules, C. elegans.

Waterborne diarrhoeal infection risk from multiple water sources and the impact of an earthquake.

In the Kathmandu Valley of Nepal, locals depend on multiple water sources due to the limited access to safe water, which is a great global concern regarding its impact on human health. This study aimed to compare the infection risk of diarrhoea from multiple water sources with different concentrations of Escherichia coli among water supply areas and evaluate the impact of changing water sources due to the Gorkha earthquake on the infection risk. The concentration of enteropathogenic E. coli was estimated in samples of piped water, jar water, groundwater, and tanker water, which were collected in the Valley. The volume of each water ingestion was determined based on a questionnaire survey and considering drinking and bathing sources. The highest estimated risk was observed for households drinking groundwater from shallow dug wells, followed by tanker water. The estimated risk implied the regional disparity due to various water sources with different quality. After the earthquake, the ratio of households drinking only jar water increased, and the estimated risk decreased. The damage on piped water supply, the decrease of tanker water availability and the decrease of residents' trust in groundwater quality presumably enhanced the consumption of jar water despite its high price.

Correction: An antibody-supermolecule conjugate for tumor-specific targeting of tumoricidal methylated ß-cyclodextrin-threaded polyrotaxanes.

Correction for 'An antibody-supermolecule conjugate for tumor-specific targeting of tumoricidal methylated ß-cyclodextrin-threaded polyrotaxanes' by Kei Nishida et al., J. Mater. Chem. B, 2020, DOI: 10.1039/d0tb00575d.

An antibody-supermolecule conjugate for tumor-specific targeting of tumoricidal methylated ß-cyclodextrin-threaded polyrotaxanes.

We previously found that acid-labile polyrotaxane containing methylated ß-cyclodextrin (Me-PRX) induces endoplasmic reticulum (ER) stress-related autophagy and autophagic cell death. Me-PRX-induced autophagic cell death occurs even in apoptosis-resistant cells; tumor-targeted Me-PRX delivery could thus be an effective cancer treatment approach. In this study, antibody-supermolecule conjugates, consisting of a tumor-specific antibody and Me-PRX, were designed to achieve a tumor-specific delivery of Me-PRX. Trastuzumab, a monoclonal antibody against HER2 expressed in various malignant tumors, was selected as a tumor-targeting antibody, and phenyl maleimide group-modified Me-PRX (Mal-Me-PRX) was conjugated to the cysteine residue of the reduced Trastuzumab to obtain a Trastuzumab-Me-PRX conjugate (Tras-Me-PRX). The cellular association of Tras-Me-PRX to HER2-expressing tumor cells was remarkably greater than that of unmodified Me-PRX. Moreover, Tras-Me-PRX effectively reduced the viability of HER2-expressing tumor cells at a lower concentration compared to the unmodified Me-PRX. In conclusion, antibody-Me-PRX conjugates are regarded as a new class of antibody-drug conjugates that would contribute to the chemotherapy of cancers.

Enhanced autophagy induction via the mitochondrial delivery of methylated ß-cyclodextrin-threaded polyrotaxanes using a MITO-Porter.

Failure of autophagy induction results in the accumulation of abnormal mitochondria to cause neurodegenerative diseases. Artificial autophagy activation via the mitochondrial delivery of polyrotaxane with autophagy induced activity is achieved using a MITO-Porter, a nanodevice for mitochondrial delivery. This strategy can be applied to innovative research and therapy.

Development of an objective water security index and assessment of its association with quality of life in urban areas of developing countries.

A composite metric assessing water security's physical dimension at the micro/ community level is lacking but is essential for setting priorities for program and policy implementations. We prepared an objective index (OI) of water security to measure the physical dimension using a model centered on household water-use behavior in developing countries' urban areas. A cross-sectional household survey (n = 1500) with multi-stage cluster design was conducted from December 2015 to February 2016 in the Kathmandu Valley, which has faced long-term, severe water shortage. A structured questionnaire probed socio-demographic characteristics, water sources, frequency and quantity of water use, cost related to water, etc. A 15-item water insecurity scale was used to measure subjective and experiential dimension of water insecurity. The World Health Organization Quality of Life - BREF was used to measure quality of life (QoL). The QoL has been considered as proxy of well-being in this study. The OI measured differential water security within small cities, the utility's service areas for instance, and identified area-specific key dimensions that need improvement. Overall, the OI and its key dimensions can be useful measures to design water-scarcity averting programs and policies, specific to a particular community's needs. The increased OI values were significantly and positively associated with better physical and psychological health and better social relationship domains of QoL suggesting health implications of water security.

Groundwater use and diarrhoea in urban Nepal: novel application of a geostatistical interpolation technique linking environmental and epidemiologic survey data.

Background: Groundwater is a common domestic water source in developing countries, but is persistently contaminated with enteropathogens. However, studies on determinants of diarrhoea have predominantly focused on piped water. This study examines the relationship between groundwater microbial quality and household diarrhoea occurrence (HDO). Methods: Considering it as a proxy of enteropathogens, this study analysed Escherichia coli concentrations in groundwater wells. Ordinary kriging, a geostatistical technique in geographic information systems, was used to interpolate the E. coli concentration to survey points that had secondary survey data (n=942). The relationship between E. coli and HDO using simple and multivariate statistical analyses in SPSS was analysed. Results: A total of 77% of households used groundwater. One-third of households were without piped-water access (PWA), and these households were significantly more likely to use groundwater than those with PWA. Of the 87 households that reported HDO, 77% were groundwater users. Of the groundwater users, the households with HDO consumed groundwater with significantly higher E. coli concentrations than the households without HDO. Of the households without PWA, the increase in the E. coli concentration increased the odds of HDO (adjusted odds ratio=3.15; 95% CI=1.07-9.22). Conclusion: It is suggested that the groundwater microbial quality is a risk factor for HDO and illustrates this by an application of an interpolation technique relevant for developing countries.