Characterization and regulation of 2D-3D convertible lipid membrane transformation.

Micro-nanomaterials that can adopt different structures are powerful tools in the fields of biological and medical sciences. We previously developed a lipid membrane that can convert between 2D nanosheet and 3D vesicle forms using cationic copolymer polyallylamine-graft-polyethylene glycol and the anionic peptide E5. The properties of the membrane during conversion have been characterized only by confocal laser scan microscopy. Furthermore, due to the 2D symmetry of the lipid nanosheet, the random folding of the lipid bilayer into either the original or the reverse orientation occurs during sheet-to-vesicle conversion, compromising the structural consistency of the membrane. In this study, flow cytometry was applied to track the conversion of more than 5000 lipid membranes from 3D vesicles to 2D nanosheets and back to 3D vesicles, difficult with microscopies. The lipid nanosheets exhibited more side scattering intensity than 3D vesicles, presumably due to free fluctuation and spin of the sheets in the suspension. Furthermore, by immobilizing bovine serum albumin as one of the representative proteins on the outer leaflet of giant unilamellar vesicles at a relatively low coverage, complete restoration of lipid membranes to the original 3D orientation was obtained after sheet-to-vesicle conversion. This convertible membrane system should be applicable in a wide range of fields. Our findings also provide experimental evidence for future theoretical studies on membrane behavior.

Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion.

Bcl2-associated athanogene-1 protein (Bag1) acts as a co-chaperone of heat shock protein 70 and heat shock cognate 70 and regulates multiple cellular processes, including cell proliferation, apoptosis, environmental stress response, and drug resistance. Since Bag1 knockout mice exhibited fetal lethality, the in vivo function of Bag1 remains unclear. In this study, we established a mouse line expressing Bag1 gene missing exon 5, which corresponds to an encoding region for the interface of heat shock protein 70/heat shock cognate 70. Despite mice carrying homoalleles of the Bag1 mutant (Bag1Δex5) expressing undetectable levels of Bag1, Bag1Δex5 homozygous mice developed without abnormalities. Bag1Δex5 protein was found to be highly unstable in cells and in vitro. We found that the growth of mouse embryonic fibroblasts derived from Bag1Δex5-homo mice was attenuated by doxorubicin and a glutathione (GSH) synthesis inhibitor, buthionine sulfoximine. In response to buthionine sulfoximine, Bag1Δex5-mouse embryonic fibroblasts exhibited a higher dropping rate of GSH relative to the oxidized glutathione level. In addition, Bag1 might mitigate cellular hydrogen peroxide levels. Taken together, our results demonstrate that the loss of Bag1 did not affect mouse development and that Bag1 is involved in intracellular GSH homeostasis, namely redox homeostasis.

Fecal DNA analysis coupled with the sighting records re-expanded a known distribution of dugongs in Ryukyu Islands after half a century.

DNA analysis of large herbivore feces samples collected from seagrass beds at two distant sites (Irabu Island in Miyako Islands and Kushi in Okinawa Island) in the Ryukyu Islands proved that some of these feces were from dugongs, which had been treated in recent studies as extinct in this region since the last stranding of a deceased individual in 2019. In addition, local knowledge of sightings of animals thought to be dugongs and confirmed cases of dugong feeding trails since 2010 were compiled to estimate its recent distribution. This is the first scientific report on the presence of this mammal in the Ryukyu Islands within the last four years, and particularly in the Miyako Islands within the last half-century. As the Ryukyu Islands are known to be the northern limit of the dugong's fragmented distribution in East Asia, conservation efforts are therefore needed.

Spontaneous Liquid Droplet-to-Gel Transition of Citrulline Polypeptide Complexed with Nucleic Acids.

Inside cells, proteins complex with nucleic acids to form liquid droplets resulting from liquid-liquid phase separation. The presence of mutated proteins can change the state of these liquid droplets to solids or gels, triggering neurodegenerative diseases. The mechanism of the liquid to solid or gel transition is still unclear. Solutions of poly(l-ornithine-co-l-citrulline) (PLOC) copolymers, which exhibit upper critical solution temperature-type behavior, change state upon cooling. In this study, we evaluated the effect of nucleic acids complexed with PLOC on phase changes. In the presence of nucleic acids, such as polyC and polyU, PLOC formed liquid droplets at low temperatures. The droplets dissolved at temperatures above the phase separation temperature. The phase separation temperature depended on the chemical structure of the nucleobase, implying that electrostatic and hydrogen bonding interactions between the nucleic acid and PLOC influenced phase separation. Furthermore, the liquid droplets spontaneously changed to gel-like precipitates due to spontaneous release of nucleic acids from the complex. The rate of the liquid droplet-to-gel transition depended on the magnitude of electrostatic and hydrogen bonding interactions between PLOC and nucleic acid. PLOC complexed with mRNA also underwent a liquid droplet-to-gel transition upon the release of mRNA. This work provides insights into the mechanism of pathogenic transitions of the cellular droplets.

Effect of microclimatic temperatures on the development period of 3 rice planthopper species (Hemiptera: Delphacidae): a phenology model based on field observations.

Most pest phenology models are temperature dependent. Generally, the air temperature at reference height is used to predict pest development, but the air temperature varies between inside and outside the crop canopy, where pests reside. Here, we sampled 3 rice planthopper species-Nilaparvata lugens (Stål), Sogatella furcifera (Horváth), and Laodelphax striatellus (Fallén)-and micrometeorological observations in paddy fields to analyze how thermal environments inside the canopy affect pest development. Seasonal variations in the population density of these species were surveyed in 3 experimental fields with 2 water temperature plots (normal and low-water temperature plots). The development periods of the 3 species were predicted individually based on pest phenology models using temperatures recorded at 6 heights (0.0-2.0 m). We calculated the root mean square error (RMSE) values from the predicted and observed development periods for each rice planthopper. The development prediction using the temperature inside the canopy was more accurate than that utilizing the temperature at the reference height (2.0 m). In the low-water temperature plot, the RMSE value for N. lugens, S. furcifera, and L. striatellus was 6.4, 5.6, and 4.1 when using the temperature at the reference height (2.0 m), respectively, and 2.8, 3.8, and 2.9 when employing the temperature inside the canopy at 0.25 m, respectively. The development prediction utilizing the air temperature at the bottom (0.25 m) of canopy, where N. lugens resides, was most effective for N. lugens among the 3 species. These findings suggest the importance of utilizing microhabitat-based temperatures to predict pest development.

Alternative method for determining leaf CO2 assimilation without gas exchange measurements: Performance, comparison and sensitivity analysis.

We present an alternative method to determine leaf CO2 assimilation rate (An ), eliminating the need for gas exchange measurements in proximal and remote sensing. This method combines the Farquhar-von Caemmerer-Berry photosynthesis model with mechanistic light reaction (MLR) theory and leaf energy balance (EB) analysis. The MLR theory estimates the actual electron transport rate (J) by leveraging chlorophyll fluorescence via pulse amplitude-modulated fluorometry for proximal sensing or sun-induced chlorophyll fluorescence measurements for remote sensing, along with spectral reflectance. The EB equation is used to directly estimate stomatal conductance from leaf temperature. In wheat and soybean, the MLR-EB model successfully estimated An variations, including midday depression, under various environmental and phenological conditions. Sensitivity analysis revealed that the leaf boundary layer conductance (gb ) played an equal, if not more, crucial role compared to the variables for J. This was primarily caused by the indirect influence of gb through the EB equation rather than its direct impact on convective CO2 exchange on the leaf. Although the MLR-EB model requires an accurate estimation of gb , it can potentially reduce uncertainties and enhance applicability in photosynthesis assessment when gas exchange measurements are unavailable.

Malachite green-derivatized cationic comb-type copolymer acts as a photoresponsive artificial chaperone.

Molecular chaperones play vital roles in various physiological reactions by regulating the folding and assembly of biomacromolecules. We have demonstrated that cationic comb-type copolymers exhibit chaperone activity for anionic biomolecules including DNA and ionic peptide via the formation of soluble interpolyelectrolyte complexes. The development of smart artificial chaperones that can be spatiotemporally controlled by a remotely guided signal would expand the functions of artificial chaperones. Herein, to enable photocontrol of chaperone activity, a cationic comb-type copolymer bearing malachite green as a photoresponsive unit was designed. We first prepared a series of carboxylic acid derivatives of malachite green identified a derivative that could be quickly and quantitatively converted to the cationic form from the nonionic form by photoirradiation. This derivative was conjugated to the cationic comb-type copolymer, poly(allylamine)-graft-poly(ethylene glycol) through a condensation reaction. Upon photoirradiation, the copolymer bearing 9 mol% malachite green enhanced the membrane disruptive activity of acidic peptide E5 and induced morphological changes in liposomes. This demonstration of photoresponsive activation of chaperoning activity of a copolymer suggests that the installation of carboxyl derivatives of malachite green will impart photoresponsiveness to various materials including biopolymers.

Cationic copolymer and crowding agent have a cooperative effect on a Na+-dependent DNAzyme.

DNAzymes are promising agents for theranostics and biosensors. Sodium dependent DNAzymes have been developed for sensing and imaging of Na+, but these DNAzymes have low catalytic activity. Herein, we demonstrate that a molecular crowded environment containing 10 to 40 wt% PEG enhanced the catalytic activity of a Na+-dependent DNAzyme, EtNa, although dextran did not. The cationic copolymer poly(L-lysine)-graft-poly(ethylene glycol) at 0.03 wt% (0.3 g L-1) enhanced the reaction rate of EtNa by 10-fold, which is similar to the acceleration induced by 15 wt% (150 g L-1) PEG. A cooperative impact of the copolymer and crowding agent was observed: the combination resulted in an impressive 46-fold acceleration effect. Thus, the use of a cationic copolymer and a crowding agent is a promising strategy to improve the activity of Na+-dependent DNAzyme-based nanomachines, biosensors, and theranostics, especially in environments lacking divalent metal ions.

Consumer reaction to indoor farming using LED lighting technology and the effects of providing information thereon.

Indoor vertical farming using artificial light has gained popularity as one solution to food problems. However, prior studies have shown that some consumers have a negative impression that crops are grown in an artificial environment. The increased use of purple Light-Emitting Diode (LED) lighting, which would make the growing environment look more artificial, may exacerbate that negative perception, leading to low acceptance of vertically farmed produce. Given that consumers are increasingly seeing indoor vertical farming directly, for example, in supermarkets and office buildings, it is important to understand how they perceive the use of purple LED lighting to grow crops and whether these perceptions can be improved by learning more about the scientific basis for artificial light cultivation. This study aimed to determine whether purple LED lighting reduces consumers' perceptions of indoor vertical farming compared to traditional white lighting, and to examine whether providing information on plant growth and artificial light changes those perceptions. We administered a web-based questionnaire to 961 Japanese respondents, and analyzed the response data using analysis of variance and an ordered probit model to explore the factors that define the likability for indoor vertical farming. The results revealed that the color of LED lighting had a limited influence on consumers' perceptions of indoor vertical farming, whereas explaining the principle of plant growth under artificial light improves their perceptions. Additionally, personal factors, such as resistance to novel food technology, trust in food safety, and awareness of indoor vertical farming, had a significant impact on the perceptions. It is crucial to expand opportunities for people to interact with artificial light cultivation and disseminate information about its scientific mechanisms.

A Cationic Copolymer Enhances Responsiveness and Robustness of DNA Circuits.

Toehold-mediated DNA circuits are extensively employed to construct diverse DNA nanodevices and signal amplifiers. However, operations of these circuits are slow and highly susceptive to molecular noise such as the interference from bystander DNA strands. Herein, this work investigates the effects of a series of cationic copolymers on DNA catalytic hairpin assembly, a representative toehold-mediated DNA circuit. One copolymer, poly(L -lysine)-graft-dextran, significantly enhances the reaction rate by 30-fold due to its electrostatic interaction with DNA. Moreover, the copolymer considerably alleviates the circuit's dependency on the length and GC content of toehold, thereby enhancing the robustness of circuit operation against molecular noise. The general effectiveness of poly(L -lysine)-graft-dextran is demonstrated through kinetic characterization of a DNA AND logic circuit. Therefore, use of a cationic copolymer is a versatile and efficient approach to enhance the operation rate and robustness of toehold-mediated DNA circuits, paving the way for more flexible design and broader application.

2D-3D-Convertible, pH-Responsive Lipid Nanosheets.

2D nanosheets self-assembled with amphiphilic molecules are promising tools for biomedical applications; yet, there are challenges to form and stabilize these nanosheets under complex physiological conditions. Here, the development of lipid nanosheets with high structural stability that can be reversibly converted to cell-sized vesicles by changes in pH within the physiological range robustly, are described. The system is controlled by the membrane disruptive peptide E5 and a cationic copolymer anchored on lipid membranes. It is envisioned that nanosheets formed using the dual anchoring peptide/cationic copolymer system can be employed in dynamic lipidic nanodevices, such as the vesosomes described here, drug delivery systems, and artificial cells.

Immune thrombocytopenic purpura in primary biliary cholangitis and localized cutaneous systemic sclerosis: case report and literature review.

Primary biliary cholangitis (PBC) is a chronic progressive cholestatic liver disease of uncertain etiology. Although PBC is frequently complicated by Sjögren's syndrome and chronic thyroiditis, it can also be associated with a variety of other autoimmune disorders. We herein describe a rare case of immune thrombocytopenic purpura (ITP) coexistence with PBC and localized cutaneous systemic sclerosis (LcSSc). A 47-year-old woman with PBC and LcSSc who was positive for antiphospholipid antibody experienced a rapid decrease in platelet count to 1.8 × 104/µL during follow-up. After clinical findings ruled out thrombocytopenia from cirrhosis, a diagnosis of ITP was made following bone marrow examination. Her human leukocyte antigen (HLA) type was HLA-DPB1*05:01, which has been associated with disease susceptibility to PBC and LcSSc, but not to ITP. A careful review of similar reports suggested that in PBC, other collagen disease complications, positive antinuclear antibody, and positive antiphospholipid antibody may all support a diagnosis of ITP. Clinicians should be vigilant for ITP when rapid thrombocytopenia is observed during the course of PBC.

Human Heme Oxygenase-1 Promoter Activity Is Mediated by Z-DNA Formation.

In recent years, it has been shown that Z-DNA formation in DNA plays functionally significant roles in nucleic acid metabolism, such as gene expression, chromosome recombination, and epigenetic regulation. The reason for the identification of these effects is mainly due to the advancement of Z-DNA detection methods in target genome regions in living cells.The heme oxygenase-1 (HO-1) gene encodes an enzyme that degrades an essential prosthetic heme, and environmental stimuli, including oxidative stress, lead to robust induction of the HO-1 gene. Many DNA elements and transcription factors are involved in the induction of the HO-1 gene, and Z-DNA formation in the thymine-guanine (TG) repetitive sequence in the human HO-1 gene promoter region is required for maximum gene induction.Here, we describe a detailed protocol for Z-DNA detection in the human HO-1 gene promoter region based on chromatin immunoprecipitation with quantitative PCR. We also provide some control experiments to consider in routine lab procedures.

Programmed Control of Fluorescence Blinking Patterns based on Electron Transfer in DNA.

Fluorescence imaging uses changes in the fluorescence intensity and emission wavelength to analyze multiple targets simultaneously. To increase the number of targets that can be identified simultaneously, fluorescence blinking can be used as an additional parameter. To understand and eventually control blinking, we used DNA as a platform to elucidate the processes of electron transfer (ET) leading to blinking, down to the rate constants. With a fixed ET distance, various blinking patterns were observed depending on the DNA sequence between the donor and acceptor units of the DNA platform. The blinking pattern was successfully described with a combination of ET rate constants. Therefore, molecules with various blinking patterns can be developed by tuning ET. It is expected that the number of targets that can be analyzed simultaneously will increase by the power of the number of blinking patterns.

Label-free detection of HPV mRNA with an artificial chaperone-enhanced MNAzyme (ACEzyme)-based electrochemical sensor.

Nucleic acid biosensors for point-of-care (POC) diagnostic applications are highly desirable. The ability to detect DNA and RNA in a simple, rapid, affordable and portable format leads to a range of important applications for early screening in the field of disease monitoring and management. Herein, we report the development of an isothermal, label-free electrochemical biosensor that was designed on the basis of target-driven MNAzyme cleavage activity. Hybridization with HPV mRNA, a model nucleic acid target, activated MNAzyme and initiated the cleavage of immobilized hairpin substrates, leading to changes in the electrochemical signal. Under optimal conditions, a detection limit of 2.6 pM was obtained with an incubation time of 60 min. Furthermore, an artificial chaperone-enhanced MNAzyme (ACEzyme) system was integrated to an electrochemical biosensor for the first time. The analytical performance of the biosensor was enhanced, and the detection time was significantly reduced by the addition of PLL-g-Dex, which exhibits nucleic acid chaperone-like activity. A detection limit of 0.88 pM was obtained with a threefold decrease in incubation time without prior amplification. The proposed biosensing platform shows the advantages of simple fabrication and operation, good selectivity in the presence of single-base mismatch, and excellent versatility in a complex mixture of total RNA. We believe that this isothermal, label-free, and protein-free nucleic acid analysis platform could provide foundations for the further development of a universal nucleic acid biosensing platform for clinical application.

Label free electrochemical DNA biosensor for COVID-19 diagnosis.

The COVID-19 pandemic has significantly increased the development of the development of point-of-care (POC) diagnostic tools because they can serve as useful tools for detecting and controlling spread of the disease. Most current methods require sophisticated laboratory instruments and specialists to provide reliable, cost-effective, specific, and sensitive POC testing for COVID-19 diagnosis. Here, a smartphone-assisted Sensit Smart potentiostat (PalmSens) was integrated with a paper-based electrochemical sensor to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A disposable paper-based device was fabricated, and the working electrode directly modified with a pyrrolidinyl peptide nucleic acid (acpcPNA) as the biological recognition element to capture the target complementary DNA (cDNA). In the presence of the target cDNA, hybridization with acpcPNA probe blocks the redox conversion of a redox reporter, leading to a decrease in electrochemical response correlating to SARS-CoV-2 concentration. Under optimal conditions, a linear range from 0.1 to 200 nM and a detection limit of 1.0 pM were obtained. The PNA-based electrochemical paper-based analytical device (PNA-based ePAD) offers high specificity toward SARS-CoV-2 N gene because of the highly selective PNA-DNA binding. The developed sensor was used for amplification-free SARS-CoV-2 detection in 10 nasopharyngeal swab samples (7 SARS-CoV-2 positive and 3 SARS-CoV-2 negative), giving a 100% agreement result with RT-PCR.

Autonomous Vesicle/Sheet Transformation of Cell-Sized Lipid Bilayers by Hetero-Grafted Copolymers.

Lipid bilayer transformations are involved in biological phenomena including cell division, autophagy, virus infection, and vesicle transport. Artificial materials to manipulate membrane dynamics play a vital role in cellular engineering and drug delivery technology that accesses the membranes of cells or liposomes. Transformation from 3D lipid vesicles to 2D nanosheets is thermodynamically prohibited because the apolar/polar interfaces between the hydrophobic bilayer edges and water are energetically unfavorable. We recently reported that cell-sized lipid vesicles (or giant vesicles) can be thoroughly transformed to 2D nanosheets by the addition of the amphiphilic E5 peptide and a cationic graft copolymer. Here, to understand the mechanisms underlying the lipid nanosheet formation, we systematically investigated the structural effects of the cationic copolymers on nanosheet formation. We found that lipid nanosheet formation is controlled in an all-or-nothing manner when the graft content of the copolymer is increased from 5.7 mol % to 7.7 mol %. This finding prompted us to obtain autonomous 2D/3D transformation system. A newly designed hetero-grafted cationic copolymers with thermoresponsive poly(N-isopropylacrylamide) grafts enables spontaneous 3D vesicle/2D nanosheet transformation in response to temperature. These findings would enable us to obtain smart nanointerfaces that trigger cell-sized lipid membrane dynamics in response to diverse stimuli and to create 2D-3D convertible lipid-based biomaterials.

Economies of scale in constructing plant factories with artificial lighting and the economic viability of crop production.

Since the introduction of LED lamps a decade ago, the plant factory with artificial lighting (PFAL) has been expected to be a savior that overcomes the food crisis, brings food safety, and enhances environmental friendliness. Despite such high expectations, the diffusion of commercial crop production in PFALs has been slow. It has been said that the main reason for this is the huge initial investment required to construct PFALs. This situation has attracted studies to access the economic feasibility of the crop production in PFALs. One thing strange in these studies is that they pay little attention to the scale of their PFALs. PFALs are factories so that they would be subject to economies of scale. If so, the scale of PFALs is an important factor that determines the economic feasibility of plant production in PFALs. However, no study has thus far attempted to examine whether economies of scale exist in the construction of PFALs. To fill this gap, this paper tries to examine, based on the data on the investment cost of PFAL construction collected from various countries and regions in the world, whether economies of scale exist in PFAL construction and, if yes, how it affects the economic viability of the plant production in PFALs by searching for the minimum scale that ensures PFAL crop production economically viable. The results show that economies of scale exist in PFAL construction, and that the production of lettuce, PFALs' most popular crop, is now well on a commercial basis with the technology level of the most advanced PFAL operators, but strawberries has not reached that stage yet. It is also shown that crop production in PFALs is highly sensitive to changes in the yield and the price of the crops: A 30% decline either in the yield or the price of lettuce would easily bring PFALs bankruptcy. It is discussed that the optimum scale of PFALs would depend not only on the economies of scale but also on the transaction costs, such as the costs of searching and keeping a sufficient number of buyers who offer good and stable crop prices.

Smart Protein Refolding System Based on UCST-Type Ureido Polymers.

We have reported that ureido polymers exhibit upper critical solution temperature (UCST)-type phase behavior in solution, which is the opposite of lower critical solution temperature (LCST)-type behavior. Furthermore, UCST-type ureido polymers undergo liquid-liquid phase separation (LLPS) upon cooling rather than the liquid-solid phase transition of the typical LCST-type polymers. In this study, ureido polymers with hydrophobic groups were prepared to evaluate the effects of cooling-induced LLPS of UCST-type polymers on refolding of proteins. When protein was heated with a ureido polymer functionalized with undecyl groups, aggregation of the protein was prevented. Subsequent cooling incubation resulted in the spontaneous release of the protein from the polymer. The released protein had enzymatic activity, suggesting that the protein refolded properly. Interestingly, efficient refolding was observed when the solution of the UCST-type ureido polymer and protein was incubated at around the phase separation temperature of the polymer, implying that cooling-induced LLPS of the polymer enhanced the release of the protein. Additionally, by centrifugation at 4 °C, the refolded protein was readily separated from the ureido polymers, which precipitated upon cooling.

Cationic Copolymer-Augmented DNA Hybridization Chain Reaction.

Various DNA assembly techniques and structures have emerged with the continuous progress of DNA nanotechnology. DNA hybridization chain reaction (HCR) is a representative example owing to isothermal and enzyme-free features. However, HCR is time consuming and is inhibited by nucleases present in biological samples. Herein, we demonstrated that a cationic copolymer, poly(l-lysine)-graft-dextran (PLL-g-Dex), significantly facilitated HCR and increased its initiator sensitivity by 40-fold. PLL-g-Dex promoted the generation of HCR products with high molecular weight by accelerating the initiation and the subsequent growth steps of HCR. Moreover, PLL-g-Dex protected the HCR system from nucleases, permitting HCR in the presence of serum components. Addition of PLL-g-Dex is a universal and efficient strategy that does not require optimization of the reactor setup or DNA sequences, thus laying a solid foundation for the wider application of HCR.