Association of a Combination of Sarcopenia and Type 2 Diabetes with Blood Parameters, Nutrient Intake, and Physical Activity: A Nationwide Population-Based Study.

This study aimed to investigate the association of sarcopenia and type 2 diabetes (T2D) with blood parameters, nutrient intake, and physical activity in older Korean adults. We divided 2952 participants into four groups: sarcopenic diabetes (SD), sarcopenia alone (S), diabetes alone (D), and non-sarcopenia and non-diabetes (NSND). Sarcopenia was defined by the appendicular skeletal muscle mass index, and T2D by fasting glucose levels or ongoing treatment. Blood samples were collected after an 8-h fast. Nutrient intake was assessed using a 24-h recall; physical activity was evaluated using a questionnaire. Compared with those in the other groups, the men in the S and SD groups showed significantly lower hemoglobin and hematocrit levels; vitamin D levels in men and parathyroid hormone levels in women were significantly lower in the SD group. Total energy, protein, and carbohydrate intakes were significantly lower in the SD and S groups than those in the D and NSND groups. Physical inactivity was significantly more common in the SD group (men: odds ratio, 1.61; women: odds ratio, 2.37) than in the NSND group. A combination of sarcopenia and diabetes as well as sarcopenia alone was associated with low levels of blood parameters, poor nutrient intake, and low physical activity.

Identification of Factors Necessary for Enabling Technology-Based Dietary Record Surveys: A Qualitative Focus Group Interview with Japanese Dietitians.

Weighed food records together with an in-person interview approach constitute the most basic methods used to estimate energy and nutrient intakes in dietary surveys. In the background of the coronavirus disease-2019 pandemic, the need for non-face-to-face dietary surveys using information and communication technology (ICT) is increasing. We aimed to evaluate ICT-based dietary record surveys and identify factors that may enable this survey method to become more widely used in the future. We conducted a non-face-to-face survey of dietary records of 44 Japanese individuals, maintained by dietitians using dietary photography and video conferencing services. We conducted a focus group interview with the six dietitians who conducted that survey. Their opinions on the factors necessary to popularize ICT-based dietary survey method were analyzed. In the focus group interview, dietitians highlighted fewer restrictions on time and place as positive aspects. Negative aspects included insufficient skills to operate computers, difficulty in hearing, and understanding facial expressions using ICT. We identified three main factors for enabling widespread use of ICT-based dietary record survey: individual skill, device and technology, and social environmental factors. This suggests that a comprehensive approach is necessary for popularizing the use of ICT in dietary surveys.

Fungal Pigment-Assisted Silver Nanoparticle Synthesis and Their Antimicrobial and Cytotoxic Potential.

The biological production of nanoparticles has taken center stage among the various generation methods available owing to its environment-friendly characteristics and reduced energy requirements. A protocol for pigment-assisted silver nanoparticle (AgNP) synthesis was established, employing an extracellular composite pigment produced by the Ascomycota Talaromyces purpurogenus (presently Talaromyces purpureogenus). The extracellular pigment can reduce the precursor silver salt into nanoparticles in the presence of sodium hydroxide and light. Transmission electron microscopy may be used to characterize the bio-generated nanoparticles, which can also be tested for their biological properties using antimicrobial and anticancer assays.

Visualization of Gene Reciprocity among Lactic Acid Bacteria in Yogurt by RNase H-Assisted Rolling Circle Amplification-Fluorescence In Situ Hybridization.

Recently, we developed an in situ mRNA detection method termed RNase H-assisted rolling circle amplification-fluorescence in situ hybridization (RHa-RCA-FISH), which can detect even short mRNA in a bacterial cell. However, because this FISH method is sensitive to the sample condition, it is necessary to find a suitable cell permeabilization and collection protocol. Here, we demonstrate its further applicability for detecting intrinsic mRNA expression using lactic acid bacteria (LAB) as a model consortium. Our results show that this method can visualize functional gene expression in LAB cells and can be used for monitoring the temporal transition of gene expression. In addition, we also confirmed that data obtained from bulk analyses such as RNA-seq or microarray do not always correspond to gene expression in individual cells. RHa-RCA-FISH will be a powerful tool to compensate for insufficient data from metatranscriptome analyses while clarifying the carriers of function in microbial consortia. By extending this technique to capture spatiotemporal microbial gene expression at the single-cell level, it will be able to characterize microbial interactions in phytoplankton-bacteria interactions.

Assay for advanced glycation end products generating intracellular oxidative stress through binding to its receptor.

Advanced glycation end products (AGEs) are a heterogenous group of glycation adducts on amino acids produced with sugars or dicarbonyls. Intracellular inflammation triggered by binding of AGEs to receptor for AGEs (RAGE) is linked to some chronic diseases. Here, we established a competitive assay format to comprehensively quantify AGEs which bound to RAGE. RAGE-binding activities of sugar- and dicarbonyl-derived AGEs were correlated with oxidative stress in cultured cells generated by the respective AGEs, suggesting that this would be a promising method for evaluating AGEs which could affect cellular functions despite limited information on individual glycation adducts.

Direct detection of mRNA expression in microbial cells by fluorescence in situ hybridization using RNase H-assisted rolling circle amplification.

Meta-analyses using next generation sequencing is a powerful strategy for studying microbiota; however, it cannot clarify the role of individual microbes within microbiota. To know which cell expresses what gene is important for elucidation of the individual cell's function in microbiota. In this report, we developed novel fluorescence in situ hybridization (FISH) procedure using RNase-H-assisted rolling circle amplification to visualize mRNA of interest in microbial cells without reverse transcription. Our results show that this method is applicable to both Gram-negative and Gram-positive microbes without any noise from DNA, and it is possible to visualize the target mRNA expression directly at the single-cell level. Therefore, our procedure, when combined with data of meta-analyses, can help to understand the role of individual microbes in the microbiota.

Biosynthesis of Silver Nanoparticles Mediated by Extracellular Pigment from Talaromyces purpurogenus and Their Biomedical Applications.

In recent years, green syntheses have been researched comprehensively to develop inexpensive and eco-friendly approaches for the generation of nanoparticles. In this context, plant and microbial sources are being examined to discover potential reducing agents. This study aims to utilize an extracellular pigment produced by Talaromyces purpurogenus as a prospective reducing agent to synthesize silver nanoparticles (AgNPs). Biosynthesized AgNPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), electron probe micro analyser (EPMA), and zeta potential. The pigment functional groups involved in the generation of AgNPs were investigated using Fourier transform infrared spectroscopy. TEM images showed that the generated nanoparticles were spherical, hexagonal, rod-shaped, and triangular-shaped with a particle size distribution from 4 to 41 nm and exhibited a surface plasmon resonance at around 410 nm. DLS and zeta potential studies revealed that the particles were polydispersed and stable (-24.8 mV). EPMA confirmed the presence of elemental silver in the samples. Biosynthesized AgNPs exhibited minimum inhibitory concentrations of 32 and 4 µg/mL against E. coli and S. epidermidis, respectively. Further, cytotoxicity of the AgNPs was investigated against human cervical cancer (HeLa), human liver cancer (HepG2), and human embryonic kidney (HEK-293) cell lines using 5-fluorouracil as a positive control. A significant activity was recorded against HepG2 cell line with a half-maximal inhibitory concentration of 11.1 µg/mL.

Microplate-based Assay for Screening of Advanced Glycation End Products Binding to Its Receptor.

Advanced Glycation End products (AGEs) are a group of amino-acid modifications produced with sugars or di-carbonyls. Some AGEs are known to affect health through binding to the receptor of AGEs (RAGE). Here, we propose a method for screening RAGE-binding AGEs by a competitive assay using purified RAGE and AGEs-specific antibody. This method has clarified that at least carboxyethyl lysine and pentosidine among methylglyoxal-derived AGEs are involved in RAGE binding, suggesting that this would be a promising method for classifying RAGE-binding AGEs.

RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection.

RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3'-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms.

Here, we demonstrated the expression of the N-glycosylated extracellular ligand binding domain of receptor for advanced glycation end products (sRAGE) in middle silk glands (MSGs) of transgenic silkworms using the GAL4/UAS system. Over 1 mg of sRAGE was obtained from one transgenic silkworm. sRAGE purified from the silkworm exhibited good stability and maintained specific ligand-binding ability. In addition, N-glycan analysis of sRAGE revealed that N-glucan completely lacked potentially allergenic fucose. Moreover, co-expression of biotin ligase (BirA) with C-terminal BioEase-tagged sRAGE in MSGs resulted in efficient biotinylation of sRAGE after addition of biotin bait. C-terminal biotinylated sRAGE could be immobilized onto a solid surface in one direction through binding to streptavidin without any loss of ability. The dissociation constant of sRAGE with fructose-BSA, a typical RAGE ligand, was 7.25 × 10-7 M, consistent with that on the mammalian cell surface. Thus, we developed a novel, innovative silkworm expression system for efficient expression of recombinant sRAGE, which could serve as a basis for the elucidation of RAGE-ligand interactions and facilitate the search for new ligands and inhibitors.

Development of a bench-top extra-cleanroom for DNA amplification.

Prevention of airborne contamination has become an important factor in biotechnology; however, conventional laminar-airflow cabinets (LAF-cabinets) are no longer sufficient as a countermeasure against nano-sized airborne contaminants in the laboratory. Here we present a bench-top extra-cleanroom classified as ISO-1 that can prevent contamination from airborne nanoparticles. This bench-top extra-cleanroom consists of a novel clean-zone-creating system that is equipped with nanofibrous, nonwoven filters. In addition, the cleanroom is also equipped with an ionizer to prevent plasticware from collecting dust by electrostatic charge attraction. This combination of features allows the cleanroom to prevent DNA contamination derived from airborne nanoparticles. Our extra-cleanroom with ionizer could be useful in various areas of biotechnology that are easily affected by airborne contaminants.

Analyses of nuclear proteins and nucleic acid structures using atomic force microscopy.

Since the inception of atomic force microscopy (AFM) in 1986, the value of this technology for exploring the structure and biophysical properties of a variety of biological samples has been increasingly recognized. AFM provides the opportunity to both image samples at nanometer resolution and also measure the forces on the surface of the sample. Here, we describe a variety of methods for studying nuclear samples including single nucleic acid molecules, higher-order chromatin structures, the nucleolus, and the nucleus. Protocols to prepare nucleic acids, nucleic acid-protein complexes, reconstituted chromatin, the cell nucleus, and the nucleolus are included, as well as protocols describing how to prepare the AFM substrate and the AFM tip. Finally, we describe how to perform conventional imaging, high-speed imaging, recognition imaging, force spectroscopy, and nanoindentation experiments.

Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose.

We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3'-5' exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

Expanding possibilities of rolling circle amplification as a biosensing platform.

Rolling circle amplification (RCA) catalyzed by φ29 DNA polymerase offers a simple method for DNA amplification in the presence of a circular DNA template and its complimentary primer. RCA continuously produces long single-strand DNA using the strand displacement activity of polymerase during DNA synthesis. This property allows one to monitor the progress of a reaction by means of electrophoresis or fluorescence measurements, and has eventually allowed the application of RCA to signal increments in the sensing of a variety of molecular species. Originally, RCA was successfully applied for the detection of specific DNA, such as single nucleotide polymorphisms. In addition, the conjugation of an antibody with a primer achieves efficient signal enhancement in antigen detection, and mRNA can also be specifically detected. Since RCA is a carry-over contamination-resistant, cost-effective, and user-friendly method of DNA amplification, RCA could be a universal technology for biosensing in fields of medical- and food-related industries.

Core histone charge and linker histone H1 effects on the chromatin structure of Schizosaccharomyces pombe.

Histones are highly conserved proteins among eukaryotes. However, yeast histones are more divergent in their sequences. In particular, the histone tail regions of the fission yeast, Schizosaccharomyces pombe, have fewer lysine residues, making their charges less positive than those of higher eukaryotes. In addition, the S. pombe chromatin lacks linker histones. How these factors affected yeast chromatin folding was analysed by biochemical reconstitution in combination with atomic force microscopy. Reconstitution of a nucleosome array showed that S. pombe chromatin has a more open structure similar to reconstituted human acetylated chromatin. The S. pombe nucleosomal array formed thinner fibers than those of the human nucleosomal array in the presence of mammalian linker histone H1. Such S. pombe fibers were more comparable to human acetylated fibers. These findings suggest that the core histone charges would determine the intrinsic characteristics of S. pombe chromatin and affect inter-nucleosomal interactions.

Gold nanoparticles as localization markers for direct and live imaging of particle absorption through a Caco-2 cell monolayer using dark-field microscopy.

Recently, efforts have been made to reduce the size of food particles containing functional ingredients, since reducing the size is expected to improve intestinal absorption. However, the absorption mechanisms have yet to be fully clarified. Therefore, a microscopy-based method for studying interactions between the particles and intestinal cells is required. We optimized the experimental conditions for observing gold nanoparticles (AuNPs) on the surface of an unfixed Caco-2 cell using dark-field microscopy (DFM). Tight junctions were clearly visible with AuNPs on the cells, producing intense scattered light under DFM. This suggests that AuNPs could be used as localization markers to visualize particle absorption through Caco-2 cells.

Direct detection of green fluorescent protein messenger RNA expressed in Escherichia coli by rolling circle amplification.

Prevailing conventional microbial detection methods depend largely on microbial cultivation in selective media that requires several days. Polymerase chain reaction (PCR)-based methods, including quantitative reverse transcription PCR, are also rapid and useful methods for identification of target microbes, although for practical use they still suffer from disadvantages such as contamination problems and cost. Here we demonstrate that RNA-primed rolling circle amplification (RPRCA) using phi29 DNA polymerase, a precircularized probe, and SYBR Green II achieved real-time detection of specific messenger RNA (mRNA) from living microbes. The precircularized DNA probe was prepared by intramolecular ligation using CircLigase and treated by exonuclease I to eliminate uncircularized oligonucleotide, thereby significantly reducing potential noise by nonspecific amplified DNA by-products that affect successive RPRCA. When in vitro transcribed green fluorescent protein (GFP) mRNA was used as a primer, RPRCA could specifically detect at least 1 fmol of this mRNA in the presence of a precircularized probe that had a sequence complementary to the 3' terminus of mRNA without reverse transcription. This method could also detect expressed GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without DNase treatment. These data suggest that RPRCA has the potential to be a direct, rapid, and convenient method for detecting microbial mRNA.

Rolling circle amplification for signal enhancement in ovalbumin detection.

We have developed a rolling circle amplification-procedure as a method to enhance signals of immunoassay (immuno-RCA) to detect one of the major food allergens, ovalbumin (OVA). Immuno-RCA in combination with a fluorescent dye and a circular DNA probe prepared by intramolecular ligation easily allowed the real-time detection of OVA, and could apparently detect signals from OVA with concentrations ranging from 10(-12) to 10(-7) g/mL. Therefore, sensitive and easily handled aspects of this method would contribute to an effective signal enhancement in immunoassay for food allergen detections.

The transient receptor potential channels TRPP2 and TRPC1 form a heterotetramer with a 2:2 stoichiometry and an alternating subunit arrangement.

There is functional evidence that polycystin-2 (TRPP2) interacts with other members of the transient receptor potential family, including TRPC1 and TRPV4. Here we have used atomic force microscopy to study the structure of the TRPP2 homomer and the interaction between TRPP2 and TRPC1. The molecular volumes of both Myc-tagged TRPP2 and V5-tagged TRPC1 isolated from singly transfected tsA 201 cells indicated that they assembled as homotetramers. The molecular volume of the protein isolated from cells expressing both TRPP2 and TRPC1 was intermediate between the volumes of the two homomers, suggesting that a heteromer was being formed. The distribution of angles between pairs of anti-Myc antibodies bound to TRPP2 particles had a large peak close to 90 degrees and a smaller peak close to 180 degrees , consistent with the assembly of TRPP2 as a homotetramer. In contrast, the corresponding angle distributions for decoration of the TRPP2-TRPC1 heteromer by either anti-Myc or anti-V5 antibodies had predominant peaks close to 180 degrees . This decoration pattern indicates a TRPP2:TRPC1 subunit stoichiometry of 2:2 and an alternating subunit arrangement.

Chromatin dynamics of unfolding and refolding controlled by the nucleosome repeat length and the linker and core histones.

Chromatin is composed of genomic DNA and histones, forming a hierarchical architecture in the nucleus. The chromatin hierarchy is common among eukaryotes despite different intrinsic properties of the genome. To investigate an effect of the differences in genome organization, chromatin unfolding processes were comparatively analyzed using Schizosaccaromyces pombe, Saccharomyces cerevisiae, and chicken erythrocyte. NaCl titration showed dynamic changes of the chromatin. 400-1000 mM NaCl facilitated beads with approximately 115 nm in diameter in S. pombe chromatin. A similar transition was also observed in S. cerevisiae chromatin. This process did not involve core histone dissociation from the chromatin, and the persistence length after the transition was approximately 26 nm for S. pombe and approximately 28 nm for S. cerevisiae, indicating a salt-induced unfolding to "beads-on-a-string" fibers. Reduced salt concentration recovered the original structure, suggesting that electrostatic interaction would regulate this discrete folding-unfolding process. On the other hand, the linker histone was extracted from chicken chromatin at 400 mM NaCl, and AFM observed the "beads-on-a-string" fibers around a nucleus. Unlike yeast chromatin, therefore, this unfolding was irreversible because of linker histone dissociation. These results indicate that the chromatin unfolding and refolding depend on the presence and absence of the linker histone, and the length of the linker DNA.