Chromatographic purification of small extracellular vesicles using an affinity column for phospholipid membranes.

OBJECTIVES: This study aimed to investigate whether chromatography using an ExoPUA column, an affinity column for phospholipid membranes, could potentially serve as an efficient, rapid, scalable, and reproducible method for purifying small extracellular vesicles (sEVs). RESULTS: We used the ExoPUA column connected to a fast-performance liquid chromatography system. One-step chromatographic purification of sEVs from culture supernatant using the ExoPUA protocol resulted in an 82 ± 16-fold increase in purity with a yield of 38 ± 5% of sEVs. The purified sEVs contained CD9, CD63, TSG101, and miRNA (miR-21), but not the endoplasmic reticulum protein Calnexin. Transmission electron microscopy indicated that the purified sEVs were intact. The purification performance of the ExoPUA protocol showed superior results in terms of yield compared to that of the differential ultracentrifugation method, the most commonly used method for purifying sEVs in laboratories, and purity compared to that of the DEAE chromatography protocol. CONCLUSION: The sEVs were effectively purified in the bind-elute mode and the ExoPUA column can be refreshed and sterilized with sodium hydroxide (NaOH), having high potential for multiple sEV purification in a scalable and industrial manner.

Improved bioluminescence-based endotoxin measurement method using a salt-resistant luciferase mutant.

Endotoxin is a component of the cell wall of gram-negative bacteria and causes fever and shock symptoms upon entering the bloodstream. We previously demonstrated that the bioluminescence-based Limulus amebocyte lysate test is highly sensitive and rapid for measuring endotoxin. However, as the firefly luciferase reaction is inhibited in the presence of sodium chloride, the endotoxin detection method did not meet the validation guidelines under medical dialysis conditions (range of 75-125% of the measured values tested in water). Here, we used a salt-resistant luciferase mutant, which met the criteria for validation of endotoxin measurement.

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride.

OBJECTIVES: Firefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study was aimed at obtaining mutant luciferase enzymes resistant to the sodium chloride inhibition. RESULTS: We first obtained two mutant luciferase enzymes whose inhibition were alleviated and determined the mutations to be Val288Ile and Glu488Val. Under medical dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride. CONCLUSIONS: The mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The luciferase CR may be extensively useful in any bioassay which includes firefly luciferase and is employed in the presence of sodium chloride.

Asbestos Detection with Fluorescence Microscopy Images and Deep Learning.

Fluorescent probes can be used to detect various types of asbestos (serpentine and amphibole groups); however, the fiber counting using our previously developed software was not accurate for samples with low fiber concentration. Machine learning-based techniques (e.g., deep learning) for image analysis, particularly Convolutional Neural Networks (CNN), have been widely applied to many areas. The objectives of this study were to (1) create a database of a wide-range asbestos concentration (0-50 fibers/liter) fluorescence microscopy (FM) images in the laboratory; and (2) determine the applicability of the state-of-the-art object detection CNN model, YOLOv4, to accurately detect asbestos. We captured the fluorescence microscopy images containing asbestos and labeled the individual asbestos in the images. We trained the YOLOv4 model with the labeled images using one GTX 1660 Ti Graphics Processing Unit (GPU). Our results demonstrated the exceptional capacity of the YOLOv4 model to learn the fluorescent asbestos morphologies. The mean average precision at a threshold of 0.5 (mAP@0.5) was 96.1% ± 0.4%, using the National Institute for Occupational Safety and Health (NIOSH) fiber counting Method 7400 as a reference method. Compared to our previous counting software (Intec/HU), the YOLOv4 achieved higher accuracy (0.997 vs. 0.979), particularly much higher precision (0.898 vs. 0.418), recall (0.898 vs. 0.780) and F-1 score (0.898 vs. 0.544). In addition, the YOLOv4 performed much better for low fiber concentration samples (<15 fibers/liter) compared to Intec/HU. Therefore, the FM method coupled with YOLOv4 is remarkable in detecting asbestos fibers and differentiating them from other non-asbestos particles.

Circulating FGF23 is not associated with cardiac dysfunction, atherosclerosis, infection or inflammation in hemodialysis patients.

Fibroblast growth factor (FGF) 23 is a bone-derived hormone regulating serum inorganic phosphate (Pi) concentration. FGF23 is also involved in the development of chronic kidney disease (CKD)-mineral and bone disorder. Serum FGF23 concentration begins to increase early in the progression of CKD and can be remarkably high in hemodialysis patients with end-stage renal disease. It has been reported that high FGF23 concentration is a risk factor for cardiac dysfunction, atherosclerosis, infection or systemic inflammation in CKD patients. FGF23 was also shown to induce cardiac hypertrophy directly acting on cardiomyocytes. However, it is still controversial whether high FGF23 is causing cardiac dysfunction, atherosclerosis, infection or systemic inflammation in CKD patients. In the current study, we investigated whether FGF23 concentration is associated with cardiac dysfunction, atherosclerosis, infection or systemic inflammation in Japanese hemodialysis patients. We recruited 119 hemodialysis patients and examined the association between serum FGF23 concentration and several parameters concerning mineral metabolism, cardiac dysfunction, atherosclerosis, infection, and systemic inflammation. Serum FGF23 concentration was independently associated with serum calcium and Pi concentration (ß = 0.276, p < 0.001; ß = 0.689, p < 0.001). However, serum FGF23 concentration was not associated with parameters of cardiac dysfunction, atherosclerosis, infection, and systemic inflammation, either. Our results do not support the hypothesis that high FGF23 in dialysis patients is the cause of cardiac dysfunction, atherosclerosis, infection or systemic inflammation.

Degradation of phosphate polymer polyP enhances lactic fermentation in mice.

In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a "molecular fossil" due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.

Stabilisation of lipid membrane-incorporated porphyrin derivative aqueous solutions and their photodynamic activities.

Lipid membrane-incorporated porphyrin derivatives (LMIPors) having three phenyl moieties and one pyridyl or pyridinium moiety at the meso-positions were more stable than LMIPors having phenyl and/or pyridyl moieties. The former two LMIPors showed high photodynamic activity toward cancer cells under photoirradiation at wavelengths over 600 nm, which are the most suitable for photodynamic therapy. The photodynamic activities were greater than that of Photofrin, which is currently the main drug employed clinically as a photosensitiser. The results represent significant progress toward the optimisation of LMIPors as photosensitisers.

Insulin sensor cells for the analysis of insulin secretion responses in single living pancreatic ß cells.

Investigation of the functions of insulin-secreting cells in response to glucose in single-living cells is essential for improving our knowledge on the pathogenesis of diabetes. Therefore, it is desired to develop a new convenient method that enables the direct detection of insulin secreted from single-living cells. Here, insulin-sensor-cells expressing a protein-based insulin-detecting probe immobilized on the extracellular membrane were developed to evaluate the insulin-secretion response in single-living pancreatic ß cells. The protein-based insulin-detecting probe (NαLY) was composed of a bioluminescent protein (nano-luc), the αCT segment of the insulin receptor, L1 and CR domains of the insulin receptor, and a fluorescent protein (YPet). NαLY exhibited a bioluminescence resonance energy transfer (BRET) signal in response to insulin; thus, cells of Hepa1-6 line were genetically engineered to express NαLY on the extracellular membrane. The cells were found to act as insulin-sensor-cells, exhibiting a BRET signal in response to insulin. When the insulin-sensor-cells and pancreatic ß cells (MIN6 cell line) were cocultured and stimulated with glucose, insulin-sensor-cells nearby pancreatic ß cells showed the spike-shaped BRET signal response, whereas the insulin-sensor-cells close to one pancreatic ß cell did not exhibit such signal response. However, all the insulin-sensor-cells showed a gradual increase in BRET signals, which were presumably attributed to the increase in insulin concentrations in the culture dish, confirming the function of these insulin-sensor-cells. Therefore, we demonstrated that heterogenetic insulin secretion in single-living pancreatic ß cells could be measured directly using the insulin sensor cells.

High photodynamic activities of water-soluble inclusion complexes of 5,15-diazaporphyrins in cyclodextrin.

Water-soluble inclusion complexes of 5,15-diazaporphyrin derivatives in the cavities of two trimethyl-ß-cyclodextrins (TMe-ß-CDxs) were synthesised. In the 2 : 1 complexes, two aryl groups of the diazaporphyrins protruded from the upper rims of two TMe-ß-CDxs. The complexes displayed high photodynamic activity under photoirradiation at wavelengths longer than 620 nm. Although the substituents on the two aryl groups protruding from TMe-ß-CDx barely affected intracellular uptake by HeLa cells, the cellular uptake of these complexes was as high as that of a TMe-ß-CDx-tetra(4-hydroxyphenyl)porphyrin complex. Furthermore, the diazaporphyrins in the complexes with TMe-ß-CDxs were able to generate high levels of singlet oxygen because of their strong absorption of light with wavelengths greater than 620 nm.

Arginine-mediated dissociation of single cells and cell sheets from a polystyrene culture dish.

Here, we report a novel non-enzymatic cell dissociation method, based on our finding that adherent cells dissociate rapidly from the polystyrene culture dish when incubated in an l- or d-arginine-containing solution. We also demonstrate the successful detachment of confluent NIH/3T3 cell monolayers from the culture dish as a cell sheet by the addition of an arginine solution.

Predictive factors of posttransplant glucose intolerance in Japanese patients with type 1 diabetes after pancreas transplantation.

Pancreas transplantation (PTx) has been performed worldwide for patients with type 1 diabetes accompanied with end-stage renal disease or uncontrollable glycemic fluctuation. Nevertheless, risk factors of posttransplant glucose intolerance, which is responsible for progress of diabetic complications, remains unclear, especially in cases without pancreatic graft function loss. Therefore, this study was conducted to search for predictive factors of future glucose tolerance in PTx recipients without pancreatic graft function loss. Subjects were selected from among 41 Japanese patients with type 1 diabetes who received PTx between 2000 and 2016 in Osaka University Hospital, and 24 subjects free from rejections and thromboses were analyzed. Several examinations to evaluate insulin secretion and insulin sensitivity within 6 months after transplantation (initial examination) were performed. Glucose tolerance was evaluated by 120-minute post-load plasma glucose level during 75-g oral glucose tolerance tests (OGTT), referred to as PGOGTT120, at the initial examination and between 1 year and 2 years posttransplantation (maintenance period). The initial examination factors that were correlated with PGOGTT120 in the maintenance period were PGOGTT120 [r = 0.52 (p = 0.01)], insulinogenic index [r = -0.65 (p < 0.01)], and the ratio of incremental area under the curve of insulin to that of plasma glucose (iAUCR) calculated from data of OGTT [r = -0.65 (p < 0.01)]. Insulinogenic index [ß = -0.28 (p = 0.02)] and iAUCR [ß = -0.29 (p = 0.02)] were still significantly correlated with PGOGTT120 in the maintenance period after adjustment for PGOGTT120 at the initial examination. In conclusion, insulinogenic index and iAUCR from OGTT performed in the early posttransplantation period were predictive factors of future glucose intolerance.

Improvement of Photodynamic Activity of Lipid-Membrane-Incorporated Fullerene Derivative by Combination with a Photo-Antenna Molecule.

The weak absorbance of pristine C60 , C70 , and fullerene derivatives at wavelengths over 600 nm hampers the use of these molecules as photosensitizers (PSs) for photodynamic therapy (PDT). The coexistence of light-harvesting antenna molecules with a fullerene derivative in lipid membrane bilayers solved this issue. By controlling the location of the C60 derivative in the lipid membrane, the liposomal dyad system for PDT improved the photodynamic activity via an efficient photoenergy transfer from antenna molecules to the fullerene derivative. The photodynamic activity was found to be much higher than those of dyad systems using pristine C60 and C70 .

Engineering of Escherichia coli to facilitate efficient utilization of isomaltose and panose in industrial glucose feedstock.

Industrial glucose feedstock prepared by enzymatic digestion of starch typically contains significant amounts of disaccharides such as maltose and isomaltose and trisaccharides such as maltotriose and panose. Maltose and maltosaccharides can be utilized in Escherichia coli fermentation using industrial glucose feedstock because there is an intrinsic assimilation pathway for these sugars. However, saccharides that contain α-1,6 bonds, such as isomaltose and panose, are still present after fermentation because there is no metabolic pathway for these sugars. To facilitate more efficient utilization of glucose feedstock, we introduced glvA, which encodes phospho-α-glucosidase, and glvC, which encodes a subunit of the phosphoenolpyruvate-dependent maltose phosphotransferase system (PTS) of Bacillus subtilis, into E. coli. The heterologous expression of glvA and glvC conferred upon the recombinant the ability to assimilate isomaltose and panose. The recombinant E. coli assimilated not only other disaccharides but also trisaccharides, including alcohol forms of these saccharides, such as isomaltitol. To the best of our knowledge, this is the first report to show the involvement of the microbial PTS in the assimilation of trisaccharides. Furthermore, we demonstrated that an L-lysine-producing E. coli harboring glvA and glvC converted isomaltose and panose to L-lysine efficiently. These findings are expected to be beneficial for industrial fermentation.

The C-Terminal Zwitterionic Sequence of CotB1 Is Essential for Biosilicification of the Bacillus cereus Spore Coat.

UNLABELLED: Silica is deposited in and around the spore coat layer of Bacillus cereus, and enhances the spore's acid resistance. Several peptides and proteins, including diatom silaffin and silacidin peptides, are involved in eukaryotic silica biomineralization (biosilicification). Homologous sequence search revealed a silacidin-like sequence in the C-terminal region of CotB1, a spore coat protein of B. cereus. The negatively charged silacidin-like sequence is followed by a positively charged arginine-rich sequence of 14 amino acids, which is remarkably similar to the silaffins. These sequences impart a zwitterionic character to the C terminus of CotB1. Interestingly, the cotB1 gene appears to form a bicistronic operon with its paralog, cotB2, the product of which, however, lacks the C-terminal zwitterionic sequence. A ΔcotB1B2 mutant strain grew as fast and formed spores at the same rate as wild-type bacteria but did not show biosilicification. Complementation analysis showed that CotB1, but neither CotB2 nor C-terminally truncated mutants of CotB1, could restore the biosilicification activity in the ΔcotB1B2 mutant, suggesting that the C-terminal zwitterionic sequence of CotB1 is essential for the process. We found that the kinetics of CotB1 expression, as well as its localization, correlated well with the time course of biosilicification and the location of the deposited silica. To our knowledge, this is the first report of a protein directly involved in prokaryotic biosilicification. IMPORTANCE: Biosilicification is the process by which organisms incorporate soluble silicate in the form of insoluble silica. Although the mechanisms underlying eukaryotic biosilicification have been intensively investigated, prokaryotic biosilicification was not studied until recently. We previously demonstrated that biosilicification occurs in Bacillus cereus and its close relatives, and that silica is deposited in and around a spore coat layer as a protective coating against acid. The present study reveals that a B. cereus spore coat protein, CotB1, which carried a C-terminal zwitterionic sequence, is essential for biosilicification. Our results provide the first insight into mechanisms required for biosilicification in prokaryotes.

Sequential cleavage of insulin receptor by calpain 2 and γ-secretase impairs insulin signalling.

AIMS/HYPOTHESIS: Soluble insulin receptor (sIR), the ectodomain of the insulin receptor (IR), has been detected in human plasma and its concentration paralleled that of blood glucose. We have previously developed an in vitro model using HepG2 liver-derived cells, which mimics changes in sIR levels in plasma from diabetic patients and shows that calcium-dependent proteases cleave IR extracellularly (a process known as shedding). The present study aimed to reveal the mechanisms of IR cleavage. METHODS: Using the in vitro model, we investigated the molecular mechanisms of IR cleavage, which is accelerated by high-glucose treatment. We also analysed the relationship between IR cleavage and cellular insulin resistance, and the correlation between plasma sIR levels and insulin sensitivity, which was assessed by the euglycaemic-hyperinsulinaemic clamp technique. RESULTS: Here, we determined that calpain 2, which is secreted into the extracellular space associated with exosomes, directly cleaved the ectodomain of the IRß subunit (IRß), which in turn promoted intramembrane cleavage of IRß by γ-secretase. IR cleavage impaired insulin signalling and the inhibition of IR cleavage (by knockdown of calpain 2 and γ-secretase), restored IR substrate-1 and Akt, independent of IR. Furthermore, the glucose-lowering drug, metformin, prevented IR cleavage accompanied by inhibition of calpain 2 release in exosomes, and re-established insulin signalling. In patients with type 2 diabetes, plasma sIR levels inversely correlated with insulin sensitivity. CONCLUSIONS/INTERPRETATION: Sequential cleavage of IR by calpain 2 and γ-secretase may contribute to insulin signalling in cells and its inhibition may be partly responsible for the glucose-lowering effects of metformin. Thus, IR cleavage may offer a new mechanism for the aetiology of insulin resistance.

Continuous Monitoring of Specific mRNA Expression Responses with a Fluorescence Resonance Energy Transfer-Based DNA Nano-tweezer Technique That Does Not Require Gene Recombination.

This letter discusses the feasibility of continuously monitoring specific mRNA expression responses in a living cell with a probe structured as a fluorescence resonance energy transfer (FRET)-based DNA nano-tweezer (DNA-NT). The FRET-based DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from an open state to a closed state in recognition of a target mRNA, resulting in the closing of the distal relation of previously modified FRET-paired fluorescent dyes and generating a FRET signal. The expressions of glucose transporters (GLUT) 1 and 4 in a mouse hepato-carcinoma (Hepa 1-6 cells) were selected as the target model. Live-cell imaging analysis of Hepa 1-6 cells with both FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those measured with the conventional mass analysis technique of semiquantitative real-time (RT) polymerase chain reaction (PCR). From these results, it is concluded that continuous monitoring of gene expression response without gene recombination is feasible with a FRET-based DNA-NT, even in a single cell manner.

A Review of Insulin-Dosing Formulas for Continuous Subcutaneous Insulin Infusion (CSII) for Adults with Type 1 Diabetes.

Dosing guidelines for patients with type 1 diabetes using continuous subcutaneous insulin infusion (CSII), which are historically based on clinical experience and retrospective studies of patients consuming an American diet, recommend that basal insulin should represent approximately 50 % of the total daily dose (TDD). Recent prospective studies in the USA and Japan conclude that the more appropriate proportion is closer to 30-40 % of TDD. In addition, currently used formulas for calculating the carbohydrate-to-insulin ratio (CIR) and correction factor (CF) may lead to underdosing of bolus insulin by as much as 12.8-50 % for a hypothetical patient. The discrepancies between traditional formulas and data from newer studies can be accounted for by the more rigorous design of the newer studies (e.g., prospective design, controlled diets, meal omission, and frequent glucose monitoring). International differences in diet composition may also be important to consider when developing dosing recommendations for CSII.

A Lactobacillus mutant capable of accumulating long-chain polyphosphates that enhance intestinal barrier function.

Inorganic polyphosphate (polyP) was previously identified as a probiotic-derived substance that enhances intestinal barrier function. PolyP-accumulating bacteria are expected to have beneficial effects on the human gastrointestinal tract. In this study, we selected Lactobacillus paracasei JCM 1163 as a strain with the potential to accumulate polyP, because among the probiotic bacteria stored in our laboratory, it had the largest amount of polyP. The chain length of polyP accumulated in L. paracasei JCM 1163 was approximately 700 phosphate (Pi) residues. L. paracasei JCM 1163 accumulated polyP when Pi was added to Pi-starved cells. We further improved the ability of L. paracasei JCM 1163 to accumulate polyP by nitrosoguanidine mutagenesis. The mutant accumulated polyP at a level of 1500 nmol/mg protein-approximately 190 times that of the wild-type strain. PolyP extracted from the L. paracasei JCM 1163 significantly suppressed the oxidant-induced intestinal permeability in mouse small intestine. In conclusion, we have succeeded in breeding the polyP-accumulating Lactobacillus mutant that is expected to enhance intestinal barrier function.

Serum carboxy-terminal telopeptide of type I collagen levels are associated with carotid atherosclerosis in patients with cardiovascular risk factors.

Carboxy-terminal telopeptide of type I collagen (ICTP) is generated through matrix metalloproteinase (MMP)-dependent type I collagen digestion, and has been widely utilized as a biomarker for bone turnover. The fact that atherosclerotic lesions are rich in both type I collagen and MMP-producing macrophages led to the hypothesis that serum ICTP concentrations may serve as a non-invasive clinical biomarker for atherosclerosis. Therefore, the association of serum ICTP concentrations with the maximum intima-media thickness (IMT) of carotid arteries, a surrogate index of systemic atherosclerosis, or brachial-ankle pulse wave velocity (baPWV) in patients with atherosclerotic risk factors was evaluated. A total of 52 male and 65 female (mean age: 62.8 yrs) patients without renal failure, malignancies or bone diseases known to affect serum ICTP concentrations were recruited. Patients with max IMTs ≥1.1 mm showed significantly higher serum ICTP concentrations compared with patients with max IMTs <1.1 mm (3.33 ± 0.97 vs 2.82 ± 0.65 ng/mL, p<0.05). Serum ICTP concentration was also positively correlated with max IMT (p<0.001) or baPWV values (p<0.05). Multivariate analyses also revealed that serum ICTP concentrations were correlated with max IMT (p<0.001; 95% CI 0.200 to 0.454). These results suggest that serum ICTP concentrations can be used as a non-invasive biomarker for systemic atherosclerosis.

Differential Counting of Asbestos Using Phase Contrast and Fluorescence Microscopy.

Considering the increasing use of various asbestos substitutes, asbestos risk management in many industries may require accurate techniques for detecting and distinguishing asbestos from non-asbestos fibers. Using fluorescently labeled asbestos-binding proteins, we previously developed a novel method for detection and counting of asbestos fibers under fluorescence microscopy (FM). This method can provide speedy, on-site detection and identification of the asbestos fibers and has higher sensitivity than phase contrast microscopy (PCM). However, current asbestos exposure limits are derived from risk assessments based on epidemiological studies that were conducted using PCM fiber counts. Therefore, the sensitivity of asbestos testing should be maintained at PCM level to properly assess compliance with these limit values. Here, we developed and tested a novel application of FM as a differential counting method that complements PCM analysis and is fully compatible with the PCM-based epidemiological data. In the combined PCM-FM method, the fluorescent asbestos-binding probe is applied prior to filter clearing. The method makes it possible to easily switch between two microscopic techniques while analyzing the same fields of view: PCM is used for counting fibers, and FM for differentiating asbestos from non-asbestos fibers. Using airborne dust samples from demolition sites in Japan, we compared PCM-FM with scanning electron microscopy (SEM)-based differential counting method. Statistical analysis indicated a slight conservative bias of PCM-FM method, combined with relatively high variability across the full range of fiber concentrations in our sample set. Using correlative microscopy, we also evaluated the specificity of FM staining, which is a potential cause of variability between the two methods. The energy-dispersive X-ray analysis indicated that ~95% of fluorescently stained fibers in the demolition site samples were correctly identified as asbestos. While further research is needed to fully clarify the causes of variability between FM- and SEM-based differential counting, PCM-FM could be used for rapid and selective detection of asbestos fibers in field samples.