Advanced biopharmaceutical manufacturing requires novel process analytical technologies for the rapid and sensitive assessment of the higher-order structures of therapeutic proteins. However, ...conventional physicochemical analyses of denatured proteins have limitations in terms of sensitivity, throughput, analytical resolution, and real-time monitoring capacity. Although probe-based sensing can overcome these limitations, typical non-specific probes lack analytical resolution and provide little to no information regarding which parts of the protein structure have been collapsed. To meet these analytical demands, we generated biosensing probes derived from artificial proteins that could specifically recognize the higher-order structural changes in antibodies at the protein domain level. Biopanning of phage-displayed protein libraries generated artificial proteins that bound to a denatured antibody domain, but not its natively folded structure, with nanomolar affinity. The protein probes not only recognized the higher-order structural changes in intact IgGs but also distinguished between the denatured antibody domains. These domain-specific probes were used to generate response contour plots to visualize the antibody denaturation caused by various process parameters, such as pH, temperature, and holding time for acid elution and virus inactivation. These protein probes can be combined with established analytical techniques, such as surface plasmon resonance for real-time monitoring or plate-based assays for high-throughput analysis, to aid in the development of new analytical technologies for the process optimization and monitoring of antibody manufacturing.
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•Biosensing-based quality control technology for therapeutic antibodies was developed.•Novel probes that specifically bound to denatured antibody domains were generated.•These probes facilitate biosensing of antibody denaturation at the protein domain level.•This technology can be applied for process optimization of antibody manufacturing.
Accumulating evidence suggests that some dietary patterns, specifically high fat diet (HFD), increase the risk of developing sporadic Alzheimer disease (AD). Thus, interventions targeting HFD-induced ...metabolic dysfunctions may be effective in preventing the development of AD. We previously demonstrated that amyloid precursor protein (APP)-overexpressing transgenic mice fed HFD showed worsening of cognitive function when compared with control APP mice on normal diet. Moreover, we reported that voluntary exercise ameliorates HFD-induced memory impairment and β-amyloid (Aβ) deposition. In the present study, we conducted diet control to ameliorate the metabolic abnormality caused by HFD on APP transgenic mice and compared the effect of diet control on cognitive function with that of voluntary exercise as well as that of combined (diet control plus exercise) treatment. Surprisingly, we found that exercise was more effective than diet control, although both exercise and diet control ameliorated HFD-induced memory deficit and Aβ deposition. The production of Aβ was not different between the exercise- and the diet control-treated mice. On the other hand, exercise specifically strengthened the activity of neprilysin, the Aβ-degrading enzyme, the level of which was significantly correlated with that of deposited Aβ in our mice. Notably, the effect of the combination treatment (exercise and diet control) on memory and amyloid pathology was not significantly different from that of exercise alone. These studies provide solid evidence that exercise is a useful intervention to rescue HFD-induced aggravation of cognitive decline in transgenic model mice of AD.
Background: Exercise and diet control are fundamental approaches to metabolic conditions caused by high fat diet (HFD).
Results: HFD-induced memory deficit and Aβ deposition were more ameliorated in the exercise- than in the diet control-induced mice.
Conclusion: Exercise was more effective than diet control in preventing HFD-induced AD development.
Significance: Exercise has the highest priority in the prevention of AD.
Abstract The pathogenesis of Alzheimer's disease (AD) is tightly associated with metabolic dysfunctions. In particular, a potential link between type 2 diabetes (T2DM) and AD has been suggested ...epidemiologically, clinically, and experimentally, and some studies have suggested that exercise or dietary intervention reduces risk of cognitive decline. However, there is little solid molecular evidence for the effective intervention of metabolic dysfunctions for prevention of AD. In the present study, we established the AD model mice with diabetic conditions through high-fat diet (HFD) to examine the effect of environmental enrichment (EE) on HFD-induced AD pathophysiology. Here, we demonstrated that HFD markedly deteriorated memory impairment and increased β-amyloid (Aβ) oligomers as well as Aβ deposition in amyloid precursor protein (APP) transgenic mice, which was reversed by exposure to an enriched environment for 10 weeks, despite the continuation of HFD. These studies provide solid evidence that EE is a useful intervention to ameliorate behavioral changes and AD pathology in HFD-induced aggravation of AD symptoms in APP transgenic mice.
Abstract Cardiac progenitor cells (CPCs) are a crucial source of cells in cardiac development and regeneration. However, reported CPCs are heterogeneous, and no gene has been identified to ...transiently mark undifferentiated CPCs throughout heart development. Here we show that Spalt-like gene 1 ( Sall1 ), a zing-finger transcription factor, is expressed in undifferentiated CPCs giving rise to both left and right ventricles. Sall1 was transiently expressed in precardiac mesoderm contributing to the first heart field (left ventricle precursors) but not in the field itself. Similarly, Sall1 expression was maintained in the second heart field (outflow tract/right ventricle precursors) but not in cardiac cells. In vitro, high levels of Sall1 at mesodermal stages enhanced cardiomyogenesis, whereas its continued expression suppressed cardiac differentiation. Our study demonstrates that Sall1 marks CPCs in an undifferentiated state and regulates cardiac differentiation. These findings provide fundamental insights into CPC maintenance, which can be instrumental for CPC-based regenerative medicine.
The aim of this study was to clarify the effect of glycine on the growth of Staphylococcus aureus and the production of staphylococcal enterotoxin A (SEA) in cooked rice. We inoculated eleven ...strains of SEA-producing S. aureus at inoculum size of 106 CFU/g into cooked rice to which 0-5% glycine was added, and then quantified the bacterial number and SEA after incubating them at 37℃. The maximal concentrations of SEA were 2.1-40.8 ng/g, 1.9-53.9 ng/g, 0.9-35.0 ng/g, and 0 (not detected) －3.7 ng/g in cooked rice to which 0.5, 1, 2, and 5% glycine was added, respectively. Most strains of S. aureus produced less SEA in cooked rice to which 2 and 5% glycine was added than in the non-added control during the early phase of growth. However, the amount of SEA in contaminated cooked rice with S. aureus will reach a sufficient level to cause food poisoning regardless of the addition or non-addition of glycine, before its growth is perceived organoleptically.
A rapid and sensitive flow-injection method with peroxyoxalate chemiluminescence (CL) detection for the determination of glucose and uric acid by using an immobilized enzyme reactor is described. CL ...produced in a flow cell by the reaction of hydrogen peroxide with bis(2, 4, 6-trichlorophenyl)oxalate and a fluorescent compound, 2, 4, 6, 8-tetrathiomorpholinopyrimido5, 4-dpyrimidine, was monitored. Linear responses between the concentration and the CL intensity for glucose and uric acid were obtained up to 3×10-4M (6nmol per 20μl injection, r=0.999) and 1×10-5M (0.2nmol per 20μl injection, r=1.000), respectively; both limits of detection were the same (5×10-7M, 10pmol per 20μl injection, S/N>3). The relative standard deviations for ten replicate measurements were 1.1% for glucose (2×10-5M, 0.4 nmol/injection) and 2.2% for uric acid (5×10-6M, 0.1nmol/injection). Good correlations between the results of serum glucose or uric acid assay by the proposed methods and by the conventional colorimetric methods (r=0.994for glucose and r=0.986 for uric acid) were obtained.
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