Small, Clickable, and Monovalent Magnetofluorescent Nanoparticles Enable Mechanogenetic Regulation of Receptors in a Crowded Live-Cell Microenvironment.

Multifunctional magnetic nanoparticles have shown great promise as next-generation imaging and perturbation probes for deciphering molecular and cellular processes. As a consequence of multicomponent integration into a single nanosystem, pre-existing nanoprobes are typically large and show limited access to biological targets present in a crowded microenvironment. Here, we apply organic-phase surface PEGylation, click chemistry, and charge-based valency discrimination principles to develop compact, modular, and monovalent magnetofluorescent nanoparticles (MFNs). We show that MFNs exhibit highly efficient labeling to target receptors present in cells with a dense and thick glycocalyx layer. We use these MFNs to interrogate the E-cadherin-mediated adherens junction formation and F-actin polymerization in a three-dimensional space, demonstrating the utility as modular and versatile mechanogenetic probes in the most demanding single-cell perturbation applications.

90-Day repeated oral toxicity test of D-allulose produced from Microbacterium foliorum.

D-allulose is considered an ideal substitute for sucrose, because it has 70% of the sweetness of sucrose and ultra-low energy. Chemical and biotechnological methods have been developed to produce Dallulose from D-fructose because D-allulose exists in extremely small quantities in nature. In this study, we performed a 90-day repeated oral dose toxicity test on rats using D-allulose produced from Microbacterium foliorum-a non-GMO species isolated from salad ginseng-in dosages of 0, 1250, 2500 and 5000 mg/kg/day. We developed a toxicity determination criterion based on the significant change caused by the administration of the substance to estimate the NOEL, NOAEL, and LOAEL of the substance applied in this study. This test found only minor compound-related changes in both male and female rats in the high dose group and no important compound-related changes. Thus, we determined the NOAEL of Dallulose in both sexes to be 5,000 mg/kg/day. This study's finding of a NOAEL of 5,000 mg/kg/day should ensure that D-allulose produced from Microbacterium foliorum is classified as a safe and ordinary substance.

Repeated dose 90-day oral toxicity test of G-7% NANA in rats: An application of new criterion for toxicity determination to test article-induced changes.

G-7% NANA is N-acetylneuraminic acid(NANA) containing 7% sialic acid isolated from glycomacropeptide (GMP), a compound of milk. Since NANA is likely to have immunotoxicity, the need to ensure safety for long-term administration has been raised. In this study, a 90-day repeated oral dose toxicity test was performed in rats using G-7% NANA in the dosages of 0, 1250, 2500 and 5000 mg/kg/day.A toxicity determination criterion based on the significant change caused by the administration of the substancewas developed for estimating NOEL, NOAEL and LOAELapplied to this study. When analyzing the immunological markers, no significant changes were observed, even if other significant changes were observed in the high dose group. In accordance with the toxicity determination criterion developed, the NOEL in male and female has been determined as 2500 mg/kg/day, and the NOAEL in females has been determined as 5000 mg/kg/day. The toxicity determination criterion, applied for the first time in the repeated dose toxicity tests, could provide a basis for distinguishing NOEL and NOAEL more clearly; nevertheless, the toxicity determination criterion needs to be supplemented by adding differentiating adverse effects and non-adverse effects based on more experiences of the repeated dose toxicity tests.

Molecular Decrowding by Tissue Expansion Allows Precise Determination of the Spatial Distribution of Synaptic Proteins at a Nanometer Scale by exTEM.

To understand how the molecular machinery of synapses works, it is essential to determine an inventory of synaptic proteins at a subsynaptic resolution. Nevertheless, synaptic proteins are difficult to localize because of the low expression levels and limited access to immunostaining epitopes. Here, we report on the exTEM (epitope-exposed by expansion-transmission electron microscopy) method that enables the imaging of synaptic proteins in situ. This method combines TEM with nanoscale resolution and expandable tissue-hydrogel hybrids for enhanced immunolabeling with better epitope accessibility via molecular decrowding, allowing successful probing of the distribution of various synapse-organizing proteins. We propose that exTEM can be employed for studying the mechanisms underlying the regulation of synaptic architecture and function by providing nanoscale molecular distribution of synaptic proteins in situ. We also envision that exTEM is widely applicable for investigating protein nanostructures located in densely packed environments by immunostaining of commercially available antibodies at nanometer resolution.

Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling.

Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand-receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme-substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.

Changes in Blood Glucose Level After Steroid Injection for Musculoskeletal Pain in Patients With Diabetes.

OBJECTIVE: To investigate changes in blood glucose level after steroid injection in patients with type 2 diabetes mellitus (DM) and factors affecting those changes. METHODS: We retrospectively studied 51 patients with type 2 DM who underwent steroid injection for shoulder and back pain. Mean fasting blood sugar (FBS) levels for 7 days before steroid injection was used as the baseline blood glucose level, which was compared with FBS levels for 14 days after steroid injection. We compared the differences in blood glucose changes between HbA1c >7% and HbA1c ≤7% groups and those between insulin and non-insulin treated groups. Demographic data, injection site, and steroid dose were analyzed. RESULTS: Compared to baseline, blood glucose significantly (p=0.012) elevated 1 day after steroid injection but not 2 days after injection. In the HbA1c >7% and insulin groups, blood glucose was significantly increased 1 day after injection compared to that in the HbA1c ≤7% (p=0.011) and non-insulin (p=0.024) groups, respectively. Higher HbA1c level before injection was significantly (p=0.003) associated with the degree of blood glucose increase 1 day after injection. No significant differences were noted in the degree of blood glucose increase according to injection site or steroid dose. CONCLUSION: Higher HbA1c level was associated with greater elevation in blood glucose 1 day after steroid injection. Careful monitoring of blood glucose is required on the first day after steroid injection in patients with poorly controlled DM.

Effect of Extracorporeal Shockwave Therapy Versus Intra-articular Injections of Hyaluronic Acid for the Treatment of Knee Osteoarthritis.

OBJECTIVE: To evaluate and compare the effects and outcomes of extracorporeal shock wave therapy (ESWT) and intra-articular injections of hyaluronic acid (HA) in patients with knee osteoarthritis (OA). METHODS: Of the 78 patients recruited for the study, 61 patients met the inclusion criteria. The enrolled patients were randomly divided into two groups: the ESWT group and the HA group. The ESWT group underwent 3 sessions of 1,000 shockwave pulses performed on the affected knee with the dosage adjusted to 0.05 mJ/mm2 energy. The HA group was administered intra-articular HA once a week for 3 weeks with a 1-week interval between each treatment. The results were measured with the visual analogue scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Lequesne index, 40-m fast-paced walk test, and stair-climb test (SCT). A baseline for each test was measured before treatment and then the effects of the treatments were measured by each test at 1 and 3 months after treatment. RESULTS: In both groups, the scores of the VAS, WOMAC, Lequesne index, 40-m fast-paced walk test, and SCT were significantly improved in a time-dependent manner (p<0.01). There were no statistically significant differences measured at 1 and 3 months after treatment between the two groups (p>0.05). CONCLUSION: The ESWT can be an alternative treatment to reduce pain and improve physical functions in patients with knee OA.