An Ultrasoft and Flexible PDMS-Based Balloon-Type Implantable Device for Controlled Drug Delivery.

Non-biodegradable implants have undergone extensive investigation as drug delivery devices to enable advanced healthcare toward personalized medicine. However, fibroblast encapsulation is one of the major challenges in all non-biodegradable implants, besides other challenges such as high initial burst, risk of membrane rupture, high onset time, non-conformal contact with tissues, and tissue damage. To tackle such challenges, we propose a novel ultrasoft and flexible balloon-type drug delivery device for unidirectional and long-term controlled release. The ultrasoft balloon-type device (USBD) was fabricated by using selective bonding between 2 polydimethylsiloxane (PDMS) membranes and injecting a fluid into the non-bonded area between them. The balloon acted as a reservoir containing a liquid drug, and at the same time, the membrane of the balloon itself acted as the pathway for release based on diffusion. The release was modulated by tuning the thickness and composition of the PDMS membrane. Regardless of the thickness and composition, all devices exhibited zero-order release behavior. The longest zero-order release and nearly zero-order release were achieved for 30 days and 58 days at a release rate of 1.16 µg/day and 1.68 µg/day, respectively. In vivo evaluation was performed for 35 days in living rats, where the USBD maintained zero-order and nearly zero-order release for 28 days and 35 days, respectively. Thanks to the employment of ultrasoft and flexible membranes and device design, the USBD could achieve minimal tissue damage and foreign body responses. It is expected that the proposed device may provide a novel approach for long-term drug delivery with new therapeutic modalities.

Epigenetic Regulation of the Expression of T Cell Stimulatory and Inhibitory Factors by Histone H3 Lysine Modification Enzymes and Its Prognostic Roles in Glioblastoma.

BACKGROUND: This study aimed to identify the specific T cell co-stimulatory and co-inhibitory factors that play prognostic roles in patients with glioblastoma. Additionally, the unique histone H3 modification enzymes that regulate the expression levels of these specific co-stimulatory and co-inhibitory factors were investigated. METHODS: The medical records of 84 patients newly diagnosed with glioblastoma at our institution from January 2006 to December 2020 were retrospectively reviewed. Immunohistochemical (IHC) staining for T cell co-stimulatory factors (CD27, CD28, CD137, OX40, and ICOS), T cell co-inhibitory factors (CTLA4, PD1, PD-L1, TIM3, and CD200R), and histone H3 lysine modification enzymes (MLL4, RIZ, EZH1, NSD2, KDM5c, JMJD1a, UTX, and JMJD5) was performed on archived paraffin-embedded tissues obtained by biopsy or resection. Quantitative real time-polymerase chain reaction (qRT-PCR) was performed for specific factors, which demonstrated causal relationships, in order to validate the findings of the IHC examinations. RESULTS: The mean follow-up duration was 27.5 months (range, 4.1-43.5 months). During this period, 76 patients (90.5%) died, and the mean OS was 19.4 months (95% confidence interval, 16.3-20.9 months). Linear positive correlations were observed between the expression levels of CD28 and JMJD1a (R2 linear = 0.982) and those of CD137 and UTX (R2 linear = 1.528). Alternatively, significant negative correlations were observed between the expression levels of CTLA4 and RIZ (R2 linear = -1.746) and those of PD-L1 and EZH1 (R2 linear = -2.118); these relationships were confirmed by qRT-PCR. In the multivariate analysis, increased expression levels of CD28 (P = 0.042), and CD137 (P = 0.009), and decreased expression levels of CTLA4 (P = 0.003), PD-L1 (P = 0.020), and EZH1 (P = 0.040) were significantly associated with longer survival. CONCLUSION: These findings suggest that the expression of certain T cell co-stimulatory factors, such as CD28 and CD 137, and co-inhibitory factors, such as CTLA4 and PD-L1 are associated with prognosis of glioblastoma patients.

Silver electroceutical technology to treat sarcopenia.

While the world is rapidly transforming into a superaging society, pharmaceutical approaches to treat sarcopenia have hitherto not been successful due to their insufficient efficacy and failure to specifically target skeletal muscle cells (skMCs). Although electrical stimulation (ES) is emerging as an alternative intervention, its efficacy toward treating sarcopenia remains unexplored. In this study, we demonstrate a silver electroceutical technology with the potential to treat sarcopenia. First, we developed a high-throughput ES screening platform that can simultaneously stimulate 15 independent conditions, while utilizing only a small number of human-derived primary aged/young skMCs (hAskMC/hYskMC). The in vitro screening showed that specific ES conditions induced hypertrophy and rejuvenation in hAskMCs, and the optimal ES frequency in hAskMCs was different from that in hYskMCs. When applied to aged mice in vivo, specific ES conditions improved the prevalence and thickness of Type IIA fibers, along with biomechanical attributes, toward a younger skMC phenotype. This study is expected to pave the way toward an electroceutical treatment for sarcopenia with minimal side effects and help realize personalized bioelectronic medicine.

Farnesol prevents aging-related muscle weakness in mice through enhanced farnesylation of Parkin-interacting substrate.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis. Reduced PGC-1α abundance is linked to skeletal muscle weakness in aging or pathological conditions, such as neurodegenerative diseases and diabetes; thus, elevating PGC-1α abundance might be a promising strategy to treat muscle aging. Here, we performed high-throughput screening and identified a natural compound, farnesol, as a potent inducer of PGC-1α. Farnesol administration enhanced oxidative muscle capacity and muscle strength, leading to metabolic rejuvenation in aged mice. Moreover, farnesol treatment accelerated the recovery of muscle injury associated with enhanced muscle stem cell function. The protein expression of Parkin-interacting substrate (PARIS/Zfp746), a transcriptional repressor of PGC-1α, was elevated in aged muscles, likely contributing to PGC-1α reduction. The beneficial effect of farnesol on aged muscle was mediated through enhanced PARIS farnesylation, thereby relieving PARIS-mediated PGC-1α suppression. Furthermore, short-term exercise increased PARIS farnesylation in the muscles of young and aged mice, whereas long-term exercise decreased PARIS expression in the muscles of aged mice, leading to the elevation of PGC-1α. Collectively, the current study demonstrated that the PARIS-PGC-1α pathway is linked to muscle aging and that farnesol treatment can restore muscle functionality in aged mice through increased farnesylation of PARIS.

Fully Automated Continuous Centrifugal Microfluidics Isolates Natural Killer Cells with High Performance and Minimal Stress.

Natural killer (NK) cells are a part of the innate immune system, providing the first line of defense against cancer cells and pathogens at an early stage. Hence, they are attracting attention as a valuable resource for allogeneic cell immunotherapy. However, NK cells exist with limited proportion in blood, and obtaining sufficient clinical-grade NK cells with highly viable and minimal stress is critical for successful immune cell therapy. Conventional purification methods via immunoaffinity or density gradient centrifugation had several limitations in yield, purity, and cellular stress, which might cause an increased risk for graft versus host disease and reduced efficacy due to NK cell malfunction, exhaustion, and apoptosis. Moreover, reducing the variations of isolation performance caused by the manual process is another unmet need for uniform quality of the living drug. Here, an automated system using an NK disc (NKD) based on continuous centrifugal microfluidics (CCM) technology was developed to isolate NK cells from whole blood with high yield, purity, reproducibility, and low stress. The CCM technology, which operates fluidic manipulation under disc rotation, enabled precise extraction of the ultra-thin target fluid layer generated by blood centrifugation. Compared to the conventional manual method, the CCM-NKD isolated NK cells with higher yield (recovery rate) and purity, while maintaining better reproducibility. Furthermore, since the CCM-NKD maintained substantially milder centrifugation conditions (120 ×g for 10 min) compared to the conventional approach (1200 ×g for 20 min), it showed reduced cellular stress and increased antioxidant capacity in the isolated NK cells. Based on the results, the CCM-NKD is expected to be a useful tool to provide highly intact and viable cell weapons for successful immune cell therapy.

Cytokine engineered NK-92 therapy to improve persistence and anti-tumor activity.

Natural killer (NK) cells are an attractive cell source in cancer immunotherapy due to their potent antitumor ability and promising safety for allogenic applications. However, the clinical outcome of NK cell therapy has been limited due to poor persistence and loss of activity in the cytokine-deficient tumor microenvironment. Benefits from exogenous administration of soluble interleukin-2 (IL-2) to stimulate the activity of NK cells have not been significant due to cytokine consumption and activation of other immune cells, compromising both efficacy and safety. Methods: To overcome these drawbacks, we developed a novel membrane-bound protein (MBP) technology to express IL-2 on the surface of NK-92 cells (MBP NK) inducing autocrine signal for proliferation without IL-2 supplementation. Results: The MBP NK cells exhibited not only improved proliferation in IL-2 deficient conditions but also stronger secretion of cytolytic granules leading to enhanced anti-tumor activity both in vitro and in vivo. Furthermore, the experiment with a spheroid solid tumor model exhibited enhanced infiltration by MBP NK cells creating higher local effector-to-target ratio for efficient tumor killing. These results suggest MBP technology can be an effective utility for NK-92 cell engineering to increase anti-tumor activity and reduce potential adverse effects, providing a higher therapeutic index in clinical applications.

Lossless Immunocytochemistry Based on Large-Scale Porous Hydrogel Pellicle for Accurate Rare Cell Analysis.

Rare cells, such as circulating tumor cells or circulating fetal cells, provide important information for the diagnosis and prognosis of cancer and prenatal diagnosis. Since undercounting only a few cells can lead to significant misdiagnosis and incorrect decisions in subsequent treatment, it is crucial to minimize cell loss, particularly for rare cells. Moreover, the morphological and genetic information on cells should be preserved as intact as possible for downstream analysis. The conventional immunocytochemistry (ICC), however, fails to meet these requirements, causing unexpected cell loss and deformation of the cell organelles which may mislead the classification of benign and malignant cells. In this study, a novel ICC technique for preparing lossless cellular specimens was developed to improve the diagnostic accuracy of rare cell analysis and analyze intact cellular morphology. To this end, a robust and reproducible porous hydrogel pellicle was developed. This hydrogel encapsulates cells to minimize cell loss from the repeated exchange of reagents and prevent cell deformation. The soft hydrogel pellicle allows stable and intact cell picking for further downstream analysis, which is difficult with conventional ICC methods that permanently immobilize cells. The lossless ICC platform will pave the way for robust and precise rare cell analysis toward clinical practice.

Electroceutical approach ameliorates intracellular PMP22 aggregation and promotes pro-myelinating pathways in a CMT1A in vitro model.

Charcot-Marie-Tooth disease subtype 1A (CMT1A) is one of the most prevalent demyelinating peripheral neuropathies worldwide, caused by duplication of the peripheral myelin protein 22 (PMP22) gene, which is expressed primarily in Schwann cells (SCs). PMP22 overexpression in SCs leads to intracellular aggregation of the protein, which eventually results in demyelination. Unfortunately, previous biochemical approaches have not resulted in an approved treatment for CMT1A disease, compelling the pursuit for a biophysical approach such as electrical stimulation (ES). However, the effects of ES on CMT1A SCs have remained unexplored. In this study, we established PMP22-overexpressed Schwannoma cells as a CMT1A in vitro model, and investigated the biomolecular changes upon applying ES via a custom-made high-throughput ES platform, screening for the condition that delivers optimal therapeutic effects. While PMP22-overexpressed Schwannoma exhibited intracellular PMP22 aggregation, ES at 20 Hz for 1 h improved this phenomenon, bringing PMP22 distribution closer to healthy condition. ES at this condition also enhanced the expression of the genes encoding myelin basic protein (MBP) and myelin-associated glycoprotein (MAG), which are essential for assembling myelin sheath. Furthermore, ES altered the gene expression for myelination-regulating transcription factors Krox-20, Oct-6, c-Jun and Sox10, inducing pro-myelinating effects in PMP22-overexpressed Schwannoma. While electroceuticals has previously been applied in the peripheral nervous system towards acquired peripheral neuropathies such as pain and nerve injury, this study demonstrates its effectiveness towards ameliorating biomolecular abnormalities in an in vitro model of CMT1A, an inherited peripheral neuropathy. These findings will facilitate the clinical translation of an electroceutical treatment for CMT1A.

Implantable Electroceutical Approach Improves Myelination by Restoring Membrane Integrity in a Mouse Model of Peripheral Demyelinating Neuropathy.

Although many efforts are undertaken to treat peripheral demyelinating neuropathies based on biochemical interventions, unfortunately, there is no approved treatment yet. Furthermore, previous studies have not shown improvement of the myelin membrane at the biomolecular level. Here, an electroceutical treatment is introduced as a biophysical intervention to treat Charcot-Marie-Tooth (CMT) disease-the most prevalent peripheral demyelinating neuropathy worldwide-using a mouse model. The specific electrical stimulation (ES) condition (50 mV mm-1 , 20 Hz, 1 h) for optimal myelination is found via an in vitro ES screening system, and its promyelinating effect is validated with ex vivo dorsal root ganglion model. Biomolecular investigation via time-of-flight secondary ion mass spectrometry shows that ES ameliorates distribution abnormalities of peripheral myelin protein 22 and cholesterol in the myelin membrane, revealing the restoration of myelin membrane integrity. ES intervention in vivo via flexible implantable electrodes shows not only gradual rehabilitation of mouse behavioral phenotypes (balance and endurance), but also restored myelin thickness, compactness, and membrane integrity. This study demonstrates, for the first time, that an electroceutical approach with the optimal ES condition has the potential to treat CMT disease and restore impaired myelin membrane integrity, shifting the paradigm toward practical interventions for peripheral demyelinating neuropathies.

A fully automated primary neuron purification system using continuous centrifugal microfluidics.

Progress in neurological research has experienced bottlenecks owing to the limited availability of purified primary neurons. Since neuronal cells are non-proliferative, it is necessary to obtain purified neurons from animal models or human patients for experimental work. However, currently available methods for purifying primary neurons are time-consuming (taking approximately 1 week), and suffer from insufficient viability and purity. Here, we report a method for rapid enrichment of neurons from the mouse embryonic dorsal root ganglion (DRG), using a fully-automated continuous centrifugal microfluidics (CCM) based neuron purification disc (NPD). Non-neuronal cells were removed via negative depletion by combining density gradient centrifugation and immunomagnetic separation. The CCM-NPD platform enables effective isolation of intact neurons within 13 min, which is approximately 800 times faster than the conventional chemical purification method. Furthermore, the neurons purified using the CCM-NPD platform showed better neurite growth, along with higher viability (93.5%) and purity (97.0%) after 1 week of culture, compared to the chemical purification method. Therefore, the proposed automated and rapid system yields purified DRG neurons with high viability and purity, while avoiding the use of harsh chemicals. We believe this system will significantly mitigate the shortage of purified primary neurons and advance neurological research.

Advantages of Adult Mouse Dorsal Root Ganglia Explant Culture in Investigating Myelination in an Inherited Neuropathic Mice Model.

A co-culture of neurons and Schwann cells has frequently been used to investigate myelin sheath formation. However, this approach is restricted to myelin-related diseases of the peripheral nervous system. This study introduces and compares an ex vivo model of adult-mouse-derived dorsal root ganglia (DRG) explant, with an in vitro co-culture of dissociated neurons from mouse embryo DRG and Schwann cells from a mouse sciatic nerve. The 2D co-culture has disadvantages of different mouse isolation for neurons and Schwann cells, animal number, culture duration, and the identification of disease model. However, 3D DRG explant neurons and myelination cells in Matrigel-coated culture are obtained from the same mouse, the culture period is shorter than that of 2D co-culture, and fewer animals are needed. In addition, it has simpler and shorter experimental steps than 2D co-culture. This culture system may prove advantageous in studies of biological functions and pathophysiological mechanisms of disease models, since it can reflect disease characteristics as traditional co-culture does. Therefore, it is suggested that a DRG explant culture is a scientifically, ethically, and economically more practical option than a co-culture system for studying myelin dynamics, myelin sheath formation, and demyelinating disease.

Continuous centrifugal microfluidics (CCM) isolates heterogeneous circulating tumor cells via full automation.

Understanding cancer heterogeneity is essential to finding diverse genetic mutations in metastatic cancers. Thus, it is critical to isolate all types of CTCs to identify accurate cancer information from patients. Moreover, full automation robustly capturing the full spectrum of CTCs is an urgent need for CTC diagnosis to be routine clinical practice. Methods: Here we report the full capture of heterogeneous CTC populations using fully automated, negative depletion-based continuous centrifugal microfluidics (CCM). Results: The CCM system demonstrated high performance (recovery rates exceeding 90% and WBC depletion rate of 99.9%) across a wide range of phenotypes (EpCAM(+), EpCAM(-), small-, large-sized, and cluster) and cancers (lung, breast, and bladder). Applied in 30 lung adenocarcinoma patients harboring epidermal growth factor receptor (EGFR) mutations, the system isolated diverse phenotypes of CTCs in marker expression and size, implying the importance of unbiased isolation. Genetic analyses of intra-patient samples comparing cell-free DNA with CCM-isolated CTCs yielded perfect concordance, and CTC enumeration using our technique was correlated with clinical progression as well as response to EGFR inhibitors. Conclusion: Our system also introduces technical advances which assure rapid, reliable, and reproducible results, thus enabling a more comprehensive application of robust CTC analysis in clinical practice.

An electroceutical approach enhances myelination via upregulation of lipid biosynthesis in the dorsal root ganglion.

As the myelin sheath is crucial for neuronal saltatory conduction, loss of myelin in the peripheral nervous system (PNS) leads to demyelinating neuropathies causing muscular atrophy, numbness, foot deformities and paralysis. Unfortunately, few interventions are available for such neuropathies, because previous pharmaceuticals have shown severe side effects and failed in clinical trials. Therefore, exploring new strategies to enhance PNS myelination is critical to provide solution for such intractable diseases. This study aimed to investigate the effectiveness of electrical stimulation (ES) to enhance myelination in the mouse dorsal root ganglion (DRG)-anex vivomodel of the PNS. Mouse embryonic DRGs were extracted at E13 and seeded onto Matrigel-coated surfaces. After sufficient growth and differentiation, screening was carried out by applying ES in the 1-100 Hz range at the beginning of the myelination process. DRG myelination was evaluated via immunostaining at the intermediate (19 daysin vitro(DIV)) and mature (30 DIV) stages. Further biochemical analyses were carried out by utilizing ribonucleic acid sequencing, quantitative polymerase chain reaction and biochemical assays at both intermediate and mature myelination stages. Imaging of DRG myelin lipids was carried out via time-of-flight secondary ion mass spectrometry (ToF-SIMS). With screening ES conditions, optimal condition was identified at 20 Hz, which enhanced the percentage of myelinated neurons and average myelin length not only at intermediate (129% and 61%) but also at mature (72% and 17%) myelination stages. Further biochemical analyses elucidated that ES promoted lipid biosynthesis in the DRG. ToF-SIMS imaging showed higher abundance of the structural lipids, cholesterol and sphingomyelin, in the myelin membrane. Therefore, promotion of lipid biosynthesis and higher abundance of myelin lipids led to ES-mediated myelination enhancement. Given that myelin lipid deficiency is culpable for most demyelinating PNS neuropathies, the results might pave a new way to treat such diseases via electroceuticals.

Hypo-trimethylation of Histone H3 Lysine 4 and Hyper-tri/dimethylation of Histone H3 Lysine 27 as Epigenetic Markers of Poor Prognosis in Patients with Primary Central Nervous System Lymphoma.

PURPOSE: This study aimed to investigate the methylation status of major histone modification sites in primary central nervous system lymphoma (PCNSL) samples and examine their prognostic roles in patients with PCNSL. MATERIALS AND METHODS: Between 2007 and 2020, 87 patients were histopathologically diagnosed with PCNSL. We performed immunohistochemical staining of the formalin-fixed paraffin-embedded samples of PCNSL for major histone modification sites, such as H3K4, H3K9, H3K27, H3K14, and H3K36. After detection of meaningful methylation sites, we examined histone modification enzymes that induce methylation or demethylation at each site using immunohistochemical staining. The meaningful immunoreactivity was validated by western blotting using fresh tissue of PCNSL. RESULTS: More frequent recurrences were found in hypomethylation of H3K4me3 (p=0.004) and hypermethylation of H3K27me2 (p<0.001) and H3K27me3 (p=0.002). These factors were also statistically related to short PFS and overall survival in the univariate and multivariate analyses. Next, histone modification enzymes inducing the demethylation of H3K4 (lysine-specific demethylase-1/2 and Jumonji AT-rich interactive domain [JARID] 1A-D]) and methylation of H3K27 (enhancer of zeste homolog [EZH]-1/2) were immu- nohistochemically stained. Among them, the immunoreactivity of JARID1A inversely associated with the methylation status of H3K4me3 (R2=-1.431), and immunoreactivity of EZH2 was directly associated with the methylation status of H3K27me2 (R2=0.667) and H3K27me3 (R2=0.604). These results were validated by western blotting in fresh PCNSL samples. CONCLUSION: Our study suggests that hypomethylation of H3K4me3 and hypermethylation of H3K27me2 and H3K27me3 could be associated with poor outcomes in patients with PCNSL and that these relationships are modified by JARID1A and EZH2.

Epigenetic Role of Histone Lysine Methyltransferase and Demethylase on the Expression of Transcription Factors Associated with the Epithelial-to-Mesenchymal Transition of Lung Adenocarcinoma Metastasis to the Brain.

PURPOSE: The objective of this study was to investigate the epigenetic role of histone lysine methylation/demethylation on the expression of epithelial-to-mesenchymal transition (EMT) associated transcriptional factors (TFs) during the metastasis of lung adenocarcinoma to the brain. METHODS: Paired samples of lung adenocarcinoma and brain metastasis (BM) were analyzed in 46 individual patients. Both samples were obtained by surgical resection or biopsy of the lung and brain. The paraffin-fixed formalin-embedded samples were obtained from the pathology archives in our institute. In samples of lung adenocarcinoma and BM, immunohistochemical staining was performed for epithelial markers, mesenchymal markers, EMT-TFs, histone lysine methyltransferase and demethylase. RESULTS: The immunoreactivity of EMT-TFs such as Slug (15.6% vs. 42.6%, p = 0.005), Twist (23.6% vs. 45.9%, p = 0.010) and ZEB1 (15.0% vs. 55.9%, p = 0.002) was increased in BM compared with that in lung adenocarcinoma. Epigenetic inducers such as H3K4 methyltransferase (MLL4, p = 0.018) and H3K36me3 demethylase (UTX, p = 0.003) were statistically increased, and epigenetic repressors such as EZH2 (H3K27 methyltransferase, p = 0.046) were significantly decreased in BM compared with those in lung adenocarcinoma. The expression of UTX-ZEB1 (R2 linear = 1.204) and MLL4-Slug (R2 linear = 0.987) was increased in direct proportion, and EZH2-Twist (R2 linear = -2.723) decreased in reverse proportion. CONCLUSIONS: The results suggest that certain histone lysine methyltransferase/demethylase, such as MLL4, UTX, and EZH2, regulate the expression of EMT-TFs such as Slug, ZEB1, and Twist epigenetically, which may thereby influence cancer metastasis from the lung to the brain.

Recent Advances of Fluid Manipulation Technologies in Microfluidic Paper-Based Analytical Devices (µPADs) toward Multi-Step Assays.

Microfluidic paper-based analytical devices (µPADs) have been suggested as alternatives for developing countries with suboptimal medical conditions because of their low diagnostic cost, high portability, and disposable characteristics. Recently, paper-based diagnostic devices enabling multi-step assays have been drawing attention, as they allow complicated tests, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), which were previously only conducted in the laboratory, to be performed on-site. In addition, user convenience and price of paper-based diagnostic devices are other competitive points over other point-of-care testing (POCT) devices, which are more critical in developing countries. Fluid manipulation technologies in paper play a key role in realizing multi-step assays via µPADs, and the expansion of biochemical applications will provide developing countries with more medical benefits. Therefore, we herein aimed to investigate recent fluid manipulation technologies utilized in paper-based devices and to introduce various approaches adopting several principles to control fluids on papers. Fluid manipulation technologies are classified into passive and active methods. While passive valves are structurally simple and easy to fabricate, they are difficult to control in terms of flow at a specific spatiotemporal condition. On the contrary, active valves are more complicated and mostly require external systems, but they provide much freedom of fluid manipulation and programmable operation. Both technologies have been revolutionized in the way to compensate for their limitations, and their advances will lead to improved performance of µPADs, increasing the level of healthcare around the world.

Multiplex Protein Imaging with Secondary Ion Mass Spectrometry Using Metal Oxide Nanoparticle-Conjugated Antibodies.

In spite of recent developments in mass spectrometry imaging techniques, high-resolution multiplex protein bioimaging techniques are required to unveil the complex inter- and intracellular biomolecular interactions for accurate understanding of life phenomena and disease mechanisms. Herein, we report multiplex protein imaging with secondary ion mass spectrometry (SIMS) using metal oxide nanoparticle (MONP)-conjugated antibodies with <300 nm spatial resolution in the low ion dose without ion beam damage because of the high secondary ion yields of the MONPs, which can provide simultaneous imaging of several proteins, especially from cell membranes. We applied our new imaging technique for the study of hippocampal tissue samples from control and Alzheimer's disease (AD) model mice; the proximity of protein clusters in the hippocampus CA1 region showed intriguing dependence on aging and AD progress, suggesting that protein cluster proximity may be helpful for understanding pathological pathways in the microscopic cellular level.

Indoprofen prevents muscle wasting in aged mice through activation of PDK1/AKT pathway.

BACKGROUND: Muscle wasting, resulting from aging or pathological conditions, leads to reduced quality of life, increased morbidity, and increased mortality. Much research effort has been focused on the development of exercise mimetics to prevent muscle atrophy and weakness. In this study, we identified indoprofen from a screen for peroxisome proliferator-activated receptor γ coactivator α (PGC-1α) inducers and report its potential as a drug for muscle wasting. METHODS: The effects of indoprofen treatment on dexamethasone-induced atrophy in mice and in 3-phosphoinositide-dependent protein kinase-1 (PDK1)-deleted C2C12 myotubes were evaluated by immunoblotting to determine the expression levels of myosin heavy chain and anabolic-related and oxidative metabolism-related proteins. Young, old, and disuse-induced muscle atrophic mice were administered indoprofen (2 mg/kg body weight) by gavage. Body weight, muscle weight, grip strength, isometric force, and muscle histology were assessed. The expression levels of muscle mass-related and function-related proteins were analysed by immunoblotting or immunostaining. RESULTS: In young (3-month-old) and aged (22-month-old) mice, indoprofen treatment activated oxidative metabolism-related enzymes and led to increased muscle mass. Mechanistic analysis using animal models and muscle cells revealed that indoprofen treatment induced the sequential activation of AKT/p70S6 kinase (S6K) and AMP-activated protein kinase (AMPK), which in turn can augment protein synthesis and PGC-1α induction, respectively. Structural prediction analysis identified PDK1 as a target of indoprofen and, indeed, short-term treatment with indoprofen activated the PDK1/AKT/S6K pathway in muscle cells. Consistent with this finding, PDK1 inhibition abrogated indoprofen-induced AKT/S6K activation and hypertrophic response. CONCLUSIONS: Our findings demonstrate the effects of indoprofen in boosting skeletal muscle mass through the sequential activation of PDK1/AKT/S6K and AMPK/PGC-1α. Taken together, our results suggest that indoprofen represents a potential drug to prevent muscle wasting and weakness related to aging or muscle diseases.

An adiponectin receptor agonist antibody stimulates glucose uptake and fatty-acid oxidation by activating AMP-activated protein kinase.

Adiponectin (Ad) is a representative adipocytokine that regulates energy homeostasis including glucose transport and lipid oxidation through activation of AMP-activated protein kinase (AMPK) pathways. Plasma levels of Ad are reduced in obesity, which contributes to type 2 diabetes. Therefore, agents that activate the Ad signaling pathway could ameliorate metabolic diseases such as type 2 diabetes. Here, we report the identification of a high-affinitive agonist antibody against Ad receptors. The antibody was selected by using phage display of human combinatorial antibody libraries. The selected antibody induced phosphorylation of the acetyl-CoA carboxylase (ACC) and AMPK in skeletal muscle cells and stimulated glucose uptake and fatty-acid oxidation (FAO) in myotubes. In addition, the antibody significantly lowered blood glucose levels during a glucose challenge in normal mice as well as basal blood glucose levels in a type 2 diabetic mouse model. Taken together, these results suggest that the agonist antibody could be a promising therapeutic agent for the treatment of metabolic syndrome such as type 2 diabetes.

Cell Seeding Technology for Microarray-Based Quantitative Human Primary Skeletal Muscle Cell Analysis.

Pipetting techniques play a crucial role in obtaining reproducible and reliable results, especially when seeding cells on small target areas, such as on microarrays, biochips or microfabricated cell culture systems. For very rare cells, such as human primary skeletal muscle cells (skMCs), manual (freehand) cell seeding techniques invariably result in nonuniform cell spreading and heterogeneous cell densities, giving rise to undesirable variations in myogenesis and differentiation. To prevent such technique-dependent variation, we have designed and fabricated a simple, low-cost pipet guidance device (PGD), and holder that works with hand-held pipettes. This work validates the accuracy and reproducibility of the PGD platform and compares its effectiveness with manual and robotic seeding techniques. The PGD system ensures reproducibility of cell seeding, comparable to that of more expensive robotic dispensing systems, resulting in a high degree of cell uniformity and homogeneous cell densities, while also enabling cell community studies. As compared to freehand pipetting, PGD-assisted seeding of C2C12 mouse myoblasts showed 5.3 times more myotube formation and likewise myotubes derived from PGD-seeded human primary skMCs were 3.6 times thicker and 2.2 times longer. These results show that this novel, yet simple PGD-assisted pipetting technique provides precise cell seeding on small targets, ensuring reproducible and reliable high-throughput cell assays.