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.

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.

Reciprocal inhibition of expression between RAV1 and BES1 modulates plant growth and development in Arabidopsis.

RAV1 (Related to ABI3/VP1) is a plant-specific B3 and AP2 domain-containing transcription factor that acts as a negative regulator of growth in many plant species. The expression of RAV1 is downregulated by brassinosteroids (BRs); large-scale transcriptome analyses have shown that the expression of RAV1 was previously targeted by BRI1-EMS-SUPPRESOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1), which are critical transcription factors for the BR-signaling process. Using RAV1-overexpressing transgenic plants, we showed that RAV1 overexpression reduced the BR signaling capacity, resulting in the downregulation of BR biosynthetic genes and BES1 expression. Furthermore, we demonstrated that BES1, not BZR1, is directly bound to the RAV1 promoter and repressed RAV1 expression, and vice versa; RAV1 is also bound to the BES1 promoter and repressed BES1 expression. This mutual inhibition was specific to RAV1 and BES1 because RAV1 exhibited binding activity to the BZR1 promoter but did not repress BZR1 expression. We observed that constitutively activated BR signaling phenotypes in bes1-D were attenuated by the repression of endogenous BES1 expression in transgenic bes1-D plants overexpressing RAV1. RNA-sequencing analysis of RAV1-overexpressing transgenic plants and bes1-D mutant plants revealed differentially expressed genes by RAV1 and BES1 and genes that were oppositely co-regulated by RAV1 and BES1. RAV1 and BES1 regulated different transcriptomes but co-regulated a specific set of genes responsible for the balance between growth and defense. These results suggested that the mutual inhibitory transcriptional activities of RAV1 and BES1 provide fine regulatory mechanisms for plant growth and development.

Rhamnogalacturonan I-rich polysaccharide isolated from fermented persimmon fruit increases macrophage-stimulatory activity by activating MAPK and NF-κB signaling.

BACKGROUND: Persimmon (Diospyros kaki) is a familiar and widespread fruit, cultivated worldwide. To date, physiological and chemical changes in fermented persimmon fruit and its active compounds have been rarely investigated. Moreover, comparative studies on the pharmacological activities of fermented persimmon fruit-derived compounds have not been reported. RESULTS: To investigate the effect of traditional fermented foods on immunostimulatory activity, non-fermented persimmon fruit (D. kaki, DK) and fermented persimmon fruit (fermented D. kaki, FDK) were prepared and further fractionated into low- and high-molecular weight fractions. FDK exhibited significantly higher activity toward the production of macrophage-stimulatory mediators compared with that of DK, and the high-molecular weight fraction (FDK-H) isolated from FDK was shown to have more potent activity than FDK. FDK-H not only increased the expression of immunostimulatory genes (TNF-α, IL-6, IL-12, and iNOS), but also stimulated the phosphorylation of both MAPK (ERK, JNK, and p38) and NF-κB (p65 and IκB) signaling molecules underlying macrophage activation. The putative chemical characteristic of FDK-H was identified as a pectic rhamnogalacturonan (RG) I-rich polysaccharide with a high molecular weight of 304 kDa containing galacturonic acid, arabinose, rhamnose, and galactose as the major monosaccharide units. CONCLUSION: The present study reveals that traditional fermentation is a useful method for increasing the macrophage-immunostimulatory activity of persimmon fruit, and the increased activity may be associated with structural modification of persimmon polysaccharides. This study may serve to identify a functional ingredient as an immunostimulatory agent, and our results may be applied to develop a new immunostimulatory product using FDK-H. © 2021 Society of Chemical Industry.

[The peak latency prolongation of the blink reflex in a patient with trigeminal neuralgia of Meckel's cave mass]. / A pislogási reflexcsúcs latenciájának megnyúlása egy Meckel-barlang-terime következtében kialakuló trigeminusneuralgia esetben.

The blink reflex test of the trigeminal nerve can provide valuable information about lesions site. However it may not find small compressive lesions. We observed peak latency prolongation of the blink reflex test in a patient with trigeminal neuralgia caused by a small Meckel's cave mass, in whom the onset latency was normal. Conclusion - We suggest peak latency of the blink reflex might be a valuable aid for discerning small mass in patients with trigeminal neuralgia. This is the first case report of compressive trigeminal neuralgia showing peak latency prolongation of the blink reflex test.

Effectiveness of liraglutide 3 mg for the treatment of obesity in a real-world setting without intensive lifestyle intervention.

OBJECTIVE: We investigated the efficacy and safety of liraglutide 3 mg daily in combination with diet and exercise 2, 4, and 6 months after initiation in real-world settings in Korea. METHODS: People first using liraglutide starting in 2018 were recruited from ten sites in Korea. Body weight and body mass index (BMI) were measured after 2, 4, and 6 months and compared with baseline values. RESULTS: The full cohort comprised 769 participants: 672 in the 2-month group, 427 in the 4-month group, and 219 in the 6-month group. The baseline mean ± standard deviation of BMI and body weight were 32.2 ± 5.1 kg/m2, and 87.5 ± 18.8 kg, respectively. Body weight and BMI decreased after initiation of liraglutide treatment: -2.94 kg and -1.08 kg/m2 at 2 months; -4.23 kg and -1.55 kg/m2 at 4 months, and -5.14 kg and -1.89 kg/m2 at 6 months (all P < 0.001). In the 6-month cohort, 52.5% and 18.3% of subjects lost ≥5% and ≥10% of body weight, respectively. After 6 months, systolic and diastolic blood pressure decreased significantly by 3.90 and 1.93 mmHg, respectively. In those with diabetes mellitus, HbA1c and fasting glucose levels decreased significantly by 1.14% and 27.8 mg/dl, respectively. Among all participants, 27.6% experienced adverse effects, including nausea (20.8%), vomiting (5.2%), diarrhoea (2.5%), and skin rash (3.6%). Documented reasons for discontinuation of treatment were lack of effect (4.4%), adverse events (4.3%), and high cost (3.1%). CONCLUSIONS: In real-world settings in Korea, daily treatment with liraglutide 3 mg was associated with clinically meaningful weight loss without serious adverse events.

Development and Validation of a Method for Determining the Quercetin-3-O-glucuronide and Ellagic Acid Content of Common Evening Primrose (Oenothera biennis) by HPLC-UVD.

Toward the standardization of common evening primrose (Oenothera biennis) sprout extract (OBS-E), we aimed to obtain indicator compounds and use a validated method. HPLC-UVD allowed simultaneous quantification of the indicator compounds quercetin-3-O-glucuronide and ellagic acid. The method was validated in terms of specificity, linearity, precision, accuracy, and limit of detection/limit of quantification (LOD/LOQ). High specificity and linearity was demonstrated, with correlation coefficients of 1.0000 for quercetin-3-O-glucuronide and 0.9998 for ellagic acid. The LOD/LOQ values were 0.486/1.472 µg/mL for quercetin-3-O-glucuronide and 1.003/3.039 µg/mL for ellagic acid. Intra-day and inter-day variability tests produced relative standard deviation for each compound of <2%, a generally accepted precision criterion. High recovery rate were also obtained, indicating accuracy validation. The OBS-E prepared using various concentrations of ethanol were then analyzed. The 50% ethanol extract had highest content of quercetin-3-O-glucuronide, whereas the 70% ethanol extract possessed the lowest. However, the ellagic acid content was highest in the 70% ethanol extract and lowest in the 90% ethanol extract. Thus, quercetin-3-O-glucuronide and ellagic acid can be used industrially as indicator compounds for O. biennis sprout products, and our validated method can be used to establish indicator compounds for other natural products.

Effects of a High-Molecular-Weight Polysaccharides Isolated from Korean Persimmon on the Antioxidant, Anti-Inflammatory, and Antiwrinkle Activity.

Persimmon (Diospyros kaki), a familiar and widespread fruit worldwide, is known to exhibit several physiological effects because of the presence of pharmacologically active compounds called phytochemicals. However, its high-molecular-weight compounds, particularly polysaccharides, have not been extensively studied. In this study, D. kaki extract (DK) was fractionated into low- and high-molecular-weight fractions (DK-L and DK-H, respectively) through ethanol fractionation, and their effects on antioxidant, anti-inflammatory, and antiwrinkle activities were investigated by an in vitro system. DK-H contained significantly higher contents of neutral sugar, uronic acid, and polyphenols compared to DK and DK-L. Furthermore, DK-H exhibited significantly improved pharmacological activities, such as antioxidant, anti-inflammatory, and antiwrinkle properties, compared to those of DK and DK-L, demonstrating that DK-H may play an important role in mediating the beneficial effects of persimmon. Sugar composition analysis and molecular characterization indicated that DK-H consisted of a galacturonic acid (GalA)-rich polysaccharide with a molecular weight of >345 kDa that mainly comprised GalA and small amounts of neutral sugar and polyphenol residues. These results suggest that the bioactive fraction DK-H is likely to be a GalA-rich pectic polysaccharide containing a small number of polyphenol residues, which may be a novel candidate in the pharmaceutical and cosmeceutical industries.

Receptor-like protein kinases RPK1 and BAK1 sequentially form complexes with the cytoplasmic kinase OST1 to regulate ABA-induced stomatal closure.

Regulation of plant water status occurs via abscisic acid (ABA)-induced stomatal closure. Open Stomata 1 (OST1) is a critical ABA signaling component regulating this process in guard cells. We previously reported that BRI1-associated receptor kinase 1 (BAK1) positively regulates ABA-induced stomatal closure by interacting with and phosphorylating OST1. Here, using Arabidopsis, we show that the receptor-like protein kinase 1 (RPK1), previously known to be induced by ABA, is a positive ABA-signaling component in guard cell movement, and interacts with OST1. ABA-inducible expression patterns were observed in RPK1 and OST1, but not in BAK1. We investigated the underlying mechanisms by which the RPK1-OST1 and BAK1-OST1 complexes interact in stomatal guard cells by monitoring the complex formation continuously using fluorescence resonance energy transfer analyses. We found that the BAK1-OST1 complex was formed earlier than the RPK1-OST1 complex in response to ABA. In vitro and semi-in vivo kinase assays revealed that a transphosphorylation event occurred in the RPK1-OST1 complex, which differs from that in the BAK1-OST1 complex, wherein only OST1 phosphorylation occurred via BAK1. ABA-insensitive 1 (ABI1) only dephosphorylated OST1 in the BAK1-OST1 complex, but dephosphorylated both RPK1 and OST1 proteins in the RPK1-OST1 complex. Our results suggest that there are multiple coordinated ABA signaling systems to regulate stomatal movement.

The current status of gender equity in medicine in Korea: an online survey about perceived gender discrimination.

BACKGROUND: Although the number of women doctors has increased in South Korea, and efforts to improve gender awareness have gained importance in recent years, the issue of gender equity in the medical field has not been fully evaluated. The aim of this study was to determine the current status of gender equity in the medical profession in Korea. METHODS: An online survey on perceived gender discrimination was conducted for 2 months, with both men and women doctors participating. The results were analyzed using descriptive statistics. RESULTS: A total of 1170 doctors responded to the survey (9.2% response rate). The survey found that 47.3% of the women respondents and 18.2% of the men had experienced gender discrimination in the resident selection process (P < 0.05), 17.2% of the women and 8.7% of the men had experienced discrimination during the fellowship application process (P < 0.05), and 36.2% of the women and 8.0% of the men had experienced discrimination during the professorship application process (P < 0.05). Both men and women cited the issue of childbirth and parenting as the number one cause of gender discrimination against women doctors. CONCLUSIONS: This study revealed the presence of perceived gender discrimination in the Korean medical society. To address discrimination, a basic approach is necessary to change the working environment so that it is flexible for women doctors, and to change the current culture where the burden of family care, including pregnancy, childbirth, and childcare, is the primary responsibility of women.

A survey of the working status of family medicine physicians in clinics and hospitals in Korea.

BACKGROUND: In the approximately 35 years since family medicine was established in South Korea, family medicine physicians have sought to expand their expertise to cover clinical fields beyond primary medicine. This study examines their working status and compares the working conditions of family medicine physicians in clinics and hospitals in Korea. METHODS: We conducted an online survey with 4057 family medicine physicians in Korea in 2016. The results were analyzed using descriptive statistics. RESULTS: Of the respondents, 572 doctors were working in clinics and 441 in hospitals. In the analysis of treatment pattern by doctors, the rate of chronic disease management was 84.7% in clinics and 93.4% in hospitals (p <  0.001), and the rate of diseases covered by national insurance was 74.8% in clinics and 76.9% in hospitals (p = 0.005). Among physicians younger than 40 years, the rate of chronic disease management and diseases covered by national insurance were 64.6 and 68.0% in clinics and 93.6 and 78.5% in hospitals, retrospectively. CONCLUSIONS: Family medicine physicians working in hospitals have higher rates of chronic disease management and diseases covered by national insurance. This discrepancy of treatment pattern became larger for doctors younger than 40 years. More in-depth studies of the treatment pattern and its tendencies between family medicine physicians in clinics and hospitals are needed in the future.

Case of the Index Patient Who Caused Tertiary Transmission of COVID-19 Infection in Korea: the Application of Lopinavir/Ritonavir for the Treatment of COVID-19 Infected Pneumonia Monitored by Quantitative RT-PCR.

Since mid-December of 2019, coronavirus disease 2019 (COVID-19) infection has been spreading from Wuhan, China. The confirmed COVID-19 patients in South Korea are those who came from or visited China. As secondary transmissions have occurred and the speed of transmission is accelerating, there are rising concerns about community infections. The 54-year old male is the third patient diagnosed with COVID-19 infection in Korea. He is a worker for a clothing business and had mild respiratory symptoms and intermittent fever in the beginning of hospitalization, and pneumonia symptoms on chest computerized tomography scan on day 6 of admission. This patient caused one case of secondary transmission and three cases of tertiary transmission. Hereby, we report the clinical findings of the index patient who was the first to cause tertiary transmission outside China. Interestingly, after lopinavir/ritonavir (Kaletra, AbbVie) was administered, ß-coronavirus viral loads significantly decreased and no or little coronavirus titers were observed.

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.

Cost-effectiveness of antiviral prophylaxis during pregnancy for the prevention of perinatal hepatitis B infection in South Korea.

BACKGROUND: In Korea, hepatitis B virus (HBV) infection accounts for approximately 65-75% of HBV-related diseases, such as chronic hepatitis and liver cancer, and mother-to-child transmission is presumed to be a major source of the infection. To tackle this issue, the Korean government launched the national Perinatal Hepatitis B Prevention Program (PHBPP) in 2002. This study analyzed the cost-effectiveness of the PHBPP with antiviral prophylaxis compared with the current PHBPP and/or universal vaccination, as well as identified the optimal strategy to eliminate mother-to-child transmission of HBV in Korea. METHODS: A decision tree model with the Markov process was developed and simulated over the lifetime of a birth cohort in Korea during the year 2014. The current PHBPP providing HBV vaccine and hepatitis B immune globulin to neonates born to HBV positive mothers was compared against two other strategies, universal vaccination of HBV and PHBPP with antiviral prophylaxis, with respect to their costs and health outcomes. The Korean National Health Insurance database was investigated to estimate the costs of HBV-related diseases and utilization of health resources. Costs were assessed from the health care system perspective and converted to 2014 US dollars. Health outcome measures were quality-adjusted life years (QALYs) and number of HBV-related diseases and deaths. Both costs and QALYs were discounted at 5%, following the recommendation of the Health Insurance Review & Assessment Service in Korea. The incremental cost-effectiveness ratio (ICER) obtained from the analysis was evaluated using the willingness-to-pay (WTP) in the Korean society. RESULTS: PHBPP with antiviral prophylaxis in Korea was cost-effective compared with the current PHBPP. An introduction of antiviral prophylaxis to pregnant women with a high viral load of HBV averted 13 HBV-related deaths per 100,000 people and saved 82 QALYs in total (ICER: $16,159/QALY). CONCLUSIONS: Considering that WTP in Korea is $29,000, PHBPP with antiviral prophylaxis appears to be a cost-effective strategy. To further decrease the burden of perinatal hepatitis B in Korea, adding antiviral prophylaxis to PHBPP is recommended.

Recent trends in the prevalence of underweight, overweight, and obesity in Korean adults: The Korean National Health and Nutrition Examination Survey from 1998 to 2014.

BACKGROUND: Recent obesity studies have reported that the rising trend in obesity has stabilized or leveled off. Our study aimed to update estimates of the recent prevalence trend in obesity based on the Korean National Health and Nutrition Examination Survey 1998-2014. METHODS: A total of 66,663 subjects were included and defined as being either underweight, overweight, or obese, in accordance with a BMI of 18.5 kg/m2 or lower, 23 kg/m2 or higher, and 25 kg/m2 or higher, respectively. RESULTS: The prevalence of underweight in KNHANES I through VI surveys was 4.7%, 3.3%, 3.4%, 3.3%, 2.7%, and 2.6%, respectively, in men (P for trend = 0.03, ß = −0.002) and 5.4%, 6.1%, 5.8%, 6.5%, 7.6%, and 7.5%, respectively, in women (P for trend = 0.04, ß = 0.003). Also for KNHANES I through VI, the respective prevalence of overweight/obesity was 50.3%, 57.2%, 62.5%, 62.3%, 61.4%, and 62.3% in men (p for trend<0.01, ß = 0.009) and 48.3%, 50.3%, 50.0%, 47.8%, 47.0%, and 45.3% in women (p for trend<0.01, ß = -0.01), respectively. CONCLUSIONS: The obesity occurrence in men was trending upward with respect to overweight/obesity and for grade 1 and 2 obesity, but not for abdominal obesity. However, the obesity trends in women were leveling off from overweight/obesity, grade 1 obesity, and abdominal obesity measures. Further studies are required with data on muscle mass and adiposity for effective obesity control policies.

Positive association between the changes in chemerin and adiponectin levels after weight reduction.

PURPOSE: Adiponectin and chemerin have been reported their associations with insulin resistance and chronic inflammation. However, the relationship between adiponectin and chemerin themselves has not been fully elucidated. Therefore, we investigated the effects of changes in adiponectin and chemerin levels after a weight intervention. MATERIALS AND METHODS: We recruited 136 healthy overweight or obese subjects from 2006 to 2009 and provided all participants lifestyle modification therapy with diet consultations over 16 weeks. We assigned the participants to take orlistat or sibutramine or to a no prescription group. We analyzed the data using paired t-tests, Pearson's partial correlation analysis, and stepwise multiple linear regression analysis. RESULTS: ∆ in chemerin was positively correlated with ∆ in adiponectin (r = 0.29, p < 0.01), and these trends were similar in the insulin-resistant (r = 0.35, p = 0.03) and insulin-sensitive (r = 0.27, p < 0.01) groups. In multiple regression analyses, Δadiponectin, ΔQUICKI (quantitative insulin-sensitivity check index), Δglucose, and ΔDBP were significantly associated with Δchemerin in the insulin-resistant group, and initial chemerin level, ΔQUICKI, ΔBMI (body mass index), and taking orlistat were associated with Δchemerin in the insulin-sensitive group. CONCLUSIONS: Changes in chemerin levels were positively associated with changes in adiponectin levels. The association between these changes might be related to chemerin's dual inflammatory and anti-inflammatory effects or insulin resistance and insulin sensitivity enhancing effects, depending on the metabolic conditions. Additional studies are needed to clarify the mechanisms that underlie the effects of adiponectin and chemerin.

Association between Sarcopenic Obesity and Bone Mineral Density in Middle-Aged and Elderly Korean.

OBJECTIVES: To identify the prevalence of osteosarcopenic obesity and to evaluate the association between sarcopenic obesity and bone mineral density in middle-aged and elderly Korean. METHODS: This study included 3,385 males and 4,064 females (age ≥50 years and in the stage of menopause) who participated in the Korean National Health and Nutrition Examination Survey (KNHANES) from 2008 to 2011. The cutoff value for sarcopenia was 7.26 kg/m2 for men and 5.45 kg/m2 for female. The fat mass was >30% for men and 40% for female; these values were used to define obesity. Based on the combination of sarcopenia and obesity status, all subjects were classified as sarcopenic obese (SO), sarcopenic nonobese (SNO), nonsarcopenic obese (NSO), or nonsarcopenic nonobese. RESULTS: The incidence rates of sarcopenia, sarcopenic obesity, and osteosarcopenic obesity were 31.5, 5.1, and 4.1%, respectively. Following adjustment, the ORs for osteoporosis were 8.67 in the SO group (95% CI 4.19-17.94), 3.85 in the SNO group (95% CI 2.61-5.67), and 1.88 in the NSO group (95% CI 0.72-4.91) in men and 2.93 in the SO group (95% CI 1.99-4.32), 1.71 in the SNO group (95% CI 1.31-2.23), and 0.58 in the NSO group (95% CI 0.43-0.78) in women. CONCLUSIONS: Sarcopenic obesity is associated with the development of osteoporosis among the middle-aged and elderly Korean population.

Method to purify and analyze heterogeneous senescent cell populations using a microfluidic filter with uniform fluidic profile.

To precisely purify and study aged (senescent) cells, we have designed, fabricated, and demonstrated a novel diamond-structure (DS) microfluidic filter. Nonuniform flow velocities within the microfilter channel can compromise microfluidic filter performance, but with this new diamond structure, further optimized via simulation, we achieve a uniform microfilter flow field, improving the throughput of size-based separation of senescent cells, as obtained by 39-passaged human dermal fibroblasts. After separating these aged cells into two groups, consisting of large- and small-sized cells, we assessed senescence by measuring lipofuscin accumulation and ß-galactosidase activity. Our results reveal that even though these senescent cells had been equivalently passaged in culture, a high degree of size distribution and senescent phenotype heterogeneity was observed. In particular, the smaller-sized cells tended to express a younger phenotype while the larger aged cells demonstrated an older phenotype. We suggest that size-based separation of senescent cells, subtyped into small- and large-sized cohorts, offers an alternative method to purify such aged cells, thereby enabling more precise study of the mechanisms of aging, autophagy impairment, and rejuvenation.

Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis.

Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.