Microfluidic Printing-Based Method for the Multifactorial Study of Cell-Free Protein Networks.

Protein networks can be assembled in vitro for basic biochemistry research, drug screening, and the creation of artificial cells. Two standard methodologies are used: manual pipetting and pipetting robots. Manual pipetting has limited throughput in the number of input reagents and the combination of reagents in a single sample. While pipetting robots are evident in improving pipetting efficiency and saving hands-on time, their liquid handling volume usually ranges from a few to hundreds of microliters. Microfluidic methods have been developed to minimize the reagent consumption and speed up screening but are challenging in multifactorial protein studies due to their reliance on complex structures and labeling dyes. Here, we engineered a new impact-printing-based methodology to generate printed microdroplet arrays containing water-in-oil droplets. The printed droplet volume was linearly proportional (R2 = 0.9999) to the single droplet number, and each single droplet volume was around 59.2 nL (coefficient of variation = 93.8%). Our new methodology enables the study of protein networks in both membrane-unbound and -bound states, without and with anchor lipids DGS-NTA(Ni), respectively. The methodology is demonstrated using a subnetwork of mitogen-activated protein kinase (MAPK). It takes less than 10 min to prepare 100 different droplet-based reactions, using <1 µL reaction volume at each reaction site. We validate the kinase (ATPase) activity of MEK1 (R4F)* and ERK2 WT individually and together under different concentrations, without and with the selective membrane attachment. Our new methodology provides a reagent-saving, efficient, and flexible way for protein network research and related applications.

Application of Engineered Zinc Finger Proteins Immobilized on Paramagnetic Beads for Multiplexed Detection of Pathogenic DNA.

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a double-stranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.

Building protein networks in synthetic systems from the bottom-up.

The recent development of synthetic biology has expanded the capability to design and construct protein networks outside of living cells from the bottom-up. The new capability has enabled us to assemble protein networks for the basic study of cellular pathways, expression of proteins outside cells, and building tissue materials. Furthermore, the integration of natural and synthetic protein networks has enabled new functions of synthetic or artificial cells. Here, we review the underlying technologies for assembling protein networks in liposomes, water-in-oil droplets, and biomaterials from the bottom-up. We cover the recent applications of protein networks in biological transduction pathways, energy self-supplying systems, cellular environmental sensors, and cell-free protein scaffolds. We also review new technologies for assembling protein networks, including multiprotein purification methods, high-throughput assay screen platforms, and controllable fusion of liposomes. Finally, we present existing challenges towards building protein networks that rival the complexity and dynamic response akin to natural systems. This review addresses the gap in our understanding of synthetic and natural protein networks. It presents a vision towards developing smart and resilient protein networks for various biomedical applications.

Orthogonal tuning of gene expression noise using CRISPR-Cas.

The control of gene expression noise is important for improving drug treatment and the performance of synthetic biological systems. Previous work has tuned gene expression noise by changing the rate of transcription initiation, mRNA degradation, and mRNA translation. However, these methods are invasive: they require changes to the target genetic components. Here, we create an orthogonal system based on CRISPR-dCas9 to tune gene expression noise. Specifically, we modulate the gene expression noise of a reporter gene in Escherichia coli by incorporating CRISPR activation and repression (CRISPRar) simultaneously in a single cell. The CRISPRar uses a single dCas9 that recognizes two different single guide RNAs (sgRNA). We build a library of sgRNA variants with different expression activation and repression strengths. We find that expression noise and mean of a reporter gene can be tuned independently by CRISPRar. Our results suggest that the expression noise is tuned by the competition between two sgRNAs that modulate the binding of RNA polymerase to promoters. The CRISPRar may change how we tune expression noise at the genomic level. Our work has broad impacts on the study of gene functions, phenotypical heterogeneity, and genetic circuit control.

Escherichia coli removal from model substrates: Underlying mechanism based on nanofluid structural forces.

Understanding the interactions between bacteria and solid surfaces that result in bacterial adhesion and removal is of immense importance for reducing foodborne illness outbreaks. A nanofluid formulation comprised of a sodium dodecyl sulfate (SDS) micellar aqueous solution in the presence of an organic acid (as a pH controller) was used to test the E. coli K12 removal from two substrates, polyvinylchloride (PVC) and partially hydrophobic glass. We investigated the bacterial removal efficacy based on the combined effect of the nanofluid's structural forces and bacterial isoelectric point. To quantify the bacteria-PVC coverage, we used fluorescence microscope. The Langmuir isotherm at the low volume fraction was applied to estimate the adsorption energy of E. coli K12. We obtained a value of about 2.5±0.2kT. This value compared favorably with the value of 2.1kT reported previously for E. coli NCTC 9002 (Vanloosdrecht et al., 1989). We applied the dynamic light scattering method to estimate the radius of the gyration of E. coli K12. The radius of the gyration was used to estimate the limit of surface area covered by the bacterium and compared it to the surface area measured from the image taken with fluorescence microscope. We found that they are in good agreement with each other. We modeled the nanofluid oscillatory structural energy against the E. coli K12 adsorption energy by applying the statistical mechanics approach. Based on the model prediction, the oscillatory interaction energy was estimated at the vertex between a bacterium and the substrate (i.e., the wedge film's interaction energy at one particle layer). The evaluated film's repulsive energy due to the oscillatory structural forces (OSF) was about 15.6±4.4kT of the 0.02M SMNF (the SDS micellar nanofluid formulation) and several times higher than the bacterial adsorption energy, 2.5±0.2kT. The OSF of the 0.06M SMNF was measured by AFM (the oscillatory decay force curve). The period and number of oscillations versus distance was annualized and used to obtain information for the effective size of the nanoparticles and nanofluid's effective volume fraction. These findings suggest that the OSF is capable of bacteria/microorganism removal from contaminated substrates.

Early diagnosis of Alzheimer's disease from elevated olfactory mucosal miR-206 level.

MicroRNA-206, which suppresses the expression of brain-derived neurotrophic factor, is known to be elevated in the brains of Alzheimer's disease (AD) patients. We performed intranasal biopsy of the olfactory epithelia of early dementia patients (n = 24) and cognitively healthy controls (n = 9). Patients with significant depression (n = 8) were analyzed separately, as their cognitive impairments were thought to be caused by their depression. Real-time PCR was performed on the biopsied tissues. The relative microRNA-206 level exhibited a 7.8-fold increase (P = 0.004) in the mild cognitive impairment group (CDR 0.5; n = 13) and a 41.5-fold increase (P < 0.001) in the CDR 1 group (n = 11). However, this level was not increased in the depression group, even in those with cognitive decline. Using the optimal cutoff value, the sensitivity/specificity for diagnosing CDR 0.5 and CDR 1 dementia were 87.5%/94.1% and 90.9%/93.3%, respectively. In ROC analysis, the AUCs were 0.942 and 0.976 in the CDR 0.5 and CDR 1 groups, respectively. The olfactory mucosal microRNA-206 level and cognitive assessment scores were significantly correlated in the non-depressed subjects with cognitive impairment. In conclusion, the olfactory mucosal microRNA-206 level can be easily measured, and it can be utilized as an excellent biomarker for the diagnosis of early AD, including mild cognitive impairment.

Seasonal Variation, Cranial Autonomic Symptoms, and Functional Disability in Migraine: A Questionnaire-Based Study in Tertiary Care.

OBJECTIVE/BACKGROUND: Seasonal variation of migraine attack frequency has been described as a phenomenon. We aimed to compare functional disability and the occurrence of cranial autonomic symptoms (CASs) in patients who reported a seasonal variation in their migraine attack frequency with those who did not. METHODS: We conducted a questionnaire-based observational study on patients with migraine without aura who visited our institution from January 2005 to December 2013. Patient demographics, headache characteristics, and accompanying symptoms were recorded, and functional disability was evaluated by Migraine Disability Assessment (MIDAS) Questionnaire. RESULTS: Of 4423 patients screened, 769 were eligible for analysis, and 104 (13.5%) of them reported seasonal variation. Several CAS features such as conjunctival injection (25.0% vs 14.0%), lacrimation (20.2% vs 10.8%), eyelid edema (20.2% vs 10.2%), forehead and facial sweating (22.1% vs 11.4%), and ptosis (23.1% vs 11.4%) were more prominent in this subset of patients. They showed higher MIDAS scores (15.4 ± 23.5) than the other migraineurs (10.4 ± 16.9), with a 1.77-fold increased risk (95% confidence interval 1.06-2.96) of severe functional disability (MIDAS score ≥21) after adjustment for age group, sex, headache frequency, intensity, and duration. The higher the number of CASs, the greater also was the proportion of patients with severe functional disability. CONCLUSIONS: Patients who reported seasonal variation in migraine also reported more CASs and had more severe functional disability. The profound functional disability in the migraineurs reporting seasonal variation or CAS also provides direction for proactive clinical management in these patients.

The First Case of Successful Transcatheter Aortic Valve Implantation Using CoreValve in Korea.

Surgical replacement of the aortic valve is the standard therapy for severe aortic valve stenosis. However, it is generally associated with increased mortality and morbidities in older individuals. Transcatheter aortic valve implantation (TAVI) is a less invasive procedure and has shown similar clinical outcomes as surgical treatment in elderly patients at high risk for conventional surgery. In this report, we describe the first case of TAVI using a CoreValve in Korea. An 84-year-old man with symptomatic severe aortic valve stenosis was successfully treated by transfemoral TAVI. The patient was discharged without any significant complications and remained free of adverse clinical event for a follow-up duration of 6 months.

The first korean patient with severe aortic stenosis and bilateral iliofemoral artery disease treated with transcatheter aortic valve implantation by transsubclavian approach.

Transcatheter aortic valve implantation (TAVI) is indicated as an alternative treatment modality to surgical aortic valve replacement for high risk patients. The standard retrograde approach through the femoral artery is not feasible in the case of unfavorable iliofemoral anatomy or severe peripheral arterial disease (PAD). However, patients with aortic stenosis (AS) have a higher prevalence of for PAD because both diseases are consequences of atherosclerotic degenerative changes. Transsubclavian, transapical, and direct access to the ascending aorta by thoracotomy are alternative routes for the TAVI procedure. In this report, we present the first Korean patient with symptomatic severe AS and bilateral iliofemoral artery disease who was successfully treated with TAVI using a CoreValve (Medtronic, Minneapolis, MN, USA) by transsubclavian approach.

Prognostic Estimation of Advanced Heart Failure With Low Left Ventricular Ejection Fraction and Wide QRS Interval.

BACKGROUND AND OBJECTIVES: Cardiac resynchronization therapy (CRT) has been known to improve the outcome of advanced heart failure (HF) but is still underutilized in clinical practice. We investigated the prognosis of patients with advanced HF who were suitable for CRT but were treated with conventional strategies. We also developed a risk model to predict mortality to improve the facilitation of CRT. SUBJECTS AND METHODS: Patients with symptomatic HF with left ventricular ejection fraction ≤35% and QRS interval >120 ms were consecutively enrolled at cardiovascular hospital. After excluding those patients who had received device therapy, 239 patients (160 males, mean 67±11 years) were eventually recruited. RESULTS: During a follow-up of 308±236 days, 56 (23%) patients died. Prior stroke, heart rate >90 bpm, serum Na ≤135 mEq/L, and serum creatinine ≥1.5 mg/dL were identified as independent factors using Cox proportional hazards regression. Based on the risk model, points were assigned to each of the risk factors proportional to the regression coefficient, and patients were stratified into three risk groups: low- (0), intermediate-(1-5), and high-risk (>5 points). The 2-year mortality rates of each risk group were 5, 31, and 64 percent, respectively. The C statistic of the risk model was 0.78, and the model was validated in a cohort from a different institution where the C statistic was 0.80. CONCLUSION: The mortality of patients with advanced HF who were managed conventionally was effectively stratified using a risk model. It may be useful for clinicians to be more proactive about adopting CRT to improve patient prognosis.

Heat-processed ginseng enhances the cognitive function in patients with moderately severe Alzheimer's disease.

OBJECTIVES: Ginseng has been reported to improve cognitive function in animals and in healthy and cognitively impaired individuals. In this study, we investigated the efficacy of a heat-processed form of ginseng that contains more potent ginsenosides than raw ginseng in the treatment of cognitive impairment in patients with moderately severe Alzheimer's disease (AD). METHODS: Forty patients with AD were randomized into one of three different dose groups or the control group as follows: 1.5 g/day (n = 10), 3 g/day (n = 10), and 4.5 g/day (n = 10) groups, or control (n = 10). The Alzheimer's Disease Assessment Scale (ADAS) and Mini-Mental State Examination (MMSE) were used to assess cognitive function for 24 weeks. RESULTS: The treatment groups showed significant improvement on the MMSE and ADAS. Patients with higher dose group (4.5 g/day) showed improvements in ADAS cognitive, ADAS non-cognitive, and MMSE score as early as at 12 weeks, which sustained for 24-week follow-up. DISCUSSION: These results demonstrate the potential efficacy of a heat-processed form of ginseng on cognitive function and behavioral symptoms in patients with moderately severe AD.

Improvement of cognitive deficit in Alzheimer's disease patients by long term treatment with korean red ginseng.

A 24-week randomized open-label study with Korean red ginseng (KRG) showed cognitive benefits in patients with Alzheimer's disease. To further determine long-term effect of KRG, the subjects were recruited to be followed up to 2 yr. Cognitive function was evaluated every 12 wk using the Alzheimer's Disease Assessment Scale (ADAS) and the Korean version of the Mini Mental Status Examination (K-MMSE) with the maintaining dose of 4.5 g or 9.0 g KRG per d. At 24 wk, there had been a significant improvement in KRG-treated groups. In the long-term evaluation of the efficacy of KRG after 24 wk, the improved MMSE score remained without significant decline at the 48th and 96th wk. ADAS-cog showed similar findings. Maximum improvement was found around week 24. In conclusion, the effect of KRG on cognitive functions was sustained for 2 yr follow-up, indicating feasible efficacies of long-term follow-up for Alzheimer's disease.

Transplantation of patient-derived adipose stem cells in YAC128 Huntington's disease transgenic mice.

Huntington's disease (HD) is a genetic neurodegenerative disorder caused by abnormal expansion of CAG in the huntingtin gene. In R6/2 HD transgenic mice, human adipose-derived stem cells (ASCs) can slow disease progression via secretion of multiple paracrine growth factors. In order to prompt autologous ASCs transplantation in HD, we isolated ASCs from subcutaneous adipose tissues from a HD patient and a normal volunteer. ASCs were grown in two different types of stem cell culture media, EGM-2MV (endothelial growth medium-2 MV) or mesenchymal culture medium (MesenPRO). Cell-surface markers CD13, CD29, CD31, CD34, and CD44 were characterized by flow cytometry. BDNF, HGF, IGF, LIF, NGF, and VEGF expressions were determined by RT-PCR. Cell surface markers for HD ASCs were similar to those for normal ASCs. HD ASCs expressed multiple growth factors, and were similar to normal ASCs, except for NGF; however, they can be altered by culture medium. ASCs were transplanted in bilateral striata of 8 month-old YAC128 mice. At 12 months of age, normal ASCs reduced striatal atrophy, while HD ASCs failed to prevent it. Compared to the control YAC128 group, no improvement in Rotarod performance was observed in any of the transplanted YAC128 mice. However, when normal ASCs were transplanted at 12 months, Rotarod performance was maintained for 4 weeks, with detectable transplanted cells in the striatum and periventricular area. In summary, cultured HD patient-derived ASCs express multiple growth factors with the same cell surface markers as those of normal ASCs in vitro. The efficacy of ASCs transplantation in YAC128 transgenic models can be modified, depending on the time window.

Protective action of the immunomodulator ginsan against carbon tetrachloride-induced liver injury via control of oxidative stress and the inflammatory response.

The aim of the present study was to evaluate immunomodulator ginsan, a polysaccharide extracted from Panax ginseng, on carbon tetrachloride (CCl(4))-induced liver injury. BALB/c mice were injected i.p. with ginsan 24 h prior to CCl(4) administration. Serum liver enzyme levels, histology, expression of antioxidant enzymes, and several cytokines/chemokines were subsequently evaluated. Ginsan treatment markedly suppressed the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and hepatic histological necrosis increased by CCl(4) treatment. Ginsan inhibited CCl(4) induced lipid peroxidation through the cytochrome P450 2E1 (CYP2E1) downregulation. The hepatoprotective effect of ginsan was attributed to induction of anti-oxidant protein contents, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) as well as restoration of the hepatic glutathione (GSH) concentration. The marked increase of proinflammatory cytokines (IL-1beta, IFN-gamma) and chemokines (MCP-1, MIP-2beta, KC) in CCl(4) treated mice was additionally attenuated by ginsan, thereby preventing leukocyte infiltration and local inflammation. Our results suggest that ginsan effectively prevent liver injury, mainly through downregulation of oxidative stress and inflammatory response.

White matter hyperintensities and cognitive dysfunction in Alzheimer disease.

The effect of white matter lesions in magnetic resonance imaging or vascular atherosclerosis on cognitive function is not fully understood in Alzheimer disease (AD). In this investigation, we examined the influence of white matter lesions on cognitive decline in AD. A total of 142 patients with AD (44 men, mean age 65.7 + 7.6 years; mean education period 7.8 + 5.0 years) were included. Patients were divided into 4 groups based on the severities of white matter hyperintensities (WMH) in brain magnetic resonance images (MRI) using Fazekas scale. Cognitive functions were determined using the Korean version of the Mini-Mental State Examination (K-MMSE) and the Clinical Dementia Rating (CDR) scale before acetylcholinesterase inhibitors were administered. Of the 142 patients, 30% (43/142) had no white matter signal abnormality (grade 0). Fourteen percentage (20/142) were grade 1, 42% (59/142) grade 2, and 14% (20/142) were grade 3. Mean K-MMSE scores declined as MRI grades increased to grade 2 and 3 compared to grade 0 (P < .01). Clinical Dementia Ratings were also aggravated by MRI grade. These results remained significant after adjusting for compounding factors affecting cognitive functions; sex, age, number of years in full-time education, hypertension, diabetes, hypercholesterolemia, smoking, and atrial fibrillation. The presence of WMHs were associated with score of MMSE and CDR impairment in patients with AD. These features could be a correctable factor hastening cognitive decline in AD.

N-acetylphytosphingosine enhances the radiosensitivity of lung cancer cell line NCI-H460.

Ceramides are well-known second messengers that induce apoptosis in various kinds of cancer cells, and their effects are closely related to radiation sensitivity. Phytoceramides, the yeast counterparts of the mammalian ceramides, are also reported to induce apoptosis. We investigated the effect of a novel ceramide derivative, N-acetylphytosphingosine (NAPS), on the radiosensitivity of NCI-H460 human lung carcinoma cells and its differential cytotoxicity in tumor and normal cells. The combination of NAPS with radiation significantly increased clonogenic cell death and caspase-dependent apoptosis. The combined treatment greatly increased Bax expression and Bid cleavage, but not Bcl-2 expression. However, there was no effect on radiosensitivity and apoptosis in BEAS2B cells, which derive from normal human bronchial epithelium. Cell proliferation and DNA synthesis were significantly inhibited by NAPS in both NCI-H460 and BEAS2B cells, but only the BEAS2B cells recovered by 48h after removal of the NAPS. Furthermore, the NCI-H460 cells underwent more DNA fragmentation than the BEAS2B cells in response to NAPS. Our results indicate that NAPS may be a potential radiosensitizing agent with differential effects on tumor vs. normal cells.

The immunomodulator ginsan induces resistance to experimental sepsis by inhibiting Toll-like receptor-mediated inflammatory signals.

Ginsan, a polysaccharide extracted from Panax ginseng, has multiple immunomodulatory effects. In this study, we show that pretreatment of ginsan (25 mug/kg) protected mice from lethality induced by Staphylococcus aureus challenge. This survival benefit was associated with enhanced bacterial clearance from circulation, spleen and kidney. The phagocytic activity of macrophages treated with ginsan was significantly enhanced against S. aureus. However, the production of proinflammatory cytokines, such as TNF-alpha, IL-1beta, IL-6, IFN-gamma, IL-12, and IL-18, was markedly down-regulated in ginsan-treated mice compared with those of control-infected mice. The expression of Toll-like receptor (TLR) 2 and the adaptor molecule MyD88, which was greatly increased in septic macrophages, was significantly reduced by ginsan treatment in vitro. Similarly, the expression of phospho-JNK1/2, phospho-p38 MAPK, and NF-kappaB was decreased in the same culture system. These results illustrate that the antiseptic activity of ginsan can be attributed to enhanced bacterial clearance, and reduced proinflammatory cytokines via the TLR signaling pathway.

The Effect of Paroxetine on the Reduction of Migraine Frequency is Independent of Its Anxiolytic Effect.

BACKGROUND AND PURPOSE: Anxiety is the most important precipitating factor of migraine attacks, and more than half of migraineurs have coexisting anxiety disorders. Paroxetine, an antidepressant, is one of the selective serotonin reuptake inhibitors (SSRIs) that has an anxiolytic effect, and is also known to be effective for migraine prophylaxis. The aim of this study was to determine the role of the anxiolytic effect of paroxetine on the prevention of migraine. METHODS: This study investigated migraineurs with a general anxiety disorder who visited the neurological clinic. The following efficacy variables were assessed at baseline and after taking paroxetine (20 for 12 weeks: headache frequency, Hamilton Anxiety Rating Scale (HAM-A), Headache Management Self-Efficacy Scale (HMSE), and Headache Disability Inventory (HDI). The correlation between the headache responsiveness to paroxetine and improvement in anxiety levels was analyzed. RESULTS: Twenty-four patients (aged 54.96+/-12.09 years, mean+/-SD) were included in this study. Paroxetine reduced headache frequency by 49.1% within 12 weeks (p<0.05 vs baseline). HAM-A and HMSE scores also showed an improvement, whereas there was no significant change in HDI score. The baseline HAM-A scores did not differ between paroxetine responders and nonresponders. In addition, the improvement in HAM-A score was not correlated with the reduction in headache frequency. CONCLUSIONS: Paroxetine decreased the headache frequency and reduced anxiety levels. However, the anxiolytic effect of paroxetine was not correlated with the migraine prevention effect. These observation indicate that the anxiolytic effect of paroxetine does not contribute strongly to its prophylactic effect on migraine frequency in migraineurs with anxiety disorder.