Effect of saline irrigation temperature difference on postoperative acute pain and hypothermia during biportal endoscopic spine surgery.

BACKGROUND: Recently, enhanced recovery after surgery (ERAS) protocols have attracted attention; they emphasize on avoiding intraoperative hypothermia while performing lumbar fusion surgery. However, none of the studies have reported the protocol for determining the temperature of saline irrigation during biportal endoscopic spine surgery (BESS) procedure. This study evaluated the effectiveness of warm saline irrigation during BESS in acute postoperative pain and inflammatory reactions. MATERIALS AND METHODS: Fifty-five patients who underwent BESS procedure were retrospectively analyzed for the incidence of perioperative hypothermia (< 36oC), postoperative inflammatory factors (white blood cells (WBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), interleukin-6 (IL-6), serum amyloid A (SAA)), and clinical outcomes (back visual analog scale (VAS) score, postoperative shivering). The patients were divided into the warm and cold saline irrigation groups. RESULTS: Hemoglobin, WBC, ESR, creatine kinase, and creatine kinase-muscle brain levels did not significantly differ between the warm and cold saline groups. The mean CRP, IL-6, and SAA levels were significantly higher in the cold saline group than in the warm saline group (p = 0.0058, 0.0028, and 0.0246, respectively); back VAS scores were also higher with a statistically significant difference until two days postoperatively (p < 0.001). During the entire procedure, the body temperature was significantly lower in the cold saline irrigation group, but the hypothermia incidence rate significantly differed 30 min after the operation was started. CONCLUSIONS: Using warm saline irrigation during BESS is beneficial for early recovery after surgery, as it is associated with reduced postoperative pain and complication rates.

Shoulder Traction as a Possible Risk Factor for C5 Palsy in Anterior Cervical Surgery: A Cadaveric Study.

Background and Objectives: Many risk factors for postoperative C5 palsy (PC5P) have been reported regarding a "cord shift" after a posterior approach. However, there are few reports about shoulder traction as a possible risk factor of anterior cervical surgery. Therefore, we assessed the stretched nerve roots when shoulder traction was applied on cadavers. Materials and Methods: Eight cadavers were employed in this study, available based on age and the presence of foramen stenosis. After dissecting the sternocleidomastoid muscle of the cadaver, the shoulder joint was pulled with a force of 2, 5, 8, 10, 15, and 20 kg. Then, the stretched length of the fifth nerve root was measured in the extra-foraminal zone. In addition, the same measurement was performed after cutting the carotid artery to accurately identify the nerve root's origin. After an additional dissection was performed so that the superior trunk of the brachial plexus could be seen, the stretched length of the fifth and sixth nerve roots was measured again. Results: Throughout the entire experiment, the fifth nerve root stretched out for an average of 1.94 mm at 8 kg and an average of 5.03 mm at a maximum force of 20 kg. In three experiments, the elongated lengths of the C5 nerve root at 8 kg and 20 kg were 1.69/4.38 mm, 2.13/5.00 mm, and 0.75/5.31 mm, respectively, and in the third experiment, the elongated length of the C6 nerve root was 1.88/5.44 mm. Conclusions: Although this was a cadaveric experiment, it suggests that shoulder traction could be the risk factors for PC5P after anterior cervical surgery. In addition, for patients with foraminal stenosis and central stenosis, the risk would be higher. Therefore, the surgeon should be aware of this, and the patient would need sufficient explanation.

Revisiting the Ramachandran plot based on statistical analysis of static and dynamic characteristics of protein structures.

Ramachandran plots, which describe protein structures by plotting the dihedral angle pairs of the backbone on a two-dimensional plane, have played an important role in structural biology over the past few decades. However, despite continued discovery of new protein structures to date, the Ramachandran plot is still constructed by only a small number of data points, and further it cannot reflect the steric information of proteins. Here, we investigated the secondary structure of proteins in terms of static and dynamic characteristics. As for static feature, the Ramachandran plot was revisited for the dataset consisting of 9,148 non-redundant high-resolution protein structures released in the protein data bank until April 1, 2022. By calculating amino acid propensities, it was found that the proportion of secondary structures with respect to residue depth is directly related to their hydrophobicity. As for dynamic feature, normal mode analysis (NMA) based on an elastic network model (ENM) was carried out for the dataset using our KOSMOS web server (http://bioengineering.skku.ac.kr/kosmos/). All ENM-based NMA results were stored in the KOSMOS database, allowing researchers to use them in various ways. In this process, it was commonly found that high B-factors appeared at the edge of the alpha helix region, which was elucidated by introducing residue depth. In addition, by investigating the change in dihedral angle, it was possible to quantitatively survey the contribution of structural change of protein on the Ramachandran plot. In conclusion, our statistical analysis of protein characteristics will provide insight into a range of protein structural studies.

PredMS: a random forest model for predicting metabolic stability of drug candidates in human liver microsomes.

MOTIVATION: Poor metabolic stability leads to drug development failure. Therefore, it is essential to evaluate the metabolic stability of small compounds for successful drug discovery and development. However, evaluating metabolic stability in vitro and in vivo is expensive, time-consuming and laborious. In addition, only a few free software programs are available for metabolic stability data and prediction. Therefore, in this study, we aimed to develop a prediction model that predicts the metabolic stability of small compounds. RESULTS: We developed a computational model, PredMS, which predicts the metabolic stability of small compounds as stable or unstable in human liver microsomes. PredMS is based on a random forest model using an in-house database of metabolic stability data of 1917 compounds. To validate the prediction performance of PredMS, we generated external test data of 61 compounds. PredMS achieved an accuracy of 0.74, Matthew's correlation coefficient of 0.48, sensitivity of 0.70, specificity of 0.86, positive predictive value of 0.94 and negative predictive value of 0.46 on the external test dataset. PredMS will be a useful tool to predict the metabolic stability of small compounds in the early stages of drug discovery and development. AVAILABILITY AND IMPLEMENTATION: The source code for PredMS is available at https://bitbucket.org/krictai/predms, and the PredMS web server is available at https://predms.netlify.app. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Treatment of Soft Tissue Defects after Minimally Invasive Plate Osteosynthesis in Fractures of the Distal Tibia: Clinical Results after Reverse Sural Artery Flap.

Introduction: Distal tibial fractures make up approximately 3% to 10% of all tibial fractures or about 1% of lower extremity fractures. MIPO is an appropriate procedure and method to achieve stable metal plate fixation and osseointegration by minimizing soft tissue damage and vascular integrity at the fracture site. MIPO to the medial tibia during distal tibial fractures induces skin irritation due to the thickness of the metal plate, which causes discomfort and pain on the medial side of the distal leg, and if severe, complications such as infection and skin defect may occur. The reverse sural flap is a well-researched approach for covering defects in the lower third of the leg, ankle, and foot. Materials and Methods: Among 151 patients with distal tibia fractures who underwent minimally invasive metal plate fixation, soft tissue was injured due to postoperative complications. We treated 13 cases with necrosis and exposed metal plates by retrograde nasogastric artery flap surgery. For these patients, we collected obligatory patient records, radiological data, and wound photographs of the treatment results and complications of reconstructive surgery. Results: In all the cases, flap survival was confirmed at the final outpatient follow-up. The exposed area of the metal plate was well coated, and there was no plate failure due to complete necrosis. Three out of four women complained of aesthetic dissatisfaction because the volume of the tunnel through which the skin mirror passed and the skin plate itself were thick. In two cases, defatting was performed to reduce the thickness of the plate while removing the metal plate. Conclusions: Metal plate exposure after distal tibial fractures have been treated with minimally invasive metal plate fusion and can be successfully treated with retrograde nasogastric artery flaps, and several surgical techniques are used during flap surgery.

Identification and New Indication of Melanin-Concentrating Hormone Receptor 1 (MCHR1) Antagonist Derived from Machine Learning and Transcriptome-Based Drug Repositioning Approaches.

Melanin-concentrating hormone receptor 1 (MCHR1) has been a target for appetite suppressants, which are helpful in treating obesity. However, it is challenging to develop an MCHR1 antagonist because its binding site is similar to that of the human Ether-à-go-go-Related Gene (hERG) channel, whose inhibition may cause cardiotoxicity. Most drugs developed as MCHR1 antagonists have failed in clinical development due to cardiotoxicity caused by hERG inhibition. Machine learning-based prediction models can overcome these difficulties and provide new opportunities for drug discovery. In this study, we identified KRX-104130 with potent MCHR1 antagonistic activity and no cardiotoxicity through virtual screening using two MCHR1 binding affinity prediction models and an hERG-induced cardiotoxicity prediction model. In addition, we explored other possibilities for expanding the new indications for KRX-104130 using a transcriptome-based drug repositioning approach. KRX-104130 increased the expression of low-density lipoprotein receptor (LDLR), which induced cholesterol reduction in the gene expression analysis. This was confirmed by comparison with gene expression in a nonalcoholic steatohepatitis (NASH) patient group. In a NASH mouse model, the administration of KRX-104130 showed a protective effect by reducing hepatic lipid accumulation, liver injury, and histopathological changes, indicating a promising prospect for the therapeutic effect of NASH as a new indication for MCHR1 antagonists.

DeepHIT: a deep learning framework for prediction of hERG-induced cardiotoxicity.

MOTIVATION: Blockade of the human ether-à-go-go-related gene (hERG) channel by small compounds causes a prolonged QT interval that can lead to severe cardiotoxicity and is a major cause of the many failures in drug development. Thus, evaluating the hERG-blocking activity of small compounds is important for successful drug development. To this end, various computational prediction tools have been developed, but their prediction performances in terms of sensitivity and negative predictive value (NPV) need to be improved to reduce false negative predictions. RESULTS: We propose a computational framework, DeepHIT, which predicts hERG blockers and non-blockers for input compounds. For the development of DeepHIT, we generated a large-scale gold-standard dataset, which includes 6632 hERG blockers and 7808 hERG non-blockers. DeepHIT is designed to contain three deep learning models to improve sensitivity and NPV, which, in turn, produce fewer false negative predictions. DeepHIT outperforms currently available tools in terms of accuracy (0.773), MCC (0.476), sensitivity (0.833) and NPV (0.643) on an external test dataset. We also developed an in silico chemical transformation module that generates virtual compounds from a seed compound, based on the known chemical transformation patterns. As a proof-of-concept study, we identified novel urotensin II receptor (UT) antagonists without hERG-blocking activity derived from a seed compound of a previously reported UT antagonist (KR-36676) with a strong hERG-blocking activity. In summary, DeepHIT will serve as a useful tool to predict hERG-induced cardiotoxicity of small compounds in the early stages of drug discovery and development. AVAILABILITY AND IMPLEMENTATION: https://bitbucket.org/krictai/deephit and https://bitbucket.org/krictai/chemtrans. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Invasive dental procedures as risk factors for postoperative spinal infection and the effect of antibiotic prophylaxis.

AIM: To identify invasive dental procedures as a risk factor for postoperative spinal infection (PSI) and evaluate the effectiveness of antibiotic prophylaxis. MATERIALS AND METHODS: We analysed 229,335 patients who underwent spinal surgery with instrumentation from 2010 to 2017, using the nationwide database. The incidence of spinal infection 2 years after surgery was determined. Invasive dental procedures as a risk factor for PSI and the effects of antibiotic prophylaxis during this period were also analysed. RESULTS: A total of 15,346 patients (6.69%) were diagnosed with PSI. It was found that advanced age, male sex, and a high Charlson Comorbidity Index were risk factors for PSI. The risk of PSI did not increase following dental procedures (adjusted hazard ratio [HR] 0.850; 95% confidence interval [CI], 0.793-0.912) and was not affected by antibiotics (adjusted HR 1.097; 95% CI, 0.987-1.218). Patients who received dental treatment as early as 3 months after spinal surgery had the lowest risk of postoperative infection (adjusted HR 0.869; 95% CI, 0.795-0.950). CONCLUSIONS: Invasive dental procedure does not increase the risk of PSI, and antibiotic prophylaxis before dental procedure was not effective in preventing spinal infection.

Development of a FOXM1-DBD Binding Assay for High-Throughput Screening Using TR-FRET Assay.

Electrophoretic mobility shift assay (EMSA) technology has been widely employed for the analysis of transcription factors such as Forkhead box protein M1 (FOXM1). However, the application of high-throughput screening (HTS) in performing, such analyses are limited as it uses time consuming electrophoresis procedure and radioisotopes. In this study, we developed a FOXM1-DNA binding domain (DBD) binding assay based on time-resolved fluorescence energy transfer (TR-FRET) that enables HTS for the inhibitors of FOXM1-DNA interaction. This assay was robust, highly reproducible and could be easily miniaturized into 384-well plate format. The signal-to-background (S/B) ratio and Z' factor were calculated as 7.46 and 0.74, respectively, via a series of optimization of the assay conditions. A pilot library screening of 1019 natural compounds was performed using the FOXM1-DBD binding assay. Five hit compounds, namely, AC1LXM, BRN5, gangaleoidin, leoidin, and roemerine were identified as the inhibitors of FOXM1. In a cell viability assay, it was demonstrated that cell proliferation of FOXM1 overexpressed cell lines was suppressed in cell lines such as MDA-MB-231 and MCF-7 by five hit compounds. These results indicate that developed FOXM1-DBD binding assay can be applied to highly efficiency HTS of compound libraries.

Is There an Association Between Psychiatric Disorders and Adolescent Idiopathic Scoliosis? A Large-database Study.

BACKGROUND: Children with adolescent idiopathic scoliosis (AIS) have reduced quality of life related to poor self-image, perhaps because of cosmetic concerns. However, there has not been a large-database epidemiologic study on the association between psychiatric disorders and scoliosis. QUESTIONS/PURPOSES: Using the Korean National Health Insurance database, we asked: (1) How common are psychiatric disorders among children with AIS? (2) After controlling for gender, age, insurance type, and residential district, are psychiatric disorders more common among children with AIS than among age-matched controls? METHODS: A retrospective analysis was conducted using sample datasets from the Health Insurance Review and Assessment Service from 2012 to 2016, which is a 10% randomly extracted sample of total inpatients and outpatients each year. The mean number of total patients in each dataset was 1,047,603 ± 34,534. The mean number of children with AIS was 7409 ± 158 for each year. The age criteria was 10 to 19 years for the matching. Mood disorders, anxiety disorders, and behavioral disorders were selected as disorders possibly associated with AIS. We identified children with AIS who had any of the disorders above, and we obtained the prevalence of these disorders based on diagnostic codes. As an exploratory analysis, clinically meaningful variables were selected among the available codes in the dataset, and a univariable logistic regression test was performed for each variable. A multivariable logistic regression test with advanced variables was performed to identify the adjusted odds ratios of psychiatric disorders in children with AIS. RESULTS: The median (range) prevalence of psychiatric disorders in children with AIS from 2012 to 2016 was 7% (6% to 7%). Compared with children who did not have AIS, and after controlling for gender, age, insurance type, and residential district, children with AIS were more likely to have psychiatric disorders in all 5 years. The adjusted ORs of psychiatric disorders in children with AIS compared with children who did not have AIS ranged from 1.47 to 1.74 (2012: OR 1.60 [95% CI 1.46 to 1.75]; p < 0.001; 2013: OR 1.73 [95% CI 1.58 to 1.89]; p < 0.001; 2014: OR 1.74 [95% CI 1.59 to 1.91]; p < 0.001; 2015: OR 1.71 [95% CI 1.56 to 1.88]; p < 0.001; 2016: OR 1.47 [95% CI 1.33 to 1.62]; p < 0.001). CONCLUSION: Considering the higher prevalence of psychiatric disorders in children with AIS compared with children who did not have AIS, children with AIS and their parents should be counseled about the increased risk of deteriorating mental health of the patients, and surgeons should provide early referral to pediatric psychiatrists. Further studies should investigate the effect of the factors related to AIS, such as curve type, Cobb angle, and treatment modality. LEVEL OF EVIDENCE: Level III, prognostic study.

Which Factors Affect the Stress of Intraoperative Orthopedic Surgeons by Using Electroencephalography Signals and Heart Rate Variability?

Can we recognize intraoperative real-time stress of orthopedic surgeons and which factors affect the stress of intraoperative orthopedic surgeons with EEG and HRV? From June 2018 to November 2018, 265 consecutive records of intraoperative stress measures for orthopedic surgeons were compared. Intraoperative EEG waves and HRV, comprising beats per minute (BPM) and low frequency (LF)/high frequency (HF) ratio were gathered for stress-associated parameters. Differences in stress parameters according to the experience of surgeons, intraoperative blood loss, and operation time depending on whether or not a tourniquet were investigated. Stress-associated EEG signals including beta 3 waves were significantly higher compared to EEG at rest for novice surgeons as the procedure progressed. Among senior surgeons, the LF/HF ratio reflecting the physical demands of stress was higher than that of novice surgeons at all stages. In surgeries including tourniquets, operation time was positively correlated with stress parameters including beta 1, beta 2, beta 3 waves and BPM. In non-tourniquet orthopedic surgeries, intraoperative blood loss was positively correlated with beta 1, beta 2, and beta 3 waves. Among orthopedic surgeons, those with less experience demonstrated relatively higher levels of stress during surgery. Prolonged operation time or excessive intraoperative blood loss appear to be contributing factors that increase stress.

Optimization of cyclic sulfamide derivatives as 11ß-hydroxysteroid dehydrogenase 1 inhibitors for the potential treatment of ischemic brain injury.

The 11ß-hydroxysteroiddehydrogenase type 1(11ß-HSD1), acortisolregenerating enzyme that amplifies tissue glucocorticoidlevels, plays an important role in diabetes, obesity, and glaucoma and is recognized as a potential therapeutic target for various disease conditions. Moreover, a recent study demonstrated that selective 11ß-HSD1 inhibitor can attenuate ischemic brain injury. This prompted us to optimize cyclic sulfamide derivative for aiming to treat ischemic brain injury. Among the synthesized compounds, 6e has an excellent in vitro activivity with an IC50 value of 1 nM toward human and mouse 11ß-HSD1 and showed good 11ß-HSD1 inhibition in ex vivo study using brain tissue isolated from mice. Furthermore, in the transient middle cerebral artery occlusion model in mice, 6e treatment significantly attenuated infarct volume and neurological deficit following cerebral ischemia/reperfusion injury. Additionally, binding modes of 6e for human and mouse 11ß-HSD1 were suggested.

Computational determination of hERG-related cardiotoxicity of drug candidates.

BACKGROUND: Drug candidates often cause an unwanted blockage of the potassium ion channel of the human ether-a-go-go-related gene (hERG). The blockage leads to long QT syndrome (LQTS), which is a severe life-threatening cardiac side effect. Therefore, a virtual screening method to predict drug-induced hERG-related cardiotoxicity could facilitate drug discovery by filtering out toxic drug candidates. RESULT: In this study, we generated a reliable hERG-related cardiotoxicity dataset composed of 2130 compounds, which were carried out under constant conditions. Based on our dataset, we developed a computational hERG-related cardiotoxicity prediction model. The neural network model achieved an area under the receiver operating characteristic curve (AUC) of 0.764, with an accuracy of 90.1%, a Matthews correlation coefficient (MCC) of 0.368, a sensitivity of 0.321, and a specificity of 0.967, when ten-fold cross-validation was performed. The model was further evaluated using ten drug compounds tested on guinea pigs and showed an accuracy of 80.0%, an MCC of 0.655, a sensitivity of 0.600, and a specificity of 1.000, which were better than the performances of existing hERG-toxicity prediction models. CONCLUSION: The neural network model can predict hERG-related cardiotoxicity of chemical compounds with a high accuracy. Therefore, the model can be applied to virtual high-throughput screening for drug candidates that do not cause cardiotoxicity. The prediction tool is available as a web-tool at http://ssbio.cau.ac.kr/CardPred .

Synthesis and SAR of 5-aryl-furan-2-carboxamide derivatives as potent urotensin-II receptor antagonists.

The synthesis and biological evaluation as potential urotensin-II receptor antagonists of a series of 5-arylfuran-2-carboxamide derivatives 1, bearing a 4-(3-chloro-4-(piperidin-4-yloxy)benzyl)piperazin-1-yl group, are described. The results of a systematic SAR investigation of furan-2-carboxamides with C-5 aryl groups possessing a variety of aryl ring substituents led to identification of the 3,4-difluorophenyl analog 1y as a highly potent UT antagonist with an IC50 value of 6 nM. In addition, this substance was found to display high metabolic stability, and low hERG inhibition and cytotoxicity, and to have an acceptable PK profile.

Optimization study towards more potent thiazolidine-2,4-dione IKK-ß modulator: Synthesis, biological evaluation and in silico docking simulation.

Inhibition of IKK-ß (inhibitor of nuclear factor kappa-B kinase subunit beta) has been broadly documentedas a promising approach for treatment of acute and chronic inflammatory diseases, cancer, and autoimmune diseases. Recently, we have identified a novel class of thiazolidine-2,4-diones as structurally novel modulators for IKK-ß. Herein, we report a hit optimization study via analog synthesis strategy aiming to acquire more potent derivative(s), probe the structure activity relationship (SAR), and get reasonable explanations for the elicited IKK-ß inhibitory activities though an in silico docking simulation study. Accordingly, a new series of eighteen thiazolidine-2,4-dione derivatives was rationally designed, synthesized, identified with different spectroscopic techniques and biologically evaluated as noteworthy IKK-ß potential modulators. Successfully, new IKK-ß potent modulators were obtained, including the most potent analog up-to-date 7m with IC50 value of 260 nM. A detailed structure activity relationship (SAR) was discussed and a mechanistic study for 7m was carried out indicating its irreversible inhibition mode with IKK-ß (Kinact value = 0.01 (min-1). Furthermore, the conducted in silico simulation study provided new insights for the binding modes of this novel class of modulators with IKK-ß.

Understanding carbon nanotube channel formation in the lipid membrane.

Carbon nanotubes (CNTs) have been considered a prominent nano-channel in cell membranes because of their prominent ion-conductance and ion-selectivity, offering agents for a biomimetic channel platform. Using a coarse-grained molecular dynamics simulation, we clarify a construction mechanism of vertical CNT nano-channels in a lipid membrane for a long period, which has been difficult to observe in previous CNT-lipid interaction simulations. The result shows that both the lipid coating density and length of CNT affect the suitable fabrication condition for a vertical and stable CNT channel. Also, simulation elucidated that a lipid coating on the surface of the CNT prevents the CNT from burrowing into the lipid membrane and the vertical channel is stabilized by the repulsion force between the lipids in the coating and membrane. Our study provides an essential understanding of how CNTs can form stable and vertical channels in the membrane, which is important for designing new types of artificial channels as biosensors for bio-fluidic studies.

Montreal cognitive assessment reflects cognitive reserve.

BACKGROUND: The Montreal Cognitive Assessment (MoCA) is known to have discriminative power for patients with Mild Cognitive Impairment (MCI). Recently Cognitive Reserve (CR) has been introduced as a factor that compensates cognitive decline. We aimed to assess whether the MoCA reflects CR. Furthermore, we assessed whether there were any differences in the efficacy between the MoCA and the Mini-Mental State Examination (MMSE) in reflecting CR. METHODS: MoCA, MMSE, and the Cognitive Reserve Index questionnaire (CRIq) were administered to 221 healthy participants. Normative data and associated factors of the MoCA were identified. Correlation and regression analyses of the MoCA, MMSE and CRIq scores were performed, and the MoCA score was compared with the MMSE score to evaluate the degree to which the MoCA reflected CR. RESULTS: The MoCA reflected total CRIq score (CRI; B = 0.076, P < 0.001), CRI-Education (B = 0.066, P <  0.001), and CRI-Working activity (B = 0.025, P = 0.042), while MMSE reflected total CRI (B = 0.044, P <  0.001) and CRI-Education (B = 0.049, P <  0.001) only. The MoCA differed from the MMSE in the reflection of total CRI (Z = 2.30). CONCLUSION: In this study, we show that the MoCA score reflects CR more sensitively than the MMSE score. Therefore, we suggest that MoCA can be used to assess CR and early cognitive decline.

Fabrication and Characterization of Finite-Size DNA 2D Ring and 3D Buckyball Structures.

In order to incorporate functionalization into synthesized DNA nanostructures, enhance their production yield, and utilize them in various applications, it is necessary to study their physical stabilities and dynamic characteristics. Although simulation-based analysis used for DNA nanostructures provides important clues to explain their self-assembly mechanism, structural function, and intrinsic dynamic characteristics, few studies have focused on the simulation of DNA supramolecular structures due to the structural complexity and high computational cost. Here, we demonstrated the feasibility of using normal mode analysis for relatively complex DNA structures with larger molecular weights, i.e., finite-size DNA 2D rings and 3D buckyball structures. The normal mode analysis was carried out using the mass-weighted chemical elastic network model (MWCENM) and the symmetry-constrained elastic network model (SCENM), both of which are precise and efficient modeling methodologies. MWCENM considers both the weight of the nucleotides and the chemical bonds between atoms, and SCENM can obtain mode shapes of a whole structure by using only a repeated unit and its connectivity with neighboring units. Our results show the intrinsic vibrational features of DNA ring structures, which experience inner/outer circle and bridge motions, as well as DNA buckyball structures having overall breathing and local breathing motions. These could be used as the fundamental basis for designing and constructing more complicated DNA nanostructures.

Development of a High-Throughput Assay for Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1 Based on Time-Resolved Fluorescence Energy Transfer.

Although enzyme-linked immunosorbent assay (ELISA) technology has been widely accepted for binding assays against the polo-box domain (PBD) of polo-like kinase-1 (Plk1), these assays have a limitation-related heterogeneous procedure, such as multiple incubations and washing steps to apply high-throughput screenings (HTSs). In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z' factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z' factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g]isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries.

A novel role of G protein-coupled receptor kinase 5 in urotensin II-stimulated cellular hypertrophy in H9c2UT cells.

Urotensin II (UII) is a neural hormone that induces cardiac hypertrophy and may be involved in the pathogenesis of cardiac remodeling and heart failure. Hypertrophy has been linked to histone deacetylase 5 (HDAC5) phosphorylation and nuclear factor κB (NF-κB) translocation, both of which are predominantly mediated by G protein-coupled receptor kinase 5 (GRK5). In the present study, we found that UII rapidly and strongly stimulated nuclear export of HDAC5 and nuclear import of NF-κB in H9c2 cells overexpressing the urotensin II receptor (H9c2UT). Hence, we hypothesized that GRK5 and its signaling pathway may play a role in UII-mediated cellular hypertrophy. H9c2UT cells were transduced with a GRK5 small hairpin RNA interference recombinant lentivirus, resulting in the down-regulation of GRK5. Under UII stimulation, reduced levels of GRK5 in H9c2UT cells led to suppression of UII-mediated HDAC5 phosphorylation and activation of the NF-κB signaling pathway. In contrast, UII-mediated activations of ERK1/2 and GSK3α/ß were not affected by down-regulation of GRK5. In a cellular hypertrophy assay, down-regulation of GRK5 significantly suppressed UII-mediated hypertrophy of H9c2UT cells. Furthermore, UII-mediated cellular hypertrophy was inhibited by amlexanox, a selective GRK5 inhibitor, in H9c2UT cells and neonatal cardiomyocytes. Our results suggest that GRK5 may be involved in a UII-mediated hypertrophic response via activation of NF-κB and HDAC5 at least in part by ERK1/2 and GSK3α/ß-independent pathways.