Towards Generating Authentic Human-Removed Pictures in Crowded Places Using a Few-Second Video.

If we visit famous and iconic landmarks, we may want to take a photo of them. However, such sites are usually crowded, and taking photos with only landmarks without people could be challenging. This paper aims to automatically remove people in a picture and produce a natural image of the landmark alone. To this end, it presents Thanos, a system to generate authentic human-removed images in crowded places. It is designed to produce high-quality images with reasonable computation cost using short video clips of a few seconds. For this purpose, a multi-frame-based recovery region minimization method is proposed. The key idea is to aggregate information partially available from multiple image frames to minimize the area to be restored. The evaluation result presents that the proposed method outperforms alternatives; it shows lower Fréchet Inception Distance (FID) scores with comparable processing latency. It is also shown that the images by Thanos achieve a lower FID score than those of existing applications; Thanos's score is 242.8, while those by Retouch-photos and Samsung object eraser are 249.4 and 271.2, respectively.

Density Multiplication of Highly Periodic Sub-5 nm Supramolecular Dendrimer Cylinders on Block Copolymer Lamellar-Assisted High-Resolution Patterns.

Density multiplication in nanopatterning is one of the most efficient techniques for increasing the resolution of the inherent patterns. Thus far, most of the density multiplication techniques integrate bottom-up (or top-down) patterning onto guide patterns prepared by the top-down approach. Although the bottom-up approach exhibits several advantages of cost-effectiveness and high resolution, very few studies have reported bottom-up patterning within a bottom-up template. In this study, the density multiplication of supramolecular cylinders into a block copolymer (BCP)-based guide lamellar pattern is demonstrated by the directed self-assembly (DSA) of a dendrimer and BCPs for the first time. Supramolecular cylinders of sub-5 nm scale are confined into trenches based on 50 and 100 nm scales of a lamellar polystyrene (PS)-poly(methyl methacrylate) (PMMA) BCP, which led to 10×-level to 20×-level density multiplication. Moreover, the orientation of the dendrimer is dependent on the dendrimer film thickness, and the corresponding mechanism is revealed. Notably, the strong guiding effect from the high-resolution guide patterns improved the ordering behavior in the highly curved pattern. Graphoepitaxy via the confinement of an ultrahigh-resolution dendrimer into the guide pattern based on BCP demonstrates promise as a density multiplication method for generating highly ordered nanostructures and complex structures.

Multi-Camera-Based Sorting System for Surface Defects of Apples.

In this paper, we addressed the challenges in sorting high-yield apple cultivars that traditionally relied on manual labor or system-based defect detection. Existing single-camera methods failed to uniformly capture the entire surface of apples, potentially leading to misclassification due to defects in unscanned areas. Various methods were proposed where apples were rotated using rollers on a conveyor. However, since the rotation was highly random, it was difficult to scan the apples uniformly for accurate classification. To overcome these limitations, we proposed a multi-camera-based apple sorting system with a rotation mechanism that ensured uniform and accurate surface imaging. The proposed system applied a rotation mechanism to individual apples while simultaneously utilizing three cameras to capture the entire surface of the apples. This method offered the advantage of quickly and uniformly acquiring the entire surface compared to single-camera and random rotation conveyor setups. The images captured by the system were analyzed using a CNN classifier deployed on embedded hardware. To maintain excellent CNN classifier performance while reducing its size and inference time, we employed knowledge distillation techniques. The CNN classifier demonstrated an inference speed of 0.069 s and an accuracy of 93.83% based on 300 apple samples. The integrated system, which included the proposed rotation mechanism and multi-camera setup, took a total of 2.84 s to sort one apple. Our proposed system provided an efficient and precise solution for detecting defects on the entire surface of apples, improving the sorting process with high reliability.

Prognostic Significance of Nonobstructive Left Main Coronary Artery Disease in Patients With and Without Diabetes: Long-Term Outcomes From the CONFIRM Registry.

BACKGROUND: Prognostic significance of non-obstructive left main (LM) disease was recently reported. However, the influence of diabetes mellitus (DM) on event rates in patients with and without non-obstructive LM disease is not well-known. METHODS: We evaluated 27,252 patients undergoing coronary computed tomographic angiography from the COroNary CT Angiography Evaluation For Clinical Outcomes: An InteRnational Multicenter (CONFIRM) Registry. Cumulative long-term incidence of all-cause mortality (ACM) was assessed between DM and non-DM patients by normal or non-obstructive LM disease (1-49% stenosis). RESULTS: The mean age of the study population was 57.6±12.6 years. Of the 27,252 patients, 4,434 (16%) patients had DM. A total of 899 (3%) deaths occurred during the follow-up of 3.6±1.9. years. Compared to patients with normal LM, those with non-obstructive LM had more pronounced overall coronary atherosclerosis and more cardiovascular risk factors. After clinical risk factors, segment involvement score, and stenosis severity adjustment, compared to patients without DM and normal LM, patients with DM were associated with increased ACM regardless of normal (HR 1.48, 95% CI 1.22-1.78, p<0.001) or non-obstructive LM (HR 1.46, 95% CI 1.04-2.04, p=0.029), while nonobstructive LM disease was not associated with increased ACM in patients without DM (HR 0.85, 95% CI 0.67-1.07, p=0.165) and there was no significant interaction between DM and LM status (HR 1.03, 95% CI 0.69-1.54, p=0.879). CONCLUSION: From the CONFIRM registry, we demonstrated that DM was associated with increased ACM. However, the presence of non-obstructive LM was not an independent risk marker of ACM, and there was no significant interaction between DM and non-obstructive LM disease for ACM.

Computational design of a thermolabile uracil-DNA glycosylase of Escherichia coli.

Polymerase chain reaction (PCR) is a powerful tool to diagnose infectious diseases. Uracil DNA glycosylase (UDG) is broadly used to remove carryover contamination in PCR. However, UDG can contribute to false negative results when not inactivated completely, leading to DNA degradation during the amplification step. In this study, we designed novel thermolabile UDG derivatives by supercomputing molecular dynamic simulations and residual network analysis. Based on enzyme activity analysis, thermolability, thermal stability, and biochemical experiments of Escherichia coli-derived UDG and 22 derivatives, we uncovered that the UDG D43A mutant eliminated the false negative problem, demonstrated high efficiency, and offered great benefit for use in PCR diagnosis. We further obtained structural and thermodynamic insights into the role of the D43A mutation, including perturbed protein structure near D43; weakened pairwise interactions of D43 with K42, N46, and R80; and decreased melting temperature and native fraction of the UDG D43A mutant compared with wild-type UDG.

Comparison of Core Body Temperatures in Patients Administered Remimazolam or Propofol during Robotic-Assisted and Laparoscopic Radical Prostatectomy.

Background and Objectives: Different types of anesthetics affect thermoregulatory mechanisms, such as the redistribution of body temperature, loss of skin heat, or inhibition of thermoregulatory vasoconstriction. Therefore, we compared remimazolam with propofol in terms of core body temperature in patients undergoing robotic-assisted and laparoscopic radical prostatectomy. Materials and methods: Ninety patients were randomly assigned to either the propofol−remifentanil (PR) group or the remimazolam−remifentanil (RR) group. The PR group (n = 45) received effect-site concentrations of 6.0 µg/mL of propofol and 4 ng/mL of remifentanil, followed by 0.9 mg/kg of 1% rocuronium and maintenance with effect-site concentrations of 2−4 µg/mL of propofol and 3 ng/mL of remifentanil. The RR group (n = 45) received remimazolam 6 mg/kg/h by continuous intravenous infusion and the effect-site concentration of 4 ng/mL of remifentanil, followed by 0.9 mg/kg of 1% rocuronium, remimazolam 1−3 mg/kg/h, and remifentanil 3 ng/mL. The primary outcome was core body temperature, and secondary outcomes included vasoconstriction threshold (°C) and time to onset of vasoconstriction (min). Results: The core body temperature in the RR group was significantly higher at 60, 80, 100, 120, 140, 160, and 180 min after induction than in the PR group (p < 0.01). The vasoconstriction threshold was significantly higher in the RR group (35.2 ± 0.4) than in the PR group (34.8 ± 0.3) (p < 0.01). The time to onset of vasoconstriction was significantly less in the RR group (150.5 ± 10.2) than in the PR group (158.5 ± 8.4) (p < 0.01). However, the incidence of intraoperative hypothermia was not significant between two groups. Conclusions: Remimazolam appears to reduce vasoconstriction threshold less than and had a faster onset of vasoconstriction, resulting in superior thermoregulatory control.

Structural and molecular insight into the pH-induced low-permeability of the voltage-gated potassium channel Kv1.2 through dewetting of the water cavity.

Understanding the gating mechanism of ion channel proteins is key to understanding the regulation of cell signaling through these channels. Channel opening and closing are regulated by diverse environmental factors that include temperature, electrical voltage across the channel, and proton concentration. Low permeability in voltage-gated potassium ion channels (Kv) is intimately correlated with the prolonged action potential duration observed in many acidosis diseases. The Kv channels consist of voltage-sensing domains (S1-S4 helices) and central pore domains (S5-S6 helices) that include a selectivity filter and water-filled cavity. The voltage-sensing domain is responsible for the voltage-gating of Kv channels. While the low permeability of Kv channels to potassium ion is highly correlated with the cellular proton concentration, it is unclear how an intracellular acidic condition drives their closure, which may indicate an additional pH-dependent gating mechanism of the Kv family. Here, we show that two residues E327 and H418 in the proximity of the water cavity of Kv1.2 play crucial roles as a pH switch. In addition, we present a structural and molecular concept of the pH-dependent gating of Kv1.2 in atomic detail, showing that the protonation of E327 and H418 disrupts the electrostatic balance around the S6 helices, which leads to a straightening transition in the shape of their axes and causes dewetting of the water-filled cavity and closure of the channel. Our work offers a conceptual advancement to the regulation of the pH-dependent gating of various voltage-gated ion channels and their related biological functions.

Productivity limits and potentials of the principles of conservation agriculture.

One of the primary challenges of our time is to feed a growing and more demanding world population with reduced external inputs and minimal environmental impacts, all under more variable and extreme climate conditions in the future. Conservation agriculture represents a set of three crop management principles that has received strong international support to help address this challenge, with recent conservation agriculture efforts focusing on smallholder farming systems in sub-Saharan Africa and South Asia. However, conservation agriculture is highly debated, with respect to both its effects on crop yields and its applicability in different farming contexts. Here we conduct a global meta-analysis using 5,463 paired yield observations from 610 studies to compare no-till, the original and central concept of conservation agriculture, with conventional tillage practices across 48 crops and 63 countries. Overall, our results show that no-till reduces yields, yet this response is variable and under certain conditions no-till can produce equivalent or greater yields than conventional tillage. Importantly, when no-till is combined with the other two conservation agriculture principles of residue retention and crop rotation, its negative impacts are minimized. Moreover, no-till in combination with the other two principles significantly increases rainfed crop productivity in dry climates, suggesting that it may become an important climate-change adaptation strategy for ever-drier regions of the world. However, any expansion of conservation agriculture should be done with caution in these areas, as implementation of the other two principles is often challenging in resource-poor and vulnerable smallholder farming systems, thereby increasing the likelihood of yield losses rather than gains. Although farming systems are multifunctional, and environmental and socio-economic factors need to be considered, our analysis indicates that the potential contribution of no-till to the sustainable intensification of agriculture is more limited than often assumed.

Effects of Female Reproductive Hormone Levels on Inadvertent Intraoperative Hypothermia during Laparoscopic Gynecologic Surgery: A Retrospective Study.

Background and Objectives: Female reproductive hormones may affect core body temperature. This study aimed to investigate the effects of female reproductive hormones on inadvertent intraoperative hypothermia in patients who underwent laparoscopic gynecologic surgery under general anesthesia. Materials and Methods: This retrospective study included 660 menstruating and menopausal female patients aged 19-65 years. The patients were divided into two groups according to the occurrence of inadvertent intraoperative hypothermia: non-hypothermia group (N = 472) and hypothermia group (N = 188). After propensity score matching, 312 patients (N = 156 in each group) were analyzed to investigate the association between intraoperative hypothermia and female reproductive hormones. As potential predictors of inadvertent hypothermia, the levels of female reproductive hormones were analyzed using binary logistic regression. Results: The association of estradiol (r = -0.218, p = 0.000) and progesterone (r = -0.235, p = 0.000) levels with inadvertent intraoperative hypothermia was significant but weakly negative before matching; however, it was significant and moderately negative after matching (r = -0.326, p = 0.000 and r = -0.485, p = 0.000, respectively). In a binary logistic analysis, the odds ratio for estradiol was 0.995 (p = 0.014, 0.993 < 95% confidence interval [CI] < 0.998) before matching and 0.993 (p = 0.000, 0.862 < 95% CI < 0.930) after matching, and that for progesterone was 0.895 (p = 0.000, 0.862 < 95% CI < 0.930) before matching and 0.833 (p = 0.014, 0.990 < 95% CI < 0.996) after matching. Conclusions: Estradiol and progesterone levels were associated with inadvertent intraoperative hypothermia. However, the odds ratio for female reproductive hormone levels was close to 1. Therefore, female reproductive hormones may not be a risk factor for hypothermia during gynecologic surgery under general anesthesia. However, a small sample size in this study limits the generalizability of the results.

Artificial Intelligence in Intracoronary Imaging.

PURPOSE OF REVIEW: This paper investigates present uses and future potential of artificial intelligence (AI) applied to intracoronary imaging technologies. RECENT FINDINGS: Advances in data analytics and digitized medical imaging have enabled clinical application of AI to improve patient outcomes and reduce costs through better diagnosis and enhanced workflow. Applications of AI to IVUS and IVOCT have produced improvements in image segmentation, plaque analysis, and stent evaluation. Machine learning algorithms are able to predict future coronary events through the use of imaging results, clinical evaluations, laboratory tests, and demographics. The application of AI to intracoronary imaging holds significant promise for improved understanding and treatment of coronary heart disease. Even in these early stages, AI has demonstrated the ability to improve the prediction of cardiac events. Large curated data sets and databases are needed to speed the development of AI and enable testing and comparison among algorithms.

Development of a live attenuated trivalent porcine rotavirus A vaccine against disease caused by recent strains most prevalent in South Korea.

Porcine rotaviruses cause severe economic losses in the Korean swine industry due to G- and P-genotype mismatches between the predominant field and vaccine strains. Here, we developed a live attenuated trivalent porcine group A rotavirus vaccine using 80 cell culture passages of the representative Korean predominant strains G8P[7] 174-1, G9P[23] PRG942, and G5P[7] K71. Vaccination with the trivalent vaccine or its individual components induced no diarrhea during the first 2 weeks post-vaccination, i.e., the vaccines were attenuated. Challenge of trivalent-vaccinated or component-vaccinated piglets with homologous virulent strain(s) did not induce diarrhea for 2 weeks post-challenge. Immunization with the trivalent vaccine or its individual components also alleviated the histopathological lesions in the small intestines caused by challenge with the corresponding original virulent strain(s). Fecal secretory IgAs specific for each of vaccine strains were detected starting at 14 days post-vaccination (dpv), and IgA levels gradually increased up to 28 dpv. Oral immunization with the trivalent vaccine or its individual components induced high levels of serum virus-neutralizing antibody by 7 dpv. No diarrhea was observed in any experimental piglets during five consecutive passages of each vaccine strain. Our data indicated that the live attenuated trivalent vaccine was safe and effective at protecting piglets from diarrhea induced by challenge exposure of homologous virulent strains. This trivalent vaccine will potentially contribute toward controlling porcine rotavirus disease in South Korea and other countries where rotavirus infections with similar G and P genotypes are problematic.

Automated classification of dense calcium tissues in gray-scale intravascular ultrasound images using a deep belief network.

BACKGROUND: IVUS is widely used to quantitatively assess coronary artery disease. The purpose of this study was to automatically characterize dense calcium (DC) tissue in the gray scale intravascular ultrasound (IVUS) images using the image textural features. METHODS: A total of 316 Gy-scale IVUS and corresponding virtual histology images from 26 patients with acute coronary syndrome who underwent IVUS along with X-ray angiography between October 2009 to September 2014 were retrospectively acquired and analyzed. One expert performed all procedures and assessed their IVUS scans. After image acquisition, the DC candidate and corresponding acoustic shadow regions were automatically determined. Then, nine image-base feature groups were extracted from the DC candidates. In order to reduce the dimensionalities, principal component analysis (PCA) was performed, and selected feature sets were utilized as an input for a deep belief network. Classification results were validated using 10-fold cross validation. RESULTS: The dimensionality of the feature map was efficiently reduced by 50% (from 66 to 33) without any performance decrease using PCA method. Sensitivity, specificity, and accuracy of the proposed method were 92.8 ± 0.1%, 85.1 ± 0.1%, and 88.4 ± 0.1%, respectively (p < 0.05). We found that the window size could largely influence the characterization results, and selected the 5 × 5 size as the best condition. We also validated the performance superiority of the proposed method with traditional classification methods. CONCLUSIONS: These experimental results suggest that the proposed method has significant clinical applicability for IVUS-based cardiovascular diagnosis.

Use of computed tomography and minimum intensity projection in the detection of lobar pneumonia mimicking lung lobe torsion in a dog.

A 10-year-old female spayed Dachshund was referred with progressive coughing for 1 month. The dog was tentatively diagnosed with right middle lung torsion based on pleural effusion, vesicular emphysema, abruptly ending bronchus in consolidated right middle lung, and no contrast enhancement of the affected lobe on radiography and computed tomography (CT). There was no evidence of torsion upon thoracotomy, and histological examination confirmed lobar pneumonia. The CT images were reevaluated using minimum intensity projection and revealed normal bronchial courses. The minimum intensity projection technique can be to assist in evaluation of the bronchial tree for dogs with suspected lung lobe torsion and other pulmonary diseases.

Randomized trial of rivaroxaban versus warfarin in the evaluation of progression of coronary atherosclerosis.

Warfarin, a vitamin K antagonist, is associated with systemic vascular calcification. We evaluated whether rivaroxaban (a direct oral factor Xa inhibitor with no interaction with vitamin K) will slow the progression in coronary plaque volumes compared with warfarin in patients with nonvalvular atrial fibrillation using coronary computed tomography angiography.

Study on Improved Flight Coefficient Estimation and Trajectory Analysis of a Flying Disc through Onboard Magnetometer Measurements.

This paper proposes a novel and accurate method for estimating the flight coefficient of a flying disc typically operating at a high rotation rate. In particular, the proposed method introduces a new algorithm that takes advantage of magnetic data measured by a miniaturized sensor module onboard a conventional disc. Since the geomagnetic field measured by the magnetic sensor mounted on the rotating body yields a general sinusoidal waveform, a frequency domain analysis is employed in computing the rotational rate. Furthermore, on the basis of the estimated rate during a whole flight period, a yaw damping derivative coefficient is derived, which enables an accurate prediction of the disc's flight trajectory. For performance verification, both a reference rotation table test and a real flight test are performed, for which a miniaturized embedded sensor module is designed and manufactured for an onboard flight test. A reference rotation test validates the performance of the proposed method. Subsequently, a flight test, in which a simulator-based trajectory is compared with the true reference trajectory, verifies that the proposed method better predicts the flight trajectory by incorporating the estimated coefficient.

Porcine Sapelovirus Uses α2,3-Linked Sialic Acid on GD1a Ganglioside as a Receptor.

UNLABELLED: The receptor(s) for porcine sapelovirus (PSV), which causes diarrhea, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs, remains largely unknown. Given the precedent for other picornaviruses which use terminal sialic acids (SAs) as receptors, we examined the role of SAs in PSV binding and infection. Using a variety of approaches, including treating cells with a carbohydrate-destroying chemical (NaIO4), mono- or oligosaccharides (N-acetylneuraminic acid, galactose, and 6'-sialyllactose), linkage-specific sialidases (neuraminidase and sialidase S), lectins (Maakia amurensislectin andSambucus nigralectin), proteases (trypsin and chymotrypsin), and glucosylceramide synthase inhibitors (dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and phospholipase C), we demonstrated that PSV could recognize α2,3-linked SA on glycolipids as a receptor. On the other hand, PSVs had no binding affinity for synthetic histo-blood group antigens (HBGAs), suggesting that PSVs could not use HBGAs as receptors. Depletion of cell surface glycolipids followed by reconstitution studies indicated that GD1a ganglioside, but not other gangliosides, could restore PSV binding and infection, further confirming α2,3-linked SA on GD1a as a PSV receptor. Our results could provide significant information on the understanding of the life cycle of sapelovirus and other picornaviruses. For the broader community in the area of pathogens and pathogenesis, these findings and insights could contribute to the development of affordable, useful, and efficient drugs for anti-sapelovirus therapy. IMPORTANCE: The porcine sapelovirus (PSV) is known to cause enteritis, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs. However, the receptor(s) that the PSV utilizes to enter host cells remains largely unknown. Using a variety of approaches, we showed that α2,3-linked terminal sialic acid (SA) on the cell surface GD1a ganglioside could be used for PSV binding and infection as a receptor. On the other hand, histo-blood group antigens also present in the cell surface carbohydrates could not be utilized as PSV receptors for binding and infection. These findings should contribute to the understanding of the sapelovirus life cycle and to the development of affordable, useful and efficient drugs for anti-sapelovirus therapy.

The relationship between physical capacity and fear avoidance beliefs in patients with chronic low back pain.

[Purpose] The aim of this study was to investigate the relationship between physical capacity and fear avoidance beliefs in patients with chronic low back pain. [Subjects and Methods] This cross sectional study included 131 male university students with chronic low back pain. All the patients completed a fear avoidance beliefs questionnaire. Each participant performed a physical capacity test, which included hand grip force, leg strength, abdominal muscle endurance, flexibility, and cardiopulmonary endurance testing. [Results] Negative correlation was observed between physical capacity (leg strength, abdominal muscle endurance) and fear avoidance beliefs regarding work. Physical capacity (hand grip force, leg strength, cardiopulmonary endurance) showed a negative correlation with fear avoidance beliefs about physical activity. Abdominal muscle endurance and cardiopulmonary endurance were predictors of fear avoidance beliefs. [Conclusion] Physical capacity showed a negative correlation with fear avoidance beliefs in patients with chronic low back pain. The results of this study suggest that physical capacity is an important factor for predicting fear avoidance beliefs in patients with chronic low back pain.

Comparison of the effects of core stabilization and chest mobilization exercises on lung function and chest wall expansion in stroke patients.

[Purpose] The main purpose of this study was to compare the effects of core stabilization and chest mobilization exercises on pulmonary function and chest expansion in chronic stroke patients. [Subjects and Methods] Thirty stroke patients were randomly divided into two groups: a core stabilization exercise group (n=15) and a chest mobilization exercise group (n=15). Each exercise was performed 3 times per week for 30 minutes for 4 weeks, and pulmonary function and chest expansion when breathing were measured for both groups. [Results] There were significant increases in both forced vital capacity and forced expiratory volume in 1 second before and after intervention. Core stabilization exercise resulted in a significant increase in peak expiratory flow, and significant increases in upper and lower chest expansion were detected with chest mobilization exercise. However, no significant difference was revealed between the two groups. [Conclusion] This study suggested that both exercises were effective in some aspects of pulmonary function while core stabilization can help increase peak expiratory flow and chest mobilization can assist with chest expansion.

Pathogenesis of Korean SapelovirusA in piglets and chicks.

Sapelovirus A (SV-A), formerly known as porcine sapelovirus as a member of a new genus Sapelovirus, is known to cause enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. We have recently identified α2,3-linked sialic acid on GD1a ganglioside as a functional SV-A receptor rich in the cells of pigs and chickens. However, the role of GD1a in viral pathogenesis remains elusive. Here, we demonstrated that a Korean SV-A strain could induce diarrhoea and intestinal pathology in piglets but not in chicks. Moreover, this Korean SV-A strain had mild extra-intestinal tropisms appearing as mild, non-suppurative myelitis, encephalitis and pneumonia in piglets, but not in chicks. By real-time reverse transcription (RT) PCR, higher viral RNA levels were detected in faecal samples than in sera or extra-intestinal organs from virus-inoculated piglets. Immunohistochemistry confirmed that high viral antigens were detected in the epithelial cells of intestines from virus-inoculated piglets but not from chicks. This Korean SV-A strain could bind the cultured cell lines originated from various species, but replication occurred only in cells of porcine origin. These data indicated that this Korean SV-A strain could replicate and induce pathology in piglets but not in chicks, suggesting that additional porcine-specific factors are required for virus entry and replication. In addition, this Korean SV-A strain is enteropathogenic, but could spread to the bloodstream from the gut and disseminate to extra-intestinal organs and tissues. These results will contribute to our understanding of SV-A pathogenesis so that efficient anti-sapelovirus drugs and vaccines could be developed in the future.

Molecular Basis of the Membrane Interaction of the ß2e Subunit of Voltage-Gated Ca(2+) Channels.

The auxiliary ß subunit plays an important role in the regulation of voltage-gated calcium (CaV) channels. Recently, it was revealed that ß2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of ß-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the ß2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the ß2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated ß2e subunit on inactivation kinetics and regulation of CaV channels by PIP2. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of ß2e (K2A/W5A) increased the PIP2 sensitivity of CaV2.2 and CaV1.3 channels by ∼3-fold compared with wild-type ß2e subunit. Together, our results suggest that membrane targeting of the ß2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-ß2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.