Synergistic enhanced rolling circle amplification based on mutS and radical polymerization for single-point mutation DNA detection.

The detection of nucleic acids in biofluids is essential for changing the paradigm of disease diagnosis. As there are very few nucleic acids present in human biofluids, a high sensitivity method is required to detect nucleic acids for disease diagnosis. The Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation is associated with non-small cell lung cancer. It is a point mutation and requires a highly selective detection technique. In this study, high sensitivity and selectivity were achieved for the detection of KRAS mutation using rolling circle amplification (RCA), atomic transfer radical polymerization (ATRP), mutS enzyme, and electrochemical sensors. Although RCA can isothermally amplify DNA, it has low selectivity for detecting single-base mismatch DNA, and its sensitivity is not suitable for circulating tumor DNA detection. The selectivity of RCA was improved by using mutS, which can bind specifically to point mutations. In addition, as a method of isothermal radical polymerization, ATRP was used to amplify the weak signal of RCA. Since RCA and ATRP reactions occur simultaneously, detection time was reduced, and the calculated detection limit was 3.09 aM. Computational and experimental analyses were conducted to verify each detection step and the combination of mutS, ATRP, and RCA. The experiment was performed using normal human serum samples for biological application, and the proposed detection method was confirmed to have excellent potential for diagnosing cancer patients.

A Review of Suicide Risk Assessment Tools and Their Measured Psychometric Properties in Korea.

While there has been a slew of review studies on suicide measurement tools until now, there were not any reviews focusing on suicide assessment tools available in Korea. This review aimed to examine the psychometric properties of tools developed in Korea or the translated versions from the original tools in their foreign language and to identify potential improvements and supplements for these tools. A literature search was done using the Korean academic information search service, Research Information Service System, to identify the suicide measures to be included in this review. Abstracts were screened to identify which measures were used to assess suicide-related factors. Based on the established inclusion and exclusion criteria, 18 tools remained and we assessed their psychometric properties. The current review indicated several major findings. First, many of the tools did not report predictive validity and even those with predictive validity were based on past suicide attempts. Second, some of the tools overlooked the interactive component for the cause of suicide. In addition, information to supplement the self-reported and clinician-administered reports by collecting reports from the subjects' families and acquaintances is needed. It is also important to develop a screening tool that examines other aspects of an individual's personal life, including unemployment, bereavement, divorce, and childhood trauma. Moreover, tools that have been studied in more diverse groups of the population are needed to increase external validity. Finally, the linguistic translation of the tools into Korean needs to consider other cultural, social, and psychological factors of the sample of interest.

Development and validation study of Game Overuse Screening Questionnaire.

The aim of this study was to develop a screening questionnaire to distinguish high-risk individuals associated with game overuse from casual internet users. Reliability, validity, and diagnostic ability were evaluated for the newly developed Game Overuse Screening Questionnaire (GOS-Q). Preliminary items were assessed by 50 addiction experts online and 30 questions were selected. A total of 158 subjects recruited from six community centers for internet addiction participated in this study. Finally, 150 people were used in the analysis after excluding eight non-respondents. GOS-Q, Young's internet addiction scale, and Korean scale for internet addiction were used to assess concurrent validity. Internal consistency and item-total correlations were favorable (α= 0.96, r= 0.47-0.82). Test-retest reliability was moderate in size (r= 0.74). GOS-Q showed superior concurrent validity, and the highest correlation with Y-Scale (r= 0.77). The construct validity was marginally supported by a six-factor model using exploratory factor analysis. The area under the Receiver Operating Characteristic curve was 0.945. The high-risk addiction group was effectively characterized by a cut-off point of 38.5, with a sensitivity of 0.87 and a specificity of 0.88. Overall, the current study supports the use of GOS-Q as a reliable screening tool in a variety of settings.

Spider silk with weaker bonding resulting in higher strength and toughness through progressive unfolding and load transfer.

The superior mechanical properties of silk is known to come partly from its hydrogen bonds, which is determined by its amino acid sequences. Hydrogen bonds are one of the main sources of strength of silk fiber, yet the toughest silk fibers have amino acids sequences that results in lesser number of hydrogen bonds than other silk fibers. In this work, we show how such silk fiber with lower number of hydrogen bonds may result in fiber with higher toughness by investigating the process of how hydrogen bond characteristics of silk are translated into its mechanical properties. From the tensile pulling tests via molecular dynamics simulations on silk fiber with varying number of hydrogen bonds, the mechanism of how weaker bonded silk results in higher strength and toughness by synergic effect with the characteristic progressive unfolding and load transfer of silk fiber is explained. The results provide new perspectives on how silk and other fibers should be designed to achieve higher toughness.

Effects of the Hydrophobicity of Key Residues on the Characteristics and Stability of Glucose Oxidase on a Graphene Surface.

Glucose oxidase (GOx) is one of the most widely investigated enzymes in the field of bioelectrochemistry. It is mainly used for the detection of glucose in solutions and enzyme-based biofuel cells. On the basis of the combination of GOx with graphene, novel nanodevices exceeding conventional limits can be developed. To develop a hybrid enzyme-graphene nanodevice with a good performance, it is important that GOx is deposited well on the graphene surface while maintaining its structure and not impeding the oxidation activity of the GOx. In this study, we propose a method to improve the stability of GOx and secure its immobility on the graphene sheet and its glucose-binding affinity by single-point mutation of GOx using molecular dynamics simulations. We confirm that the structural stability, immobility, and substrate binding affinity of GOx can be modified by changing the hydrophobicity of a key residue. We demonstrate that biosensors or biofuel cells can be redesigned and their properties can be improved by using molecular dynamics simulation.

Highly sensitive and selective detection of single-nucleotide polymorphisms using gold nanoparticle MutS enzymes and a micro cantilever resonator.

Mutant DNAs are important markers useful for the diagnosis of human disease. Single-nucleotide polymorphisms (SNPs) represent the most common types of DNA mutations. As there is only a one base pair change in a single nucleotide between the SNP and the wild-type DNA, it is difficult to distinguish the SNPs. In this report, a highly sensitive and selective detection and discrimination of SNPs is performed using MutS, gold nanoparticles (AuNP) and a resonator. A single mismatched base exists between the SNP mutation and the probe DNA on the resonator, and MutS binds to the DNA at the location of the mismatch. As MutS is attached to AuNP (MutS-AuNP), both MutS and AuNP are adsorbed onto the resonator. The detection is based on the resonance frequency shift of the resonator following the adsorption of MutS-AuNP on the resonator. Highly sensitive detection of DNA mutations was achieved using AuNPs that act as mass amplifiers, and the obtained limit of detected was 100 fM. Additionally, our proposed method detected mutations in the presence of as little as 0.1% wild-type, and discrimination of specific mutations was also achieved. The results obtained from our proposed method suggest its potential for diagnosing cancer patients.

Differences in anxiety sensitivity factors between anxiety and depressive disorders.

BACKGROUND: Anxiety sensitivity (AS) refers to the tendency to fear physical sensations associated with anxiety due to concerns about potential physical, social, or cognitive consequences. Many previous studies were limited by the use of the anxiety sensitivity index (ASI) or the ASI-revised (ASI-R), which are both measurements with unitary or unstable structures. No recent study that has utilized the ASI-3 examined the relations between AS dimensions and depression. Thus, we examined multiple relationships between AS and anxiety disorders and depression using the ASI-3. METHODS: The total sample consisted of 667 outpatients, diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders fourth text revision as assessed by a structured clinical interview. There were eight patient groups: multiple anxiety disorder, major depressive disorder (MDD), panic disorder (PD), social phobia (SP), obsessive-compulsive disorder (OCD), posttraumatic stress disorder (PTSD), generalized anxiety disorder (GAD), and anxiety disorder not otherwise specified (AD NOS). We conducted one-way analysis of variances and post hoc tests to compare the ASI-3 total and subscale scores across the groups. RESULTS: The physical concern score was higher in patients with PD than patients with MDD, SP, OCD, or GAD. The social concern score was higher in the SP group than those with MDD, PD, GAD, and AD NOS. Patients with GAD and PTSD showed higher cognitive concern scores than the patients with PD. CONCLUSION: Results partially replicated the relationship between PD and physical concern, between SP and social concern, and between GAD and cognitive concern examining the relationships between AS dimensions and anxiety disorders.

Mechanically inferior constituents in spider silk result in mechanically superior fibres by adaptation to harsh hydration conditions: a molecular dynamics study.

Spider silk exhibits mechanical properties such as high strength and toughness that are superior to those of any man-made fibre (Bourzac 2015 Nature519, S4-S6 (doi:10.1038/519S4a)). This high strength and toughness originates from a combination of the crystalline (exhibiting robust strength) and amorphous (exhibiting superb extensibility) regions present in the silk (Asakura et al 2015 Macromolecules48, 2345-2357 (doi:10.1021/acs.macromol.5b00160)). The crystalline regions comprise a mixture of poly-alanine and poly-glycine-alanine. Poly-alanine is expected to be stronger than poly-glycine-alanine, because alanine exhibits greater interactions between the strands than glycine (Tokareva et al 2014 Acta Biomater.10, 1612-1626 (doi:10.1016/j.actbio.2013.08.020)). We connect this characteristic sequence to the interactions observed upon the hydration of spider silk. Like most proteinaceous materials, spider silks become highly brittle upon dehydration, and thus water collection is crucial to maintaining its toughness (Gosline et al 1986 Endeavour10, 37-43 (doi:10.1016/0160-9327(86)90049-9)). We report on the molecular dynamic simulations of spider silk structures with different sequences for the crystalline region of the silk structures, of wild-type (WT), poly-alanine, and poly-glycine-alanine. We reveal that the characteristic sequence of spider silk results in the ß-sheets being maintained as the degree of hydration changes and that the high water collection capabilities of WT spider silk sequence prevent the silk from becoming brittle and weak in dry conditions. The characteristic crystalline sequence of spider dragline silk is therefore relevant not for maximizing the interactions between the strands but for adaption to changing hydration conditions to maintain an optimal performance even in harsh conditions.

Mechanical features of various silkworm crystalline considering hydration effect via molecular dynamics simulations.

Silk materials are receiving significant attention as base materials for various functional nanomaterials and nanodevices, due to its exceptionally high mechanical properties, biocompatibility, and degradable characteristics. Although crystalline silk regions are composed of various repetitive motifs with differing amino acid sequences, how the effect of humidity works differently on each of the motifs and their structural characteristics remains unclear. We report molecular dynamics (MD) simulations on various silkworm fibroins composed of major motifs (i.e. (GAGAGS)n, (GAGAGA)n, and (GAGAGY)n) at varying degrees of hydration, and reveal how each major motifs of silk fibroins change at each degrees of hydration using MD simulations and their structural properties in mechanical perspective via steered molecular dynamics simulations. Our results explain what effects humidity can have on nanoscale materials and devices consisting of crystalline silk materials.

Structural analysis of oligomeric and protofibrillar Aß amyloid pair structures considering F20L mutation effects using molecular dynamics simulations.

Aß amyloid proteins are involved in neuro-degenerative diseases such as Alzheimer's, Parkinson's, and so forth. Because of its structurally stable feature under physiological conditions, Aß amyloid protein disrupts the normal cell function. Because of these concerns, understanding the structural feature of Aß amyloid protein in detail is crucial. There have been some efforts on lowering the structural stabilities of Aß amyloid fibrils by decreasing the aromatic residues characteristic and hydrophobic effect. Yet, there is a lack of understanding of Aß amyloid pair structures considering those effects. In this study, we provide the structural characteristics of wildtype (WT) and phenylalanine residue mutation to leucine (F20L) Aß amyloid pair structures using molecular dynamics simulation in detail. We also considered the polymorphic feature of F20L and WT Aß pair amyloids based on the facing ß-strand directions between the amyloid pairs. As a result, we were able to observe the varying effects of mutation, polymorphism, and protofibril lengths on the structural stability of pair amyloids. Furthermore, we have also found that opposite structural stability exists on a certain polymorphic Aß pair amyloids depending on its oligomeric or protofibrillar state, which can be helpful for understanding the amyloid growth mechanism via repetitive fragmentation and elongation mechanism. Proteins 2017; 85:580-592. © 2016 Wiley Periodicals, Inc.

Steered molecular dynamics analysis of the role of cofilin in increasing the flexibility of actin filaments.

Cofilin is one of the most essential regulatory proteins and participates in the process of disassembling actin filaments. Cofilin induces conformational changes to actin filaments, and both the bending and torsional rigidity of the filament. In this study, we investigate the effects of cofilin on the mechanical properties of actin filaments using computational methods. Three models defined by their number of bound cofilins are constructed using coarse-grained MARTINI force field, and they are then extended with steered molecular dynamics simulation. After obtaining the stress-strain curves of the models, we calculate their Young's moduli and other mechanical properties that have not yet been determined for actin filaments. We analyze the cause of the different behaviors of the three models based on their atomistic geometrical differences. Finally, it is demonstrated that cofilin binding causes changes in the distances, angles, and stabilities of the residues in actin filaments.

Biophysical characterization of cofilin-induced extension-torsion coupling in actin filaments.

Cofilin makes the actin filament flexible and thermally unstable by disassembling the filament and inducing bending and torsional compliance. Actin monomers bound to cofilin are able to chemically and mechanically interact in response to external forces. In this study, we performed two molecular dynamics tensile tests for actin and cofilactin filaments under identical conditions. Surprisingly, cofilactin filaments were found to be twisted, generating shear stress caused by torsion. Additionally, analysis by plane stress assumption indicated that the extension-torsion coupling effect increases the amount of principal stress by 10%. Using elasticity and solid mechanics theories, our study elucidates the role of cofilin in the disassembly of actin filaments under tensile forces.

Morphology and mechanical properties of multi-stranded amyloid fibrils probed by atomistic and coarse-grained simulations.

Amyloid fibrils are responsible for pathogenesis of various diseases and exhibit the structural feature of an ordered, hierarchical structure such as multi-stranded helical structure. As the multi-strandedness of amyloid fibrils has recently been found to be highly correlated with their toxicity and infectivity, it is necessary to study how the hierarchical (i.e. multi-stranded) structure of amyloid fibril is formed. Moreover, although it has recently been reported that the nanomechanics of amyloid proteins plays a key role on the amyloid-induced pathogenesis, a critical role that the multi-stranded helical structure of the fibrils plays in their nanomechanical properties has not fully characterized. In this work, we characterize the morphology and mechanical properties of multi-stranded amyloid fibrils by using equilibrium molecular dynamics simulation and elastic network model. It is shown that the helical pitch of multi-stranded amyloid fibril is linearly proportional to the number of filaments comprising the amyloid fibril, and that multi-strandedness gives rise to improving the bending rigidity of the fibril. Moreover, we have also studied the morphology and mechanical properties of a single protofilament (filament) in order to understand the effect of cross-ß structure and mutation on the structures and mechanical properties of amyloid fibrils. Our study sheds light on the underlying design principles showing how the multi-stranded amyloid fibril is formed and how the structure of amyloid fibrils governs their nanomechanical properties.

Effects of lysine residues on structural characteristics and stability of tau proteins.

Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aß-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau protein structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures.

Influence of Aromatic Residues on the Material Characteristics of Aß Amyloid Protofibrils at the Atomic Scale.

Amyloid fibrils, which cause a number of degenerative diseases, are insoluble under physiological conditions and are supported by native contacts. Recently, the effects of the aromatic residues on the Aß amyloid protofibril were investigated in a ThT fluorescence study. However, the relationship between the material characteristics of the Aß protofibril and its aromatic residues has not yet been investigated on the atomic scale. Here, we successfully constructed wild-type (WT) and mutated types of Aß protofibrils by using molecular dynamics simulations. Through principle component analysis, we established the structural stability and vibrational characteristics of F20L Aß protofibrils and compared them with WT and other mutated models such as F19L and F19LF20L. In addition, structural stability was assessed by calculating the elastic modulus, which showed that the F20L model has higher values than the other models studied. From our results, it is shown that aromatic residues influence the structural and material characteristics of Aß protofibrils.

Cofilin reduces the mechanical properties of actin filaments: approach with coarse-grained methods.

An actin filament is an essential cytoskeleton protein in a cell. Various proteins bind to actin for cell functions such as migration, division, and shape control. ADF/cofilin is a protein that severs actin filaments and is related to their dynamics. Actin is known to have excellent mechanical properties. Binding cofilin reduces its mechanical properties, and is related to the severing process. In this research, we applied a coarse-grained molecular dynamics simulation (CGMD) method to obtain actin filaments and cofilin-bound actin (cofilactin) filaments. Using these two obtained models, we constructed an elastic network model-based structure and conducted a normal mode analysis. Based on the low-frequency normal modes of the filament structure, we applied the continuum beam theory to calculate the mechanical properties of the actin and cofilactin filaments. The CGMD method provided structurally accurate actin and cofilactin filaments in relation to the mechanical properties, which showed good agreement with the established experimental results.