Molecular Diagnostic System Using Engineered Fusion Protein-Conjugated Magnetic Nanoparticles.

To effectively control the spread of new infectious diseases, there is a need for highly sensitive diagnostic methods to detect viral nucleic acids rapidly. This study outlines a universal and simple detection strategy that uses magnetic nanoparticles (MNPs) and a novel MagR-MazE fusion protein for molecular diagnostics to facilitate sensitive detection. This study has engineered a novel MNP conjugate that can be generated easily, without using many chemical reagents. The technique is a nucleic acid detection method, using MagR-MazE fusion protein-conjugated MNPs, where the results can be visualized with the naked eye, regardless of the oligonucleotide sequences of the target in the lateral flow assay. This method could sensitively detect polymerase chain reaction (PCR) products of 16S ribosomal RNA (rRNA) and the 2019-nCoV-N-positive control gene in 5 min. It shows a low limit of detection (LoD) of 0.013 ng/µL for dsDNA. It is simpler and more rapid, sensitive, and versatile than other techniques, making it suitable for point-of-care testing. The proposed detection system and MNP conjugation strategy using a fusion protein can be widely applied to various fields requiring rapid on-site diagnosis.

Structural basis for the inhibition of PDK2 by novel ATP- and lipoyl-binding site targeting compounds.

Pyruvate dehydrogenase kinase (PDK) controls the activity of pyruvate decarboxylase complex (PDC) by phosphorylating key serine residues on the E1 subunit, which leads to a decreased oxidative phosphorylation in mitochondria. Inhibition of PDK activity by natural/synthetic compounds has been shown to reverse the Warburg effect, a characteristic metabolism in cancer cells. PDK-PDC axis also has been associated with diabetes and heart disease. Therefore, regulation of PDK activity has been considered as a promising strategy to treat related diseases. Here we present the X-ray crystal structure of PDK2 complexed with a recently identified PDK4 inhibitor, compound 8c, which has been predicted to bind at the lipoyl-binding site and interrupt intermolecular interactions with the E2-E3bp subunits of PDC. The co-crystal structure confirmed the specific binding location of compound 8c and revealed the remote conformational change in the ATP-binding pocket. In addition, two novel 4,5-diarylisoxazole derivatives, GM10030 and GM67520, were synthesized and used for structural studies, which target the ATP-binding site of PDK2. These compounds bind to PDK2 with a sub-100nM affinity as determined by isothermal titration calorimetry experiments. Notably, the crystal structure of the PDK2-GM10030 complex displays unprecedented asymmetric conformation of human PDK2 dimer, especially in the ATP-lids and C-terminal tails.

Human Behavior Analysis by Means of Multimodal Context Mining.

There is sufficient evidence proving the impact that negative lifestyle choices have on people's health and wellness. Changing unhealthy behaviours requires raising people's self-awareness and also providing healthcare experts with a thorough and continuous description of the user's conduct. Several monitoring techniques have been proposed in the past to track users' behaviour; however, these approaches are either subjective and prone to misreporting, such as questionnaires, or only focus on a specific component of context, such as activity counters. This work presents an innovative multimodal context mining framework to inspect and infer human behaviour in a more holistic fashion. The proposed approach extends beyond the state-of-the-art, since it not only explores a sole type of context, but also combines diverse levels of context in an integral manner. Namely, low-level contexts, including activities, emotions and locations, are identified from heterogeneous sensory data through machine learning techniques. Low-level contexts are combined using ontological mechanisms to derive a more abstract representation of the user's context, here referred to as high-level context. An initial implementation of the proposed framework supporting real-time context identification is also presented. The developed system is evaluated for various realistic scenarios making use of a novel multimodal context open dataset and data on-the-go, demonstrating prominent context-aware capabilities at both low and high levels.

Protective roles of highly conserved motif 1 in tardigrade cytosolic-abundant heat soluble protein in extreme environments.

Tardigrades are remarkable microscopic animals that survive harsh conditions such as desiccation and extreme temperatures. Tardigrade-specific intrinsically disordered proteins (TDPs) play an essential role in the survival of tardigrades in extreme environments. Cytosolic-abundant heat soluble (CAHS) protein, a key TDP, is known to increase desiccation tolerance and to protect the activity of several enzymes under dehydrated conditions. However, the function and properties of each CAHS domain have not yet been elucidated in detail. Here, we aimed to elucidate the protective role of highly conserved motif 1 of CAHS in extreme environmental conditions. To examine CAHS domains, three protein constructs, CAHS Full (1-229), CAHS ∆Core (1-120_184-229), and CAHS Core (121-183), were engineered. The highly conserved CAHS motif 1 (124-142) in the CAHS Core formed an amphiphilic α helix, reducing the aggregate formation and protecting lactate dehydrogenase activity during dehydration-rehydration and freeze-thaw treatments, indicating that CAHS motif 1 in the CAHS Core was essential for maintaining protein solubility and stability. Aggregation assays and confocal microscopy revealed that the intrinsically disordered N- and C-terminal domains were more prone to aggregation under our experimental conditions. By explicating the functions of each domain in CAHS, our study proposes the possibility of using engineered proteins or peptides derived from CAHS as a potential candidate for biological applications in extreme environmental stress responses.

Interaction of replication protein A with two acidic peptides from human Bloom syndrome protein.

Bloom syndrome protein (BLM) is one of five human RecQ helicases which maintain genomic stability. Interaction of BLM with replication protein A (RPA) stimulates the DNA unwinding ability of BLM. The interaction is expected to be crucial in the DNA damage response. Although this stimulation of BLM by RPA is of particular importance in cancer cells, the precise binding surfaces of both proteins are not well understood. In this study, we show by fluorescence polarisation anisotropy that both acidic surface peptides of BLM specifically bind to the RPA70N domain of RPA. Our NMR analysis and docking models show that the basic cleft region of RPA70N is the binding site for both peptides and that the acidic peptide/basic cleft interaction governs RPA-BLM binding.

Self-arrangement of nanoparticles toward crystalline metal oxides with high surface areas and tunable 3D mesopores.

We demonstrate a new design concept where the interaction between silica nanoparticles (about 1.5 nm in diameter) with titania nanoparticles (anatase, about 4 nm or 6 nm in diameter) guides a successful formation of mesoporous titania with crystalline walls and controllable porosity. At an appropriate solution pH (~1.5, depending on the deprotonation tendencies of two types of nanoparticles), the smaller silica nanoparticles, which attach to the surface of the larger titania nanoparticles and provide a portion of inactive surface and reactive surface of titania nanoparticles, dictate the direction and the degree of condensation of the titania nanoparticles, resulting in a porous 3D framework. Further crystallization by a hydrothermal treatment and subsequent removal of silica nanoparticles result in a mesoporous titania with highly crystalline walls and tunable mesopore sizes. A simple control of the Si/Ti ratio verified the versatility of the present method through the successful control of mean pore diameter in the range of 2-35 nm and specific surface area in the ranges of 180-250 m(2) g(-1). The present synthesis method is successfully extended to other metal oxides, their mixed oxides and analogues with different particle sizes, regarding as a general method for mesoporous metal (or mixed metal) oxides.

Endoscopic and Clinical Factors Affecting the Prognosis of Colorectal Endoscopic Submucosal Dissection-Related Perforation.

BACKGROUND/AIMS: Although colorectal endoscopic submucosal dissection (ESD)-related perforation is not uncommon, the factors affecting clinical outcomes after perforation have not been investigated. This study was designed to investigate the factors influencing the clinical course of ESD-related colon perforation. METHODS: Forty-three patients with colorectal ESD-related perforation were evaluated. The perforations were classified as endoscopic or radiologic perforations. The patients' medical records and endoscopic pictures were analyzed. RESULTS: The clinical outcomes were assessed by the duration of nil per os, intravenous antibiotics administration, and hospital stays, which were 2.7±1.5, 4.9±2.3, and 5.1±2.3 days, respectively. Multivariate analyses revealed that a larger tumor size, ESD failure, specific endoscopists, and abdominal pain were independently related to a poorer outcome. The time between perforation and clipping was 15.8±25.4 minutes in the endoscopic perforation group. The multivariate analysis of this group indicated that delayed clipping, specific endoscopists, and abdominal pain were independently associated with poorer outcomes. CONCLUSIONS: Tumor size, ESD failure, abdominal pain, and the endoscopist were factors that affected the clinical outcomes of patients with colorectal ESD-related perforation. The time between the perforation and clipping was an additional factor influencing the clinical course of endoscopic perforation. Decreasing this time period may improve outcomes.