Optimized High-Yield Purification of Obesity-Associated Melanocortin 4 Receptor.

BACKGROUND: Obesity has emerged as a global public health challenge associated with increased risk of hyperlipidemia and hypertension. It contributes to high sympathetic activity and increased catecholamine levels. The hypothalamic melanocortin system is known to regulate the energy homeostasis. The role of melanocortin 4 receptor (MC4R) has been demonstrated pharmacologically and in animal studies, which showed that severe obesity in MC4R knockout mice was caused by increased food intake and decreased energy consumption. Over 70 multiple different mis- -sense and nonsense mutations in hMC4R have been found at a high frequency of 2-8% in severe early onset or hereditary obesity. The single amino acid variation (D90N) located in the second transmembrane domain (TM2) of MC4R results in accelerated growth and childhood onset obesity. Interestingly, the functional characterization of D90N hMC4R mutant TM2 (m-hMC4R-TM2) revealed normal cell surface expression and binding with agonist similar to the hMC4R wild-type TM2 (wt-hMC4R-TM2) but loss of signal transduction mediated via Gs/adenylyl cyclase activation. It is essential to delineate the three-dimensional structure of MC4Rs in order to elucidate their functional aspects. OBJECTIVE: In this study, we demonstrate the optimized expression and isolation of wt/m-hMC4R-TM2 proteins under different chemical cleavage reaction times and purification procedures via SDS precipitation. The solid-state NMR spectroscopy was carried out to study the structure of wt/m-hMC4R- TM2 protein in the anisotropic phospholipid bicelles. METHODS: The KSI-wt/m-hMC4R-TM2 fusion proteins developed in cell culture with LB medium. In order to isolate the expressed fusion protein from the cell, ultrasonication, Ni-NTA affinity chromatography, dialysis, and lyophilization techniques were used. Then, to obtain a protein with higher purity and higher yield, the CNBr chemical cleavage time was subdivided into 30 minutes, 1 h, 2 h, 3 h, and 4 h. Purification process was performed using FPLC, and 100 mM KCl and dialysis were used to remove the SDS. CD spectrometer, MALDI-TOF, solution-state NMR, and solid-state NMR were used to confirmed purity and structure of the wt/m-hMC4R-TM2. RESULTS: The precipitation method was used to remove the SDS bound to proteins as KCl-SDS. We optimized the 2 h cleavage reaction times for both wt-hMC4R-TM2 and m-hMC4R-TM2 depending on the purity based on mass spectra and 1H-15N HSQC spectra and the yield after final purification. The 1D 1H-15N CP (Cross polarization) solid-state NMR spectra suggest that the wt/m-hMC4R- TM2 undergo rotational diffusion around a perpendicular axis along the bilayer normal. CONCLUSION: We expressed wt/m-hMC4R-TM2 in E.coli and optimized the isolation and purification process, especially CNBr chemical cleavage time. The efficiency of KCl-SDS precipitation was confirmed via MALDI-TOF MS and the pure proteins obtained using this method were characterized by CD spectroscopy and solution-state NMR. The results of 1H-15N HSQC spectra in solution- state NMR also show the probability for structural studies. The 1D 1H-15N CP solid-state NMR spectra indicate that most of the residues in both the wt/m-hMC4R-TM2 peptides are integrated into the membrane.

NMR structural studies and mechanism of action of Lactophoricin analogs as antimicrobial peptides.

Antimicrobial peptides (AMPs) are effective alternatives to conventional antibiotics. They protect the host from the constant invasion of a broad range of infectious microorganisms. AMPs have been at the forefront of the response to multidrug-resistant microbial strains and appear to be ideal drug candidates. Lactophoricin (LPcin), naturally produced from bovine milk, is a typical cationic antimicrobial peptide. Three analog peptides, including LPcin-YK5, LPcin-YK8, and LPcin-YK11, with enhanced antimicrobial activity compared to the wild-type LPcin, were designed and expressed in our laboratory. We investigated the structure and antimicrobial mechanisms of action of the three novel antimicrobial peptide analogs derived from LPcin using solution NMR and solid-state NMR spectroscopy in membrane environments. Our results revealed that the three LPcin analogs exhibited helical structures with different tilt angles on the phospholipid membrane surface. We proposed three-dimensional conformations and antibacterial mechanisms of action of the three peptide analogs in the phospholipid bilayers using two-dimensional solid-state separated local field NMR experiments.

Magnetic Resonance Imaging Observations of the Conus Medullaris in a Korean Population.

STUDY DESIGN: Retrospective study (level of evidence: level 3). PURPOSE: To study the anatomy of the conus medullaris in Koreans. OVERVIEW OF LITERATURE: The anatomical position of the conus medullaris is well-documented in anatomy textbooks; however, the shape of the conus in the canal rarely described. Furthermore, to our knowledge, no study in Korea has not yet assessed the shape of the conus as well as its position in the canal via cadavaric dissection and/or magnetic resonance imaging (MRI). METHODS: MRI findings of 189 Korean patients aged 2-94 years (93 men and 94 women) were assessed. No subjects from other ethnicities were included. The method proposed by Arai and colleagues was used to assess the termination point and shape of the conus in the canal. The position of the intervertebral disc trisection of the vertebral body closest to the tip of the conus was recorded at the canal level. RESULTS: The tip of the conus medullaris was positioned from the upper T12 body to the L2-L3 disc, mostly in L1 bodies (52.4%), followed by the L2 bodies (22.5%), the L1-L2 disc, and the L2-L3 disc (1.1%). The shape of the conus was classified as type A in 74 (39.6%), type B in 58 (31%), and type C in 55 patients (29.4%). The conus did not terminate at the L3 body in any patient. In the first decade ones (five children) conus positioned rather lowly from L1 bodies to L2-L3 disc, and no type A conus shape, and mostly type B (80%). CONCLUSIONS: The conus medullaris was positioned mostly in the lower one-third of L1 and it in the first decades terminated lowly. No type A in the first decade one, and type B was mostly frequently formed which was followed by type C.

Statistical analyses on inverted perovskite solar cells employing non-fullerene-based small molecule as a cathode interfacial layer.

In this data article, we present the influences of the solvent, concentration, and spin rates of 3,9-bis(2-methylene-(3-(1,1-dicyanomethylene)-indanone))-5,5,11,11-tetrakis(4-hexylphenyl)-dithieno[2,3-d:2',3'-d']-s-indaceno[1,2-b:5,6-b']dithiophene) (ITIC) material on the performances of perovskite solar cells (PSCs). The device parameters such as open-circuit voltage (Voc), short circuit current (Jsc), fill factor (FF), and power conversion efficiency (PCE) were measured with Keithley 2400 source meter unit under 100 mW/cm2 (AM 1.5 G). The data in this article describe the optimization of ITIC-based PSCs and are directly related to our research article "Non-fullerene-based small molecules as an efficient n-type electron transporting layers in inverted organic-inorganic halide perovskite solar cells" (Noh et al., Submitted for publication) [1].

Antimicrobial Activity of Antimicrobial Peptide LPcin-YK3 Derived from Bovine Lactophoricin.

We previously reported on lactophoricin (LPcin), a cationic α-helical antimicrobial peptide derived from bovine milk, which has antimicrobial effects on Candida albicans as well as Gram-positive and Gram-negative bacteria. In this study, we designed the LPcin-YK3 peptide, a shorter analog of LPcin, and investigated its antimicrobial activity. This peptide, consisting of 15 amino acids with + 3 net charges, was an effective antimicrobial agent against the on the Gram-positive strain, Staphylococcus aureus (MIC: 0.62 µg/ml). In addition, the hemolytic activity assay revealed that the peptide was not toxic to mouse and human erythrocytes up to 40 µg/ml. We also used circular dichroism spectroscopy to confirm that peptide in the presence of lipid has α-helical structures and later provide an overview of the relationship between each structure and antimicrobial activity. This peptide is a member of a new class of antimicrobial agents that could potentially overcome the problem of bacterial resistance caused by overuse of conventional antibiotics. Therefore, it could be used as a therapeutic or natural additive, particularly in the cosmetics industry.

Design and Engineering of Antimicrobial Peptides Based on LPcin-YK3, an Antimicrobial Peptide Derivative from Bovine Milk.

We have previously derived a novel antimicrobial peptide, LPcin-YK3(YK3), based on lactophoricin and have successfully studied and reported on the relationship between its structure and function. In this study, antimicrobial peptides with improved antimicrobial activity, less cytotoxicity, and shorter length were devised and characterized on the basis of YK3, and named YK5, YK8, and YK11. The peptide design was based on a variety of knowledge, and a total of nine analog peptides consisted of one to three amino acid substitutions and C-terminal deletions. In detail, tryptophan substitution improved the membrane perturbation, lysine substitution increased the net charge, and excessive amphipathicity decreased. The analog peptides were examined for structural characteristics through spectroscopic analytical techniques, and antimicrobial susceptibility tests were used to confirm their activity and safety. We expect that these studies will provide a platform for systematic engineering of new antibiotic peptides and generate libraries of various antibiotic peptides.

Photo-cross-linked perylene diimide derivative materials as efficient electron transporting layers in inverted polymer solar cells.

We present an efficient and stable interfacial material based on a water-soluble perylene diimide derivative functionalized with ionic and methacrylate groups (abbreviated as PDIM), which can be stabilized by the photo-polymerization of diacrylate groups at both ends of the side chain in the PDIM. The characteristics of the photo-cross-linked PDIM films were examined using absorption spectra, cyclic voltammetry, work function, and surface morphology. The feasibility of the photo-cross-linked PDIM films as a novel electron transporting layer (ETL) in polymer solar cells (PSCs) was also investigated. The PTB7-Th:PC71BM-based PSC using the PDIM as the ETL achieved the excellent power conversion efficiency of 9.44% similar to the conventional polyethylenimine ethoxylated (PEIE) and better than ZnO. Furthermore, the PSC with the PDIM films exhibited a similar lifetime to that of the PEIE-based device. This approach suggests that the photo-cross-linked PDIM film could be regarded as a promising interfacial material for fabricating highly efficient PSCs.

NMR Structural Studies of Antimicrobial Peptides: LPcin Analogs.

Lactophoricin (LPcin), a component of proteose peptone (113-135) isolated from bovine milk, is a cationic amphipathic antimicrobial peptide consisting of 23 amino acids. We designed a series of N- or C-terminal truncated variants, mutated analogs, and truncated mutated analogs using peptide-engineering techniques. Then, we selected three LPcin analogs of LPcin-C8 (LPcin-YK1), LPcin-T2WT6W (LPcin-YK2), and LPcin-T2WT6W-C8 (LPcin-YK3), which may have better antimicrobial activities than LPcin, and successfully expressed them in E. coli with high yield. We elucidated the 3D structures and topologies of the three LPcin analogs in membrane environments by conducting NMR structural studies. We investigated the purity of the LPcin analogs and the α-helical secondary structures by performing (1)H-(15)N 2D HSQC and HMQC-NOESY liquid-state NMR spectroscopy using protein-containing micelle samples. We measured the 3D structures and tilt angles in membranes by conducting (15)N 1D and 2D (1)H-(15)N SAMMY type solid-state NMR spectroscopy with an 800 MHz in-house-built (1)H-(15)N double-resonance solid-state NMR probe with a strip-shield coil, using protein-containing large bicelle samples aligned and confirmed by molecular-dynamics simulations. The three LPcin analogs were found to be curved α-helical structures, with tilt angles of 55-75° for normal membrane bilayers, and their enhanced activities may be correlated with these topologies.

Linearly configured BOCDA system using a differential measurement scheme.

We experimentally demonstrate a linearly configured Brillouin optical correlation domain analysis (BOCDA) system enhanced by a differential measurement scheme. On-off control of the pump phase modulation with an intentional loss at the end of a fiber under test is applied for the acquisition of a Brillouin gain spectrum. This application leads to a four-fold enhancement of the spatial resolution and doubling of the measurement range in comparison with the former system under the same modulation parameters.

Differential measurement scheme for Brillouin optical correlation domain analysis.

We newly propose and experimentally demonstrate a differential measurement scheme for Brillouin optical correlation domain analysis, where the difference between Brillouin gain spectra constructed by a normal and a phase-modulated Brillouin pump waves are analyzed to measure local Brillouin frequencies in optical fibers. In experiments, a five-fold enhancement in the spatial resolution is obtained compared to an ordinary BOCDA system under the same modulation parameters, as a result of the improved dynamic range by the suppression of background noises.

Bidirectional measurement for Brillouin optical correlation domain analysis.

We propose and experimentally demonstrate a bidirectional measurement for Brillouin optical correlation domain analysis as a novel and simple way of the performance enhancement. Brillouin gain and loss spectra of two adjacent correlation peaks are simultaneously and independently analyzed by applying midpoint attenuation in a fiber under test, which doubles both the speed and the range of the distributed measurement.

In situ gas sensing using a remotely detectable probe with replaceable insert.

We demonstrate a spectroscopic gas sensor using an optical fiber probe with a replaceable insert. The probe consists of a hollow-core photonic bandgap fiber (HC-PBGF) with a core diameter of 10.9 µm and a glass tube where a 2-µm hollow core fiber (HCF) with a gold coated end facet can be inserted. The HCF is designed to function as both a gate where gases can enter the HC-PBGF and a mirror reflecting the guided light back to the HC-PBGF. The opposite distal end of the probe is also designed to be able to regulate the gas pressure within the HC-PBGF for a high gas flow rate, while still transmitting the reflected light to the analysis instrument. The remote sensing probe, we believe, has much potential for detecting gases in hazardous environments.

Variable-frequency lock-in detection for the suppression of beat noise in Brillouin optical correlation domain analysis.

We propose and experimentally demonstrate a novel lock-in detection method to avoid a beat noise in Brillouin optical correlation domain analysis (BOCDA) which appears in the sweep of the sensing position and deteriorates the measurement accuracy by distorting the acquired Brillouin gain spectrum. In our analysis, the origin of the beat noise is shown to be the fluctuation of the Brillouin gain induced by the chopping of the intensity-modulated pump wave, and the optimal relation between the modulation and the lock-in frequencies is developed as an effective solution to circumvent the beat noise.