Search

Recent improvements in mRNA display have enabled the selection of peptides that incorporate non‐natural amino acids, thus expanding the chemical diversity of macrocycles beyond what is accessible in nature. Such libraries have incorporated non‐natural amino acids at the expense of natural amino acids by reassigning their codons. Here we report an alternative approach to expanded amino‐acid diversity that preserves all 19 natural amino acids (no methionine) and adds 6 non‐natural amino acids, resulting in the highest sequence complexity reported to date. We have applied mRNA display to this 25‐letter library to select functional macrocycles that bind human STING, a protein involved in immunoregulation. The resulting STING‐binding peptides include a 9‐mer macrocycle with a dissociation constant (K D) of 3.4 nM, which blocks binding of cGAMP to STING and induces STING dimerization. This approach is generalizable to expanding the amino‐acid alphabet in a library beyond 25 building blocks., In vitro selection of macrocyclic peptides from a library that uses a 25‐amino‐acid alphabet has allowed the identification of peptides that block binding of human STING to its natural ligand and induce dimerization of STING monomers. Our method splits multiple degenerate codons and reassigns them to non‐natural amino acids.

γ-Aminobutyric acid type A (GABAA) receptors in the brain are located in the outer membranes of brain cells where the concentration of cholesterol is high. Of the 25 available high-resolution structures available for GABAA receptors, none were determined in the presence of cholesterol, but four include resolved molecules of cholesterol hemisuccinate (CHS). Here, a molecular docking procedure is used to sweep the transmembrane (TM) surfaces of the receptors for cholesterol binding sites. Cholesterol docking poses determined in this way match 89% of the resolved CHS when CHS molecules deemed unlikely to represent typical bound cholesterols are excluded. The receptors are pentameric, and their TM surfaces consist of a set of five facets, each including pairs of TM helices from two adjacent subunits. Each facet contains hydrophobic hollows running from one side of the membrane to the other, within which are six potential binding sites for cholesterol, three on each side of the membrane. High-resolution structures of GABAA receptors with bound neurosteroids show that neurosteroids bind in these cholesterol binding sites, so the binding of neurosteroids and cholesterol will be competitive.

Aims: This study aims to elucidate the structural difference and biochemical properties of bioactive compounds of microalgal biomasses. Background : The structural difference and biochemical properties of bioactive compounds termed as water-soluble macromolecules (WSMs) are interested in evaluating their biological activities. Methods: This study was performed to elucidate the structural difference and biochemical properties of bioactive compounds termed as water-soluble macromolecules (WSMs) isolated from defatted microalgal biomasses of Botryococcus braunii and Dunaliella tertiolecta. Results: The compositional analysis of both WSMs revealed that WSM-Bb is a hetero-macromolecule consisting of various monosaccharides, whereas WSM-Dt was characterized as a homo-macromolecule that mainly consists of glucose. Interestingly, WSM-Bb showed the significant tyrosinase inhibitory activity with the increase of both the concentration and reaction time. Whereas, there was no significant inhibitory activity observed by WSM-Dt. Conclusion: Inhibitory action of WSM-Bb toward both tyrosinase and tyrosine in the either simultaneous or separate reaction may be mainly due to the physical affinity of WSM-Bb. These results emphasize the identification of the primary components of these WSMs and their relevance with the antioxidant function.

Repeated column chromatography of solvent fractions extracted from the flowers of Lilium Asiatic hybrids ensured purification of three benzyl glycosides I – III, which were reliably identified based on the analysis of various spectroscopic data. Compounds I – III (20 μg/mL) reduced nuclear factor-kappa B (NF-kB) (p-p65/p-65) by 9.64 ± 0.66%, 30.98 ± 1.08%, and 54.04 ± 1.12%, respectively. Compounds II and III markedly inhibited the expression of vascular cell adhesion molecule 1 (VCAM-1) in tumor necrosis factor (TNF)-α-stimulated human aortic smooth muscle cells (HASMCs) by 38.68 ± 2.52% and 37.9 ± 2.76%, respectively. Compound III significantly suppressed U937 monocytes attachment to the TNF-α-stimulated HASMCs.

The extra hepatic delivery of antisense oligonucleotides (ASOs) remains a challenge and hampers the widespread application of this powerful class of therapeutic agents. In that regard, pancreatic beta cells are a particularly attractive but challenging cell type because of their pivotal role in diabetes and the fact that they are refractory to uptake of unconjugated ASOs. To circumvent this, we have expanded our understanding of the structure activity relationship of ASOs conjugated to Glucagon Like Peptide 1 Receptor (GLP1R) agonist peptide ligands. We demonstrate the key role of the linker chemistry and its optimization to design maleimide based conjugates with improved in vivo efficacy. In addition, truncation studies and scoping of a diverse set of GLP1R agonists proved fruitful to identify additional targeting ligands efficacious in vivo including native hGLP1(7-36)NH2. Variation of the carrier peptide also shed some light on the dramatic impact of subtle sequence differences on the corresponding ASO conjugate performance in vivo, an area which clearly warrant further investigations. We have confirmed the remarkable potential of GLP1R agonist conjugation for the delivery of ASOs to pancreatic beta cell by effectively knocking down islet amyloid polypeptide (IAPP) mRNA, a potential proapoptotic target, in mice.

The structures of seven compounds based on guanidinium cations, isolated sulfate anions, and additional organic cations and solvent molecules are reported. All seven compounds, (C(NH2)3)2(SO4) (1), (C(NH2)3)3(C5H6N)(SO4)2 (2), (C(NH2)3)3(C5H6N)(SO4)2·H2O (3), (C(NH2)3)3(C5H6N)(SO4)2·CH3OH (4), (C(NH2)3)11(C5H6N)(SO4)6·2.5C2H5OH (5), (C(NH2)3)3(C6H16N)(SO4)2 (6), and (C(NH2)3)2(C20H26N2O2)(SO4)2 (7) crystallize without a center of symmetry, and are built of related motifs of six-membered rings via hydrogen bonding of three guanidinium cations and three sulfate anions. These six-membered rings form extended sheets and frameworks through additional hydrogen bonding interactions. The presence of additional cations and solvent molecules in varying ratios add structural diversity by modifying the guanidinium sulfate frameworks, but retaining the acentricity of the structures. The study reveals a remarkable tendency for these guanidinium sulfate frameworks to crystallize without a center of symmetry, and furthermore, in polar or chiral space groups. This provides a potential pathway for the use of hydrogen bonding interactions to design structures having interesting physical or nonlinear optical properties.

A 31-year-old previously healthy, pregnant woman presented with acute, unilateral, painless paracentral vision loss in the left eye. The results of neuroimaging studies were normal. Hypercoagulable workup was negative, and fundus examination showed no retinal emboli and no retinal vascular abnormalities. The patient had well-controlled blood pressure and did not have eclampsia/preeclampsia. Although a presumptive referral diagnosis of "optic neuritis" was made, optical coherence tomography (OCT) of the macula showed a hyperreflective band involving the inner nuclear layer, consistent with paracentral acute middle maculopathy (PAMM). Furthermore, OCT angiography (OCT-A) showed flow attenuation of the outer retinal capillary plexus, further supporting a diagnosis of PAMM in pregnancy. Clinicians should be aware of the benefit of OCT-A as a complement to macular OCT in the evaluation of acute monocular vision loss mimicking retrobulbar optic neuropathy, particularly when signs and findings of other retinal vasculopathy are absent. PAMM should be considered in pregnant patients with acute visual changes.

Objective: To investigate the relationship between the monocyte-to-high-density lipoprotein cholesterol ratio (MHR) and clinical manifestations in patients with systemic sclerosis (SSc). Method: Th...

We examined the effect of ice‐binding protein derived from Leucosporidium (LeIBP) on the cryopreservation of bull semen and compared it with that derived from previously reported Antifreeze Protein III (AFPIII). Six concentrations of LeIBP (10–1 ~ 104 μg/ml) and AFPIII (10–1 ~ 104 μg/ml) were added to the bull semen extender, respectively. Sperm kinematic parameters were measured to examine sperm toxicity and cryopreserved sperm quality. Measures of antioxidant activity such as superoxide dismutase (SOD), reduced glutathione/oxidative glutathione (GSH/GSSG), and total antioxidant capacity (TAC) were analysed to identify the effect of LeIBP on sperm quality. In addition, sperm viability was analysed using a flow cytometer and fluorescence microscope by SYBR14/PI staining. The results showed that the LeIBP groups (0.1, 1 and 10 μg/ml) were less toxic, and the quality of the sperm were dramatically improved in the extenders containing 0.1 μg/ml LeIBP among concentrations of LeIBP and AFPIII. The SOD activity of LeIBP was greater than that of AFPIII and control. In addition, sperm viability was enhanced in the LeIBP‐treated group. In summary, LeIBP is a useful cryoprotective adjuvant for bull sperm cryopreservation, and the most efficient concentration of LeIBP is 0.1 μg/ml., Supplementation with LeIBP can allow for a greater cryoprotective effect and viability in bull sperm.

Flexible, thin, and lightweight supercapacitors have been regarded as important power sources for portable and wearable electronics; however, these are usually limited by relatively low areal or vo...

Inwardly rectifying, voltage-gated, two-pore domain, and related K+ channels are located in eukaryotic membranes rich in cholesterol. Here, molecular docking is used to detect specific binding sites ("hot spots") for cholesterol on K+ channels with characteristics that match those of known cholesterol binding sites. The transmembrane surfaces of all available high-resolution structures for K+ channels were swept for potential binding sites. Cholesterol poses were found to be located largely in hollows between protein ridges. A comparison between cholesterol poses and resolved phospholipids suggests that not all cholesterol molecules binding to the transmembrane surface of a K+ channel will result in displacement of a phospholipid molecule from the surface. Competition between cholesterol binding and binding of anionic phospholipids essential for activity could explain some of the effects of cholesterol on channel function.

Behçet's disease (BD) is a recurrent, multisystemic, inflammatory blood vessel disorder that can result in mouth, genital, and skin ulcers; arthritis; and eye and intestinal inflammation. We describe a 21-year-old Korean female patient with intestinal BD refractory to conventional medical treatment and biologic drugs. The patient was initially treated with high-dose steroids and sulfasalazine. Two months later, a skin rash occurred as a side effect of sulfasalazine. Therefore, infliximab (IFX) was administered, and disease activity decreased. However, IFX also induced a skin rash; hence, the patient was switched to adalimumab. After 12 months, the patient experienced a relapse of intestinal BD. Hence, treatment was initiated using a combination of methotrexate and adalimumab; however, this treatment was ineffective. Methotrexate was discontinued and replaced with 5‑aminosalicylic acid while maintaining adalimumab, and no recurrence has been observed to date. We report this novel strategy involving the use of anti-tumor necrosis factor‑α agents for patients with resistant BD; however, further large cohort studies are required to verify its usefulness.

Antibiotic resistance is a significant and growing public health problem. This work investigated the use of two different carbon nanomaterials, single-walled carbon nanotubes (SWNTs) and nanographe...

The Reactor Experiment for Neutrino Oscillation (RENO) experiment has been taking data using two identical liquid scintillator detectors of 44.5 tons since August 2011. The experiment has observed the disappearance of reactor neutrinos in their interactions with free protons, followed by neutron capture on hydrogen. Based on 1500 live days of data taken with 16.8 GW$_{th}$ reactors at the Hanbit Nuclear Power Plant in Korea, the near (far) detector observes 567690 (90747) electron antineutrino candidate events with a delayed neutron capture on hydrogen. This provides an independent measurement of $\theta_{13}$ and a consistency check on the validity of the result from n-Gd data. Furthermore, it provides an important cross-check on the systematic uncertainties of the n-Gd measurement. Based on a rate-only analysis, we obtain sin$^{2}$2$\theta _{13}$= 0.087 $\pm$ 0.008 (stat.) $\pm$ 0.014 (syst.)., Comment: 26 pages, 19 figures, 6 tables

We present a strictly nonblocking $4$ × $4$ electrooptic silicon photonic switch fabric with on-chip gain. The switch integrates 12 Mach-Zehnder cells in a 3-stage topology equipped with fast electrooptic phase shifters, and thermooptic phase trimmers. To compensate for the losses of the fabric, a 4-channel GaInAsP/InP semiconductor optical amplifier array is flip-chip attached into an etched cavity in the silicon photonic chip with butt-coupled waveguide interfaces. The chip is wirebonded to a CMOS driver that provides push-pull drive to each elementary Mach-Zehnder cell. We demonstrate an optical switch assembly with net neutral insertion loss in the C-band together with nanosecond-scale reconfiguration time.

Rapid and efficient nucleic acid (NA) extraction and concentration are required for point-of-care analysis in order to prevent an epidemic/pandemic disease outbreak. Typical silica-based NA extraction methods have limitations such as being time-consuming, requiring human intervention, and resulting in a low recovery yield. In this study, we have developed a pathogenic DNA extraction device based on electrokinetic separation incorporated with a silicon nitride (SiNx) nanofilter, which expedites the DNA extraction procedure with advantages of being convenient, efficient, and inexpensive. This DNA extraction device consists of a computer numerical control (CNC) milled-Teflon gadget with a cis-chamber as a cell lysate reservoir and a trans-chamber as a elution solution reservoir, with the SiNx nanofilter being inserted between the two chambers. The SiNx nanofilter was fabricated using a photolithographic method in conjunction with nanoimprinting. Approximately 7.2 million nanopores of 220 nm diameter were located at the center of the nanofilter. When a DC electric field is applied through the nanopores, DNA is transferred from the cis-chamber to the trans-chamber to isolate the DNA from the cell debris. To demonstrate the DNA extraction performance, we measured the absorbances at 260 and 280 nm and performed a real-time polymerase chain reaction (real-time PCR) using the recovered DNA to verify its feasibility for downstream genetic analysis. Moreover, the DNA extraction device was successfully operated using a 1.5 V alkaline battery, which verifies the portability of the device for point-of-care testing. Such an advanced DNA extraction system can be utilized in various fields including clinical analysis, pathogen detection, forensic analysis, and on-site detection.

We evaluated imaging alteration associated with porphyry copper style mineralisation using aeromagnetic data in the Highland Valley Copper (HVC) district as part of the Natural Sciences and Enginee...

Intricate regulatory networks govern the net balance of cholesterol biosynthesis, uptake and efflux; however, the mechanisms surrounding cholesterol homeostasis remain incompletely understood. Here, we develop an integrative genomic strategy to detect regulators of LDLR activity and identify 250 genes whose knockdown affects LDL-cholesterol uptake and whose expression is modulated by intracellular cholesterol levels in human hepatic cells. From these hits, we focus on MMAB, an enzyme which catalyzes the conversion of vitamin B12 to adenosylcobalamin, and whose expression has previously been linked with altered levels of circulating cholesterol in humans. We demonstrate that hepatic levels of MMAB are modulated by dietary and cellular cholesterol levels through SREBP2, the master transcriptional regulator of cholesterol homeostasis. Knockdown of MMAB decreases intracellular cholesterol levels and augments SREBP2-mediated gene expression and LDL-cholesterol uptake in human and mouse hepatic cell lines. Reductions in total sterol content were attributed to increased intracellular levels of propionic and methylmalonic acid and subsequent inhibition of HMGCR activity and cholesterol biosynthesis. Moreover, mice treated with antisense inhibitors of MMAB display a significant reduction in hepatic HMGCR activity, hepatic sterol content and increased expression of SREBP2-mediated genes. Collectively, these findings reveal an unexpected role for the adenosylcobalamin pathway in regulating LDLR expression and identify MMAB as an additional control point by which cholesterol biosynthesis is regulated by its end product., The mechanisms governing cholesterol homeostasis remain incompletely understood. Here, the authors develop an integrative genomic strategy to identify MMAB, and enzyme in the adenosylcobalamin pathway, as a regulator of hepatic LDLR activity and cholesterol biosynthesis.

A bacteria-capturing platform is a critical function of accurate, quantitative, and sensitive identification of bacterial pathogens for potential usage in the detection of foodborne diseases. Despite the development of various nanostructures and their surface chemical modification strategies, relative to the principal physical contact propagation of bacterial infections, mechanically robust and nanostructured platforms that are available to capture bacteria remain a significant problem. Here, a three-dimensional (3D) hierarchically structured polyaniline nanoweb film is developed for the efficient capture of bacterial pathogens by hand-touching. This unique nanostructure ensures sufficient mechanical resistance when exposed to compression and shear forces and facilitates the 3D interfacial interactions between bacterial extracellular organelles and polyaniline surfaces. The bacterial pathogens (Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus) are efficiently captured through finger-touching, as verified by the polymerase chain reaction (PCR) analysis. Moreover, the real-time PCR results of finger-touched cells on a 3D nanoweb film show a highly sensitive detection of bacteria, which is similar to those of the real-time PCR using cultured cells without the capturing step without any interfering of fluorescence signal and structural deformation during thermal cycling. Graphic Abstract

We aimed to increase the processing area of the roll-to-roll (R2R) nanoimprint lithography (NIL) process for high productivity, using a long roller. It is common for a long roller to have bending deformation, geometric errors and misalignment. This causes the non-uniformity of contact pressure between the rollers, which leads to defects such as non-uniform patterning. The non-uniformity of the contact pressure of the conventional R2R NIL system was investigated through finite element (FE) analysis and experiments in the conventional system. To solve the problem, a new large-area R2R NIL uniform pressing system with five multi-backup rollers was proposed and manufactured instead of the conventional system. As a preliminary experiment, the possibility of uniform contact pressure was confirmed by using only the pressure at both ends and one backup roller in the center. A more even contact pressure was achieved by using all five backup rollers and applying an appropriate pushing force to each backup roller. Machine learning techniques were applied to find the optimal combination of the pushing forces. In the conventional pressing process, it was confirmed that pressure deviation of the contact area occurred at a level of 44%, when the improved system was applied, pressure deviation dropped to 5%.

C-type natriuretic peptide (CNP) is a pluripotent molecule produced by the endothelium and myocardium with therapeutic potential in pulmonary hypertension, myocardial infarction, and heart failure. However, the native peptide’s short elimination half-life of under 3 minutes limits its use clinically. To improve the pharmacokinetic profile of CNP we recombinantly expressed the CNP peptide as a fusion with an elastin-like polypeptide (ELP). ELPs are biopolymers that undergo reversible temperature-dependent phase separation and form a subcutaneous depot upon injection. CNP-ELP fusion proteins were expressed in E. coli , purified, and showed in vitro potency 2-4 fold less than native CNP. When administered subcutaneously in mice CNP-ELP showed a significantly enhanced PK profile due to the slow release of the fusion into the circulation with detectable fusion protein 24 hours after a single injection. In addition, the CNP-ELP increased growth velocity in mice significantly greater than that of native peptide indicating an in vivo activation of the CNP receptor, NPR-B. These findings represent a new method of administering a long acting CNP peptide for treatment of different cardiovascular diseases.

BACKGROUND Tocilizumab (Actemra) is a humanized anti-interleukin-6 receptor antibody that has been used as a steroid-sparing agent in giant cell arteritis (GCA). Although the clinical effects are well described in GCA, the cost-effectiveness of the use of tocilizumab in GCA is ill defined. The purpose of this study was to determine the cost-effectiveness of tocilizumab in GCA compared with prednisone alone. METHODS A retrospective study of 32 patients with biopsy-proven GCA comparing prednisone alone (16 patients) and prednisone with tocilizumab (16 patients) was performed. The cost for tocilizumab therapy for 26 weeks with mild and severe side effects (Groups 1 and 2, respectively) and for 52 weeks with mild and severe side effects (Group 3 and 4, respectively) was compared with estimated costs of mild and severe steroid-induced side effects (Groups 5 and 6, respectively). Statistical analysis between groups was conducted using independent sample t tests. RESULTS Three out of the 4 group combinations of tocilizumab with prednisone demonstrated a statistically significant (P < 0.05) difference in cost compared with prednisone alone for GCA. Group 2 (26-week tocilizumab therapy with severe steroid-induced side effects), with no statically significant difference in price when compared with steroid therapy alone and far fewer side effects, demonstrated the potential use of tocilizumab in GCA therapy. As expected, longer treatment duration with tocilizumab was associated with greater cost. With respect to side effect severity, the number of side effects of steroid therapy was inversely associated with difference in cost between tocilizumab therapy and steroid side effect treatment. CONCLUSION This study demonstrates that combination therapy of tocilizumab and prednisone is significantly more expensive than steroids alone with or without accounting for the cost of steroid-induced side effects in treated GCA. The difference in cost between the 2 therapy types is directly related to tocilizumab therapy duration and inversely related to the number or severity of steroid side effects. Patients with GCA who require a shorter duration of steroid therapy and are at risk for a high number of side effects from steroid use may be potential candidates for tocilizumab therapy, from an economic perspective.

The gut microbiome influences nutrient processing as well as host physiology. Plasma lipid levels have been associated with the microbiome, although the underlying mechanisms are largely unknown, and the effects of dietary lipids on the gut microbiome in humans are not well-studied. We used a compilation of four studies utilizing non-human primates (Chlorocebus aethiops and Macaca fascicularis) with treatments that manipulated plasma lipid levels using dietary and pharmacological techniques, and characterized the microbiome using 16S rDNA. High-fat diets significantly reduced alpha diversity (Shannon) and the Firmicutes/Bacteroidetes ratio compared to chow diets, even when the diets had different compositions and were applied in different orders. When analyzed for differential abundance using DESeq2, Bulleidia, Clostridium, Ruminococcus, Eubacterium, Coprocacillus, Lachnospira, Blautia, Coprococcus, and Oscillospira were greater in both chow diets while Succinivibrio, Collinsella, Streptococcus, and Lactococcus were greater in both high-fat diets (oleic blend or lard fat source). Dietary cholesterol levels did not affect the microbiome and neither did alterations of plasma lipid levels through treatments of miR-33 antisense oligonucleotide (anti-miR-33), Niemann–Pick C1-Like 1 (NPC1L1) antisense oligonucleotide (ASO), and inducible degrader of LDLR (IDOL) ASO. However, a liver X receptor (LXR) agonist shifted the microbiome and decreased bile acid levels. Fifteen genera increased with the LXR agonist, while seven genera decreased. Pseudomonas increased on the LXR agonist and was negatively correlated to deoxycholic acid, cholic acid, and total bile acids while Ruminococcus was positively correlated with taurolithocholic acid and taurodeoxycholic acid. Seven of the nine bile acids identified in the feces significantly decreased due to the LXR agonist, and total bile acids (nmol/g) was reduced by 62%. These results indicate that plasma lipid levels have, at most, a modest effect on the microbiome, whereas bile acids, derived in part from plasma lipids, are likely responsible for the indirect relationship between lipid levels and the microbiome.

Supported by an abundance of experimental and genetic evidence, angiopoietin-like protein 3 (ANGPTL3) has emerged as a promising therapeutic target for cardiovascular disease. ANGPTL3 is primarily produced by the liver and is a potent modulator of plasma lipids and lipoproteins. Experimental models and subjects with loss-of-function Angptl3 mutations typically present with lower levels of HDL-C than noncarriers. The effect of ANGPTL3 on HDL-C is typically attributed to its function as an inhibitor of the enzyme endothelial lipase. The ability to facilitate reverse cholesterol transport (RCT), the transport of cholesterol from peripheral tissues back to the liver, is a proposed antiatherogenic property of HDL. However, the effect of ANGPTL3 inhibition on RCT remains unclear. Here, we performed a series of dose-response and RCT studies using an Angptl3 antisense oligonucleotide (ASO) in mouse models with varying plasma lipid profiles ranging from moderately to severely hyperlipidemic. Angptl3 ASO-mediated reduction in HDL-C was limited to the model with moderate lipidemia, where the majority of plasma cholesterol was associated with HDL. Surprisingly, regardless of the effect on HDL-C, treatment with the Angptl3 ASO enhanced RCT in all models tested. The observations from the RCT assays were confirmed in HDL clearance studies, where mice treated with the Angptl3 ASO displayed increased plasma clearance and hepatic uptake of labeled HDL. The results from our studies suggest that inhibition of ANGPTL3 not only reduces levels of proatherogenic lipids but also improves HDL-mediated RCT.

A novel technique for the absolute stereochemical determination of alcohols has been developed that uses crystallization of guanidinium salts of organosulfates. The simple one-pot, two-step process leverages facile formation of guandinium organosulfate single crystals for the straightforward determination of the absolute stereochemistry of enantiopure alcohols by means of X-ray crystallography. The strong hydrogen bonding network drives the stability of the crystal lattice and allows for a diverse range of organic alcohol substrates to be analyzed.

Proinflammatory cytokines in the tumor microenvironment can promote tumor growth, yet their value as therapeutic targets remains underexploited. We validated the functional significance of the cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling axis in lung adenocarcinoma (LUAD) and generated a high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1, thereby inhibiting its oncogenic effects. eCNTFR-Fc inhibits tumor growth in multiple xenograft models and in an autochthonous, highly aggressive genetically engineered mouse model of LUAD, driven by activation of oncogenic Kras and loss of Trp53. Abrogation of CLCF1 through eCNTFR-Fc appears most effective in tumors driven by oncogenic KRAS. We observed a correlation between the effectiveness of eCNTFR-Fc and the presence of KRAS mutations that retain the intrinsic capacity to hydrolyze guanosine triphosphate, suggesting that the mechanism of action may be related to altered guanosine triphosphate loading. Overall, we nominate blockade of CLCF1-CNTFR signaling as a novel therapeutic opportunity for LUAD and potentially for other tumor types in which CLCF1 is present in the tumor microenvironment.

Nucleic acid (NA) extraction and purification are one of the crucial steps for NA-based molecular diagnosis. However, the currently developed methods are still suffering from many issues including long process time, complicated steps, requirement of trained personnel and potential inhibition caused by chaotropic agents and/ or residual reagents. Herein, a surface-modified NA extraction microchip (SNC) is newly fabricated by introducing poly(2-dimethylaminomethyl styrene) (pDMAMS) film engaged directly on the microchip surface via initiated chemical vapor deposition (iCVD) process. The positively charged SNC inner surface could directly capture the negatively charged NA efficiently and its performance is confirmed by fluorescence microscopy and X-ray photoelectron spectroscopy. The developed SNC exhibits the deoxyribonucleic acid (DNA) capture efficiency higher than 92% regardless of initial DNA concentration in range of 20 ng/µL to 0.01 ng/µL. With this versatile DNA-capturing surface, the genomic DNAs of Escherichia Coli O157:H7 (E. coli O157:H7) is successfully extracted directly from cell lysate in the SNC with higher than 90% of efficiency within 30 min. The extraction time can be reduced to at least of 10 min maintaining extraction efficiency higher than 50%. Furthermore, the genomic DNAs are directly extracted using the SNC without loss from various real samples including juice, milk and blood serum. The proposed SNC enables us to perform an one-step NA extraction for molecular diagnosis and has the potential to be integrated into a micro-total analysis in the fields of point-of-care diagnosis.

We report potentiometric performances of ion-to-electron transducer based on reduced graphene oxide (RGO) for application of all-solid-state potassium ion sensors. A large surface area and pore structure of RGO are obtained by a hydrothermal self-assembly of graphene oxide. The extensive electrochemical characterization of RGO solid contact at the interface of ion-selective membrane and gold electrode shows that the potassium ion-selective electrode based on RGO had a high sensitivity (53.34 mV/log[K+]), a low detection of limit (− 4.24 log[K+], 0.06 mM) a good potential stability, and a high resistance to light and gas interferences. The potentiometric K+-sensor device was fabricated by combining of screen-printed electrodes and a printed circuit board. The K+-sensor device accurately measures the ion concentration of real samples of commercial sports drinks, coke and orange juice, and then transfers the collected data to a mobile application through a Bluetooth module. The screen-printed ion sensors based on RGO solid contact show a great potential for real-time monitoring and point-of-care devices in human health care, water-treatment process, and environmental and chemical industries.

Transsynaptic retrograde degeneration (TSRD) of the visual pathways is the loss of retinal ganglion cells occurring upstream from a posterior visual pathway lesion. We present a case of a 54-year-old woman with an epidermoid cyst at the right temporo-occipital junction, in whom retinal optical coherence tomography angiography showed a reduction of the superficial retinal capillary density consistent with TSRD. While this reduction has been described in chronic optic neuropathies and pregeniculate lesions, to our knowledge, this is the first case report showing how a reduction in the superficial retinal capillary density occurs also for a postgeniculate lesion.

As part of the Natural Sciences and Engineering Research Council of Canada–Canada Mining Innovation Council (NSERC-CMIC) Mineral Exploration Footprints project, three selected magnetic inve...

Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.

We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues.

A plasmid-borne factor (hypothetical protein) was identified in three Shiga toxin-producing Escherichia coli O113:H21 strains using antibiotic-induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS), post-source decay (PSD) and top-down proteomic analysis. The plasmid-encoded factor was identified from three pairs of complementary fragment ions (b/y) which were also used to obtain a more accurate calculation of the mass of the protein than that possible from MS analysis. A software application tool was developed to rapidly search amino acid sequences from whole genome sequencing-derived data to match the observed protein mass and fragment ions on the assumption that the fragment ions are the result of polypeptide backbone cleavage on the C-terminal side of aspartic acid and glutamic acid residues, i.e. aspartic acid effect. Manual inspection and top-down proteomic analysis confirmed the correctness of the identification of this protein using this new application tool. Although the function of the plasmid-borne factor is not known, its antibiotic-induced expression may suggest a role in antimicrobial stress. This approach was also used to identify three highly conserved bacterial cold-shock proteins: CspC, CspE, CsbD from one of the STEC strains. In addition to polypeptide backbone cleavage, metastable CsbD showed multiple small dissociative losses perhaps suggesting ammonia loss from the four arginine residues in its sequence. This phenomenon had been previously observed for metastable ubiquitin which also has four arginine residues.

There is growing evidence of thyroid eye disease association with nutritional deficiencies including selenium and vitamin D. We conducted a retrospective chart review of all patients with clinical diagnosis of TED seen at our clinic from 2016 to 2017. Thirty-five patients met inclusion criteria and had serum 25-hydroxyvitamin D levels available, and 19 had selenium levels available. 7/35 (20%) patients had vitamin D deficiency, and 11 (31%) had vitamin D insufficiency, but none had selenium deficiency. Although both selenium and vitamin D supplementation have been recommended for TED, further investigation is necessary to justify supplementation for patients with TED.

Rapid and efficient detection of pathogenic bacteria from food is critical to prevent epidemic food poisoning. However, the isolation of pathogenic bacteria from spoiled food is hampered by the lack of proper cell cultivation and/or isolation methods. Most of currently used methods suffer from complex, time-consuming culturing steps, low scalability, and high operation cost. Herein, we developed an alternative approach for the isolation of pathogenic bacteria directly from food using a surface-modified, highly porous sponge via initiated chemical vapor deposition (iCVD) process. A hydrophobic polymer, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetra-siloxane) (pV4D4), was deposited conformally on amphiphilic 3-dimensional (3D) melamine sponge to incorporate hydrophobicity as well as oleophilicity to the porous sponge surface, which is appropriate for absorbing oil component selectively from food extracts. Furthermore, the surface-modified sponge was capable of the isolation of Escherichia coli O157:H7 (E. coli O157:H7) from heterogeneous mixture with oil/water/food particles with undistinguisible efficiency compare to artificial model system. The surface-modified sponge developed in this study will be a novel platform for oil/water separation and isolation of foodborne pathogens directly from heterogeneous mixture to enhance the efficiency of molecular diagnostics.

Many metabolic phenotypes in cancer cells are also characteristic of proliferating non-transformed mammalian cells, and attempts to distinguish between phenotypes resulting from oncogenic perturbation from those associated with increased proliferation are limited. Here, we examined the extent to which metabolic changes corresponding to oncogenic KRAS expression differed from those corresponding to epidermal growth factor (EGF)-driven proliferation in human mammary epithelial cells (HMECs). Removal of EGF from culture medium reduced growth rates and glucose/glutamine consumption in control HMECs despite limited changes in respiration and fatty acid synthesis, while the relative contribution of branched-chain amino acids to the TCA cycle and lipogenesis increased in the near-quiescent conditions. Most metabolic phenotypes measured in HMECs expressing mutant KRAS were similar to those observed in EGF-stimulated control HMECs that were growing at comparable rates. However, glucose and glutamine consumption as well as lactate and glutamate production were lower in KRAS-expressing cells cultured in media without added EGF, and these changes correlated with reduced sensitivity to GLUT1 inhibitor and phenformin treatment. Our results demonstrate the strong dependence of metabolic behavior on growth rate, and provide a model to distinguish the metabolic influences of oncogenic mutations and non-oncogenic growth.

Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes have diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. The structural basis of the mammalian mitochondrial ribosome assembly is currently not well understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving seven assembly factors. We discover that the NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by the MRM2 methyltransferase and quality control interactions with the conserved mitochondrial GTPase MTG2 that contacts the sarcin-ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit., Nature Communications, 12 (1), ISSN:2041-1723

Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. Structural basis of the mammalian mitochondrial ribosome assembly is currently not understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving 7 assembly factors. We discover that NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by MRM2 methyltransferase and quality control interactions with a conserved mitochondrial GTPase MTG2 that contacts the sarcin ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.

Objective: Reelin and its receptor Apoer2 (apolipoprotein E receptor 2) play a prominent role in endothelial cell dysfunction by promoting leukocyte–endothelial cell adhesion, an important component of the inflammatory process underlying atherosclerosis. We, therefore, hypothesized that pharmacological depletion of circulating reelin represents a novel therapeutic strategy to impede the progression of atherosclerosis. Approach and Results: In vitro studies demonstrated that human plasma induced monocyte adhesion to endothelial cells, while reelin-depleted plasma had no effect on monocyte adhesion. Signaling analysis revealed that reelin activated a Dab2, PI3K (phosphoinositide 3-kinase), Akt (protein B kinase), and NF-κB (nuclear factor κB) cascade to promote the expression of adhesion markers (E-selectin, ICAM-1 [intercellular adhesion molecule 1], and VCAM-1 [vascular cell adhesion molecule 1]). Intravital microscopy confirmed decreased leukocyte-endothelial adhesion in mice treated with reelin antisense oligonucleotide. In vascular smooth muscle cells, reelin induced Stat3 phosphorylation to promote cell proliferation, which is another hallmark of atherosclerotic plaque progression. To investigate if reelin pharmaceutical depletion protects against atherosclerosis, low-density lipoprotein receptor–deficient ( Ldlr −/− ) mice fed with high-cholesterol diet were treated with either reelin antisense oligonucleotide or neutralizing antibody (CR-50) to systemically deplete circulating reelin. In both treatments, atherosclerotic plaque progression was markedly attenuated. These in vivo results suggest that reelin depletion decreases vascular adhesion and inhibits the recruitment of monocytes and consequently prevents plaque progression. Conclusions: These findings suggest that reelin inhibition may provide a novel therapeutic approach to counteract leukocyte or monocyte adhesion as well as extravasation and inhibit the progression of atherosclerosis. This strategy may also be relevant for other diseases that involve leukocyte or monocyte extravasation as a central pathological mechanism, such as multiple sclerosis or arthritis.

Three pathogenic human coronaviruses have emerged within the last 20 years, with SARS-CoV-2 causing a global pandemic. Although therapeutic antibodies targeting the SARS-CoV-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report an antibody binding an epitope conserved in the pre-fusion core of MERS-CoV, SARS-CoV and SARS-CoV-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes spike from SARS-CoV, SARS-CoV-2 and variants of concern in in vitro pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, suggesting 3A3 prevents the S2 conformational rearrangements required for conversion to the spike post-fusion state and virus-host cell fusion. This work defines a conserved vulnerable site on the SARS-CoV-2 S2 domain and guides the design of pan-protective spike immunogens.