Optimized Cholesterol-siRNA Chemistry Improves Productive Loading onto Extracellular Vesicles

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Synthetic lipophilic antioxidant BO‐653 suppresses HCV replication

The influence of the intracellular redox state on the hepatitis C virus (HCV) life cycle is poorly understood. This study demonstrated the anti‐HCV activity of 2,3‐dihydro‐5‐hydroxy‐2,2‐dipentyl‐4,6‐di‐tert‐butylbenzofuran (BO‐653), a synthetic lipophilic antioxidant, and examined whether BO‐653's antioxidant activity is integral to its anti‐HCV activity. The anti‐HCV activity of BO‐653 was investigated in HuH‐7 cells bearing an HCV subgenomic replicon (FLR3‐1 cells) and in HuH‐7 cells infected persistently with HCV (RMT‐tri cells). BO‐653 inhibition of HCV replication was also compared with that of several hydrophilic and lipophilic antioxidants. BO‐653 suppressed HCV replication in FLR3‐1 and RMT‐tri cells in a concentration‐dependent manner. The lipophilic antioxidants had stronger anti‐HCV activities than the hydrophilic antioxidants, and BO‐653 displayed the strongest anti‐HCV activity of all the antioxidants examined. Therefore, the anti‐HCV activity of BO‐653 was examined in chimeric mice harboring human hepatocytes infected with HCV. The combination treatment of BO‐653 and polyethylene glycol‐conjugated interferon‐α (PEG‐IFN) decreased serum HCV RNA titer more than that seen with PEG‐IFN alone. These findings suggest that both the lipophilic property and the antioxidant activity of BO‐653 play an important role in the inhibition of HCV replication. J. Med. Virol. 85:241–249, 2013. © 2012 Wiley Periodicals, Inc.     

Studies on physicochemical and nutritional properties of aerial parts of Cassia occidentalis L.

In the present, work chemical composition and nutritional value of aerial parts of Cassia occidentalis L. was studied. The aerial parts of C. occidentalis possess favorable physicochemical properties with good nutritional value, such as high energy value, crude fibers, and vitamin levels. The X-ray fluorescence spectrophotometry data revealed that the sample is rich in minerals, especially in Fe, Ca, K, and Mn. Further, minerals such as Mg, Zn, Cu, Na, P, and S are present in good amount and depicted the nutritional value of the selected material. The plant sample is rich in phytochemicals such as flavonoids, alkaloids, lignin, tannins, and phenols. The presence of phytochemical constituents was confirmed by gas chromatography–mass spectrometry profile and high-performance thin layer chromatography fingerprinting techniques. The findings stimulate the on-farm cultivation of C. occidentalis on a large scale to relieve the iron deficiency in local community, and it can be used as a dietary supplement to treat anemia.     

Effects of Fiber Angle on the Tensile Properties of Partially Delignified and Densified Wood

Partial delignification and densification provide a pathway to significant improvement in the mechanical performance of wood. In order to elucidate potential effects of this treatment on the mechanical anisotropy of wood, partially delignified and densified spruce wood veneers were characterized at varying degrees of off-axis alignment. While the tensile strength and the modulus of elasticity (MOE) were clearly improved in parallel to the axis of wood fibers, this improvement quickly leveled off at misalignment angles ≥30°. For transverse tensile strength, the performance of alkaline-treated and densified wood was even inferior to that of untreated wood. Microscopic examination revealed the presence of microscopic cracks in treated wood, which are assumed to be responsible for this observation. It is concluded that impaired transverse tensile properties are a weakness of partially delignified and densified wood and should be considered when a potential usage in load-bearing applications is intended.     

Impairment of chemical clearance is relevant to the pathogenesis of refractory reflux oesophagitis

BackgroundThe pathophysiological mechanisms underlying proton pump inhibitor-refractory reflux oesophagitis has been scarcely studied.AimsTo assess impedance-pH parameters relevant to the pathogenesis of refractory reflux oesophagitis.MethodsCases referred for heartburn/regurgitation refractory to high-dosage proton pump inhibitors between January 2008 and December 2012 were reviewed and subdivided into refractory oesophagitis (29 patients, 72% males, median age 50 years), healed oesophagitis (18 patients, 67% males, median age 54 years), and non-erosive reflux disease (49 patients, 53% males, median age 42 years). On-therapy impedance-pH tracings were blindly re-analysed by one observer to assess gastric and oesophageal acid exposure time and chemical clearance as expressed by the post-reflux swallow-induced peristaltic wave index.ResultsThe median gastric and oesophageal acid exposure time did not differ among the three groups (35%, 34%, 41% and 1.2%, 0.7%, 0.8%, respectively; P > 0.05 for all comparisons). A normal oesophageal acid exposure time was found in two thirds of patients with refractory oesophagitis. The post-reflux swallow-induced peristaltic wave index was significantly lower in refractory oesophagitis (16%) than in healed oesophagitis (30%) and non-erosive reflux disease (29%) (P = 0.003).ConclusionsRefractory reflux oesophagitis is characterized by impairment of chemical clearance. Adequate acid suppression is found in the majority of patients who would likely not benefit from further proton pump inhibitor dose escalation.     

Re-epithelialization of the Buccal Mucosa after Alkaline Chemical Injury

Alkaline conditions in the oral cavity may be caused by a variety of stimuli, including tobacco products, antacids, alkaline drinking water and bicarbonate toothpaste. However, the effects of an alkaline pH on the oral mucosa had not been elucidated. The purpose of this study was to investigate how basal keratinocytes are actively involved in re-epithelialization after alkaline chemical injury. We generated epithelial defects in the oral mucosa of mice by applying an alkaline chemical, and the localization of cytokeratin 13, cytokeratin 14, PCNA and p63 was investigated during the re-epithelialization process. PCNA- and p63-positive staining was seen in basal cells covering the wound surface at 1 day after the chemical injury. Cytokeratin 14-positive and PCNA-negative basal keratinocytes were localized in a few layers of the wound epithelium during epithelial outgrowth. Cytokeratin 14-positive and PCNA-positive basal keratinocytes, indicating proliferation, were localized over the entire layer of the epithelium at the wound margin. These results imply that basal keratinocytes at the wound margin migrate to the wound surface, provoke differentiation and keratinization during epithelial outgrowth and that epithelial cells are supplied from the wound margin to the epithelial outgrowth after alkaline chemical injury.     

Facile Production of Polypyrrole Nanofibers Using a Freeze‐Drying Method

Conjugated polymer nanofibers with lengths of tens of micrometers and diameters of hundreds of nanometers are prepared via the assembly of polypyrrole (PPy) nanoparticles in water using a freeze‐drying method. The structure and uniformity of the PPy nanofibers are critically affected by the processing conditions, such as the freezing temperature and the concentration of the latex dispersion. These nanofibers are subsequently applied as vapor sensors for efficient detection of organic explosive chemicals.

     

Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts

Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein’s folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein’s functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.All proteins are biosynthesized as linear chains, and most need to fold into 3D structures to function. Studies on protein folding in buffers have revealed that a kinetic competition typically exists between protein folding, misfolding, and aggregation. It is the role of the protein homeostasis or proteostasis network in each subcellular compartment to regulate this competition and keep the folded and functional proteome within the physiological concentration range, while minimizing misfolding and aggregation in the face of stresses (1–4). It remains a challenge to discern how the proteostasis network affects the folding of proteins into biologically active conformations required for function in vivo (5).Current methodologies allow for quantification of the partitioning of a protein of interest (POI) between soluble and aggregated states but cannot determine the proportion of the soluble population that is properly folded and functional. Published folding probes have the potential to report on the folded fraction in cells or cell lysate (6–9); however, the extent to which they shift folding equilibria and quantify the folded and functional fraction faithfully has not been studied. Herein, we create POI folding probes by adapting the principle of activity-based protein profiling (10) to quantify the soluble folded and functional fraction of a particular protein in a cell lysate. We seek folding probes that bind to and selectively react with only the folded and functional state of a POI in a cell, leaving the nonfunctional states and other cellular proteins unmodified (Fig. 1A).Open in a separate windowFig. 1.A small molecule folding probe strategy to quantify the soluble folded and functional fraction of a POI in a cell lysate. (A) Overview of the general strategy to selectively covalently label a folded and functional POI without labeling its nonfunctional conformations and other cellular proteins. (B) The experimental scheme to quantify the ratio of the soluble POI that is functional (R_f).Fluorescent folding probes for the de novo-designed enzyme, retroaldolase (RA) (11), and fluorogenic folding probes (12) for the nonenzyme protein, transthyretin (TTR), were developed and scrutinized. We show that destabilized mutant RA and TTR proteins partition into folded and functional as well as misfolded soluble conformations and that this partitioning is sensitive to proteostasis network perturbations. Experiments show that a snapshot of the distribution between folded and functional vs. soluble and misfolded conformational states can be preserved during the small molecule folding probe labeling period, provided that the cellular chaperone holdase activity is sufficient, achieved by rapid ATP depletion in parallel with cell lysis. Sufficient chaperone/chaperonin holdase activity minimizes changes in the folded and functional concentration associated with probe binding and reaction with the POI and renders the relative folding and conjugation rates much less influential.