A pH-Responsive Virus-Like Particle as a Protein Cage for a Targeted Delivery.

A stimuli-responsive protein self-assembly offers promising utility as a protein nanocage for biotechnological and medical applications. Herein, the development of a virus-like particle (VLP) that undergoes a transition between assembly and disassembly under a neutral and acidic pH, respectively, for a targeted delivery is reported. The structure of the bacteriophage P22 coat protein is used for the computational design of coat subunits that self-assemble into a pH-responsive VLP. Subunit designs are generated through iterative computational cycles of histidine substitutions and evaluation of the interaction energies among the subunits under an acidic and neutral pH. The top subunit designs are tested and one that is assembled into a VLP showing the highest pH-dependent structural transition is selected. The cryo-EM structure of the VLP is determined, and the structural basis of a pH-triggered disassembly is delineated. The utility of the designed VLP is exemplified through the targeted delivery of a cytotoxic protein cargo into tumor cells in a pH-dependent manner. These results provide strategies for the development of self-assembling protein architectures with new functionality for diverse applications.

Construction and Functionalization of a Clathrin Assembly for a Targeted Protein Delivery.

Protein assemblies have drawn much attention as platforms for biomedical applications, including gene/drug delivery and vaccine, due to biocompatibility and functional diversity. Here, the construction and functionalization of a protein assembly composed of human clathrin heavy chain and light chain for a targeted protein delivery, is presented. The clathrin heavy and light chains are redesigned and associated with each other, and the resulting triskelion unit further self-assembled into a clathrin assembly with the size of about 28 nm in diameter. The clathrin assembly is dual-functionalized with a protein cargo and a targeting moiety using two different orthogonal protein-ligand pairs through one-pot reaction. The functionalized clathrin assembly exhibits about a 900-fold decreased KD value for a cell-surface target due to avidity compared to a native targeting moiety. The utility of the clathrin assembly is demonstrated by an efficient delivery of a protein cargo into tumor cells in a target-specific manner, resulting in a strong cytotoxic effect. The present approach can be used in the creation of protein assemblies with multimodality.

Total intravenous anesthesia without muscle relaxant for pulmonary wedge resection in a patient with amyotrophic lateral sclerosis: a case report.

Muscle relaxants may exacerbate the symptoms of amyotrophic lateral sclerosis (ALS). Furthermore, ALS patients often experience respiratory muscle weakness. Herein, we report the case of a 63-year-old man with ALS who underwent pulmonary wedge resection using total intravenous anesthesia without muscle relaxant and single lumen endotracheal tube. After an unremarkable surgical procedure, the patient was transferred to the intensive care unit after extubation. The patient did not experience any worsening of ALS symptoms over the one-year follow-up period. Our experience shows that total intravenous anesthesia without muscle relaxants can be used as an anesthetic method for lung surgery in ALS patients. We report this case along with a brief literature review.

Preparation of amine-functionalized lignins for the selective adsorption of Methylene blue and Congo red.

Research on low-cost bio-adsorbents for the removal of harmful substances from effluents has recently attracted significant attention. In this study, three types of amino-silane-modified lignins (ASLs) with primary, secondary, and tertiary amine groups were prepared, and their adsorption behavior toward cationic and anionic dyes was investigated. Chemical structural analyses indicated that the three amino-silane reagents resulted in different molecular self-assembly structures on the lignin surface. The ASLs exhibited enhanced thermal stabilities and increased surface areas with different surface charges in different pH ranges. Owing to the high density of primary, secondary, and tertiary amine groups, the ASLs exhibited excellent adsorption capacities for cationic and anionic dyes. Additionally, they selectively adsorb anionic and cationic dyes according to the pH conditions. The ASL with primary amine had the highest adsorption capacity for Methylene blue and Congo red, reaching 187.27 and 293.26 mg·g-1, respectively, followed by ASLs with the secondary amine and tertiary amine. All adsorption processes followed the Langmuir and Temkin isotherms and had pseudo-second-order kinetics. The hypothesized adsorption mechanism mainly involves electrostatic interaction, NH-π interaction, hydrogen bonding interaction and π-π interaction.

Dendrimer-Like Supramolecular Assembly of Proteins with a Tunable Size and Valency Through Stepwise Iterative Growth.

The assembly of proteins in a programmable manner provides insight into the creation of novel functional nanomaterials for practical applications. Despite many advances, however, a rational protein assembly with an easy scalability in terms of size and valency remains a challenge. Here, a simple bottom-up approach to the supramolecular protein assembly with a tunable size and valency in a programmable manner is presented. The dendrimer-like protein assembly, simply called a "protein dendrimer," is constructed through a stepwise and alternate addition of a building block protein. Starting from zeroth-generation protein dendrimer (pG0 ) of 27 kDa, the protein dendrimer is sequentially grown to pG1 , pG2 , pG3 , to pG4 with a molecular mass of 94, 216, 483, and 959 kDa, respectively. The valency of the protein dendrimers at the periphery increases by a factor of two after each generation, allowing a tunable valency and easy functionalization. The protein dendrimers functionalizes with a targeting moiety and a cytotoxic protein cargo shows a typical feature of multi-valency in the avidity and a highly enhanced cellular cytotoxicity, exemplifying their utility as a protein delivery platform. The present approach can be effectively used in the creation of protein architectures with new functions for biotechnological and medical applications.

Synthesis of lignin-modified cellulose nanocrystals with antioxidant activity via Diels-Alder reaction and its application in carboxymethyl cellulose film.

Multifunctional polymers derived from renewable organic substances have received significant attention. In this work, the Diels-Alder "click" reaction was used to synthesize a renewable copolymer with the mechanical strength of cellulose nanocrystals (CNCs) and the natural antioxidant activity of lignin. Chemical structural analysis and molecular weight results confirmed that lignin was successfully attached to the CNCs. Phenolic hydroxyl groups were introduced into CNCs, resulting in good antioxidant activity with an IC50 value of 1.49 mg/mL, although a slight decrease in the crystallinity index and thermal properties was observed. Additionally, carboxymethyl cellulose (CMC) films containing lignin-modified CNCs were prepared by solution casting. The lignin-modified CNCs endowed film with antioxidant activity and also contributed to increasing the tensile strength by 70%. This indicated that the lignin-modified CNCs with good antioxidant activities and mechanical strength have promising applications in multifunctional materials.

A Novel Biosorbent From Hardwood Cellulose Nanofibrils Grafted With Poly(m-Aminobenzene Sulfonate) for Adsorption of Cr(VI).

Cellulose from different lignocellulosic biomass can be used to prepare various materials. In this work, the cellulose nanofibrils were produced from hardwood bleached kraft pulp. Then, a novel biosorbent from cellulose nanofibrils grafted with poly(m-aminobenzene sulfonate) (PABS) was prepared for effective detoxification and adsorption of Cr(VI) in an aqueous medium. 6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanofibrils (TOCNF) with a high aspect ratio was used as an adsorbent matrix. PABS, an amine-rich conductive polymer, was grafted onto TOCNF via a successive two-step reaction. The analyses of Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS) confirmed the successful grafting reaction between TOCNF and PABS. The biosorbent from TOCNF-bonded PABS with the nitrogen content of 7.0% was synthesized. It exhibited excellent Cr(VI) adsorption capacity at a solution pH below 3, and almost 100% Cr(VI) can be removed. The adsorption of Cr(VI) on the biosorbent was described by a pseudo-second-order model and obeyed the Langmuir model. The Cr(VI) adsorption capacity of the biosorbent from TOCNF-bonded PABS was almost 10 times higher than that of TOCNF. It was interesting to note that part of Cr(VI) ions had been reduced to Cr(III) during the adsorption process. It indicated that the biosorbent from TOCNF grafted with PABS could detoxify and adsorb Cr(VI) synchronously.

Upregulation of DJ-1 expression in melanoma regulates PTEN/AKT pathway for cell survival and migration.

Cutaneous melanoma is known to be one of the most dangerous skin cancers because of its metastatic functions. Today, it is essential to investigate specific biomarkers for the target treatment in many diseases including cancers. DJ-1 protein, also known as Parkinson disease 7, has various functions associated with cancer progression including cell survival and migration. Phosphatase and tensin homolog (PTEN) is a tumor suppressor that regulates the PI3K/AKT signaling pathway and its mutations have been reported to frequently occur in many cancers such as thyroid, breast and skin. Recently, DJ-1 has been identified as a negative regulator of PTEN in many human cancer cells. However, the impacts and relationship of DJ-1 and PTEN have not been studied yet in melanoma. To confirm the expression of DJ-1 and PTEN in melanoma compared to normal skin tissues and find out functions of DJ-1 in melanoma cells, Western blot analysis and immunohistochemical staining were used. Transfection of G361 cells with DJ-1-specific small interfering RNA was performed to figure out the roles of DJ-1 and the relationship between DJ-1 and PTEN in melanoma cells. In our study, the DJ-1 protein was significantly increased with loss of PTEN protein in melanoma compared to that in normal skin. Inhibition of DJ-1 in G361 cells induced apoptosis, and suppressed cell survival and migration. Furthermore, suppression of DJ-1 in G361 cells increased the expression of cleaved caspase-3, cleaved PARP, Bax, p53, and Daxx as well as PTEN, while it decreased expression of survivin, caspase-3, and PARP. Also, downregulated DJ-1 inhibited phosphorylation of AKT in G361 cells. Collectively, DJ-1 overexpression could affect the proliferative and invasive capabilities of melanoma cells via regulating the PTEN/AKT pathway and apoptosis-related proteins. This study suggests that DJ-1 may be a potential target for the treatment of melanoma.

Effects of finish rolling temperature and yield ratio on variations in yield strength after pipe-forming of API-X65 line-pipe steels.

Flattened plates often show the lower or higher yield strength than initial leveled plates because tensile and compressive strains are repeatedly experienced at outer and inner walls during the pipe-forming and flattening, but reasons for the yield-strength variation after the pipe-forming are not sufficiently verified yet. In this study, ten line-pipe steels were fabricated by controlling alloying elements and finish rolling temperatures (FRTs), and the yield strength of pipe-flattened steel plates was predicted by using cyclic simulation tests, based on competing contributions of Bauschinger effect (BE) and strain hardening (SH) effect quantified from yield drop (YD) and yield rise (YR) parameters, respectively. High-FRT-treated steels (H steels) showed the lower BE and the higher SH than low-FRT-treated steels (L steels), thereby resulting in the smaller yield-strength reduction. This lower BE in the H steels was caused by the lower total boundary density, while the higher SH was caused by the higher fraction of granular bainite. According to the SH analyses between the YR parameters obtained from cyclic simulation tests and the yield ratios obtained from ordinary tensile tests, the decrease in yield-strength reduction with decreasing yield ratio was not attributed to the increase in ordinary tensile SH but to the increase in YR parameter.

One-step silanization and amination of lignin and its adsorption of Congo red and Cu(II) ions in aqueous solution.

In this work, silanized and aminated lignin (SAL) was synthesized in one step and its adsorption of Congo red and Cu(II) ions in aqueous solution was explored. Lignin was subjected to amine-silanization with (3-aminopropyl) triethoxysilane (APTES). Structural characterization substantiated successful amine-silanzation of lignin and formation of multi-layer APTES intermolecular crosslinked structure. The prepared SAL (nitrogen content = 6.1%) exhibited enhanced molecular weight, thermal stability, and water- and organic solvent-resistance properties. Additionally, the present of the porous structure of particle surface and an increase in the specific surface area and zeta potential promoted the accessibility of contaminants to the effective adsorption sites of SAL. Adsorption experiments showed that both Congo red and Cu(II) ion could be completely removed at original pH value, and their adsorption involved electrostatic attraction and complexation, respectively. The adsorption isotherms and kinetics were well described by the Langmuir and pseudo-second-order equations, respectively. The results showed that SAL is a promising adsorbent for the treatment of effluents.

Overexpression of Nrf2 promotes colon cancer progression via ERK and AKT signaling pathways.

PURPOSE: We investigated the expression of Nrf2 in colorectal cancer and its correlation with clinicopathological characteristics as well as mechanisms and roles of Nrf2 expression including cell signaling pathway, survival, proliferation, and migration. METHODS: Nrf2 expression was measured in 12 and 30 different colorectal cancer (CRC) tissues by western blot (WB) and immunohistochemistry (IHC), respectively. SW480 cells were used for cell proliferation and cell migration tests. The correlation between the expression of Nrf2 and clinicopathologic parameters were evaluated using the chi-square or Fisher exact test. Data are expressed as the mean ± standard deviation for 3 independent experiments. P < 0.05 was considered statistically significant. RESULTS: Analysis of WB demonstrated that Nrf2 proteins were increased in CRC tissues, and decreased in normal tissues. IHC staining showed that the Nrf2 expression was elevated in CRC tissues, compared to matched normal tissues. When SW480 cells were suppressed with small interfering RNA of Nrf2, cell viability was inhibited, and cell apoptosis was increased. These results were found along with suppression of the phosphorylated form of extracellular signal-regulated kinase 1/2 and AKT. CONCLUSION: This study suggests that overexpression of Nrf2 may be related to carcinogenesis and progression of CRC.

Yield-strength prediction of flattened steel pipes by competing Bauschinger effect and strain hardening during pipe-forming.

Since flattened steel sheets often show the unexpectedly lower or higher yield strength than leveled sheets, unceasing efforts have been made to accurately predict the yield strength in pipe-forming industries. In the present investigation, the yield strength of line-pipe or casing-pipe steels was predicted by competing Bauschinger effect and strain hardening occurred during the pipe-forming. Yield drop (YD) and yield rise (YR) parameters were newly defined from cyclic simulation analyses of outer and inner walls of pipes to express more reasonably the Bauschinger effect and strain hardening. The YD increased abruptly until the pre-strain of about 1%, and then saturated, while the YR increased linearly with increasing pre-strain. By combining the YD and YR, the variation in yield strength (Δσ) showed a down-and-up behavior as the Bauschinger effect and strain hardening were dominant at low and high pre-strains, respectively, and plausibly explained the relationship of Δσ and piping strain used in pipe-forming industries. According to the microstructural analyses related to the down-and-up Δσ behavior, the polygonal ferrite reduced the yield-strength reduction in the low pre-strain range, whereas the granular bainite or pearlite expanded it. This yield strength prediction coupled with microstructural analyses provide a good idea for designing and reliably predicting the yield strength of in various steel pipes.

Platinum Supported Nitrogen-Doped Carbon Nanotubes/ZIF-8 Derived Carbon Composite Electrodes for a Methanol Oxidation.

Pt-supported on ZIF-8 derived porous carbon (CZIF8) and nitrogen-doped multi-walled carbon nanotubes (NCNT) composites was prepared by pyrolysis process and chemical reduction. The electrochemical characterization and morphological analysis of composites was measured by cyclic voltammetry (CV), chronoamperometry, transmission electron microscope (TEM), and fourier transform infrared spectroscopy (FT-IR). These results indicate that the Pt-NCNT@CZIF8 nanocomposite showed electrochemically superior properties to methanol oxidation reaction and the higher electrochemical surface area (ECSA). Also, ZIF-8 derived carbon and modified the CNTs was thought to enhance the effective area of the platinum deposition site.

Characterization of solvent-fractionated lignins from woody biomass treated via supercritical water oxidation.

Crude supercritical lignin (SCL) extracted from hardwood (Quercus mongolica) treated via supercritical water (SCW) oxidation was subjected to sequential fractionation with four organic solvents; five lignin fractions (F1-F4 and FIN) were thus obtained. The molecular weight (MW) of the fractionated lignins gradually increased as fractionation proceeded. However, the content of methoxyl groups and phenolic hydroxyl groups tended to decrease with increasing molecular weight of the lignins. The functional groups of SCL and the fractionated lignins were very similar based on Fourier-transform infrared analysis. The syringyl/guaiacyl ratio (S/G ratio) of the fractionated lignins increased with an increase in the MW. The thermal stability decreased with decreasing MW of the fractionated lignins, and all fractions except for F1 had a maximum degradation temperature of around 360 °C. The glass transition temperature (Tg) of the fractions increased from 83 °C to 137 °C with increasing MW.

Pro-oxidant status and Nrf2 levels in psoriasis vulgaris skin tissues and dimethyl fumarate-treated HaCaT cells.

Reactive oxygen species (ROS) contribute to pathogenesis of many inflammatory skin diseases, including psoriasis. The aim of this study is to compare antioxidant protein expression in psoriasis vulgaris (PV) skin tissues with that in normal skin tissues in vivo and to evaluate the effects of dimethyl fumarate (DMF), used for the treatment of psoriasis, on ROS generation and apoptosis in a human keratinocyte cell line HaCaT. Compared with normal skin tissues, PV skin tissues showed increased protein oxidation as well as down-regulation of Nrf2 and its regulatory proteins such as HO-1 and AKR1C3. Using HaCaT cells to model DMF-induced pro-oxidant effects in the skin cells, we found that DMF treatment induced increased ROS levels and apoptotic cell death, as signified by increased proportion of cells with Annexin V-PE(+) staining and a sub-G0/G1 peak in the cell cycle. Preceding these changes, DMF treatment resulted in up-regulation of Nrf2, HO-1, and AKR1C3 proteins in these cells. Collectively, increased oxidative stress and impaired cellular anti-oxidant enzyme systems may participate in the pathogenesis of PV. DMF may exert an additive therapeutic efficacy in PV by attenuating the redox burden and subsequent oxidative damage to normal keratinocytes through activation of Nrf2 pathway relative to PV.

Cariporide Enhances the DNA Damage and Apoptosis in Acid-tolerable Malignant Mesothelioma H-2452 Cells.

The Na+/H+ exchanger is responsible for maintaining the acidic tumor microenvironment through its promotion of the reabsorption of extracellular Na+ and the extrusion of intracellular H+. The resultant increase in the extracellular acidity contributes to the chemoresistance of malignant tumors. In this study, the chemosensitizing effects of cariporide, a potent Na+/H+-exchange inhibitor, were evaluated in human malignant mesothelioma H-2452 cells preadapted with lactic acid. A higher basal level of phosphorylated (p)-AKT protein was found in the acid-tolerable H-2452AcT cells compared with their parental acid-sensitive H-2452 cells. When introduced in H-2452AcT cells with a concentration that shows only a slight toxicity in H-2452 cells, cariporide exhibited growth-suppressive and apoptosis-promoting activities, as demonstrated by an increase in the cells with pyknotic and fragmented nuclei, annexin V-PE(+) staining, a sub-G0/G1 peak, and a G2/M phase-transition delay in the cell cycle. Preceding these changes, a cariporide-induced p-AKT down-regulation, a p53 up-regulation, an ROS accumulation, and the depolarization of the mitochondrial-membrane potential were observed. A pretreatment with the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 markedly augmented the DNA damage caused by the cariporide, as indicated by a much greater extent of comet tails and a tail moment with increased levels of the p-histone H2A.X, p-ATMSer1981, p-ATRSer428, p-CHK1Ser345, and p-CHK2Thr68, as well as a series of pro-apoptotic events. The data suggest that an inhibition of the PI3K/AKT signaling is necessary to enhance the cytotoxicity toward the acid-tolerable H-2452AcT cells, and it underlines the significance of proton-pump targeting as a potential therapeutic strategy to overcome the acidic-microenvironment-associated chemotherapeutic resistance.

Minimum effective local anesthetic volume for surgical anesthesia by subparaneural, ultrasound-guided popliteal sciatic nerve block: A prospective dose-finding study.

Because of its rapid onset time, recent years have seen an increase in the use of ultrasound (US)-guided popliteal sciatic nerve block (PSNB) via subparaneural injection for induction of surgical anesthesia. Moreover, in below-knee surgery, combined blocks, as opposed to sciatic nerve block alone, have become more common. These combined blocks often require a large volume of local anesthetic (LA), thus increasing the risk of local-anesthetic systemic toxicity (LAST). Thus, to decrease the risk of LAST, it is important to know the minimum effective volume (MEV) required for an adequate block. We, therefore, aimed to determine the MEV of ropivacaine 0.75% for induction of surgical anesthesia by the method of US-guided popliteal sciatic nerve block via subparaneural injection.Thirty patients underwent a US-guided PSNB with ropivacaine 0.75% at a 20-mL starting volume. Using a step-up/step-down method, we determined injection volumes for consecutive patients from the preceding patient's outcome. When an effective block was achieved within 40 minutes after injection, the next patient's volume was decreased by 2 mL. If the block failed, the next patient's volume was increased by 2 mL. The sensory and motor blockade was graded according to a 4-point scale. The block was considered a success if a combination of anesthesia and paresis (a score of 3 for both the sensory and motor nerves) was achieved within 40 minutes. The primary outcome measure was the MEV resulting in a successful subparaneural block of the sciatic nerve in 50% of patients (MEV50). Additionally, the data were processed with a probit regression analysis to determine the volume required to produce a complete sciatic nerve block in 90% of subjects (ED90).The MEV50 of 0.75% ropivacaine is 6.14 mL (95% confidence interval, 4.33-7.94 mL). The ED90 by probit analysis for a subparaneural injection was 8.9 mL (95% CI, 7.09-21.75 mL).The 6.14-mL MEV50 of ropivacaine 0.75% represents a 71% reduction in volume compared with neurostimulation techniques and a 14.7% reduction in volume compared with US-guided PSNB using the alternative perineural injection technique.

Assessment of pupillary light reflex using a smartphone application.

In unconscious patients, pupillary light reflex is an indicator of brain damage. In the current study, a smartphone application was developed for the purpose of measuring pupillary light reflex with an aim to determine the agreement between pupillary light reflex measurements using a smartphone application (APP) and a penlight (PEN). The APP acquires five sequential photographs using the camera flash in order to stimulate the pupil. The initial image is captured prior to the flash, and the subsequent image is obtained while the flash is on. The remaining three images are captured whilst the flash is off. Pupillary right reflex was assessed in 30 healthy subjects using a PEN. After 10 min, the examiners inspected the images of light reflex acquired from the same subjects using the APP, and completed the corresponding questionnaire containing details of pupil size and degree of response. Agreement between the two assessment methods was determined by calculating bias, limits of agreement, and the intraclass correlation (ICC) coefficient. A statistically significant difference was not observed between the two methods regarding pupil size and degree of response. Bias was 0.1 mm and limits of agreement were ±1.5 mm, as compared with PEN. ICC was 0.93 (95% confidence interval, 0.89-0.96). Therefore, it may be concluded that the results of pupillary light reflex assessed by PEN and APP display no significant difference. Furthermore, the APP provides advantages such as portability, objectivity and the possibility of being used as objective medical evidence.