Straw lignin degradation by lignin peroxidase from Irpex lacteus cooperated with enzymes and small molecules.

OBJECTIVES: Maximizing the utility value of enzymes was achieved by exploring the effects of small molecules on the efficiency of lignin degradation by lignin peroxidase. METHODS: Using wheat straw as raw material and taking lignin degradation rate as index, it was found that laccase, glucose oxidase, malonic acid, citric acid, ZnSO4, CaCl2 could promote the lignin degradation by the lignin peroxidase from Irpex lacteus, respectively. Moreover, glucose oxidase, malonic acid and CaCl2 had obvious synergy effects on lignin degradation by the lignin peroxidase. RESULTS: The optimal conditions of lignin degradation were obtained by response surface experiment: 4% glucose oxidase, 0.74% malonic acid and 3.29% CaCl2 were added for synergistic degradation at 37 â„ƒ with 50% of water content. After 72 h quickly enzymatic hydrolysis, the degradation rate of lignin was 45.84%. CONCLUSIONS: A new green and efficient method for lignin removal from straw was obtained, which provided a reference for the efficient utilization of straw and lignin peroxidase.

Identification and Characterization of the HbPP2C Gene Family and Its Expression in Response to Biotic and Abiotic Stresses in Rubber Tree.

Plant PP2C genes are crucial for various biological processes. To elucidate the potential functions of these genes in rubber tree (Hevea brasiliensis), we conducted a comprehensive analysis of these genes using bioinformatics methods. The 60 members of the PP2C family in rubber tree were identified and categorized into 13 subfamilies. The PP2C proteins were conserved across different plant species. The results revealed that the HbPP2C genes contained multiple elements responsive to phytohormones and stresses in their promoters, suggesting their involvement in these pathways. Expression analysis indicated that 40 HbPP2C genes exhibited the highest expression levels in branches and the lowest expression in latex. Additionally, the expression of A subfamily members significantly increased in response to abscisic acid, drought, and glyphosate treatments, whereas the expression of A, B, D, and F1 subfamily members notably increased under temperature stress conditions. Furthermore, the expression of A and F1 subfamily members was significantly upregulated upon powdery mildew infection, with the expression of the HbPP2C6 gene displaying a remarkable 33-fold increase. These findings suggest that different HbPP2C subgroups may have distinct roles in the regulation of phytohormones and the response to abiotic and biotic stresses in rubber tree. This study provides a valuable reference for further investigations into the functions of the HbPP2C gene family in rubber tree.

Old Dog New Tricks: PLGA Microparticles as an Adjuvant for Insulin Peptide Fragment-Induced Immune Tolerance against Type 1 Diabetes.

Poly[lactic-co-(glycolic acid)] (PLGA) is arguably one of the most versatile synthetic copolymers used for biomedical applications. In vivo delivery of multiple substances including cells, pharmaceutical compounds, and antigens has been achieved by using PLGA-based micro-/nanoparticles although, presently, the exact biological impact of PLGA particles on the immune system remains controversial. Type 1 diabetes (T1D) is one subtype of diabetes characterized by the attack of immune cells against self-insulin-producing pancreatic islet cells. Considering the autoimmune etiology of T1D and the recent use of PLGA particles for eliciting desired immune responses in various aspects of immunotherapy, for the present study, a combination of Ins29-23 peptide (a known autoantigen of T1D) and PLGA microparticles was selected for T1D prevention assessment in nonobese diabetic (NOD) mice, a well-known animal model with spontaneous development of T1D. Thus, inoculation of PLGA microparticles + Ins29-23 completely prevented T1D development, significantly better than untreated controls and mice treated by either PLGA microparticles or Ins29-23 per se. Subsequent mechanistic investigation further revealed a facilitative role of PLGA microparticles in immune tolerance induction. In summary, our data demonstrate an adjuvant potential of PLGA microparticles in tolerance induction and immune remodulation for effective prevention of autoimmune diseases such as T1D.

Nanoparticles with CD44 Targeting and ROS Triggering Properties as Effective in Vivo Antigen Delivery System.

Currently, development of subunit vaccine based on recombinant antigens or peptides has gradually become an important alternative option for traditional vaccine. However, induction of potent immune response with desired efficacy remains a major challenge. The nanoparticle-based antigen delivery system has been considered a potential carrier system to improve the efficacy of subunit vaccine. In the present study, we have designed an immune-stimulatory delivery system by conjugating three-armed PLGA to PEG via the peroxalate ester bond which is sensitive to hydrogen peroxide (H2O2), a major reactive oxygen species (ROS). Hyaluronic acid (HA), a ligand for CD44 receptors was also modified onto the outer shell of the 3s-PLGA-PEG nanoparticles to promote immune cell uptake. For in vitro and in vivo immune response assessment, a model antigen ovalbumin (OVA) was enclosed within the core of the 3s-PLGA-PEG nanoparticles to form 3s-PLGA-PO-PEG/HA nanoparticles (PHO NPs). Our results showed that the PHO NPs enhanced dendritic cell maturation, antigen uptake, and antigen presentation in vitro, likely due to enhanced lysosomal escape. In vivo experiments further revealed that the PHO nanovaccine robustly promoted OVA-specific antibody production and T cell response accompanied by modest stimulation of memory T cells. In summary, the ROS-responsive PHO NPs with modified HA may be an effective vehicle antigen delivery system to promote antigen-induced immune response.

Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase.

BACKGROUND: Pyruvate decarboxylase (PDC) is a well-known pathway for ethanol production, but has not been demonstrated for high titer ethanol production at temperatures above 50 °C. RESULT: Here we examined the thermostability of eight PDCs. The purified bacterial enzymes retained 20% of activity after incubation for 30 min at 55 °C. Expression of these PDC genes, except the one from Zymomonas mobilis, improved ethanol production by Clostridium thermocellum. Ethanol production was further improved by expression of the heterologous alcohol dehydrogenase gene adhA from Thermoanaerobacterium saccharolyticum. CONCLUSION: The best PDC enzyme was from Acetobactor pasteurianus. A strain of C. thermocellum expressing the pdc gene from A. pasteurianus and the adhA gene from T. saccharolyticum was able to produce 21.3 g/L ethanol from 60 g/L cellulose, which is 70% of the theoretical maximum yield.

Metaproteomics reveals enzymatic strategies deployed by anaerobic microbiomes to maintain lignocellulose deconstruction at high solids.

Economically viable production of cellulosic biofuels requires operation at high solids loadings-on the order of 15 wt%. To this end we characterize Nature's ability to deconstruct and utilize mid-season switchgrass at increasing solid loadings using an anaerobic methanogenic microbiome. This community exhibits undiminished fractional carbohydrate solubilization at loadings ranging from 30 g/L to 150 g/L. Metaproteomic interrogation reveals marked increases in the abundance of specific carbohydrate-active enzyme classes. Significant enrichment of auxiliary activity family 6 enzymes at higher solids suggests a role for Fenton chemistry. Stress-response proteins accompanying these reactions are similarly upregulated at higher solids, as are ß-glucosidases, xylosidases, carbohydrate-debranching, and pectin-acting enzymes-all of which indicate that removal of deconstruction inhibitors is important for observed undiminished solubilization. Our work provides insights into the mechanisms by which natural microbiomes effectively deconstruct and utilize lignocellulose at high solids loadings, informing the future development of defined cultures for efficient bioconversion.

Mechanism of a long-term controlled drug release system based on simple blended electrospun fibers.

BACKGROUND: Drug delivery systems based on electrospun fibers have been under development for many years. However, studies of controllable long-term drug release from electrospun membrane systems and the underlying release mechanisms have seldom been reported. METHODS: In this study, electrospun membrane drug delivery systems consisting of the antibiotic ciprofloxacin hydrochloride and FDA-approved polymers are fabricated. Different second-component polymers are introduced to change the properties of a poly(d,l-lactide-co-glycolide) (PLGA) matrix, thereby altering the drug release behavior. On the basis of observations of morphology, cumulative release profiles, and determinations of release duration, the drug release kinetics and critical characteristics influencing drug release behavior are discussed. RESULTS: It is found that the drug release profiles can be divided into three stages according to the rate of drug release. Stage I is controlled by fiber swelling and diffusion according to Fick's second law. Stage II is controlled by diffusion through a fused membrane structure, which results in very slow drug release. Stage III is controlled by polymer degradation and involves release of the remaining drug. CONCLUSIONS: The results of this study of release mechanisms should provide a basis for adjustments of drug release dosage and duration, thereby contributing to the development of drug delivery systems satisfying clinical requirements.

Protein delivery nanosystem of six-arm copolymer poly(ε-caprolactone)-poly(ethylene glycol) for long-term sustained release.

BACKGROUND: To address the issue of delivery of proteins, a six-arm copolymer, six-arm poly (ε-caprolactone)-poly(ethylene glycol) (6S-PCL-PEG), was synthesized by a simple two-step reaction. Thereafter, the application of 6S-PCL-PEG as a protein carrier was evaluated. MATERIALS AND METHODS: A six-arm copolymer, six-arm poly(ε-caprolactone) (6S-PCL), was synthesized by ring-opening polymerization, with stannous octoate as a catalyst and inositol as an initiator. Then, poly(ethylene glycol) (PEG) was linked with 6S-PCL by oxalyl chloride to obtain 6S-PCL-PEG. Hydrogen-1 nuclear magnetic resonance spectrum, Fourier-transform infrared spectroscopy, and gel-permeation chromatography were conducted to identify the structure of 6S-PCL-PEG. The biocompatibility of the 6S-PCL-PEG was evaluated by a cell counting kit-8 assay. Polymeric nanoparticles (NPs) were prepared by a water-in-oil-in-water double emulsion (W1/O/W2) solvent evaporation method. The size distribution and zeta potential of NPs were determined by dynamic light scattering. Transmission electron microscopy was used to observe the morphology of NPs. Drug-loading capacity, encapsulation efficiency, and the release behavior of ovalbumin (OVA)-loading NPs were tested by the bicinchoninic acid assay kit. The stability and activity of OVA released from NPs were detected and the uptake of NPs was evaluated by NIH-3T3 cells. RESULTS: All results indicated the successful synthesis of amphiphilic copolymer 6S-PCL-PEG, which possessed excellent biocompatibility and could formulate NPs easily. High drug-loading capacity and encapsulation efficiency of protein NPs were observed. In vitro, OVA was released slowly and the bioactivity of OVA was maintained for over 28 days. CONCLUSION: 6S-PCL-PEG NPs prepared in this study show promising potential for use as a protein carrier.

Improved vaccine-induced immune responses via a ROS-triggered nanoparticle-based antigen delivery system.

Subunit vaccines that are designed based on recombinant antigens or peptides have shown promising potential as viable substitutes for traditional vaccines due to their better safety and specificity. However, the induction of adequate in vivo immune responses with appropriate effectiveness remains a major challenge for vaccine development. More recently, the implementation of a nanoparticle-based antigen delivery system has been considered a promising approach to improve the in vivo efficacy for subunit vaccine development. Thus, we have designed and prepared a nanoparticle-based antigen delivery system composed of three-armed PLGA, which is conjugated to PEG via the peroxalate ester bond (3s-PLGA-PO-PEG) and PEI as a cationic adjuvant (PPO NPs). It is known that during a foreign pathogen attack, NADPH, an oxidase, of the host organism is activated and generates an elevated level of reactive oxygen species, hydrogen peroxide (H2O2) primarily, as a defensive mechanism. Considering the sensitivity of the peroxalate ester bond to H2O2 and the cationic property of PEI for the induction of immune responses, this 3s-PLGA-PO-PEG/PEI antigen delivery system is expected to be both ROS responsive and facilitative in antigen uptake without severe toxicity that has been reported with cationic adjuvants. Indeed, our results demonstrated excellent loading capacity and in vitro stability of the PPO NPs encapsulated with the model antigen, ovalbumin (OVA). Co-culturing of bone marrow dendritic cells with the PPO NPs also led to enhanced dendritic cell maturation, antigen uptake, enhanced lysosomal escape, antigen cross-presentation and in vitro CD8+ T cell activation. In vivo experiments using mice further revealed that the administration of the PPO nanovaccine induced robust OVA-specific antibody production, upregulation of splenic CD4+ and CD8+ T cell proportions as well as an increase in memory T cell generation. In summary, we report here a ROS-triggered nanoparticle-based antigen delivery system that could be employed to promote the in vivo efficacy of vaccine-induced immune responses.

Cellulosic ethanol: status and innovation.

Although the purchase price of cellulosic feedstocks is competitive with petroleum on an energy basis, the cost of lignocellulose conversion to ethanol using today's technology is high. Cost reductions can be pursued via either in-paradigm or new-paradigm innovation. As an example of new-paradigm innovation, consolidated bioprocessing using thermophilic bacteria combined with milling during fermentation (cotreatment) is analyzed. Acknowledging the nascent state of this approach, our analysis indicates potential for radically improved cost competitiveness and feasibility at smaller scale compared to current technology, arising from (a) R&D-driven advances (consolidated bioprocessing with cotreatment in lieu of thermochemical pretreatment and added fungal cellulase), and (b) configurational changes (fuel pellet coproduction instead of electricity, gas boiler(s) in lieu of a solid fuel boiler).

Pre-instillation of tumor microparticles enhances intravesical chemotherapy of nonmuscle-invasive bladder cancer through a lysosomal pathway.

Nonmuscle-invasive bladder cancer (NMIBC) is treated with transurethral resection followed by intravesical chemotherapy. However, drug-resistant tumorigenic cells cannot be eliminated, leading to half of the treated cancers recur with increased stage and grade. Innovative approaches to enhance drug sensitivity and eradicate tumorigenic cells in NMIBC treatment are urgently needed. Here, we show that pre-instillation of tumor cell-derived microparticles (T-MP) as natural biomaterials markedly enhance the inhibitory effects of intravesical chemotherapy on growth and hematuria occurrence of orthotropic bladder cancer in mice. We provide evidence that T-MPs enter and increase the pH value of lysosomes from 4.6 to 5.6, leading to the migration of drug-loaded lysosomes along microtubule tracks toward the nucleus and discharging the drugs whereby for the entry of the nucleus. We propose that T-MPs may function as a potent sensitizer for augmenting NMIBC chemotherapy with unprecedented clinical benefits.

Synthesis of three-arm block copolymer poly(lactic-co-glycolic acid)-poly(ethylene glycol) with oxalyl chloride and its application in hydrophobic drug delivery.

PURPOSE: Synthesis of star-shaped block copolymer with oxalyl chloride and preparation of micelles to assess the prospect for drug-carrier applications. MATERIALS AND METHODS: Three-arm star block copolymers of poly(lactic-co-glycolic acid) (3S-PLGA)-polyethylene glycol (PEG) were synthesized by ring-opening polymerization, then PEG as the hydrophilic block was linked to the terminal hydroxyl of 3S-PLGA with oxalyl chloride. Fourier-transform infrared (FT-IR) spectroscopy, gel-permeation chromatography (GPC), hydrogen nuclear magnetic resonance (1H-NMR) spectra, and differential scanning calorimetry were employed to identify the structure and properties of 3S-PLGA-PEG. Rapamycin (RPM)-loaded micelles were prepared by solvent evaporation, and pyrene was used as the fluorescence probe to detect the critical micelle concentration of the copolymer. The particle size, distribution, and ζ-potential of the micelles were determined by dynamic light scattering, and the morphology of the RPM-loaded micelles was analyzed by transmission electron microscopy. High-performance liquid chromatography was conducted to analyze encapsulation efficiency and drug-loading capacity, as well as the release behavior of RPM-loaded micelles. The biocompatibility of material and the cytostatic effect of RPM-loaded micelles were investigated by Cell Counting Kit 8 assay. RESULTS: FT-IR, GPC, and 1H-NMR suggested that 3S-PLGA-PEG was successfully synthesized. The RPM-loaded micelles prepared with the 3S-PLGA-PEG possessed good properties. The micelles had good average diameter and encapsulation efficiency. For in vitro release, RPM was released slowly from 3S-PLGA-PEG micelles, showing that 3S-PLGA-PEG-RPM exhibited a better and longer antiproliferative effect than free RPM. CONCLUSION: In this study, we first used oxalyl chloride as the linker to synthesize 3S-PLGA-PEG successfully, and compared with reported literature, this method shortened the reaction procedure and improved the reaction yield. The micelles prepared with this material proved suitable for drug-carrier application.

The Effectiveness and Safety of Thermocoagulation Radiofrequency Treatment of the Ophthalmic Division (V1) and/or Maxillary (V2) and Mandibular (V3) Division in Idiopathic Trigeminal Neuralgia: An Observational Study.

BACKGROUND: Trigeminal neuralgia (TN) is a pain appearing in the ophthalmic (V1), maxillary (V2), and mandibular (V3) trigeminal branches. Pharmacologic treatment is the first line for TN; however, many patients prefer to receive minimally invasive treatment rather than medicine because of intolerable side effects. Thermocoagulation radiofrequency (TRF) is a minimally invasive treatment that has been shown to effectively treat the maxillary (V2) and mandibular (V3) divisions, but the safety of TRF treatment of the ophthalmic (V1) division has been controversial. OBJECTIVE: This study was to observe the effectiveness and safety of TRF treatment of the ophthalmic (V1) division of trigeminal branches in idiopathic TN patients. STUDY DESIGN: An observational study. SETTING: All of patients received temperature controlled TRF, the effectiveness and safety of TRF was assessed by VAS and complications. METHODS: Eighty patients with ophthalmic division (V1) or ophthalmic division (V1) combined with maxillary (V2) or mandibular (V3) divisions of idiopathic TN were treated with step-increased temperature TRF for 6 minutes. At a pulse width of 20 ms, the temperature was titrated up 2 degrees from 60 degrees to 66 degrees every 60 seconds, and then another 66 degrees or 68 degrees for 2 minutes. Meanwhile, the tip of the cannula was turned 180 degrees with each temperature titration. Patients were assessed for pain relief and corneal reflex, numbness, and masticatory muscle weakness at one week, one month, and 3 months after the procedure. RESULTS: Eighty patients were successfully treated with temperature controlled TRF for ophthalmic (V1) division. Excellent pain relief was achieved in 79 of 80 patients (98.75%) after one week, one month, and 3 months, and 78 of 80 patients (97.5%) patients experienced tolerable numbness. Only one patient lost the corneal reflex, 14 experienced a corneal reflex that was mildly decreased, and 2 patients felt a foreign body sensation in the ipsilateral eye after TRF, but there were no corneal ulcers, incidences of blindness, or other complications. LIMITATIONS: This study is limited by being an observation study and a non-prospective trial with a short-term follow-up period. CONCLUSION: Temperature controlled TRF to the ophthalmic division (V1) of the semilular ganglion is effectiveness and safe in TN. KEY WORDS: Thermocoagulation radiofrequency, pulsed radiofrequency, trigeminal neuralgia, ophthalmic division, trigeminal ganglion, pain, numbness, corneal reflex.

Hydrogen peroxide-responsive micelles self-assembled from a peroxalate ester-containing triblock copolymer.

A novel ABA triblock copolymer by using peroxalate esters as linkages was synthesized. In water, this amphiphilic copolymer self-assembled into micelles which were used as drug delivery carriers. When stimulated with hydrogen peroxide, the micelles disassembled to release drugs since the responsive peroxalate ester linkages would be cleaved after the reaction with hydrogen peroxide.

Humeral brown tumor as first presentation of primary hyperparathyroidism caused by ectopic parathyroid adenomas: report of two cases and review of literature.

Two cases of brown tumor of the humerus caused by ectopic parathyroid adenomas were presented, which to our knowledge has not been previously documented in the international literature. There are two highlights in these two cases. First, brown tumors of the long bones may commonly involve femur and tibia, rarely involve humerus in association with primary hyperparathyroidism. Second, ectopic parathyroid adenomas of our patient had an unusual location of this disorder. We explored the role of ultrasound, MIBI scintigraphy as well as FNAB (fine needle aspiration biopsy) in diagnosis of brown tumor especially simultaneously occurrence of ectopic parathyroid adenomas and the importance of a thorough diagnostic work-up. The contemporary diagnosis and treatment options will be emphasized.