Recombinant Thrombopoietin Effectively Shortens the Time to Response and Increases Platelet Counts in Elderly Patients with Severe Immune Thrombocytopenia.

BACKGROUND: This study was conducted to investigate the short-term efficacy and safety of rhTPO for the management of severe ITP in the elderly as first-line treatment. METHODS: A total of 54 elderly patients with severe ITP were studied, including 39 patients treated with a combination regimen of rhTPO plus standard treatment (glucocorticoid; rhTPO group) and 15 patients treated with glucocorticoid treatment alone (control group). The response rate, time to initial response, peak platelet counts, and time to peak platelet counts were compared, and clinical characteristics correlated with the efficacy of rhTPO were analyzed. The efficacy of rhTPO in the elderly is comparable to the non-elderly in terms of the OR, CR, time to initial response, and peak platelet counts. RESULTS: There were no differences in the overall response (OR) and the complete response (CR) in the rhTPO group compared to the control group. The time to initial response in the rhTPO group was shorter than that in the control group (p = 0.032). In patients without intravenous immunoglobulin (IVIg) and platelet transfusion, the peak platelet counts in the rhTPO group were higher than those in the control group (p = 0.003). CONCLUSIONS: Standard glucocorticoid treatment plus rhTPO effectively shortens the time to response and increases platelet counts in the elderly with severe ITP.

Rituximab plus cladribine versus R-CHOP in frontline management of marginal zone lymphoma in China: a propensity-score matched multicenter study.

Marginal zone lymphoma (MZL) is an uncommon subtype of non-Hodgkin lymphoma (NHL). Combination of rituximab and cladribine (R-2CdA) is a potential option for indolent NHL (iNHL) and mantle cell lymphoma (MCL) patients. The goal of this multicenter retrospective study was to assess the efficacy and safety of R-2CdA in MZL to support consensus-reaching in first-line therapy in advanced-stage patients. We searched electronic medical records databases of eight centers in China. Between November 2014 and December 2019, 183 symptomatic advanced MZL patients (42 treated with R-2CdA and 141 with rituximab plus cyclophosphamide, adriamycin, vincristine, and prednisone [R-CHOP]) were identified. After propensity score matching (PSM) (1:1) to adjust for clinical characteristics, 39 patients from each treatment arm were selected. The overall response rate (ORR) (84.6% vs. 94.9%, P = 0.263) and complete response rate (59.0% vs. 66.7%, P = 0.487) were comparable between two protocols. Neither progression-free survival (PFS), including the 5-year PFS (67.7% vs. 56.1%, P = 0.352), nor overall survival was improved by R-2CdA versus R-CHOP. However, R-2CdA was more tolerable than R-CHOP in MZL patients regarding grade 3/4 hematological adverse events (odds ratio [OR] 0.565, 95% confidence interval [CI] neutropenic fever (OR 0.795, 95% CI 0.678-0.932), and infections (OR 0.800, 95% CI 0.640-1.000). Overall, our study demonstrated that R-2CdA is potentially as effective as but safer than R-CHOP in advanced MZL.

Lysozyme regulates the extracellular polymer of activated sludge and promotes the formation of electroactive biofilm.

The formation of electroactive biofilm from activated sludge on electrode surface is a key step to construct a bio-electrochemical system, yet it is greatly limited by the poor affinity between the bacteria and the electrode interface. Herein, we report a new method to promote the formation of electroactive biofilm by regulating the extracellular polymeric substance (EPS) content in activated sludge with lysozyme. The investigation of the effect of lysozyme treatment on the content of extracellular polymers and the biofilm formation of electroactive bacteria suggests that lysozyme can improve the permeability of the positive bacterial cell membrane and thus increase the EPS content in the activated sludge. The characterizations of electrochemical activity, surface morphology and community structure of the anode biofilm indicate that increasing EPS content promotes the adhesion of the mixed bacteria in the activated sludge on the electrode and results in denser biofilms with better conductivities. The microbial fuel cell (MFC) inoculated with the sludge of high EPS content exhibits the power density up to 2.195 W/m2, much higher than that inoculated with the untreated sludge (1.545 W/m2). The strategy of adjusting EPS content in activated sludge with a biological enzyme can effectively enhance the ability of the bacterial community to form biofilms and exhibits great application potentials in the construction of high efficiency bio-electrochemical systems.

Expression of an alkaline feruloyl esterases from thermophilic Chaetomium thermophilum and its boosting effect on delignification of pulp.

Exploration of feruloyl esterase (FAE) with the resistance to heat and alkali conditions in biobleaching process to improve the separation efficiency of lignocellulose is the key to achieving green papermaking. Herein, we expressed FAEB of C. thermophilum and obtained a thermostable alkaline FAE that can effectively promote the removal of lignin from pulp. The faeB gene was successfully obtained through genomic Blast strategy and high-efficiency expressed under the control of strong alcohol oxidase promoter in Pichia pastoris. The recombinant CtFAEB has an optimal temperature of 65 °C and pH of 7.0. After treated at 65 °C for 1 h, CtFAEB can still retain 63.21 % of its maximum activity, showing a good thermal stability. In addition, the recombinant CtFAEB has broad pH stability and can retain about 56 % of the maximum activity even at pH 11.0. Compared with the effect of mesophilic FAE, pretreatment with thermostable CtFAEB can promote the delignification by laccase and alkaline hydrogen peroxide from the pulp at 70 °C and pH 9.0. Alignment of the protein sequences of CtFAEB and mesophilic FAE suggested that the percentage of amino acids that easily form alpha helix in CtFAEB increases, which enhances its structural rigidity and thereby improves its thermal stability and alkali tolerance. Our study provides an effective method to obtain thermostable and alkaline FAEs, which will promote its application in biobleaching and other biorefining industries.

TRIM11 promotes lymphomas by activating the ß-catenin signaling and Axin1 ubiquitination degradation.

OBJECTIVE: Lymphoma, a malignant tumor, is mainly characterized by painless lymph node enlargement and hepatosplenomegaly. At present, lymphoma is mainly treated by radiation, chemical drugs, bone marrow transplantation and surgery. However, due to the high degree of heterogeneity, lymphomas are highly different in terms of treatment intensity and prognosis. This study is designed to investigate the function of tripartite motif-containing 11 (TRIM11) in lymphomas. METHODS: The expression of TRIM11 in lymphoma tissues and multiple lymphoma cell lines was respectively detected by microarray immunohistochemistry, real-time PCR and Western blotting. After TRIM11 knockdown, overexpression, or ß-catenin inhibitor XAV939 treatment, proliferation, apoptosis and cell cycle progression, as well as expression of related-genes were detected. Next, Co-Immunoprecipitation (Co-IP) and ubiquitination detection were performed. RESULTS: Elevated expression of tripartite motif-containing 11 (TRIM11) was observed in lymphoma tissues and multiple lymphoma cell lines (Raji, Jurkat, U937 and Hut78). Knockdown of TRIM11 in lymphoma cells significantly suppressed cell proliferation and prevented cell cycle progression from entering S or G2 phase. Concurrently, the expression of ß-catenin, Cyclin D1 and c-Myc proteins in TRIM11-silenced lymphoma cells was decreased, while Axin1 was increased. In addition, TRIM11 overexpression had an opposite effect to TRIM11 knockdown, and a ß-catenin inhibitor, XAV939, potently attenuated the induction of TRIM11 on lymphoma cells. Co-IP assay showed the interaction of TRIM11 and Axin1, and TRIM11 knockdown inhibited Axin1 ubiquitination degradation. CONCLUSIONS: Together all, the results suggested that TRIM11 may be an oncogene in lymphomas, which involving the activation of the ß-catenin signaling and the ubiquitination degradation of Axin1.

Cloning, Characterization, and Functional Expression of a Thermostable Type B Feruloyl Esterase from Thermophilic Thielavia Terrestris.

Feruloyl esterases (FAEs) have great potential applications in paper and breeding industry. A new thermo-stable feruloyl esterase gene, TtfaeB was identified from the thermophilic fungus Thielavia terrestris h408. Deduced protein sequence shares the identity of 67% with FAEB from Neurospora crassa. The expression vector pPIC9K-TtfaeB was successfully constructed and electro-transformed into GS115 strain of Pichia pastoris. One transformant with high feruloyl esterase yield was obtained through plate screening and named TtFAEB1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TtFAEB1 showed a distinct protein band appearing at the position of about 35-kDa, indicating that TtfaeB gene has been successfully expressed in P. pastoris. The recombinant TtFAEB was purified by affinity chromatography and the specific activity of purified TtFAEB was 6.06 ± 0.72 U/mg. The optimal temperature and pH for purified recombinant TtFAEB was 60 °C and 7.0, respectively. TtFAEB was thermostable, retaining 96.89 and 84.16% of the maximum activity after being treated for 1 h at 50 °C and 60 °C, respectively. Additionally, the enzyme was stable in the pH range 4.5-8.0. The homology model of TtFAEB showed that it consists of a single domain adopting a typical α/ß-hydrolase fold and contains a catalytic triad formed by Ser117, Asp201, and His260. TtFAEB in association with xylanase from Trichoderma reesei could release 77.1% of FA from destarched wheat bran. The present results indicated that the recombinant TtFAEB with excellent enzymatic properties is a promising candidate for potential applications in biomass deconstruction and biorefinery.

Influence of Randomly Inserted Feruloyl Esterase A on ß-Glucosidase Activity in Trichoderma reesei.

As a well-known industrial fungus for cellulase production, the strain RUT-C30 of Trichoderma reesei was selected to produce the feruloyl esterase A (FAEA) by a random integration protocol. The strong promoter of cellobiohydrolase 1 (cbh1) gene was used to drive the expression of FAEA. Using double-joint PCR protocol, Pcbh1-faeA-TtrpC expression cassette was successfully constructed and co-transformed into RUT C30 strain of T. reesei. One transformant with high feruloyl esterase yield (3.44 ± 0.16 IU/mL) was obtained through plate screening and named TrfaeA1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TrfaeA1 showed a distinct protein band appearing at the position of about 34 kDa, indicating that faeA gene has been successfully expressed in T. reesei. Compared with that in original RUT C30 strain, ß-glucosidase production in transformant TrfaeA1 was significantly increased by about 86.4%, reaching 63.2 IU/mL due to the random insertion of faeA. Moreover, the total secretion protein and filter paper activities of the transformant TrfaeA1 were also improved by up to 5.5 and 4.3%, respectively. The present results indicated that the random insertion strategy could be an effective and feasible method to improve and optimize the cellulase system of filamentous fungi.

Characterization and Strain Improvement of a Hypercellulytic Variant, Trichoderma reesei SN1, by Genetic Engineering for Optimized Cellulase Production in Biomass Conversion Improvement.

The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG) activity but lower ß-glucosidase (BGL) activity than those of the others. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30.0% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65.0% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion.

Role of decidual CD14(+) macrophages in the homeostasis of maternal-fetal interface and the differentiation capacity of the cells during pregnancy and parturition.

OBJECTIVE: Decidual macrophages (dMΦs) have been implicated in fetal tolerance, but little information is known regarding their differentiation capacity and interactions with T cells. The present study aimed to investigate the immunological characteristics of dMΦs at mid and term pregnancy. METHODS: The dMΦs were analyzed for their phenotypes and cytokine production by flow cytometry and ELISA, respectively. The transendothelial trafficking model was implemented to allow the dMΦs to differentiate. The differentiated cells from dMΦs were also measured for their phenotypes and cytokine production with same methods. The capacity of dMΦs or the differentiated cells from dMΦs to stimulate allogeneic T lymphocyte proliferation was evaluated by T lymphocyte stimulation assays. T cell differentiation was determined by flow cytometry. RESULTS: The dMΦs in the mid-pregnancy (Mid-dMΦs) resembled the M2 phenotype. The differentiated cells from Mid-dMΦs had little stimulatory capacity on T cell proliferation and favored regulatory T cell differentiation. The dMΦs at term differentiated into dendritic (DC)-like cells, stimulating T cell activation, proliferation, and differentiation into IFN-γ-producing T cellsdecidual CONCLUSIONS: The present study suggests that the differences in phenotypes and cytokine production between Mid- and Term-dMΦs relate to their different roles in the homeostasis of the maternal-fetal interface. Mid-dMΦs differentiate into DC-like cells with immunosuppressive properties, playing an important role in maintaining homeostasis required for a successful pregnancy. Term-dMΦs differentiate into DC-like cells with immunostimulatory properties, likely involved in the activation of labor. The different differentiation capacities of dMΦs in the varied pregnancy stages may be due to the placental microenvironment.

[Cloning of feruloyl esterase gene from Aspergillus niger h408 and high-efficient expression in Pichia pastoris].

OBJECTIVE: To achieve the high-efficiency expression of feruloyl estrase gene (AnfaeA) from Aspergillus niger h408 in Pichia pastoris and characterize the recombinant feruloyl esterase (FAE). METHODS: Using gene splicing by overlap extension (SOE), we cloned AnfaeA gene from A. niger h408 and subcloned into T vector for sequencing analysis. The expression vector pPIC9K-Anfae was constructed by the ligation of the Anfae A gene into the shuttle vector pPIC9K. The plasmid pPIC9K-Anfae was linearized and then electrotransformed into P. pastoris GS115. The recombinant strain with high level of FAE activity was obtained through plate screening. Effects of pH and temperature on recombinant FAE were determined by ultraviolet (UV) methods. RESULTS: We have successfully cloned and high-efficiently expressed the AnfaeA gene (GenBank: KF911349) from A. niger h408 in P. pastoris GS115. The sequencing result showed that the length of Anfae A was 783bp. The gene contained an Open Reading Frame encoding 260 amino acids and was similar to feruloyl esterase A from A. niger by homology analysis. The deduced amino acids contained a typical active lid and catalytic triad of lipase. The SDS-PAGE result indicated that molecular weight of the recombinant FAE was about 30 kDa and the activity of the recombinant enzyme was 24.72 U/mL. The specific activity of the recombinant FAE was 40.84 U/mg. Compared with A. niger h408, the recombinant enzyme activity increased about to 1100 times. The optimal temperature and pH for recombinant FAE was 50 degrees C and 5.0, respectively. Recombinant FAE showed nearly 80% of its maximal activity at 60 degrees C and was active in the pH range 4.0-9.0. CONCLUSIONS: The high-efficient expression of AnfaeA gene in P. pastoris provided a prerequisite for achieving industrial application in feed and paper-making industry. In addition, the results established the experimental basis for further improvement of recombinant feruloyl esterase by directed evolution.

First evidence for the anti-inflammatory activity of fucoxanthin in high-fat-diet-induced obesity in mice and the antioxidant functions in PC12 cells.

Obesity, characterized as a state of low-level inflammation, is a powerful determinant influencing the development of insulin resistance and progression to type 2 diabetes. The purpose of the present study was to investigate the anti-inflammatory activity of fucoxanthin in experimental high-fat-diet-induced obesity in mice and antioxidant activity in PC12 cells under oxidative stress situation. The anti-inflammatory potential of fucoxanthin in the regulation of maleic dialdehyde (MDA), polymorphonuclear cells (PMNs), interleukin-1ß (IL-1ß), inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), and cyclooxygenase-2 (COX-2) was determined by ELISA. Fucoxanthin significantly inhibited obesity-induced upregulation of the production of IL-1ß, TNF-α, iNOS, and COX-2. Moreover, fucoxanthin suppressed MDA and infiltration of PMNs. The protective effects were associated with lack of hypertrophy and crown-like structures in mammary gland. At the same time, fucoxanthin showed an advantage of antioxidant activity in PC12 cells under oxidative stress situation. These results suggest that supplementation of fucoxanthin is a promising strategy for blocking macrophage-mediated inflammation and inflammation-induced obesity and its associated complications.

Surfactant-free synthesis of Fe3O4@PANI and Fe3O4@PPy microspheres as adsorbents for isolation of PCR-ready DNA.

Magnetic separation with composite microspheres presents an alternative strategy for applications in biomedical and bioengineering fields. However, the synthesis of core-shell structured magnetic composites universally assumes the surfactant-directing and/or silica-assisting polymerization approach to modify and stabilize the magnetic cores. In this paper, we report on the surfactant-free synthesis of well-defined core-shell structured Fe(3)O(4)@PANI and Fe(3)O(4)@PPy microspheres with high magnetization. The temperature dependence of magnetization of the samples was examined as a function of temperature between 3 and 300 K in an applied field of 500 Oe. It was found that the blocking temperature (T(B)) values of the composite spheres are well above the room temperature. The small variation in magnetization as the temperature changes renders the composite spheres a suitable candidate when used at elevated temperatures. Also, the genomic DNA can be effectively isolated from Aspergillus niger (A. niger) cells with the composite microspheres, using a PEG-NaCl binding buffer and a phosphate eluting buffer. The magnetic isolation of genomic DNA with the composite microspheres was shown to be superior to the conventional phenol-chloroform extraction, which was confirmed by agarose gel eletrophoresis and polymerase chain reaction (PCR) diagnosis. The Fe(3)O(4)@PANI and Fe(3)O(4)@PPy microspheres presented here have great potential in enzyme immobilization, drug delivery, catalysis, and sensors.

Determination of trace elements in anti-influenza virus mushrooms.

We have determined the trace element composition of anti-influenza virus mushrooms using atomic absorption spectrophotometer. The elements present in greater concentration in Ganoderma lucidum samples are selenium, iron, and zinc, with selenium being the element with the highest concentration of all, at 416 ± 38.5 mg/kg; in Cordyceps militaris samples are iron, selenium, and zinc, with iron being the element with the highest concentration of all, at 291 ± 20.9 mg/kg; in Kuehneromyces mutabilis samples are selenium, iron, and manganese, with selenium being the element with the highest concentration of all, at 203 ± 9.8 mg/kg; in Inonotus hispidus samples are zinc, selenium, and iron, with zinc being the element with the highest concentration of all, at 194 ± 16.9mg/kg; in the Collybia maculata samples are iron, selenium, and zinc, with iron being the element with the highest concentration of all, at 274 ± 22.2 mg/kg, respectively. The average metal concentrations in mushrooms decreases in the order: selenium > iron > zinc > chromium > manganese > copper > magnesium > lead. After the mice were administered (orally) with mushroom extracts for 8 weeks and inoculated intranasally with viral suspension, element levels in serum were also measured. Highly significantly increased values of Se, Zn, and Mg in the serum of mice supplemented with anti-influenza virus mushrooms were a characteristic finding. Se, Zn, and Mg present in mushrooms may play a direct or indirect role in their anti-influenza virus nature. They may provide prophylactic protection against influenza infection via stimulation of host innate immune response.

Determination of trace elements in three mushroom samples of basidiomycetes from Shandong, China.

We have determined the trace element composition of three mushrooms of Basidiomycetes, used in traditional Chinese medicine using atomic absorption spectrophotometer (AAS). Metal concentrations in mushrooms were 203-401 mg/kg for iron, 22-51 mg/kg for manganese, 84-116 mg/kg for zinc, 24.1-41.3 mg/kg for copper, 1.6-5.6 mg/kg for lead, 3.3-4.4 mg/kg for chromium, 9.3-11.5 mg/kg for nickel, 0 mg/kg for vanadium, and 55.3-71 mg/kg for magnesium. The trace metal concentrations in mushrooms are hardly affected by the ecosystem and soil where they grew, as well as by the mushroom species and trace metal species. The results can be used to set new standards to control the quality of the three mushrooms of Basidiomycetes-Ganoderma lucidum, Coprinus comatus, and Grifola frondosa.

Purification and characterization of the cold-active alkaline protease from marine cold-adaptive Penicillium chrysogenum FS010.

An extracellular cold-active alkaline serine protease from Penicillium chrysogenum FS010 has been purified. The purification procedure involved: ammonium sulfate precipitation, DEAE ion-exchange chromatography and sephadex G-100 gel chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of 41,000 +/- 1,000 Da. The protease is stable in a pH range of 7.0-9.0 and has a maximum activity at pH 9.0. Compared with other industrial proteases, the enzyme shows a high hydrolytic activities at lower temperatures and a high sensitivity at a temperature over 50 degrees C. The isoelectric point of the enzyme is approximate to 6.0. Enzymatic activity is enhanced by the addition of divalent cations such as Mg(2+) and Ca(2+) and inhibited by addition of Cu(2+)and Co(2+). PMSF and DFP are its specific inhibitors. The application of the cold-active alkaline protease is extremely extensive, and widely used in detergents, feed, food, leather and many other industries.

Cloning, sequencing and expression analysis of the first cellulase gene encoding cellobiohydrolase 1 from a cold-adaptive Penicillium chrysogenum FS010.

A cellobiohydrolase 1 gene (cbh1) was cloned from Penicillium chrysogenum FS010 by a modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). DNA sequencing shows that cbh1 has an open reading frame of 1590 bp, encoding a putative protein of 529 amino acid residues. The deduced amino acid sequence revealed that CBHI has a modular structure with a predicted molecular mass of 56 kDa and consists of a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region. The putative gene product is homologous to fungal cellobiohydrolases in Family 7 of the glycosyl hydrolases. A novel cbh1 promoter (1.3 kb) was also cloned and sequenced, which contains seven putative binding sites (5'-SYGGRG-3') for the carbon catabolite repressor CRE1. Effect of various carbon sources to the cbh1 transcription of P. chrysogenum was examined by Northern analysis, suggesting that the expression of cbh1 is regulated at transcriptional level. The cbh1 gene in cold-adaptive fungus P. chysogenum was expressed as an active enzyme in Saccharomyces cerevisiae H158. The recombinant CBHI accumulated intracellularly and could not be secreted into the medium.

Novel cold-adaptive Penicillium strain FS010 secreting thermo-labile xylanase isolated from Yellow Sea.

A novel cold-adaptive xylanolytic Penicillium strain FS010 was isolated from Yellow sea sediments. The marine fungus grew well from 4 to 20 degrees; a lower (0 degrees) or higher (37 degrees) temperature limits its growth. The strain was identified as Penicillium chrysogenum. Compared with mesophilic P. chrysogenum, the cold-adaptive fungus secreted the cold-active xylanase (XYL) showing high hydrolytic activities at low temperature (2-15 degrees) and high sensitivity to high temperature (>50 degrees). The XYL gene was isolated from the cold-adaptive P. chrysogenum FS010 and designated as xyl. The deduced amino acid sequence of the protein encoded by xyl showed high homology with the sequence of glycoside hydrolase family 10. The gene was subcloned into an expression vector pGEX-4T-1 and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase in Escherichia coli BL21. The expression product was purified and subjected to enzymatic characterization. The optimal temperature and pH for recombinant XYL was 25 degrees and 5.5, respectively. Recombinant XYL showed nearly 80% of its maximal activity at 4 degrees and was active in the pH range 3.0-9.5.