Effect of ammonium acetate on alcohol fermentation in cassava-alcohol fermentation process.

The cassava-alcohol fermentation process employing cassava requires nitrogen source to maximize yields by a commercial strain of S. cerevisiae TG1348. In this study, a factorial experimental design was used to assess a suitable nitrogen source for growth and fermentative performance of S. cerevisiae in cassava-ethanol fermentation. The alcohol fermentation time was about 39 h for urea and ammonium acetate, which was 48 h for ammonium chloride and ammonium sulphate. The fermentation time was reduced by 19 % when using urea and ammonium acetate as nitrogen source. Ammonium acetate leaded to the highest alcohol yield, which was 4% higher than for ammonium sulphate. In addition, byproduct formation differed obviously between the nitrogen sources. The glycerol yields were similar for urea, ammonium sulphate and ammonium chloride but were 24 % lower for ammonium acetate. However, glycerol yield for ammonium carbonate was higher than for other nitrogen sources. Clearly, in batch cultures the ammonium acetate not only increased ethanol generation, but also decreased glycerol generation. In order to understand why ammonium acetate promotes alcohol fermentation, acetic acid was added to different nitrogen sources. The weight loss effect of ammonium sulphate adding acetic acid and ammonium acetate as nitrogen source was the same. The fermentation time was shortened by adding acetic acid. And pH was increased by addition of acetic acid when ammonium sulfate and urea were used as nitrogen sources. The results showed that the acetate root plays an important role in ammonium acetate. The results of this study could facilitate the development of new strategies to control fermentation performance.

Comparisons of urea or ammonium on growth and fermentative metabolism of Saccharomyces cerevisiae in ethanol fermentation.

This work was mainly about the understanding of how urea and ammonium affect growth, glucose consumption and ethanol production of S. cerevisiae, in particular regarding the basic physiology of cell. The basic physiology of cell included intracellular pH, ATP, NADH and enzyme activity. Results showed that fermentation time was reduced by 19% when using urea compared with ammonium. The maximal ethanol production rate using urea was 1.14 g/L/h, increasing 30% comparing with the medium prepared with ammonium. Moreover, urea could decrease the synthesis of glycerol from glucose by 26% comparing with ammonium. The by-product of acetic acid yields decreased from 40 mmol/mol of glucose (with urea) to 24 mmol/mol of glucose (with ammonium). At the end of ethanol fermentation, cell number and pH were greater with urea than ammonium. Comparing with urea, ammonium decreased the intracellular pH by 14% (from 7.1 to 6.1). Urease converting urea into ammonia resulted in a more than 50% lower of ATP when comparing with ammonium. The values of NADH/DCW were 0.21 mg/g and 0.14 mg/g respectively with urea and ammonium, suggesting a 33% lower NADH. The enzyme activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was 0.0225 and 0.0275 U/mg protein respectively with urea and ammonium, which was consistent with the yields of glycerol.

The N- and C-terminal carbohydrate recognition domains of Haemonchus contortus galectin bind to distinct receptors of goat PBMC and contribute differently to its immunomodulatory functions in host-parasite interactions.

BACKGROUND: Hco-gal-m is a tandem-repeat galectin isolated from the adult worm of Haemonchus contortus. A growing body of studies have demonstrated that Hco-gal-m could exert its immunomodulatory effects on host peripheral blood mononuclear cells (PBMC) to facilitate the immune evasion. Our previous work revealed that C-terminal and N-terminal carbohydrate recognition domains (CRD) of Hco-gal-m had different sugar binding abilities. However, whether different domains of Hco-gal-m account differently for its multiple immunomodulatory functions in the host-parasite interaction remains to be elucidated. RESULTS: We found that the N-terminal CRD of Hco-gal-m (MNh) and the C-terminal CRD (MCh) could bind to goat peripheral blood mononuclear cells by distinct receptors: transmembrane protein 63A (TMEM63A) was a binding receptor of MNh, while transmembrane protein 147 (TMEM147) was a binding receptor of MCh. In addition, MCh was much more potent than MNh in inhibiting cell proliferation and inducing apoptosis, while MNh was much more effective in inhibiting NO production. Moreover, MNh could suppress the transcription of interferon-γ (IFN-γ), but MCh not. CONCLUSIONS: Our data suggested that these two CRDs of Hco-gal-m bind to distinct receptors and contributed differently to its ability to downregulate host immune response. These results will improve our understanding of galectins from parasitic nematodes contributing to the mechanism of parasitic immune evasion and continue to illustrate the diverse range of biological activities attributable to the galectin family.

Protective Efficacy of Coccidial Common Antigen Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) against Challenge with Three Eimeria Species.

Coccidiosis is an intestinal disorder of poultry and often caused by simultaneous infections of several Eimeria species. GAPDH is one of the immunogenic common antigens among Eimeria tenella, E. acervulina, and E. maxima identified in our previous study. The present study was performed to further evaluate its immunogenicity and protective efficacy. The genes of GAPDH cloned from E. acervulina and E. maxima were named as EaGAPDH and EmGAPDH, respectively. The immunogenicity of recombinant proteins of EaGAPDH and EmGAPDH were analyzed by Western blot. The transcription and expression of pVAX-EaGAPDH and pVAX-EmGAPDH in the injected muscles were detected by reverse transcription PCR (RT-PCR) and Western blot, respectively. GAPDH-induced changes of T lymphocytes subpopulation, cytokines production, and antibody were determined using flow cytometry, quantitative real-time PCR (qPCR), and ELISA, respectively. Finally, the protective efficacies of pVAX-EaGAPDH and pVAX-EmGAPDH were evaluated by vaccination and challenge experiments. The results revealed that the recombinant GAPDH proteins reacted with the corresponding chicken antisera. The EaGAPDH genes were successfully transcribed and expressed in the injected muscles. Vaccination with pVAX-EaGAPDH and pVAX-EmGAPDH significantly increased the proportion of CD4+ and CD8+ T lymphocytes, the cytokines productions of IFN-γ, IL-2, IL-4 et al., and IgG antibody levels compared to controls. The vaccination increased the weight gains, decreased the oocyst outputs, alleviate the enteric lesions compared to controls, and induced moderate anti-coccidial index (ACI). In conclusion, the coccidial common antigen of GAPDH induced significant humoral and cellular immune response and effective protection against E. tenella, E. acervulina, E. maxima, and mixed infection of the three Eimeria species.

Identification of common immunodominant antigens of Eimeria tenella, Eimeria acervulina and Eimeria maxima by immunoproteomic analysis.

Clinical chicken coccidiosis is mostly caused by simultaneous infection of several Eimeria species, and host immunity against Eimeria is species-specific. It is urgent to identify common immunodominant antigen of Eimeria for developing multivalent anticoccidial vaccines. In this study, sporozoite proteins of Eimeria tenella, Eimeria acervulina and Eimeria maxima were analyzed by two-dimensional electrophoresis (2DE). Western bot analysis was performed on the yielded 2DE gel using antisera of E. tenella E. acervulina and E. maxima respectively. Next, the detected immunodominant spots were identified by comparing the data from MALDI-TOF-MS/MS with available databases. Finally, Eimeria common antigens were identified by comparing amino acid sequence between the three Eimeria species. The results showed that analysis by 2DE of sporozoite proteins detected 629, 626 and 632 protein spots from E. tenella, E. acervulina and E. maxima respectively. Western bot analysis revealed 50 (E. tenella), 64 (E. acervulina) and 57 (E. maxima) immunodominant spots from the sporozoite 2DE gels of the three Eimeria species. The immunodominant spots were identified as 33, 27 and 25 immunodominant antigens of E. tenella, E. acervulina and E. maxima respectively. Fifty-four immunodominant proteins were identified as 18 ortholog proteins among the three Eimeria species. Finally, 5 of the 18 ortholog proteins were identified as common immunodominant antigens including elongation factor 2 (EF-2), 14-3-3 protein, ubiquitin-conjugating enzyme domain-containing protein (UCE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In conclusion, our results not only provide Eimeria sporozoite immunodominant antigen map and additional immunodominant antigens, but also common immunodominant antigens for developing multivalent anticoccidial vaccines.

Novel process combining anaerobic-aerobic digestion and ion exchange resin for full recycling of cassava stillage in ethanol fermentation.

A novel cleaner ethanol production process has been developed. Thin stillage is treated initially by anaerobic digestion followed by aerobic digestion and then further treated by chloride anion exchange resin. This allows the fully-digested and resin-treated stillage to be completely recycled for use as process water in the next ethanol fermentation batch, which eliminates wastewater discharges and minimizes consumption of fresh water. The method was evaluated at the laboratory scale. Process parameters were very similar to those found using tap water. Maximal ethanol production rate in the fully-recycled stillage was 0.9g/L/h, which was similar to the 0.9g/L/h found with the tap water control. The consumption of fresh water was reduced from 4.1L/L (fresh water/ethanol) to zero. Compared with anaerobically-aerobically digested stillage which had not been treated with resin, the fermentation time was reduced by 28% (from 72h to 52h) and reached the level achieved with tap water. This novel process can assist in sustainable development of the ethanol industry.

Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.

BACKGROUND: Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines. METHODS: With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed. RESULTS: Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 106 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 107 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite. CONCLUSIONS: Our results provide a cDNA expression library for further screening of T cell stimulating or inhibiting antigens of E. maxima. Moreover, our results provide six candidate protective antigens for developing new vaccines against E. maxima.

Effect of acetic acid in recycling water on ethanol production for cassava in an integrated ethanol-methane fermentation process.

Recently, the integrated ethanol-methane fermentation process has been studied to prevent wastewater pollution. However, when the anaerobic digestion reaction runs poorly, acetic acid will accumulate in the recycling water. In this paper, we studied the effect of low concentration of acetic acid (≤25 mM) on ethanol fermentation at different initial pH values (4.2, 5.2 or 6.2). At an initial pH of 4.2, ethanol yields increased by 3.0% and glycerol yields decreased by 33.6% as the acetic acid concentration was increased from 0 to 25 mM. Raising the concentration of acetic acid to 25 mM increased the buffering capacity of the medium without obvious effects on biomass production in the cassava medium. Acetic acid was metabolized by Saccharomyces cerevisiae for the reason that the final concentration of acetic acid was 38.17% lower than initial concentration at pH 5.2 when 25 mM acetic acid was added. These results confirmed that a low concentration of acetic acid in the process stimulated ethanol fermentation. Thus, reducing the acetic acid concentration to a controlled low level is more advantageous than completely removing it.

Ethanol fermentation characteristics of recycled water by Saccharomyces cerevisiae in an integrated ethanol-methane fermentation process.

An process of integrated ethanol-methane fermentation with improved economics has been studied extensively in recent years, where the process water used for a subsequent fermentation of carbohydrate biomass is recycled. This paper presents a systematic study of the ethanol fermentation characteristics of recycled process water. Compared with tap water, fermentation time was shortened by 40% when mixed water was employed. However, while the maximal ethanol production rate increased from 1.07g/L/h to 2.01g/L/h, ethanol production was not enhanced. Cell number rose from 0.6×10(8) per mL in tap water to 1.6×10(8) per mL in mixed water but although biomass increased, cell morphology was not affected. Furthermore, the use of mixed water increased the glycerol yield but decreased that of acetic acid, and the final pH with mixed water was higher than when using tap water.

PGMA-Based Star-Like Polycations with Plentiful Hydroxyl Groups Act as Highly Efficient miRNA Delivery Nanovectors for Effective Applications in Heart Diseases.

Poly(glycidyl methacrylate)-based star-like polycations with rich hydrophilic hydroxyl groups can efficiently transfer miRNA into primary cardiac fibroblasts for effective applications in cardiac diseases, such as inhibition of cardiac fibrosis and hypertrophy.

The Molecular Characterization and Immunity Identification of Microneme 3 of Eimeria acervulina.

The gene of Eimeria acervulina microneme protein 3 (EaMIC3) was cloned and characterized. According to the conserved sequence, the 3'- and 5'-ends of EaMIC3 were amplified by the rapid amplification of cDNA ends (RACE). The full length cDNA of this gene was obtained by overlapping the sequences of 3'- and 5'-extremities and amplification by reverse transcription PCR. The sequence analysis revealed that the opening reading frame (ORF) of EaMIC3 was 2,607 bp and encoded a protein of 868 amino acids with 93.04 kDa. Western blotting assay showed that the recombinant protein was successfully recognized by the sera of chickens experimentally infected with E. acervulina, whereas the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of EaMIC3. Immunofluorescence analysis indicated that EaMIC3 was expressed in the sporozoites and merozoites stages of E. acervulina. Animal challenge experiments demonstrated that the recombinant protein of EaMIC3 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented anticoccidial index (ACI) more than 165. Moreover, EaMIC3 protein produced significantly high level of IgG antibody, IFN-γ, IL-10, IL-17 TGF-ß, CD4+ , and CD8+ .

Minimally invasive perventricular device closure of ventricular septal defect: a comparative study in 80 patients.

OBJECTIVE: To evaluate the efficacy of minimally invasive perventricular device closure of ventricular septal defect (VSD). METHODS: Between September 2011 and February 2013, we collected 40 patients who underwent perventricular closure via a small lower sternal incision (minimally invasive group), aged 15.5±3.5 years (12 months to 32 years) with a body weight of 24.2±7.5 kg (10.8-58.0 kg). The mean size of VSD was 5.6±0.5 mm (2-14 mm). Another 40 patients were included as the surgical group, receiving the conventional surgical repair of VSD. The device of the minimally invasive group was released under the guidance of transesophageal echocardiography. Success rate, cardiac indicators, and clinical outcomes of the 2 groups were compared. RESULTS: The patients in the surgical group and those in the minimally invasive group showed similar results in success rate (both 97.5%). The procedure time, intensive care unit stay, hospital stay, and postoperative recovery time in the minimally invasive group were significantly shorter than those in the surgical group (58±21 minutes versus 145±26 minutes, 2±1 days versus 8±3 days, 5±1 days versus 16±6 days, 3±1 days versus 90±20 days, all P<0.05). The minimally invasive group had a higher incidence of conduction anomalies (17.5% versus 2.5%, P<0.05). In the follow-up period of 3-12 months, there was no new residual shunt, noticeable aortic regurgitation, significant arrhythmias, or device failure except for new complications in the surgical group. CONCLUSIONS: The success rate of minimally invasive perventricular device closure of VSD under transesophageal echocardiography guidance is similar to that of conventional surgical repair, but the short-term outcomes of the minimally invasive approach is much better. Long-term follow-up is necessary to confirm the effectiveness of this technique.

Biocleavable graphene oxide based-nanohybrids synthesized via ATRP for gene/drug delivery.

Graphene oxide (GO) has been proven to be promising in many biomedical fields due to its biocompatibility, unique conjugated structure, easily tunable surface functionalization and facile synthesis. In this work, a flexible two-step method was first developed to introduce the atom transfer radical polymerization (ATRP) initiation sites containing disulfide bonds onto GO surfaces. Surface-initiated ATRP of (2-dimethyl amino)ethyl methacrylate (DMAEMA) was then employed to tailor the GO surfaces in a well-controlled manner, producing a series of organic-inorganic hybrids (termed as SS-GPDs) for highly efficient gene delivery. Under reducible conditions, the PDMAEMA side chains can be readily cleavable from the GO backbones, benefiting the resultant gene delivery process. Moreover, due to the conjugated structure of the graphene basal plane, SS-GPD can attach and absorb aromatic, water insoluble drugs, such as 10-hydroxycamptothecin (CPT), producing SS-GPD-CPT. The MTT assay and the simultaneous double-staining procedure revealed that SS-GPD-CPT possessed a high potency of killing cancer cells in vitro. With a high aqueous solubility and coulombic interaction with cell membrane, SS-GPDs may have great potential in gene/drug delivery fields.

A biocleavable pullulan-based vector via ATRP for liver cell-targeting gene delivery.

Pullulan due to its specificity for liver has been widely exploited for biomedical applications. In this work, a tailor-made biocleavable pullulan-based gene vector (PuPGEA) with good hemocompatibility was successfully proposed via atom transfer radical polymerization (ATRP) for efficient liver cell-targeting gene delivery. A two-step method involving the reaction of hydroxyl groups of pullulan with cystamine was developed to introduce reduction-sensitive disulfide-linked initiation sites of ATRP onto pullulan. The poly(glycidyl methacrylate) (PGMA) side chains prepared subsequently via ATRP were functionalized with ethanolamine (EA) to produce the resultant biocleavable comb-shaped PuPGEA vectors consisting of nonionic pullulan backbones and disulfide-linked cationic EA-functionalized PGMA (PGEA) side chains with plentiful secondary amine and nonionic hydroxyl units. The cationic PGEA side chains can be readily cleavable from the pullulan backbones of PuPGEA under reducible conditions. Due to the liver targeting performance of pullulan backbones, such PuPGEA vectors exhibited much higher gene transfection efficiency and cellular uptake rates in HepG2 cell lines than in Hella cell lines. In addition, in vitro transfection efficiency and uptake mechanism of polyplex in HepG2 cells were evaluated in the presence of different endocytosis inhibitors, indicating that the asialoglycoprotein receptor was involved in transfection process of hepatocytes. More importantly, in comparison with gold standard polyethylenimine (PEI, ∼25 kDa), PuPGEA vectors possessed excellent hemocompatibility without causing undesirable hemolysis. Properly grafting short bioreducible PGEA polycation side chains from a liver cell-targeting pullulan backbone is an effective means to produce new hemocompatible polysaccharide-based gene delivery vectors.

Thermo-sensitive graphene oxide-polymer nanoparticle hybrids: synthesis, characterization, biocompatibility and drug delivery.

We report the covalent interaction mediated assembly of thermo-sensitive polymer nanoparticles (PNPs) on functionalized graphene oxide (GO) nanosheets to create novel GO-PNP hybrids for drug delivery. To this end, thermo-sensitive PNPs with an average diameter of about 50 ± 12 nm were first synthesized with the free radical polymerization reaction, and GO nanosheets were noncovalently modified with a bifunctional linker to provide reactive sites for the binding of PNPs. Finally, GO-PNP hybrids were successfully synthesized by the covalent interaction mediated assembly of PNPs on GO nanosheets. Multi-characterization techniques were utilized to identify the formation of PNPs, the modification of GO nanosheets, and the formation of GO-PNP hybrids. Cell culture experiment with the mouse osteoblast-like MC3T3-E1 cells indicates that the synthesized GO-PNP hybrids have satisfactory biocompatibility. The loading efficiency of drug molecules (Adriamycin, ADR) with GO-PNP (∼87%) is close to that with GO (∼91%), but significantly higher than that with PNPs (∼46%). The release efficiency of GO-PNP hybrids with the highest surface coverage of PNPs (∼85 PNPs per µm2) is about 22%, which is very close to that of PNPs (∼25%) and significantly higher than that of GO (∼11%). Our study indicates that this thermo-sensitive GO-PNP hybrid, when considering the drug loading and release comprehensively, has better performance than both PNPs and GO and thus can be used as a novel nanocarrier for temperature-controllable drug release. The GO-PNP hybrids with and without ADR were applied to kill cancer cells in vitro and the result shows that the GO-PNP hybrid with ADR has an obvious effect on killing cancer cells, and its performance is obviously better than both GO and PNPs. It is expected that this new hybrid material based on GO and PNPs will have great potential for in vivo applications such as to kill target cancer cells by modifying with specific antibodies.

Constitutive expression of a salinity-induced wheat WRKY transcription factor enhances salinity and ionic stress tolerance in transgenic Arabidopsis thaliana.

The isolation and characterization of TaWRKY79, a wheat class II WRKY transcription factor, is described. Its 1297 bp coding region includes a 987 bp long open reading frame. TaWRKY79 was induced by stressing seedlings with either NaCl or abscisic acid (ABA). When a fusion between an 843 bp segment upstream of the TaWRKY79 coding sequence and GUS was introduced into Arabidopsis thaliana, GUS staining indicated that this upstream segment captured the sequence(s) required to respond to ABA or NaCl treatment. When TaWRKY79 was constitutively expressed as a transgene in A. thaliana, the transgenic plants showed an improved capacity to extend their primary root in the presence of either 100 mM NaCl, 10 mM LiCl or 2 µM ABA. The inference was that TaWRKY79 enhanced the level of tolerance to both salinity and ionic stress, while reducing the level of sensitivity to ABA. The ABA-related genes ABA1, ABA2 ABI1 and ABI5 were all up-regulated in the TaWRKY79 transgenic plants, suggesting that the transcription factor operates in an ABA-dependent pathway.

Allogeneic peripheral blood stem cell transplantation in the treatment of severe aplastic anemia and severe infection.

OBJECTIVE: To investigate the efficacy of allogeneic peripheral blood stem cell transplantation (PBSCT) in the treatment of severe aplastic anemia (SAA) and severe infection. METHODS: A patient with SAA and pseudomonas aeruginosa septicemia was treated with PBSCT from an HLA-identical sibling with cyclophosphamide (CY) and total body irradiation (TBI) for conditioning. The patient was infused with 20.3 x 10(8)/kg mononuclear cells including 61.0 x 10(6)/kg CD34(+) cells following the conditioning regimen. RESULTS: Twelve days after PBSCT, the absolute neutrophil count (ANC) of 1.0 x 10(9)/L was achieved, with platelet count > 50 x 10(9)/L at twenty days. The donor origin of engraftment was confirmed by polymerase chain reaction (PCR) analysis of short tandem repeats at the end of the first, sixth and twelfth month. The patient's body temperature dropped to normal level when her ANC reached 0.5 x 10(9)/L on day 10, and the bacterial culture of blood sample became negative subsequently. Symptoms and signs of acute or chronic graft versus host disease (GVHD) were not observed in 30 months after PBSCT. CONCLUSIONS: Hematopoiesis was reconstituted shortly after PBSCT. The combination of CY and TBI and the infusion of sufficient peripheral blood stem cells may contribute to the successful engraftment. PBSCT may be considered as the first choice when hematopoietic stem cell transplantation is needed for SAA patients complicated with severe infection.