BAT6026, a novel anti-OX40 antibody with enhanced antibody dependent cellular cytotoxicity effect for cancer immunotherapy.

OX40 (CD134), a member of the TNF receptor superfamily, is a widely studied costimulatory immune checkpoint. Several OX40 agonistic antibodies are in the clinical stage for cancer treatment, among which PF-04518600 is the leader and currently in phase II trial. It has been recognized that one potential mode of action for anti-OX40 antibodies is the deletion of intratumoral Tregs. Thus, a novel human anti-OX40 antibody, BAT6026, was generated with enhanced antibody dependent cellular cytotoxicity (ADCC) via fucose deletion to strengthen its Treg depletion activity. This characteristic of BAT6026 differentiates it from other previously reported anti-OX40 antibodies in the field of tumor therapy. The affinity of BT6026 to OX40 was 0.28nM, approximately 8 times stronger than that of PF-04518600. BAT6026 effectively competed for the binding of ligand OX40L to OX40, whereas PF-04518600 only partially competed. Moreover, compared to PF-04518600, BAT6026 activated T cells more effectively when clustered by FcγRs engagement and stimulated SEB-pretreated PBMCs to secrete IL-2 cytokines in vitro. In addition, BAT6026 demonstrated stronger anti-tumor activity than PF-04518600 in an OX40-humanized mouse MC38 tumor model. BAT6026 also showed a significantly synergistic effect on tumor inhibition when combined treatment with PD-1 antibody. Analysis of tumor-infiltrating T cells revealed that BAT6026 treatment significantly reduced Treg cells and increased CD8+ T cells in tumor. Preclinical safety assessment in non-human primates demonstrated a good safety profile for BAT6026. Together these data warrant further development of BAT6026 into clinical trials for patients with cancer.

Identification of a JAK/STAT pathway receptor domeless from Pacific white shrimp Litopenaeus vannamei.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway was known to participate in dozens of immune responses in organisms. Domeless, first identified in Drosophila melanogaster, is a unique receptor involved in invertebrate JAK/STAT pathway. In this study, a cytokine receptor (LvDOME) was identified in Litopenaeus vannamei. The LvDOME cDNA was 5178bp in length with an Open Reading Frame (ORF) of 4191bp. LvDOME contained two cytokine binding modules (CBMs) and three fibronectin-type-III-like (FNIII) domains, similar to most vertebrate IL-6 receptors. LvDOME was expressed highest in shrimp muscle and could be up-regulated in the late stage of white spot syndrome virus (WSSV) infection. LvDOME could significantly enhance the activity of the WSSV wsv069 gene promoter through acting on the STAT-binding motif, suggesting LvDOME could activate the JAK/STAT pathway. Moreover, knockdown of LvDOME resulted in lower cumulative mortality of shrimps and less WSSV copies, suggesting LvDOME may be hijacked by WSSV to benefit virus replication. To our knowledge, this is the first report on the receptor of JAK/STAT pathway in shrimp.

Performance of a biotrickling filter in the removal of waste gases containing low concentrations of mixed VOCs from a paint and coating plant.

The performance of a field-scale biotrickling filter (BTF) in the removal of waste gases containing low concentrations of mixed volatile organic compounds was evaluated. Results showed that acetone and methyl ethyl ketone (MEK) were more easily removed than toluene and styrene. The removal efficiency for acetone and MEK reached over 99% on days 28 and 25 of the operation, whereas those for toluene and styrene were 80 and 63% on day 38. The maximum individual elimination capacities for styrene, toluene, acetone, and MEK were 10.2, 2.7, 4.7, and 8.4 g/m(3) h, respectively. These values were achieved at inlet loading rates of 13.9, 3.3, 4.8, and 8.5 g/m(3) h, respectively, at an empty bed retention time of 14 s. the removal efficiency for styrene and toluene rapidly increased from 67% and 83% to 86% and over 99%, respectively, when the concentration of ammonia nitrogen (N-NH(4) (+)) and phosphates (P) in the nutrients increased from 350 to 840 mg/l and 76 to 186 mg/l. When the BTF was restarted after a four-day shutdown, the removal efficiency for toluene was restored to over 99% within a week. However, that for styrene was not restored to its previous level before the shutdown. No noticeable adverse effect on acetone and MEK removal was observed. Denaturing gradient gel electrophoresis results for the bacterial community in the BTF during VOC removal showed that proteobacterial phylum was dominant, and the changes of nutrient concentration and shutdown periods may have played a role in the community structure differences.

Performances of two biotrickling filters in treating H2S-containing waste gases and analysis of corresponding bacterial communities by pyrosequencing.

Two identical biotrickling filters named BTFa and BTFb were run in parallel to examine their performances in removing hydrogen sulfide. BTFa was filled with ceramic granules, and BTFb was filled with volcanic rocks. The results showed that BTFb was more robust than BTFa under acidic conditions. At empty bed residence times (EBRTs) of 20 and 15 s, the removal efficiency of BTFa was close to 100%. At EBRTs of 10 and 5 s, the removal efficiency of BTFa slightly decreased. The removal efficiencies of BTFa decreased by different degrees at the end of each stage, dropping to 94%, 81%, 60%, and 71%, respectively. However, the H(2)S removal efficiency in BTFb consistently reached 99% throughout the experiment. Pyrosequencing analyses indicated that members of Thiomonas dominated in both BTFs, but the relative abundance of Acidithiobacillus was higher in BTFb than in BTFa.

Disruption of lactate dehydrogenase through homologous recombination to improve bioethanol production in Thermoanaerobacterium aotearoense.

To enhance ethanol production in Thermoanaerobacterium aotearoense, the lactate dehydrogenase (ldh) gene, which is responsible for lactic acid production in a key branch pathway, was successfully disrupted via homologous recombination. ldh-up and ldh-down were designed and amplified based on JW/SL-YS485-AY 278026, and they were subsequently used as homologous fragments with an inserted erythromycin resistance gene to construct the targeted vector based on pBLUESCRIPT II SK(+). Southern hybridization and PCR-based assay definitely confirmed that the ldh gene in the Δldh mutant was disrupted by the insertion of the erythromycin resistance gene. Compared with the wild type, the Δldh mutant exhibited increases of 31.0% and 31.4% in cell yield under glucose and xylose cultivation, respectively, probably because knocking out the ldh gene results in increased acetate and ATP levels. Knockout of lactate dehydrogenase produced 2.37- and 2.1-fold increases in the yield of ethanol (mole/mole substrate) under glucose and xylose cultivation, respectively. Moreover, no lactic acid was detected in Δldh mutant fermentation mixtures (detection limit of HPLC: 0.5 mM), but lactic acid was readily detected for growth of the wild-type strain on both glucose and xylose, with final concentrations up to 59.24 mM and 56.06 mM, respectively. The success of this process thoroughly demonstrates the methodological possibility of gene knockout through homologous recombination in Thermoanaerobacterium.

Control and optimization of Clostridium tyrobutyricum ATCC 25755 adhesion into fibrous matrix in a fibrous bed bioreactor.

The great performance of a fibrous bed bioreactor (FBB) is mainly dependent on the cell adhesion and immobilization into the fibrous matrix. Therefore, understanding the mechanism and factors controling cell adhesion in the fibrous matrix is necessary to optimize the FBB setup and further improve the fermentability. The adhesion behavior of a strain of Clostridium tyrobutyricum isolated from an FBB was studied, which was proven to be affected by the different environmental conditions, such as growth phase of cells, pH, ionic strength, ionic species, and composition of media. Our results also suggested that electrostatic interactions played an important role on bacteria adhesion into the fibrous matrix. This study demonstrated that the compositions of fermentation broth would have a significant effect on cell adhesion. Consequently, a two-stage glucose supply control strategy was developed to improve the performance of FBB with higher viable cell density in the operation of the FBB setup.

Enhanced butyric acid tolerance and bioproduction by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor.

Repeated fed-batch fermentation of glucose by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor (FBB) was successfully employed to produce butyric acid at a high final concentration as well as to adapt a butyric-acid-tolerant strain. At the end of the eighth fed-batch fermentation, the butyric acid concentration reached 86.9 ± 2.17 g/L, which to our knowledge is the highest butyric acid concentration ever produced in the traditional fermentation process. To understand the mechanism and factors contributing to the improved butyric acid production and enhanced acid tolerance, adapted strains were harvested from the FBB and characterized for their physiological properties, including specific growth rate, acid-forming enzymes, intracellular pH, membrane-bound ATPase and cell morphology. Compared with the original culture used to seed the bioreactor, the adapted culture showed significantly reduced inhibition effects of butyric acid on specific growth rate, cellular activities of butyric-acid-forming enzyme phosphotransbutyrylase (PTB) and ATPase, together with elevated intracellular pH, and elongated rod morphology.

Preparation and characteristics of interferon-alpha poly(lactic-co-glycolic acid) microspheres.

By a double emulsion solvent evaporation method, interferon-alpha (IFN-alpha) microspheres were prepared with poly(lactide-co-glycolide) (PLGA) and their characteristics, such as morphology, drug loading, encapsulation efficiency, in vitro release and degradation were evaluated. The IFN-alpha microspheres were prepared by different viscosities from 0.17-1.13 dL g(-1) and concentrations between 5-25% of PLGA, which not only affected the drug loading and encapsulation efficiency of IFN-alpha microspheres, but also strongly influenced the in vitro release. With smooth and porous surface, the drug loading and encapsulation efficiency of the microspheres prepared by 15% 0.89 dL g(-1) PLGA were 7.736% and 77.38%, respectively. The DSC curve of microspheres indicated IFN-alpha was loaded inside the microspheres. The degradation of microspheres was homogeneous and the mass loss was over 80% in 6 weeks. The release profile of microspheres showed a sustained fashion and the IFN-alpha released from microspheres maintained its bioactivity for 7 days.

Phosphoenolpyruvate-dependent phosphorylation of sucrose by Clostridium tyrobutyricum ZJU 8235: evidence for the phosphotransferase transport system.

The uptake and metabolism of sucrose, the major sugar in industrial cane molasses, by Clostridium tyrobutyricum ZJU 8235 was investigated and this study provided the first definitive evidence for phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity in butyric acid-producing bacteria. Glucose was utilized preferentially to sucrose when both substrates were present in the medium. The PEP-dependent sucrose: PTS was induced by growing C. tyrobutyricum on sucrose (but not glucose) as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that both soluble and membrane components were required for bioactivity. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose-grown cultures. Based on these findings, a pathway of sucrose metabolism in this organism was proposed that includes the forming of sucrose-6-phosphate via the PTS and its further degradation into glucose-6-phosphate and fructose-6-phosphate.

Production of butyric acid from glucose and xylose with immobilized cells of Clostridium tyrobutyricum in a fibrous-bed bioreactor.

Butyric acid has many applications in chemical, food, and pharmaceutical industries. In the present study, Clostridium tyrobutyricum ATCC 25755 was immobilized in a fibrous-bed bioreactor to evaluate the performance of butyrate production from glucose and xylose. The results showed that the final concentration and yield of butyric acid were 13.70 and 0.46 g g(-1), respectively, in batch fermentation when 30 g L(-1) glucose was introduced into the bioreactor. Furthermore, high concentration 10.10 g L(-1) and yield 0.40 g g(-1) of butyric acid were obtained with 25 g L(-1) xylose as the carbon source. The immobilized cells of C. tyrobutyricum ensured similar productivity and yield from repeated batch fermentation. In the fed-batch fermentation, the final concentration of butyric acid was further improved to 24.88 g L(-1) with one suitable glucose feeding in the fibrous-bed bioreactor. C. tyrobutyricum immobilized in the fibrous-bed bioreactor would provide an economically viable fermentation process to convert the reducing sugars derived from plant biomass into the final bulk chemical (butyric acid).

Immunoassay for cadmium detection and quantification.

OBJECTIVE: To detect cadmium in environmental and food samples by graphite furnace atomic absorption spectroscopy (GFAAS) and inductively coupled plasma atomic emission spectroscopy (ICPAES). METHODS: An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed based on a cadmium-specific monoclonal antibody. IC-ELISA for cadmium in environmental and food samples was evaluated. RESULTS: IC-ELISA showed an IC50 of 45.6 microg/L with a detection limit of 1.95 microg/L for cadmium, and showed a mean recovery ranging 97.67%-107.08%. The coefficient of variations for intra- and interassay was 3.41%-6.61% and 4.70%-9.21%, respectively. The correlation coefficient between IC-ELISA and GFAAS was 0.998. CONCLUSION: IC-ELISA can detect and quantify cadmium residue in environmental or food samples.

Degradation of o-chloronitrobenzene as the sole carbon and nitrogen sources by Pseudomonas putida OCNB-1.

A bacterial strain that utilized o-chloronitrobenzene (o-CNB) as the sole carbon, nitrogen and energy sources was isolated from an activated sludge collected from an industrial waste treatment plant. It was identified as Pseudomonas putida based on its morphology, physiological, and biochemical characteristics with an automatic biometrical system and the 16S rRNA sequence analysis. Microcosm study showed that the biodegradation of o-CNB was optimized at culture medium pH 8.0 and 32 degrees C. At these conditions, the strain degraded 85% of o-CNB at a starting concentration of 1.1 mmol/L in 42 h. o-Chloroaniline was identified as the major metabolite with high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The study showed that o-CNB degradation by Pseudomonas putida OCNB-1 was initiated by aniline dioxyenase, nitrobenzene reductase and catechol-1,2-dioxygenase.

Butyric acid fermentation in a fibrous bed bioreactor with immobilized Clostridium tyrobutyricum from cane molasses.

Butyrate fermentation by immobilized Clostridium tyrobutyricum was successfully carried out in a fibrous bed bioreactor using cane molasses. Batch fermentations were conducted to investigate the influence of pH on the metabolism of the strain, and the results showed that the fermentation gave a highest butyrate production of 26.2 g l(-1) with yield of 0.47 g g(-1) and reactor productivity up to 4.13 g l(-1)h(-1) at pH 6.0. When repeated-batch fermentation was carried out, long-term operation with high butyrate yield, volumetric productivity was achieved. Several cane molasses pretreatment techniques were investigated, and it was found that sulfuric acid treatment gave better results regarding butyrate concentration (34.6+/-0.8 g l(-1)), yield (0.58+/-0.01 g g(-1)), and sugar utilization (90.8+/-0.9%). Also, fed-batch fermentation from cane molasses pretreated with sulfuric acid was performed to further increase the concentration of butyrate up to 55.2 g l(-1).

Development of direct competitive enzyme-linked immunosorbent assay for the determination cadmium residue in farm produce.

Cadmium, a toxic heavy metal, poses a significant threat to human health. Currently, the methods for detecting cadmium residue in farm produce need expensive equipment, intensive labor, and much time to finish one detection. In this study, a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) based on a cadmium-chelate-specific monoclonal antibody has been developed. The DC-ELISA showed an IC(50) of 2.30 microg/L with a detection limit of 0.20 microg/L for cadmium. The assay has been demonstrated to be highly specific since the monoclonal antibody showed little or no cross-reactivity with all tested metal chelates which include Cd(2+), Pb(2+), Hg(2+), Zn(2+), Na(+), Ca(2+), Fe(3+), Mg(2+), Mn(2+), Cu(2+), Al(3+), Co(2+), Cr(2+), Ni(2+), Sn(2), and K(+). The assay showed that a mean recovery ranged from 100.47% to 103.86%, and the coefficients of variations for intra- and inter-assay were 1.73-7.14% and 3.63-6.81%, respectively. Then, several farm produces including wheat flour, apple juice, rice flour, and tea were analyzed for cadmium residue with DC-ELISA and graphite furnace atomic absorption spectroscopy (GFAAS). The correlation coefficient between the DC-ELISA and GFAAS was 0.99. It was demonstrated that the DC-ELISA can be used as a simple and economic method to detect and quantitate cadmium residue in farm produce.

Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses.

Due to the environmental concerns and the increasing price of oil, bioethanol was already produced in large amount in Brazil and China from sugarcane juice and molasses. In order to make this process competitive, we have investigated the suitability of immobilized Saccharomyces cerevisiae strain AS2.1190 on sugarcane pieces for production of ethanol. Electron microscopy clearly showed that cell immobilization resulted in firm adsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure, and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supported-biocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sugarcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and fewer yeast cells leaked into fermentation broth. Ethanol concentrations (about 89.73-77.13 g/l in average value), and ethanol productivities (about 59.53-62.79 g/l d in average value) were high and stable, and residual sugar concentrations were low in all fermentations (0.34-3.60 g/l) with conversions ranging from 97.67-99.80%, showing efficiency (90.11-94.28%) and operational stability of the biocatalyst for ethanol fermentation. The results of this study concerning the use of sugarcane as yeast supports could be promising for industrial fermentations.

[Advances in heavy metal ions immunoassay].

Heavy metal leftover on farm and stock products has become a big threat to human. It is necessary to develop some fast and efficient detection methods. Heavy metal immunoassays are new methods for detection of heavy metal ions. Compared to the traditional chemical methods, immunoassays are not only fast, cheap, simple, but also reasonably portable, highly sensitive and selective. It can be used as preliminary screening for rapid determination of heavy metal ions. Except chemical chelators, phytochelatin and metallothionein can also be used for preparing immunogen, both of them can chelate heavy metal ions to carrier protein. There are two prototype assays: polyclonal antibody immunoassay and monoclonal antibody immunoassay. The former includes fluorescence polarization immunoassay; the latter includes indirectly competitive ELISA, one-step competitive immunoassay and KinExA immunoassay. Among these assays, indirectly competitive ELISA which was used for determining heavy metal ions in the early days was easy to be interfered and showed false positive. Fluorescence polarization immunoassay which used polyclonal antibody for determining heavy metal ions was simple and cheap. KinExA instrument could be functioned as an immunosensor for environmental samples. One-step immunoassay which avoided to the addition of second antibody and chromogenic substrate was simple and sensitive. Colloidal gold enhanced immunochromatography assay is a semi-quantitation for determining heavy metal ions. As an adjunctive way for chemical methods, it has the potential application in rapid determination of heavy metal ions.

Nutritive significance of crystalline inclusion proteins of Photorhabdus luminescens in Steinernema nematodes.

Phase I cells of Photorhabdus luminescens produce two types of intracellular crystalline inclusion proteins designated CipA and CipB. The genes encoding CipA and CipB proteins from P. luminescens H06 were expressed respectively in Escherichia coli and these cells were used to feed the axenic first juveniles (J1) of three Steinernema nematode isolates in liquid cultures and on agar plates. In liquid cultures, the axenic J1 juveniles of all three test Steinernema nematode isolates were able to produce next dauer juveniles (DJs) in the E. coli cultures with at least one of the expressed Cip proteins, but unable to develop beyond the next J1 stage without expressed Cip proteins. For each target nematode isolate, addition of the supernatant of the bacterial culture of its Xenorhabdus symbiont to the tested liquid cultures did not induce the formation of DJs. However, on LB agar plates with different test E. coli cultures, all J1 juveniles of the three Steinernema strains finally developed into next DJs. It seemed that the metabolite pathway of the test bacteria in both culture systems was different. The presence of the Cip proteins has a significant influence on the DJ formation of the Steinernema nematodes in liquid culture system.

[Analysis of chloramphenicol, thiamphenicol and florfenicol in chicken by high performance liquid chromatography with electrospray ionization mass spectrometry].

The method for the analysis of three chloramphenicols including chloramphenicol, thiamphenicol and florfenicol in chickens was developed by high performance liquid chromatography combined with electrospray ionization mass spectrometry (HPLC-ESI-MS-MS). The mass spectrometer was operated in the negative ion mode using multiple reaction monitoring for qualitative and quantitative analysis of these compounds at the same time. Only 1 g of sample was needed, and no solid phase extraction (SPE) procedure was adopted. The advantages of the method are simple operation, less organic chemicals consumed, and shorter operation time. The limits of detection (LOD) were 0.010 microg/kg, and the limits of quantitation (LOQ) were 0.100 microg/kg. The linear plots were obtained between 0.050 and 1.00 microg/L. Overall recoveries were between 69.0% and 92.8% with relative standard deviations between 6.3% and 12.9%.

Catalytic antibody therapy against the insecticide carbaryl.

Catalytic antibodies have been studied widely, but little is known about their applicability as therapeutic reagents in vivo. Here we report that carbaryl, a widely used broad-spectrum carbamate insecticide that is toxic to animals and humans, is hydrolyzed by polyclonal catalytic antibodies induced in vivo by a phosphate immunogen. To test the efficacy of the in vivo-induced polyclonal antibodies, we immunized mice with the phosphate immunogen and assayed their sensitivity to carbaryl by determining the ED(50) value, the dose that produces lowest-grade tremors in 50% of animals. We found that the ED(50) for immunized mice was 43% higher than that for nonimmunized mice and that this increase in ED(50) probably resulted from the hydrolysis of carbaryl by the catalytic antibodies in vivo. Our results suggest that polyclonal catalytic antibodies can be used as therapeutic reagents in vivo.

[Construction of a set of secreting expression vectors for Saccharomyces cerevisiea].

The DNA fragment ecoding the Signal peptide of inulinase of Kluyveromyces smarxianu was synthesized chemically. This fragment was cloned in-frame in the expression vector pYES2 of Saccharomyces cerevisiae, resulting in a set of new secreting expression vectors pYES2 I, pYES2 II, pYES2 III. The L-Asparaginase gene (ASN) of E. coli and alpha-acetylactate decarboxylase gene (ALDC) of B. brevis which were amplified by PCR and cloned into the new vectors respectively were transformed into Saccharomyces cerevisia, and most of enzyme activities were secreted into the medium. The new secreting expression vectors still have excellent segregational stability even after growth for 100 h in the absence of selective pressure.