Robust trypsin coating on electrospun polymer nanofibers in rigorous conditions and its uses for protein digestion

An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC‐TR), which crosslinks additional trypsin molecules onto covalently attached trypsin (CA‐TR). EC‐TR showed better stability than CA‐TR in rigorous conditions, such as at high temperatures of 40 and 50°C, in the presence of organic co‐solvents, and at various pH's. For example, the half‐lives of CA‐TR and EC‐TR were 1.42 and 231 h at 40°C, respectively. The improved stability of EC‐TR can be explained by covalent linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40°C by using both CA‐TR and EC‐TR in digesting a model protein, enolase. EC‐TR showed better performance and stability than CA‐TR by maintaining good performance of enolase digestion under recycled uses for a period of 1 week. In the same condition, CA‐TR showed poor performance from the beginning and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes. Biotechnol. Bioeng. 2010;107: 917–923. © 2010 Wiley Periodicals, Inc.     

Stability and reproducibility of low-temperature anaerobic biological wastewater treatment

The reproducibility of low-temperature anaerobic biological wastewater treatment trials was evaluated. Two identical anaerobic expanded granular sludge bed bioreactors were used to treat synthetic volatile fatty acid-based industrial wastewater under ambient conditions (18-20 degrees C) and to investigate the effect of various environmental perturbations on reactor performance and microbial community dynamics, which were assessed by chemical oxygen demand removal or effluent volatile fatty acid determination and terminal restriction fragment length polymorphism analysis, respectively. Methanogenic activity was monitored using specific methanogenic activity assays. Reactor performance and microbial community dynamics were each well replicated between Reactor 1 and Reactor 2. Archaeal dynamics, in particular, were associated with reactor operating parameters. Terminal restriction fragment length polymorphism data suggested dynamic acetoclastic and hydrogenophilic methanogenic populations and were in agreement with temporal specific methanogenic activity data. Putative psychrophilic populations were observed in anaerobic bioreactor sludge for the first time.     

Screen-less expanded bed column: new approach for the recovery and purification of a malaria transmission blocking vaccine candidate from Pichia pastoris

An experimental malaria transmission blocking vaccine antigen, Pfs25H, expressed and secreted from Pichia pastoris was recovered and purified using a screenless expanded bed column equipped with a rotating fluid distribution system. This column was able to accommodate feed stock, containing 30% biomass, at a flow rate of 300–400 cm/h without affecting column stability. This capability is three times higher than the capability of the expanded bed column currently in use, which is equipped with a perforated plate fluid distribution system; this design could accommodate biomass concentrations of only up to 10%. The screen-less design did not affect the binding capacity, purification level or process yield and, therefore, shorten the process. Purified Pfs25H of 6.4 g were recovered from 37 l of Pichia pastoris culture in one step.     

Impact of long-term partial aeration on the removal of 2,4,6-trichlorophenol in an initially methanogenic fluidized bed bioreactor

A fluidized bed bioreactor (FBBR) was operated for more than 1000 days under two regimes, Methanogenic (M) and Methanogenic-Aerobic (M-A), to remove 2,4,6-trichlorophenol (TCP) and phenol (Phe) from a synthetic wastewater, containing different amounts of TCP and Phe, using different aeration flow-rates (0, 2.13, and 1.06 NL O(2)/L.day). M conditions (80:20 mg/L of TCP:Phe, 0 NL O(2)/L.day) showed similar TCP and Phe removal (>95%). Nevertheless accumulation of 4-chlorophenol (4CP) up to 16 mg/L and Phe up to 4 mg/L was observed, while in M-A conditions (80:20 mg/L of TCP:Phe, 2.13 NL O(2)/L.day) TCP and Phe removal achieved 99.9(+)% and after 70 days no accumulation of intermediates were detected. The increase of TCP and Phe in the influent under M-A conditions from 80:20 to 120:30 mg/L of TCP:Phe did not negatively affect the removal of TCP, intermediates and Phe; in fact, they were similar to those in previous M-A conditions. The decrease in the oxygen flow rate from 2.13 to 1.06 NL O(2)/L.day had no negative effect on pollutant removals, which were as high as in previous two M-A conditions. The specific methanogenic activity of bioparticles of the fluidized bed decreased with long-term partial aeration, starting from 1.097 mmol CH(4)/h.g(TKN) in the M regime (day 60) to <0.02 mmolCH(4)/h.g(TKN) at day 1050, suggesting aerobic regime in the bioreactor rather than an M-A regime. In conclusion, complete removal of TCP and less chlorinated intermediates could be achieved in an initially methanogenic FBBR under conditions of partial aeration, although long-term operation seemed to negatively affect the methanogenic activity of biomass. It is also likely that after extended aeration the microbial community was finally enriched with strains with the ability to attack 2,4,6-TCP under aerobic conditions. This report represents the first evidence of a long exposure to oxygen of an anaerobic microbial consortium that efficiently remove TCP.     

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release--the impacts on the hypothalamic-pituitary-adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2. Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release. CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective. We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic-pituitary-adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.     

An organic coating keeps orb‐weaving spiders (Araneae,Araneoidea, Araneidae) from sticking to their own capture threads

More than 95% of orb‐weaving spider species ensure prey capture success by producing viscous threads equipped with gluey droplets. However, this trap may bear serious risks for the web‐inhabiting spider as well. The obvious question, how a spider avoids getting stuck in its own capture spiral, has gained little scientific attention up till now. In 1905, the French naturalist Jean‐Henry Fabre concluded from anecdotal observation that orb‐weaving spiders protect themselves by a fatty surface coating. Here, we test this hypothesis by indirectly measuring the force necessary to detach an autotomized spider’s leg from the capture spiral of its own web (here called ‘index of adhesion’, IOA). Three groups of legs, each of the species Araneus diadematus Clerck, 1757 and Larinioides sclopetarius (Clerck, 1757), were tested. One was left untreated, one was washed with distilled water (H₂O), and one was washed with the organic solvent carbon disulphide (CS₂). In both species, we found a weak IOA between the spider leg and the gluey capture spiral in untreated and water‐washed legs without significant differences between the two. The IOA approximately doubled, when spider legs had been washed with carbon disulphide prior to measurement, that is, the CS₂‐washed legs stuck significantly more strongly than the untreated and water‐washed legs. These results provide indirect evidence for a protective anti‐adhesive organic coating on the spider’s body surface and so support Fabre’s hypothesis.     

Biosorption and desorption of lanthanum(III) and neodymium(III) in fixed-bed columns with Sargassum sp.: perspectives for separation of rare earth metals

Rare earth (RE) metals are essentials for the manufacturing of high-technology products. The separation of RE is complex and expensive; biosorption is an alternative to conventional processes. This work focuses on the biosorption of monocomponent and bicomponent solutions of lanthanum(III) and neodymium(III) in fixed-bed columns using Sargassum sp. biomass. The desorption of metals with HCl 0.10 mol L(-1) from loaded biomass is also carried out with the objective of increasing the efficiency of metal separation. Simple models have been successfully used to model breakthrough curves (i.e., Thomas, Bohart-Adams, and Yoon-Nelson equations) for the biosorption of monocomponent solutions. From biosorption and desorption experiments in both monocomponent and bicomponent solutions, a slight selectivity of the biomass for Nd(III) over La(III) is observed. The experiments did not find an effective separation of the RE studied, but their results indicate a possible partition between the metals, which is the fundamental condition for separation perspectives.     

High-throughput methods for miniaturization and automation of monoclonal antibody purification processes

In the last decade, high-throughput downstream process development techniques have entered the biopharmaceutical industry. As chromatography is the standard downstream purification method, several high-throughput chromatographic methods have been developed and applied including miniaturized chromatographic columns for utilization on liquid handling stations. These columns were used to setup a complete downstream process on a liquid handling station for the first time. In this article, a monoclonal antibody process was established in lab-scale and miniaturized afterwards. The scale-down methodology is presented and discussed. Liquid handling in miniaturized single and multicolumn processes was improved and applicability was demonstrated by volume balances. The challenges of absorption measurement are discussed and strategies were shown to improve volume balances and mass balances in 96-well microtiter plates. The feasibility of miniaturizing a complete downstream process was shown. In the future, analytical bottlenecks should be addressed to gain the full benefit from miniaturized complete process development.     

鬼臼毒素涂膜剂治疗子宫颈HPV 感染的疗效观察

目的:了解鬼臼毒素涂膜剂治疗子宫颈HPV感染的疗效。方法:126例临床诊断证明为子宫颈HPV感染的患者,随机分为两组,试验组63例采用0.5%鬼臼毒素涂膜剂治疗,对照组63例单独采用0.5%鬼臼毒素酊治疗。结果:①痊愈率:试验组与对照组的痊愈率分别为90.5%、73.00%,两组比较具有统计学学差异(P<0.05);②复发率:试验组与对照组第12、24周的复发率分别为9.52%、28.57%和9.52%、36.51%,两个时段的两组比较均具有统计学差异(P<0.05);③不良反应:经随访观察,两组发生了红斑、水肿、灼热、疼痛、瘙痒、溃疡、渗出等不良反应,其中两组比较试验组红斑、水肿、溃疡、渗出的发生率低于对照组,具有统计学差异(P<0.05)。结论:鬼臼毒素涂膜剂能有效治疗子宫颈HPV感染,并控制复发,副作用小,比采用鬼臼毒素酊治疗的效果好。     

Human fine body hair enhances ectoparasite detection

Although we are relatively naked in comparison with other primates, the human body is covered in a layer of fine hair (vellus and terminal hair) at a relatively high follicular density. There are relatively few explanations for the evolutionary maintenance of this type of human hair. Here, we experimentally test the hypothesis that human fine body hair plays a defensive function against ectoparasites (bed bugs). Our results show that fine body hair enhances the detection of ectoparasites through the combined effects of (i) increasing the parasite's search time and (ii) enhancing its detection.