Formate-removing inoculum dominated by Methanobacterium congolense supports succinate production from crude glycerol fermentation.

We developed a formate-removing methanogenic inoculum (FRI) to facilitate succinate production from crude glycerol by Escherichia coli. FRI converted formate to methane, thereby enabling glycerol fermentation without additional electron acceptors under neutral pH. FRI was selectively enriched from sludge from the anaerobic digester of the Seonam sewage treatment plant (Seoul); this process was assessed via Illumina sequencing and scanning electron microscopy imaging. Methanobacterium congolense species occupied only 0.3% of the archaea community in the sludge and was enriched to 99.5% in complete FRI, wherein succinate-degrading bacteria were successfully eliminated. Co-culture with FRI improved glycerol fermentation and yielded 7.3 mM succinate from 28.7 mM crude glycerol, whereby FRI completely converted formate into methane. This study is the first to demonstrate methane production by M. congolense species, using formate. M. congolense-dominated FRI can serve as a renewable facilitator of waste feedstock fermentation and enable the production of commercially important compounds.

Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors.

PURPOSE: The cyclin-dependent kinase 1 (Cdk1) and cyclin B complex performs important roles in the transition from the G2 to M phase in the cell cycle through removal of inhibitory phosphates on Cdk1, and Cdc25B, which is a dual-specific phosphatase, mediates these dephosphorylation events. However, measuring Cdc25B activity by existing methods is hampered by inadequate nonspecific substrates and the need to use a radiolabeled isotope. The present study aimed to develop an improved method with which to properly measure Cdc25B activity using a novel nonradioisotopic assay and Cdc25B overexpression cell lines. MATERIALS AND METHODS: A nonradioisotopic Cdk1 kinase assay, based on Western blotting for retinoblastoma protein and histone H1, was used to analyze Cdc25B activity. Also, stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines were constructed using the tetracycline-regulated expression system and were applied as a tool for screening for inhibitors of Cdc25B. RESULTS: The present study developed and optimized a nonradioisotopic assay method to properly measure Cdc25B activity. Furthermore, we constructed stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines for the establishment of a strong assay system with which to evaluate the specificity of Cdc25B inhibitors under conditions similar to the intracellular environment. These methods were confirmed as useful tools for measuring Cdc25B activity. CONCLUSION: The nonradioisotopic Cdk1 kinase assay and Cdc25B overexpression cell lines developed in this study can be conveniently used as tools for screening inhibitors of Cdc25B phosphatase as anticancer drugs.

Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source.

Escherichia coli has been a primary host for the prokaryotic production of antibody fragments (Fabs) and has contributed to several successes in the pharmaceutical industry. Nevertheless, the requirement of disulfide bonds often results in low-yield fermentation and a lack of cost-effectiveness. Despite the improved production of functional Fabs by fermentation below 30 °C, the limited cellular growth needs further work. To address these issues, we investigated the effect of nitrogen supply on the cellular growth and the Fab productivity. We used the anti-human VEGF-A Fab as a model that exhibited poor expression at 37 °C regardless of the amount of nitrogen supplied during fermentation. In stark contrast, the expression yield of soluble Fab with a gross nitrogen supply of 6.91 g/L of broth throughout the fermentation at 25 °C was 332 mg/L. Furthermore, and increased nitrogen supply of 10.9 g/L significantly improved the yield of active form by 59.7% and the cellular growth rate by 39.3%. These results indicate that overdosing of a nitrogen source at low temperature is critical to Fab productivity in E. coli.

Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs.

Glycosylation of the hemagglutinin (HA) and neuraminidase (NA) of the influenza provides crucial means for immune evasion and viral fitness in a host population. However, the time-dependent dynamics of each glycosylation sites have not been addressed. We monitored the potential N-linked glycosylation (NLG) sites of over 10,000 HA and NA of H1N1 subtype isolated from human, avian, and swine species over the past century. The results show a shift in glycosylation sites as a hallmark of 1918 and 2009 pandemics, and also for the 1976 "abortive pandemic". Co-segregation of particular glycosylation sites was identified as a characteristic of zoonotic transmission from animal reservoirs, and interestingly, of "reverse zoonosis" of human viruses into swine populations as well. After the 2009 pandemic, recent isolates accrued glycosylation at canonical sites in HA, reflecting gradual seasonal adaptation, and a novel glycosylation in NA as an independent signature for adaptation among humans. Structural predictions indicated a remarkably pleiotropic influence of glycans on multiple HA epitopes for immune evasion, without sacrificing the receptor binding of HA or the activity of NA. The results provided the rationale for establishing the ecological niche of influenza viruses among the reservoir and could be implemented for influenza surveillance and improving pandemic preparedness.

Purification of antibody fragments for the reduction of charge variants using cation exchange chromatography.

Recently, antibody fragments have been studied as therapeutic agents because they lack Fc effector function while having affinity similar to their original monoclonal antibody and can be produced using E. coli. Antibody fragments can be purified using affinity chromatography in the capture step, although they need a polishing step because of product-related impurities, mainly charge variants. Unlike monoclonal antibodies, few studies exist regarding the separation of charge variants in antibody variants. In this study, an efficient separation of charge variant method was assessed using a cation exchange chromatography resin with salt and a pH gradient. The SP ImpRes resin and pH gradient exhibited the most effective separation potency using combinations of resin and the separation method. The antibody fragment that did not undergo the charge variant separation process exhibited a difference in the tertiary structure of the protein and in vivo pharmacokinetics. However, the antibody fragment was similar to the reference protein when the charge variant separation process was performed. These results are expected to support efficient charge variant separation of antibody fragments and to be applied to the industrial production of therapeutic antibody fragments.

Fluctuations around mean walking behaviors in diluted pedestrian flows.

Understanding and modeling the dynamics of pedestrian crowds can help with designing and increasing the safety of civil facilities. A key feature of a crowd is its intrinsic stochasticity, appearing even under very diluted conditions, due to the variability in individual behaviors. Individual stochasticity becomes even more important under densely crowded conditions, since it can be nonlinearly magnified and may lead to potentially dangerous collective behaviors. To understand quantitatively crowd stochasticity, we study the real-life dynamics of a large ensemble of pedestrians walking undisturbed, and we perform a statistical analysis of the fully resolved pedestrian trajectories obtained by a yearlong high-resolution measurement campaign. Our measurements have been carried out in a corridor of the Eindhoven University of Technology via a combination of Microsoft Kinect 3D range sensor and automatic head-tracking algorithms. The temporal homogeneity of our large database of trajectories allows us to robustly define and separate average walking behaviors from fluctuations parallel and orthogonal with respect to the average walking path. Fluctuations include rare events when individuals suddenly change their minds and invert their walking directions. Such tendency to invert direction has been poorly studied so far, even if it may have important implications on the functioning and safety of facilities. We propose a model for the dynamics of undisturbed pedestrians, based on stochastic differential equations, that provides a good agreement with our field observations, including the occurrence of rare events.

Iterative phase-shifting algorithm immune to random phase shifts and tilts.

An iterative phase-shifting algorithm based on the least-squares principle is developed to overcome the random piston and tilt wavefront errors generated from the phase shifter. The algorithm iteratively calculates the phase distribution and the phase-shifting map to minimize the sum of squared errors in the interferograms. The performance of the algorithm is evaluated via computer simulations and validated by the Fizeau interferometer measurements. The results show that the proposed algorithm has a fast convergence rate and satisfactory phase-estimation accuracy, improving the measurement precision of the phase-shifting interferometers with significant phase-shifter errors.

Enhanced in and out-coupling of InGaAs slab waveguides by periodic metal slit arrays.

We studied the in- and the out-coupling efficiencies of photons with a thin InGaAs slab covered by periodic gold nano-slit arrays, by measuring transmission and photoluminescence (PL) spectra. While the maximum in-coupled photons into the InGaAs slab waveguide were found at dip positions in transmission spectra, the mostly out-coupled photons were observed as peaks in PL spectra. For different periods of slit arrays and incident angles we discussed spectral positions of transmission dips and efficiency of the in-coupling influenced by the absorption coefficient of InGaAs. In PL spectra we measured overall enhanced PL intensities from the InGaAs slab covered by slit arrays compared to that of a bare InGaAs, where the peak positions are determined by the period of slit arrays as well. Our findings are important for designing semiconductors both as an optically passive waveguide and active light emitter.

Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus.

A human influenza A virus X-31 (high-yielding strain) was cold-adapted for possible future use as live attenuated vaccine. Mutant influenza viruses were selected during successive serial passage in embryonated hens' eggs at progressively lower sub-optimal temperature (30, 27 degrees C followed by 24 degrees C). The cold-passaged mutant exhibited both temperature-sensitivity (ts) and cold-adapted (ca) phenotypes. The pathogenicity and immunogenicity of X-31 ca virus were studied in mice following intranasal inoculation. The mice did not show clinical signs even at high titer infection. Immunization of mice with X-31 ca virus elicited high titers of neutralizing antibody and provided complete protection against homologous and heterologous virus challenges. To assess the genetic stability, the X-31 ca virus was passaged at 37 degrees C in MDCK cells or inoculated into mice. Revertant virus was not found in the lungs of any of the mice and the supernatants of the MDCK culture. We conclude that the X-31 ca candidate vaccine virus exhibits the desired level of attenuation, immunogenicity, and protective efficacy required for live attenuated vaccine and merits further evaluation at clinical level.