Search

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.

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.

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.

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.

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.