Mechanistic modeling, simulation, and optimization of mixed-mode chromatography for an antibody polishing step.

Mixed-mode chromatography combines features of ion-exchange chromatography and hydrophobic interaction chromatography and is increasingly used in antibody purification. As a replacement for flow-through operations on traditional unmixed resins or as a pH-controlled bind-and-elute step, the use of both interaction modes promises a better removal of product-specific impurities. However, the combination of the functionalities makes industrial process development significantly more complex, in particular the identification of the often small elution window that delivers the desired selectivity. Mechanistic modeling has proven that even difficult separation problems can be solved in a computer-optimized manner once the process dynamics have been modeled. The adsorption models described in the literature are also very complex, which makes model calibration difficult. In this work, we approach this problem with a newly constructed model that describes the adsorber saturation with the help of the surface coverage function of the colloidal particle adsorption model for ion-exchange chromatography. In a case study, a model for a pH-controlled antibody polishing step was created from six experiments. The behavior of fragments, aggregates, and host cell proteins was described with the help of offline analysis. After in silico optimization, a validation experiment confirmed an improved process performance in comparison to the historical process set point. In addition to these good results, the work also shows that the high dynamics of mixed-mode chromatography can produce unexpected results if process parameters deviate too far from tried and tested conditions.

Development of a method to rapidly assess resistance/susceptibility of Micro-Tom tomatoes to Tomato yellow leaf curl virus via agroinoculation of cotyledons.

OBJECTIVE: Tomato yellow leaf curl virus (TYLCV) is one of the pathogens severely damaging tomato crops. Therefore, methods to treat or prevent TYLCV infection need to be developed. For this purpose, a method to conveniently and quickly assess infection of tomatoes by TYLCV is desired. In the present study, we established a quick method to evaluate TYLCV infection using cotyledons of Micro-Tom, a miniature tomato cultivar. RESULTS: First, we constructed a binary plasmid harboring 1.5 copies of the TYLCV genome and transformed Agrobacterium with the plasmid. By injecting agroinoculum from the resulting transformant into the branches of Micro-Tom, we confirmed the susceptibility of Micro-Tom to TYLCV. To shorten the evaluation process of TYLCV infection further, we agroinoculated cotyledons of Micro-Tom 10 days after sowing seeds. We consistently observed typical symptoms of TYLCV infection on true leaves 10 days after agroinoculation. Molecular analysis detected TYLCV progeny DNA in all leaves demonstrating symptoms 6 days after agroinoculation. Therefore, our new protocol enabled assessment of TYLCV infection within 20 days after sowing seeds. Thus, agroinoculation of Micro-Tom cotyledons will accelerate the process of screening TYLCV-resistant Micro-Toms and enable screening of larger numbers of plants more quickly, contributing to the development of TYLCV-resistant tomatoes.

Publisher Correction: Decomposition and oxidation of methionine and tryptophan following irradiation with a nonequilibrium plasma jet and applications for killing cancer cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Decomposition and oxidation of methionine and tryptophan following irradiation with a nonequilibrium plasma jet and applications for killing cancer cells.

We present evidence for the decomposition and oxidation of amino acids in aqueous solution following irradiation with a nonequilibrium plasma jet. Of 15 amino acids tested in cell culture medium, plasma irradiation induced a marked chemical change in methionine and tryptophan due to the effective production of reactive oxygen species by plasma-water interaction. We also report that plasma-treated methionine and tryptophan aqueous solutions can kill cancer cells, greatly decreasing the viability of human endometrial carcinoma (HEC-1) cancer cells due to the presence of decomposition or oxidation products generated from the amino acid. Plasma-treated methionine and tryptophan aqueous solutions also induced an anti-cancer effect on cancer-initiating cells.

Plasma-enhanced reactive linear sputtering source for formation of silicon-based thin films.

In this study, an inductively coupled plasma (ICP)-enhanced reactive sputter deposition system with a rectangular target was developed as a linear plasma source for roll-to-roll deposition processes. The longitudinal distribution of the film thickness indicated the feasibility of uniformity control via the control of the power deposition profile of the assisted ICPs. The characteristics of Si films were investigated in terms of the film thickness uniformity and film crystallinity. The results of Raman and X-ray diffraction measurements indicated the crystallization of the Si film with a crystallinity as high as 73%-78% in all the samples of the longitudinal position.

Atmospheric-Pressure Plasma Interaction with Soft Materials as Fundamental Processes in Plasma Medicine.

Molecular-structure variation of organic materials irradiated with atmospheric pressure He plasma jet have been investigated. Optical emission spectrum in the atmospheric-pressure He plasma jet has been measured. The spectrum shows considerable emissions of He lines, and the emission of O and N radicals attributed to air. Variation in molecular structure of Polyethylene terephthalate (PET) film surface irradiated with the atmospheric-pressure He plasma jet has been observed via X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR). These results via XPS and FT-IR indicate that the PET surface irradiated with the atmospheric-pressure He plasma jet was oxidized by chemical and/or physical effect due to irradiation of active species.

Plasma Interaction with Organic Molecules in Liquid as Fundamental Processes in Plasma Medicine.

Investigation of plasma-organic materials interaction in aqueous solution with atmospheric pressure plasmas have been carried out. Degradation of methylene blue (MB) in aqueous solution via atmospheric pressure He plasma exposure through gas/liquid interface have been investigated. The optical emission spectrum shows considerable emissions of He lines and the emission of O, OH and N radicals attributed to dissociation of water (H2O) and air has been confirmed. Structure variation of MB in solution treated with the atmospheric-pressure He plasma has been measured by Fourier transform infrared spectroscopy (FT-IR). The results obtained from FT-IR analysis show degradation of MB in solution treated with the atmospheric-pressure He plasma. The pH effect of MB degradation was investigated using controlled pH solutions by an ultraviolet-visible (UV-Vis) spectroscopy and FT-IR. The results show no effect of MB degradation on pH. The results exhibit that the atmospheric pressure plasmas exposure has made it possible to degrade organic materials in solution due to irradiated radicals from plasma through plasma/liquid interface.

Atmospheric-Pressure Gas-Breakdown Characteristics with a Radio-Frequency Voltage.

We present He atmospheric-pressure gas-breakdown phenomena with radio frequency (RF) voltages in the frequency region from a few tens MHz to 100 MHz. The gas-breakdown voltage for RF and very high frequency (VHF) discharges is considerably lower than that for the DC discharge, and the gas-breakdown voltage is effectively reduced to be as low as 160 V in VHF region. The discharge characteristics drastically change with increasing discharge-voltage frequency, and strong emisson is highly loclized in front of the power- and ground-electrode in the VHF discharges. The lowering gas-brakdown voltage and the localized emission-profile are well explained by the effect of the charged-particle confinement in a micro dishcarge-space.

Dynamic Properties of Helium Atmospheric Dielectric-Barrier-Discharge Plasma Jet.

We present here experiments on helium atmospheric dielectric-barrier discharge jet in open air. A long stable plasma plume is realized at high applied voltage and high gas flow rate. Optical emission measurements show that the plasma plume consists of two part: a plume head with high energy electrons and a tail part with low energy electrons. The plasma plume propagates away from the quartz-tube outlet with about 30-80 km/sec along the helium gas flow channel. The propagation velocity of plasma plume is in the time scale of electron drift velocity, and the electric field plays an important role as a driving force of the plasma plume propagation.

Inhibition of binding of tomato yellow leaf curl virus rep to its replication origin by artificial zinc-finger protein.

Previously we demonstrated that inhibition of replication-associated protein (Rep) binding to its replication origin by artificial zinc-finger proteins (AZPs) is a powerful method to prevent plant virus infection in vivo. In the present study, we applied the AZP technology to Tomato yellow leaf curl virus (TYLCV), which is a limiting factor in tomato cultivation worldwide. First, we determined 5'-ATCGGTGT ATCGGTGT-3' in the 195-bp intergenic region of the TYLCV-Israel strain, a strain reported first among TYLCV strains, as the Rep-binding site by gel shift assays. We then constructed a 6-finger AZP that bound to a 19-bp DNA including the Rep-binding site. We demonstrated that the binding affinity of the AZP was >1,000-fold greater than that of Rep and that the AZP inhibited Rep binding completely in vitro. Because the binding capability of the AZP was same as that of the AZP previously designed for geminivirus-resistant Arabidopsis thaliana, we predict that the present AZP will prevent TYLCV infection in vivo.

Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease.

To refine zinc-finger nuclease (ZFN) technology, we constructed a sandwiched ZFN, in which a DNA cleavage enzyme was sandwiched with two artificial zinc-finger proteins (AZPs). Because the sandwiched ZFN is designed to cleave the DNA between the two AZP-binding sites, the sandwiched ZFN is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To prove the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two 3-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with control nucleases that possess a single AZP.

Construction of plants resistant to TYLCV by using artificial zinc-finger proteins.

Previously, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to tomato yellow leaf curl virus (TYLCV) causing serious damage to an important agricultural crop, tomato. An AZP was designed to block binding of the TYLCV replication protein (Rep) to the replication origin. The designed AZP had much higher affinities towards the replication origin than did the Rep, and efficiently blocked Rep binding in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. The current status of the construction of the AZP-expressing transgenic plants will be reported.

Enhanced cleavage of double-stranded DNA by artificial zinc-finger nuclease sandwiched between two zinc-finger proteins.

To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with nucleases that possess a single AZP. Thus, AZP-sandwiched nucleases will further refine ZFN technology.

Generation of plants resistant to tomato yellow leaf curl virus by using artificial zinc-finger proteins.

Previously, we designed an artificial zinc-finger protein (AZP) for blocking a replication protein (Rep) of beet severe curly top virus (BSCTV) from binding to its replication origin and demonstrated that transgenic Arabidopsis plants expressing the AZP are completely resistant to the virus infection. Here we applied the AZP technology to tomato yellow leaf curl virus (TYLCV) infective to an important agricultural crop, tomato. We designed and constructed an AZP binding to the direct repeat to block the TYLCV Rep binding. In gel shift assays, we confirmed that the designed AZP has a higher affinity to the replication origin than that of Rep and that the AZP effectively inhibited the Rep binding to its replication origin in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. We will discuss properties of the AZP-transgenic plants against TYLCV infection.

Inhibition of tomato yellow leaf curl virus replication by artificial zinc-finger proteins.

Previously, we designed an artificial zinc-finger protein (AZP) for blocking a replication protein (Rep) of beet severe curly top virus (BSCTV) from binding to its replication origin and demonstrated that transgenic Arabidopsis plants expressing the AZP are completely resistant to the virus infection. Here we applied the AZP technology to tomato yellow leaf curl virus (TYLCV) infective to an important agricultural crop, tomato. We designed an AZP binding to the direct repeat to block the TYLCV Rep binding and confirmed in gel shift assays that the designed AZP has a higher affinity to the replication origin than that of Rep. Furthermore, we demonstrated in competitive binding assays that the AZP effectively inhibited the Rep binding in vitro. We discuss properties of the AZP for inhibition of TYLCV replication in detail.