The FG-repeat asymmetry of the nuclear pore complex is dispensable for bulk nucleocytoplasmic transport in vivo

Nucleocytoplasmic transport occurs through gigantic proteinaceous channels called nuclear pore complexes (NPCs). Translocation through the NPC is exquisitely selective and is mediated by interactions between soluble transport carriers and insoluble NPC proteins that contain phenylalanine-glycine (FG) repeats. Although most FG nucleoporins (Nups) are organized symmetrically about the planar axis of the nuclear envelope, very few localize exclusively to one side of the NPC. We constructed Saccharomyces cerevisiae mutants with asymmetric FG repeats either deleted or swapped to generate NPCs with inverted FG asymmetry. The mutant Nups localize properly within the NPC and exhibit exchanged binding specificity for the export factor Xpo1. Surprisingly, we were unable to detect any defects in the Kap95, Kap121, Xpo1, or mRNA transport pathways in cells expressing the mutant FG Nups. These findings suggest that the biased distribution of FG repeats is not required for major nucleocytoplasmic trafficking events across the NPC

Measurement of the Intertablet Coating Uniformity of a Pharmaceutical Pan Coating Process With Combined Terahertz and Optical Coherence Tomography In-Line Sensing.

We present in-line coating thickness measurements acquired simultaneously using two independent sensing modalities: terahertz pulsed imaging (TPI) and optical coherence tomography (OCT). Both techniques are sufficiently fast to resolve the coating thickness of individual pharmaceutical tablets in-situ during the film coating operation and both techniques are direct structural imaging techniques that do not require multivariate calibration. The TPI sensor is suitable to measure coatings greater than 50 μm and can penetrate through thick coatings even in the presence of pigments over a wide range of excipients. Due to the long wavelength, terahertz radiation is not affected by scattering from dust within the coater. In contrast, OCT can resolve coating layers as thin as 20 μm and is capable of measuring the intra-tablet coating uniformity as well as the inter-tablet coating thickness distribution within the coating pan. ¬-However, the OCT technique is less robust when it comes to the compatibility with excipients, dust and potentially the maximum coating thickness that can be resolved. Using a custom built laboratory scale coating unit, the coating thickness measurements were acquired independently by the TPI and OCT sensors throughout a film coating operation. Results of the in-line TPI and OCT measurements were compared against one another and validated with off-line TPI and weight gain measurements. Compared to other process analytical technology (PAT) sensors, such as near-infrared and Raman spectroscopy, the TPI/OCT sensors can resolve the inter-tablet thickness distribution based on sampling a significant fraction of the tablet populations in the process. By combining two complementary sensing modalities it was possible to seamlessly monitor the coating process over the range of film thickness from 20 μm to greater than 250 μm.The authors would like to acknowledge the financial support from UK EPSRC Research Grant EP/L019787/1 and EP/L019922/1. The authors acknowledge BASF for providing the materials used in this study, Colorcon Ltd. (Dartford, UK) for coating process recommendations, Hüttlin GmbH (Bosch Packaging Technology, Schopfheim, Germany) for advice on the coating unit design and the staff of the electronics and mechanical workshops in Department of Chemical Engineering and Biotechnology at University of Cambridge. HL also acknowledges travel support from Joy Welch Educational Charitable Trust

Measurement of the Intertablet Coating Uniformity of a Pharmaceutical Pan Coating Process With Combined Terahertz and Optical Coherence Tomography In-Line Sensing.

We present in-line coating thickness measurements acquired simultaneously using 2 independent sensing modalities: terahertz pulsed imaging (TPI) and optical coherence tomography (OCT). Both techniques are sufficiently fast to resolve the coating thickness of individual pharmaceutical tablets in situ during the film coating operation, and both techniques are direct structural imaging techniques that do not require multivariate calibration. The TPI sensor is suitable to measure coatings greater than 50 μm and can penetrate through thick coatings even in the presence of pigments over a wide range of excipients. Due to the long wavelength, terahertz radiation is not affected by scattering from dust within the coater. In contrast, OCT can resolve coating layers as thin as 20 μm and is capable of measuring the intratablet coating uniformity and the intertablet coating thickness distribution within the coating pan. However, the OCT technique is less robust when it comes to the compatibility with excipients, dust, and potentially the maximum coating thickness that can be resolved. Using a custom-built laboratory scale coating unit, the coating thickness measurements were acquired independently by the TPI and OCT sensors throughout a film coating operation. Results of the in-line TPI and OCT measurements were compared against one another and validated with off-line TPI and weight gain measurements. Compared with other process analytical technology sensors, such as near-infrared and Raman spectroscopy, the TPI and OCT sensors can resolve the intertablet thickness distribution based on sampling a significant fraction of the tablet populations in the process. By combining 2 complementary sensing modalities, it was possible to seamlessly monitor the coating process over the range of film thickness from 20 μm to greater than 250 μm.The authors would like to acknowledge the financial support from UK EPSRC Research Grant EP/L019787/1 and EP/L019922/1. The authors acknowledge BASF for providing the materials used in this study, Colorcon Ltd. (Dartford, UK) for coating process recommendations, Hüttlin GmbH (Bosch Packaging Technology, Schopfheim, Germany) for advice on the coating unit design and the staff of the electronics and mechanical workshops in Department of Chemical Engineering and Biotechnology at University of Cambridge. HL also acknowledges travel support from Joy Welch Educational Charitable Trust

Studying pharmaceutical tablets mixing process inside a perforated pan-coater using in-line terahertz sensing

In-line terahertz sensing has been demonstrated to measure the coating thickness of individual pharmaceutical tablets during coating operation. As coating uniformity is highly dependent on tablet mixing, this study presents our research progress to date on using in-line terahertz sensing to investigate the effects of baffle design, drum rotational speed and batch size, on tablet mixing inside a laboratory-sized perforated pan coater

Studying the pharmaceutical film coating process with terahertz sensing, optical coherence tomography and numerical modelling

Terahertz in-line sensing was successfully demonstrated on a production scale setting for measuring the coating thickness of individual pharmaceutical tablets during the film coating process. This paper reports on our recent research progress to combine terahertz in-line sensing, optical coherence tomography and numerical modelling in a lab scale setting to better understand the pharmaceutical film coating process

Targeted genome editing across species using ZFNs and TALENs

Evolutionary studies necessary to dissect diverse biological processes have been limited by the lack of reverse genetic approaches in most organisms with sequenced genomes. We established a broadly applicable strategy using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for targeted disruption of endogenous genes and cis-acting regulatory elements in diverged nematode species

Optimising Terahertz Waveform Selection of a Pharmaceutical Film Coating Process Using Recurrent Network

In-line terahertz pulsed imaging (TPI) has been utilised to measure the film coating thickness of individual tablets during the coating process in a production-scale pan coater. A criteria-based waveform selection algorithm (WSA) was developed to select terahertz signals reflected from the surface of coating tablets and determine the coating thickness. Since the WSA uses many criteria thresholds to select terahertz waveforms of sufficiently high quality, it could reject some potential candidate tablet waveforms that are close but do not reach the threshold boundary. On the premise of the availability of large datasets, we aim to improve the efficiency of WSA with machine learning. This paper presents a recurrent neural network approach to optimise waveform selection. In comparison with the conventional method of WSA, our approach allows more than double the number of waveforms to be selected while maintain great agreement with off-line thickness measurement. Moreover, the processing time of waveform selection decreases so that it can be applied for real-time coating monitoring in the pharmaceutical industry, which leads more advancement on the quality control for the pharmaceutical film coating

Terahertz waveform selection of a pharmaceutical film coating process using a recurrent network

Waveform selection plays an important role in the processing of in-line terahertz measurements of pharmaceutical tablet coating processes. This paper presents an approach to optimise waveform selection by utilising an artificial recurrent neural network and transfer learning. The results show that the averaged coating thickness gradually increases throughout the coating process. In comparison with the conventional method, our approach allows more than double the number of waveforms to be selected without compromising on the accuracy when compared against off-line measurements. Moreover, the processing time of waveform selection decreases so that it can be applied for real-time coating monitor in the pharmaceutical industry

Genetic engineering of human ES and iPS cells using TALE nucleases

Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator–like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869)National Institutes of Health (U.S.) (Grant RO1-HD045022)Howard Hughes Medical Institut