The widely used 0.2/0.22 µm polymer sterile filters were developed for small molecule and protein sterile filtration but are not well-suited for the production of large nonprotein biological therapeutics, resulting in significant yield loss and production cost increases. Here, we report on the development of membranes with isoporous sub-0.2 μm rectangular prism pores using silicon micromachining to produce microslit silicon nitride (MSN) membranes. The very high porosity (~33%) and ultrathin (200 nm) nature of the 0.2 µm MSN membranes results in a dramatically different structure than the traditional 0.2/0.22 µm polymer sterile filter, which yielded comparable performance properties (including gas and hydraulic permeance, maximum differential pressure tolerance, nanoparticle sieving/fouling behavior). The results from bacteria retention tests, conducted according to the guidance of regulatory agencies, demonstrated that the 0.2 µm MSN membranes can be effectively used as sterile filters. It is anticipated that the results and technologies presented in this study will find future utility in the production of non-protein biological therapeutics and in other biological and biomedical applications. K E Y W O R D Sbiological therapeutics, silicon nanomembranes, sterile filtration
Advanced oxidation (AO) is widely used as a pre‐treatment and/or polishing step for the treatment of wastewater from industrial processes and the destruction of particular contaminants in water sources. It has a high treatment efficacy for many different compounds and thus is ideally suited as a treatment technology for specialized facilities that receive shipments of wastewater from networks of industrial, manufacturing, and commercial facilities. The primary challenge is how to optimize the process because bulk measurements of organic content (e.g. TOC) give no information about the specific composition and specialized advanced analytical techniques (e.g. LC‐MS) are unsuitable due to the complex composition. In this study, a novel combination of design‐of‐experiments (DOE) methods and LC‐OCD analysis was used with actual wastewater samples in order to optimize the AO treatment conditions in terms of chemical reagent concentrations, develop statistical models of the process, and identify potential mechanisms of COD removal. A significant variation in organic content removal was obtained over the range of conditions tested in the DOE method. For example, the percent removal of organic contaminants in the one wastewater sample varied from a low of 36 % to a high of 82 %. Most importantly, it was found that the treatment performance differed quite significantly for wastewater samples of different composition. The results presented in our study prove the need to dynamically optimize the AO treatment conditions for wastewater sources of different origins. Furthermore, by the application of the LC‐OCD analysis a step‐by‐step mechanism of COD removal was postulated.
The discovery of CRISPR-Cas9 has revolutionized molecular biology, greatly accelerating the introduction of genetic modifications into organisms and facilitating the development of novel therapeutics and diagnostics. For many applications, guide RNA and Cas9 protein are expressed, combined, and purified to produce a ribonucleic enzyme complex that is then added into a diagnostic device or delivered into cells.The objective of this work was to develop an ultrafiltration process for the selective purification of Cas9 ribonucleoprotein by removal of excess guide RNA. A His-tagged Streptococcus pyogenes Cas9 protein was produced in Escherichia coli, purified by metal affinity chromatography, and complexed with a 40 kDa (124 nucleotide) single guide RNA. Ultrafiltration experiments were first performed on solutions containing either guide RNA or Cas9 protein to identify the effect of filtration conditions and membrane pore size on the selectivity. Shear-induced aggregation of the Cas9 led to significant fouling under some conditions. A diafiltration process was then developed using a Biomax ® 300 kDa polyethersulfone membrane to selectively remove excess guide RNA from a solution containing Cas9-bound guide RNA and free guide RNA.These results demonstrate the potential of using ultrafiltration for the removal of excess RNA during the production of functional ribonucleoprotein complexes.
Kelp forests are among the most abundant coastal marine habitats but are vulnerable to the impacts of climate change. Between 2014 and 2016, an unprecedented heatwave and associated changes in trophic dynamics threatened kelp forests across the Northeast Pacific, with impacts documented from Mexico to Alaska. However, responses have varied substantially and remain poorly characterized across large stretches of coast, especially British Columbia (B.C.), which represents a significant percentage of the range of floating kelp species in the Northeast Pacific. Here, we explore variation in floating kelp (Macrocystis, Nereocystis) persistence pre- and post-heatwave across a >675 km latitudinal gradient, asking whether B.C. kelp forests are of conservation concern. We assembled and analyzed available quantitative kelp data, comparing snapshots of kelp extent before (1994 - 2007) and after (2018 - 2021) the heatwave in 11 regions spanning a range of temperature and sea otter occupancy statuses, and contextualizing these with time series analyses, where available (n = 7 regions). We provide strong evidence that kelp forests have declined in many regions but with evidence of refugia at both local and regional scales. Kelp forest persistence was negatively correlated with summer sea temperatures in southern B.C., where temperatures varied by ~6 degrees C across sites, at times exceeding species thermal tolerances. Kelp dynamics in northern regions appeared instead to be modulated by top-down control by urchins and sea otters. Our results demonstrate that B.C. kelp forest have been substantially reduced in recent years but that regional and local-scale factors influence the resilience of forests to large-scale perturbations.
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