Multiscale stability of an intertidal kelp (Postelsia palmaeformis) near its northern range edge through a period of prolonged heatwaves.

BACKGROUND AND AIMS: Climate change, including gradual changes and extreme weather events, is driving widespread species losses and range shifts. These climatic changes are felt acutely in intertidal ecosystems, where many organisms live close to their thermal limits and experience the extremes of both marine and terrestrial environments. A recent series of multiyear heatwaves in the northeast Pacific Ocean might have impacted species even towards their cooler, northern range edges. Among them, the high intertidal kelp Postelsia palmaeformis has traits that could make it particularly vulnerable to climate change, but it is critically understudied. METHODS: In 2021 and 2022, we replicated in situ and aerial P. palmaeformis surveys that were conducted originally in 2006 and 2007, in order to assess the state of northern populations following recent heatwaves. Changes in P. palmaeformis distribution, extent, density and morphometrics were assessed between these two time points over three spatial scales, ranging from 250 m grid cells across the entire 167 km study region, to within grid cells and the individual patch. KEY RESULTS: We found evidence consistent with population stability at all three scales: P. palmaeformis remained present in all 250 m grid cells in the study region where it was previously found, and neither the extent within cells nor the patch density changed significantly between time points. However, there was evidence of slight distributional expansion, increased blade lengths and a shift to earlier reproductive timing. CONCLUSIONS: We suggest that apparent long-term stability of P. palmaeformis might be attributable to thermal buffering near its northern range edge and from the wave-exposed coastlines it inhabits, which may have decreased the impacts of heatwaves. Our results highlight the importance of multiscale assessments when examining changes within species and populations, in addition to the importance of dispersal capability and local conditions in regulating the responses of species to climate change.

Development of isoporous microslit silicon nitride membranes for sterile filtration applications.

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.

Purification of Cas9-RNA complexes by ultrafiltration.

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.

Optimization of microorganism preservation conditions for the development of an acute toxicity bioassay for biocides.

Biocides, also referred to as 'microbicides' or 'inhibitors', are widely used in industrial processes (e.g. utility water in cooling towers) to control and/or eliminate the growth of microorganisms. Because of their inherent toxicity, their presence in various sources (e.g. river sediments, potable water) can negatively affect ecosystems. Currently available biocide detection techniques are not suitable for 'point-of-use' applications since they are tedious, complicated, and often require experienced personnel to operate. To address this concern, we sought to develop a simple-to-use toxicity bioassay based on a model microorganism (E. coli) after short (<30 min) exposure to known biocides that can be stored at room temperature (preferably) or in the fridge. Based on recent work and our expertise in polymer-based preservation of biomolecules, we leveraged this knowledge to improve E. coli preservation for biocide detection purposes. A design-of-experiments strategy was used to evaluate 16 different preservation conditions from 5 process parameters (i.e. 25-1 fractional factorial). It was found that pullulan, a sugar-based polymer, improved E. coli culturability by an order of magnitude after three months of storage. Also, it was found that storing E. coli in the fridge in Milli-Q water was favorable for maintaining a high level of culturability. Finally, the toxicity of three common biocides (Cetyltrimethylammonium bromide (CTAB), ProClin™ 300, and Grotan® BK) was evaluated using a fluorescence-based assay across all 16 preservation conditions. The response of the preserved E. coli was biocide specific and at certain conditions did not vary during the entire three-month storage period.