A 3D-printed flow-cell for on-grid purification of electron microscopy samples directly from lysate.

While recent advances in cryo-EM, coupled with single particle analysis, have the potential to allow structure determination in a near-native state from vanishingly few individual particles, this vision has yet to be realised in practise. Requirements for particle numbers that currently far exceed the theoretical lower limits, challenges with the practicalities of achieving high concentrations for difficult-to-produce samples, and inadequate sample-dependent imaging conditions, all result in significant bottlenecks preventing routine structure determination using cryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks by developing affinity purification of samples on-grid; at once obviating the need to produce large amounts of protein, as well as more directly controlling the variable, and sample-dependent, process of grid preparation. In this proof-of-concept study, we demonstrate a further practical step towards this paradigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification from raw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-cell device can be interfaced directly with routinely-used laboratory equipment such as liquid chromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16") connectors, and can be used to allow binding of samples to affinity grids in a controlled environment prior to the extensive washing required to remove impurities. Furthermore, by designing a device which can be 3D printed and coupled to routinely used laboratory equipment, we hope to increase the accessibility of the techniques presented herein to researchers working towards single-particle macromolecular structures.

Recombinant human lubricin for prevention of postoperative intra-abdominal adhesions in a rat model.

BACKGROUND: Postoperative intra-abdominal adhesions are a major cause of morbidity and mortality and contribute to a heavy burden on health care resources. At present, numerous introduced adhesion prevention products have demonstrated some benefit but none are consistently effective. The aim of this study was to examine the effectiveness of recombinant human lubricin in preventing intra-abdominal adhesion formation. MATERIALS AND METHODS: A total of 62 male Wistar Albino rats were randomly assigned to the study. Six rats were used to the initial pilot study and 56 rats were randomized into four groups: (1) control cecal abrasion; (2) treatment cecal abrasion with 0.5 mg/mL lubricin solution; (3) control cecal enterotomy and primary closure; and (4) treatment cecal enterotomy and primary closure with 0.5 mg/mL lubricin solution. Rats were sacrificed at 3 d and 21 d postoperatively for the pilot and main studies, respectively. Macroscopic and microscopic adhesion severity was graded by blinded investigators. RESULTS: For the pilot study, all six rats successfully reached the end point indicating safety of the lubricin gel. In the main randomized study, adhesions in the treated cecal abrasion group were significantly reduced both macroscopically (P = 0.001) and microscopically (fibrosis P = 0.009, inflammation P < 0.0001), when compared with the control group. In the cecal enterotomy group, adhesions were reduced for the treatment group in macroscopic (P = 0.011) and microscopic grading (fibrosis P = 0.500, inflammation P = 0.206) compared with the control group. CONCLUSIONS: Recombinant human lubricin significantly reduced both macroscopic and microscopic intra-abdominal adhesions in the cecal abrasion group. The cecal enterotomy group showed modest macroscopic adhesion reduction. Future study using higher concentration of lubricin solution are needed to investigate its toxicity and more profound antiadhesion properties in significant operations.

Correction: Reusable snorkel masks adapted as particulate respirators.

[This corrects the article DOI: 10.1371/journal.pone.0249201.].

Reusable snorkel masks adapted as particulate respirators.

INTRODUCTION: During viral pandemics, filtering facepiece (FFP) masks together with eye protection form the essential components of personal protective equipment (PPE) for healthcare workers. There remain concerns regarding insufficient global supply and imperfect protection offered by currently available PPE strategies. A range of full-face snorkel masks were adapted to accept high grade medical respiratory filters using bespoke-designed 3D-printed connectors. We compared the protection offered by the snorkel to that of standard PPE using a placebo-controlled respirator filtering test as well as a fluorescent droplet deposition experiment. Out of the 56 subjects tested, 42 (75%) passed filtering testing with the snorkel mask compared to 31 (55%) with a FFP3 respirator mask (p = 0.003). Amongst the 43 subjects who were not excluded following a placebo control, 85% passed filtering testing with the snorkel versus to 68% with a FFP3 mask (p = 0.008). Following front and lateral spray of fluorescence liquid particles, the snorkel mask also provided superior protection against droplet deposition within the subject's face, when compared to a standard PPE combination of FFP3 masks and eye protection (3.19x108 versus 6.81x108 fluorescence units, p<0.001). The 3D printable adaptors are available for free download online at https://www.ImperialHackspace.com/COVID-19-Snorkel-Respirator-Project/. CONCLUSION: Full-face snorkel masks adapted as particulate respirators performed better than a standard PPE combination of FFP3 mask and eye protection against aerosol inhalation and droplet deposition. This adaptation is therefore a promising PPE solution for healthcare workers during highly contagious viral outbreaks.