Search

Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the crystal cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that crystals in a suspended drop could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the SARS‑CoV‑2 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting systematic preferred orientations, unlocking new possibilities for structure determination by MicroED., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Milk-derived extracellular vesicles (M-EVs) are low-cost, can be prepared in large quantities, and can cross the gastrointestinal barrier for oral administration. However, the composition of milk is complex, and M-EVs obtained by different extraction methods may affect their oral delivery. Based on this, a new method for extracting M-EVs based on cryogenic freezing treatment (Cryo-M-EVs) is proposed and compared with the previously reported acetic acid treatment (Acid-M-EVs) method and the conventional ultracentrifugation method (Ulltr-M-EVs). The new method simplifies the pretreatment step and achieves 25-fold and twofold higher yields than Acid-M-EVs and Ulltr-M-EVs. And it is interesting to note that Cryo-M-EVs and Acid-M-EVs have higher cellular uptake efficiency, and Cryo-M-EVs present the best transepithelial transport effect. After oral administration of the three M-EVs extracted by three methods in mice, Cryo-M-EVs effectively successfully cross the gastrointestinal barrier and achieve hepatic accumulation, whereas Acid-M-EVs and Ultr-M-EVs mostly reside in the intestine. The M-EVs obtained by the three extraction methods show a favorable safety profile at the cellular as well as animal level. Therefore, when M-EVs obtained by different extraction methods are used for oral drug delivery, their accumulation properties at different sites can be utilized to better deal with different diseases., (© 2024 Wiley‐VCH GmbH.)

In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids., (open access.)

The enhanced interest in greater convenience foods has recently led to the expansion of minimally processed potato products. This study investigated the effects of isochoric freezing on pre-peeled potato cubes, including quality attributes (microstructure, texture, and color), nutritional value (ascorbic acid (AA) content, total phenolic content, and antioxidant capacity), and polyphenol oxidase activity. Isochoric freezing (-3 °C/30 MPa) was compared with isobaric freezing (-3 °C/0.1 MPa) and individual quick freezing followed by frozen storage at -20 °C for 4 weeks. The isochoric sample had lower drip loss and volume shrinkage as well as better preserved texture and microstructure than the other samples. All freezing methods caused an increase in total phenolic content and antioxidant capacity, but a decrease in AA content. Also, all freezing methods caused browning of the thawed potatoes, but isochoric freezing delayed its onset for more than 1 week. PRACTICAL APPLICATION: Results showed that isochoric freezing of pre-peeled and cut potatoes caused less freeze damage than isobaric and individual quick freezing, which might find application in the commercial preservation of minimally processed food products., (© 2020 Institute of Food Technologists®.)

Cryogenic transmission electron microscopy of high-pressure freezing (HPF) samples is a well-established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic-scanning electronic microscope (Cryo-SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo-SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM-studies using HPF., (© 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.)

The objective of this study was to evaluate the effects of stepwise dry/wet-aging and freezing method on quality attributes of beef loins. Paired loins (M. Longissimus lumborum) from eight carcasses were assigned to either stepwise dry/wet-aging (carcass dry-aging for 10days then further wet-aging for 7days in vacuum bags) or carcass dry-aging only for 17days. Then, each loin was divided into three sections for freezing (never-frozen, blast or cryogenic freezing). Stepwise dry/wet-aged loin had lower purge/drip loss and shear force than conventionally dry-aged loin (P<0.05), but similar color and sensory characteristics (P>0.05). The cryogenic freezing resulted in a significant decrease in shear force values and a significant improvement in water-holding capacity (WHC). These findings indicate that the stepwise dry/wet-aging coupled with cryogenic freezing could provide beneficial impacts to the local meat industry by providing equivalent quality attributes as conventional dry-aging and improving WHC of frozen/thawed meat, while reducing the time needed for dry-aging., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Plastics continue to be a challenge for recovering materials at the end-of-life for vehicles. However, it may be possible to improve the recovery of plastics by exploiting material characteristics, such as shape, or by altering their behavior, such as through temperature changes, in relation to recovery processes and handling. Samples of a 2009 Dodge Challenger front fascia were shredded in a laboratory-scale hammer mill shredder. A 2 × 2 factorial design study was performed to determine the effect of sample shape (flat versus curved) and sample temperature (room temperature versus cryogenic temperature) on the size of the particles exiting from the shredder. It was determined that sample shape does not affect the particle size; however, sample temperature does affect the particle size. At cryogenic temperatures, the distribution of particle sizes is much narrower than at room temperature. Having a more uniform particle size could make recovery of plastic particles, such as these more efficient during the recycling of end-of-life vehicles. Samples of Chrysler minivan headlights were also shredded at room temperature and at cryogenic temperatures. The size of the particles of the two different plastics in the headlights is statistically different both at room temperature and at cryogenic temperature, and the particles are distributed narrowly. The research suggests that incremental changes in end-of-life vehicle processing could be effective in aiding materials recovery.

Foodborne pathogens such as Listeria monocytogenes could pose a health risk on frozen ready-to-eat (RTE) shrimp as the pathogen could grow following thawing. In this study, antimicrobial-coating treatments alone, or in combination with cryogenic freezing, were evaluated for their ability to inhibit the growth of Listeria innocua, a surrogate for L. monocytogenes, on RTE shrimp. Cooked RTE shrimp were inoculated with L. innocua at 3 population levels and treated with coating solutions consisting of chitosan, allyl isothiocyanate (AIT), or lauric arginate ester (LAE). The treated shrimp were then stored at -18 °C for 6 d before being thawed at 4, 10, or 22 °C for either 24 or 48 h. Results revealed that antimicrobial coatings achieved approximately 5.5 to 1 log CFU/g reduction of L. innocua on RTE shrimp after the treatments, depending on the inoculated population levels. The coating-treated shrimp samples had significantly (P < 0.05) less L. innocua than controls at each thawing temperature and time. Cryogenic freezing in combination with coating treatments did not achieve synergistic effects against L. innocua. Antimicrobial coatings can help to improve product safety by reducing Listeria on RTE shrimp., (Journal of Food Science © 2013 Institute of Food Technologists® No claim to original US government works.)