An Observation of a Very High Swelling of Bromovirus Members at Specific Ionic Strengths and pH.

Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV) are naked plant viruses with similar characteristics; both form a T = 3 icosahedral protein capsid and are members of the bromoviridae family. It is well known that these viruses completely disassemble and liberate their genome at a pH around 7.2 and 1 M ionic strength. However, the 1 M ionic strength condition is not present inside cells, so an important question is how these viruses deliver their genome inside cells for their viral replication. There are some studies reporting the swelling of the CCMV virus using different techniques. For example, it is reported that at a pH~7.2 and low ionic strength, the swelling observed is about 10% of the initial diameter of the virus. Furthermore, different regions within the cell are known to have different pH levels and ionic strengths. In this work, we performed several experiments at low ionic strengths of 0.1, 0.2, and 0.3 and systematically increased the pH in 0.2 increments from 4.6 to 7.4. To determine the change in virus size at the different pHs and ionic strengths, we first used dynamic light scattering (DLS). Most of the experiments agree with a 10% capsid swelling under the conditions reported in previous works, but surprisingly, we found that at some particular conditions, the virus capsid swelling could be as big as 20 to 35% of the original size. These measurements were corroborated by atomic force microscopy (AFM) and transmission electron microscopy (TEM) around the conditions where the big swelling was determined by DLS. Therefore, this big swelling could be an easier mechanism that viruses use inside the cell to deliver their genome to the cell machinery for viral replication.

Effects of substrate temperature on patterns produced by dried droplets of proteins.

Rapid diagnosis provides better clinical management of patients, helps control possible outbreaks, and increases survival. The study of deposits produced by the evaporation of droplets is a useful tool in the diagnosis of some health problems. With the aim to improve diagnostic time in clinical practice where we use the evaporation of droplets, we explored the effects of substrate temperature on pattern formation of dried droplets in globular protein solutions. Three deposit groups were observed: "functional" patterns (from 25 to 37 ∘C), "transition" patterns (from 44 to 50 ∘C), and "eye" patterns (from 58 to 63 ∘C). The dried droplets of the first two groups show a ring structure ("coffee-ring") that confines a great diversity of aggregates such as needle-like structures, tiny blade-shape crystals, highly symmetrical crystallization patterns, and amorphous salt aggregates. In contrast, the "eye" patterns are deposits with a large inner aggregate surrounded by a coffee ring, and they can appear from the evaporation of droplets in protein binary mixtures and blood serum. Interestingly, the unfolding proteins correlates with the formation of "eye" patterns. We measured stain diameter, "coffee-ring" thickness, radial density profile, and entropy computed by GLCM-statistics to quantify the structural differences among deposit groups. We found that "functional" patterns are structurally indistinguishable among them, but they are clearly different from elements of the other deposit groups. An exponential decay function describes pattern formation time as a function of substrate temperature, which is independent from protein concentration. Patterns formation at 32 ∘C takes place up to 63% less time and preserves the structural characteristics of dried droplets in proteins formed at room temperature. Therefore, we argue that droplet evaporation at this substrate temperature could be an excellent candidate to make a more efficient diagnosis based on droplet evaporation of biofluids.

Leveraging the Coffee Ring Effect for a Defect-Free Electroformation of Giant Unilamellar Vesicles.

We took advantage of the microflow hydrodynamics in the evaporation of sessile droplets to increase the height uniformity of thin lipid films for the subsequent electroformation of defect-free giant unilamellar vesicles (GUV). By serially casting progressively larger liposome suspension droplets on the same spot of an indium-tin-oxide (ITO) electrode, we managed to leverage the coffee ring effect (CRE) in the evaporation of each droplet to generate a smeared multilayer film of uniform thickness. This multidroplet technique of lipid film formation outperformed the traditional single-droplet deposition, improving the final quality of electroformed GUV samples. The proposed film formation technique constitutes a solvent-free method that results in a dramatic reduction (∼20×) in the appearance of undesirable structures like nonspherical (NSV), multilamellar (MLV), and multivesicular (MVV) vesicles.

Texture analysis of protein deposits produced by droplet evaporation.

The deposit patterns derived from droplet evaporation allow current development of medical tests and new strategies for diagnostic in patients. For such purpose, the development and implementation of algorithms capable of characterizing and differentiating deposits are crucial elements. We report the study of deposit patterns formed by the droplet evaporation of binary mixtures of proteins containing NaCl. Optical microscopy reveals aggregates such as tip arrow-shaped, dendritic and semi-rosette patterns, needle-like and scalloped lines structures, as well as star-like and prism-shaped salt crystals. We use the first-order statistics (FOS) and gray level co-occurrence matrix (GLCM) to characterize the complex texture of deposit patterns. Three significant findings arise from this analysis: first, the FOS and GLCM parameters structurally characterize protein deposits. Secondly, they conform to simple exponential laws that change as a function of the NaCl concentration. Finally, the parameters are capable of revealing the different structural changes that occur during the droplet evaporation.

Pattern formation of stains from dried drops to identify spermatozoa motility.

We study how cell motility affects the stains left by the evaporation of droplets of a biofluid suspension containing mouse spermatozoa. The suspension, which contains also a high concentration of salts usually needed by motile cells, forms, upon drying, a crystallized pattern. We examine the structural characteristics of such patterns by optical microscopy. The analysis reveals that cell motility affects the formation of elongated crystals with lateral tips, as well as the creation of interlocked aggregates. We prove that a lacunarity algorithm based on polar symmetry, distinguishes among deposits generated by motile and non-motile cells with an accuracy greater than 95%.