Protein Mixture Segregation at Coffee-Ring: Real-Time Imaging of Protein Ring Precipitation by FTIR Spectromicroscopy

During natural drying process, all solutions and suspensions tend to form the so-called “coffee-ring” deposits. This phenomenon, by far, has been interpreted by the hydrodynamics of evaporating fluids. However, in this study, by applying Fourier transform infrared imaging (FTIRI), it is possible to observe the segregation and separation of a protein mixture at the “ring”, hence we suggest a new way to interpret “coffee-ring effect” of solutions. The results explore the dynamic process that leads to the ring formation in case of model plasma proteins, such as BGG (bovine γ globulin), BSA (bovine serum albumin), and Hfib (human fibrinogen), and also report fascinating discovery of the segregation at the ring deposits of two model proteins BGG and BSA, which can be explained by an energy kinetic model, only. The investigation suggests that the coffee-ring effect of solute in an evaporating solution drop is driven by an energy gradient created from change of particle–water–air interfacial energy configuration

Effect of Ingested Tungsten Oxide (WO_<i>x</i>) Nanofibers on Digestive Gland Tissue of Porcellio scaber (Isopoda, Crustacea): Fourier Transform Infrared (FTIR) Imaging

Tungsten nanofibers are recognized as biologically potent. We study deviations in molecular composition between normal and digestive gland tissue of WO_<i>x</i> nanofibers (nano-WO_<i>x</i>) fed invertebrate Porcellio scaber (Iosopda, Crustacea) and revealed mechanisms of nano-WO_<i>x</i> effect <i>in vivo</i>. Fourier Transform Infrared (FTIR) imaging performed on digestive gland epithelium was supplemented by toxicity and cytotoxicity analyses as well as scanning electron microscopy (SEM) of the surface of the epithelium. The difference in the spectra of the Nano-WO_<i>x</i> treated and control cells showed up in the central region of the cells and were related to lipid peroxidation, and structural changes of nucleic acids. The conventional toxicity parameters failed to show toxic effects of nano-WO_<i>x</i>, whereas the cytotoxicity biomarkers and SEM investigation of digestive gland epithelium indicated sporadic effects of nanofibers. Since toxicological and cytological measurements did not highlight severe effects, the biochemical alterations evidenced by FTIR imaging have been explained as the result of cell protection (acclimation) mechanisms to unfavorable conditions and indication of a nonhomeostatic state, which can lead to toxic effects

Cryopreservation affects platelet macromolecular composition over time after thawing and differently impacts on cancer cells behavior in vitro

Cryopreservation affects platelets’ function, questioning their use for cancer patients. We aimed to investigate the biochemical events that occur over time after thawing to optimize transfusion timing and evaluate the effect of platelet supernatants on tumor cell behavior in vitro. We compared fresh (Fresh-PLT) with Cryopreserved platelets (Cryo-PLT) at 1 h, 3 h and 6 h after thawing. MCF-7 and HL-60 cells were cultured with Fresh- or 1 h Cryo-PLT supernatants to investigate cell proliferation, migration, and PLT-cell adhesion. We noticed a significant impairment of hemostatic activity accompanied by a post-thaw decrease of CD42b+ , which identifies the CD62P−-population. FTIR spectroscopy revealed a decrease in the total protein content together with changes in their conformational structure, which identified two sub-groups: 1) Fresh and 1 h Cryo-PLT; 2) 3 h and 6 h cryo-PLT. Extracellular vesicle shedding and phosphatidylserine externalization (PS) increased after thawing. Cryo-PLT supernatants inhibited cell proliferation, impaired MCF-7 cell migration, and reduced ability to adhere to tumor cells. Within the first 3 hours after thawing, irreversible alterations of biomolecular structure occur in Cryo-PLT. Nevertheless, Cryo-PLT should be considered safe for the transfusion of cancer patients because of their insufficient capability to promote cancer cell proliferation, adhesion, or migration. What is the context?Transfusion of Fresh platelets (Fresh-PLT) with prophylaxis purposes is common in onco-hematological patients.Cryopreservation is an alternative storage method that allows to extend platelet component shelf life and build supplies usable in case of emergency.It is well established that cryopreservation affects platelet function questioning their use in onco-hematological patients.It is still unknown how platelet impairment, induced by cryopreservation, occurs over time after thawing, nor how the by-products of PLT deterioration may impact on cancer cell behavior. Transfusion of Fresh platelets (Fresh-PLT) with prophylaxis purposes is common in onco-hematological patients. Cryopreservation is an alternative storage method that allows to extend platelet component shelf life and build supplies usable in case of emergency. It is well established that cryopreservation affects platelet function questioning their use in onco-hematological patients. It is still unknown how platelet impairment, induced by cryopreservation, occurs over time after thawing, nor how the by-products of PLT deterioration may impact on cancer cell behavior. What is new?In this study, we deeply characterized the functional and morphological changes induced by cryopreservation on platelets by comparing Fresh-PLT with Cryo-PLT at 1 h, 3 h and 6 h after thawing. Afterwards, we evaluated the effect of PLT supernatants on cancer cell behavior in vitro.The data presented show that within 3 hours after thawing Cryo-PLT undergo to irreversible macromolecular changes accompanied by increase of peroxidation processes and protein misfolding.After thawing the clot formation is reduced but still supported at all-time points measured, combined with unchanged phosphatidylserine expression and extracellular vesicles release over time.Cryo-PLT supernatants do not sustain proliferation and migration of cancer cells. In this study, we deeply characterized the functional and morphological changes induced by cryopreservation on platelets by comparing Fresh-PLT with Cryo-PLT at 1 h, 3 h and 6 h after thawing. Afterwards, we evaluated the effect of PLT supernatants on cancer cell behavior in vitro. The data presented show that within 3 hours after thawing Cryo-PLT undergo to irreversible macromolecular changes accompanied by increase of peroxidation processes and protein misfolding. After thawing the clot formation is reduced but still supported at all-time points measured, combined with unchanged phosphatidylserine expression and extracellular vesicles release over time. Cryo-PLT supernatants do not sustain proliferation and migration of cancer cells. WHAT is the impact?Cryo-PLT may be considered a precious back-up product to be used during periods of Fresh-PLT shortage to prevent bleeding in non-hemorrhagic patients.It is desirable to make it logistically feasible to transfuse cryopreserved platelets within 1 hour of thawing to maintain the platelets in their best performing condition. Cryo-PLT may be considered a precious back-up product to be used during periods of Fresh-PLT shortage to prevent bleeding in non-hemorrhagic patients. It is desirable to make it logistically feasible to transfuse cryopreserved platelets within 1 hour of thawing to maintain the platelets in their best performing condition.</p

Scanning and transmission electron micrographs of biofilms, cells and hami.

<p>Left panels: MSI, right panel: SM. A: Scanning electron micrograph of MSI biofilm, showing SM1 euryarchaeal cells with defined distances and cell-cell connections. Bar: 1 µm. B: Scanning electron micrograph of SM biofilm, showing SM1 euryarchaeal cells with defined distances and fine-structured cell-cell connections. In-between: Bacterial filamentous and rod-shaped cells. Bar: 1 µm. C: Scanning electron micrograph of dividing SM1 euryarchaeal cell (MSI) with cell surface appendages. Bar: 200 nm. D: Scanning electron micrograph of dividing SM1 euryarchaeal cell (SM) with cell surface appendages. Bar: 200 nm. E: Transmission electron micrograph of cell surface appendages (hami) of SM1 euryarchaeal cells from the MSI biofilm. The hami carry the nano-grappling hooks, but besides that appear bare (square), without prickles (Moissl et al 2005). Bar: 100 nm. F: Transmission electron micrograph of cell surface appendages and matrix of SM1 euryarchaeal cells from the SM biofilm. The hami reveal the typical ultrastructure, with nano-grappling hooks and barbwire-like prickle region (square, Moissl et al 2005). Bar: 100 nm.</p

Quantification of archaeal and bacterial signatures via qPCR, FISH and SR-FTIR (values in brackes give standard deviation).

<p>*data from Probst et al., 2013.</p><p>ND: Not Determined.</p

Detailed community profiling using PhyloChip G3 and SR-FTIR.

<p>A: Ordination analysis of PhyloChip G3 data based on weighted UniFrac measure of eOTU abundances followed by non-metric multidimensional scaling (NMDS). Stress for NMDS of archaeal eOTUs (#37): 0.0088. Stress for NMDS of bacterial eOTUs (#1300): 0.0223. B: Heatmap displaying significantly different families found between the two biofilm types, MSI-BF and SM-BF by PhyloChip G3 assay. Significance is based on aggregated HybScores of eOTUs on family level followed by a Welch-test. For false discovery detection please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099801#pone.0099801.s006" target="_blank">Fig. S6</a>. C: Ordination analysis of SR-FTIR data based on a linear discriminant analysis and principal component analysis (PCA-LDA) in the spectral region of 2800–3100 cm⁻¹ on the archaea spectra extracted from the maps from the three different locations. On the right there is the plot of PCA-LDA loadings. PCA-LDA1 explains for the 93.4% of the variance, PCA-LDA2 for 5.3% and PCA-LDA3 for 0.9%. Arrows point to the infrared signals used to explain the difference between the samples: 2975 cm⁻¹, 2965 cm⁻¹, 2924 cm⁻¹ and 2850 cm⁻¹. D: PCA-LDA in the spectral regions of 900–1280 cm⁻¹ and 2800–3100 cm⁻¹ on SR-FTIR spectra of the bacteria “pixels” from the chemical maps of the samples at the three different locations. On the right there is a plot of PCA-LDA loadings in the two spectral region of interest. PCA-LDA1 explains for the 54.5% of the variance, PCA-LDA2 for 28.6% and PCA-LDA3 for 7.3%. Arrows point to the main infrared signals used to explain the difference between the samples: 2958 cm⁻¹, 2925 cm⁻¹, 2870 cm⁻¹ and 2850 cm⁻¹, in the second panel 1250 cm⁻¹, 1110 cm⁻¹, 1080 cm⁻¹ and 1045 cm⁻¹.</p

Scanning electron micrograph of filamentous bacterium and surrounded and cocooned by the SM1 euryarchaeal cells (SM-BF).

<p>Bar: 1 µm.</p

The conversion of biofilm to string-of-pearls community in the spring water originating from the subsurface.

<p>A: Biofilm. B: Intermediate transition state. C: String-of-pearls community. Row 1: Schematic drawings. Orange: SM1 euryarchaeal cocci, Green: Filamentous, sulfide-oxidizing bacteria. Row 2: Photographs and scanning electron micrograph (2B) of different stages. Row 3: FISH images of different stages (for MSI samples please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099801#pone.0099801-Probst1" target="_blank">[15]</a>; Archaea orange (CY3), Bacteria green (RG)). A: SM-BF, showing high dominance of Archaea. B: Attachment of archaea to filamentous bacteria. C: String-of-pearls communities with large archaeal colony and bacterial mantle. Arrows point to archaeal microcolonies, manteled by filamentous bacteria. It is proposed that attachment of SM1 Euryarchaeota to filamentous bacteria (B) mediates the transition from biofilm (A) to the string-of-pearls community (C). Scale bars: A3: 10 µm, B2: 1 µm B3: 10 mm, C3: 25 µm.</p