Application of atomic force microscopy in the analysis of time since deposition (TSD) of red blood cells in bloodstains: A forensic analysis.

Bloodstains can provide important information about a criminal act. These biological traces, when analyzed at murder sites, for example, can determine the dynamics of a criminal offense, the identity of a suspect, and the time at which a crime was committed. Determine the time since deposition (TSD) of these blood traces may be the first clue for the police investigators to estimate the time-lapse of a murder. During a criminal attack, the blood spilled from an injury begins the process of degradation and aging from the moment it leaves the human body and comes into contact with the physical environment. The biophysical properties (morphology and elasticity) of red blood cells (RBCs) undergo several changes when outside the human body, which can be analyzed using microscopic techniques such as atomic force microscopy (AFM). Aiming to apply the AFM/force spectroscopy techniques in the analysis of criminal traces, the present study investigated the TSD for blood smears by analyzing possible changes in the RBCs of a group of voluntary donors. Also, we investigated whether there was any difference in TSD analysis after blood smears deposition onto three different surfaces (glass, metal, or ceramic); and finally, we evaluated force×distance curves obtained from deformation of the membrane surface of RBCs as a function of time. The qualitative results apparently showed that there is no perceptible difference in the structure of RBCs when AFM images were analyzed by simple visual comparison over 28 days (T0-T5). Nevertheless, our quantitative results, measured by AFM, demonstrated the increasing trend of the measurements, such as average height (µm), perimeter (µm), area (µm2) and volume (µm3) of these cells during that period. Additionally, the type of surface of bloodstain deposition should be considered during analyses for the TSD, and the results obtained on glass, metal, or ceramic supports showed significant differences. Therefore, the use of force spectroscopy to obtain force×distance curves for the forensic science approach has been shown to have applicability for the calculation of TSD in the RBCs present in the blood smears. In spite of the promising observations obtained, the use of AFM in crime scenes still requires the expansion and development of more studies for a definitive evaluation of the TSD for blood spots.

Protein extraction method for the proteomic study of Zymomonas mobilis during production of ethanol and levans.

Zymomonas mobilis has aroused considerable interest owing to its rapid metabolism and efficiency in producing ethanol and by-products such as levans, sorbitol, and gluconic acid from simple sugars. We performed a proteomic analysis of Z. mobilis UFPEDA241 to provide a global profile of regulatory proteins. The choice of the methods of extraction and cell lysis are fundamental steps and of great importance for the detection and identification of intra- and extracellular proteins of a proteome. Strains were subjected to protein extraction methods using three different reagents: TRIzol, lysis buffer, and phenol. The optimum method was taken to be the one that produced the greatest quantity and quality of proteins in one dimension for further analysis in two dimensions during the production of ethanol and levans over 72 h. The results showed that the greatest amount of protein was obtained by the phenol method (1.44 ± 0.07 mg/mL), which was significantly different (P < 0.05) to the TRIzol (1.11 ± 0.01 mg/mL), and lysis buffer (0.93 ± 0.01 mg/mL) methods (both with P > 0.05). Fermentation at 20°C produced the highest level of levans, and using two-dimensional electrophoresis and mass spectrometry it was possible to identify 34 differentially expressed spots.