RESUMO
This research expands previous studies in which color contrast between ridges and furrows of powder-enhanced latent fingermarks was explored as a possible aging parameter. The main goal is to test the sensitivity of the technique across a predetermined set of factors. In this case, experiment factors have included two donors who deposited sebaceous- and eccrine-rich fingermarks onto ceramic tile and polystyrene plastic. These were developed with either black carbon or titanium dioxide powder (TiO2 ) over eight time periods (0-72 days) and aged under three light conditions (direct light, shade, and darkness). The mean intensity (MI) and intensity amplitude (IA) metrics of color were collected from each image for statistical analyses. Results show that color contrast is affected significantly by substrate, secretion, and powder types, with an interaction effect between the substrate and powder type on both MI and IA metrics. The degree of light exposure did not have a noticeable impact on distinguishing aging patterns of fingermarks by neither powder methods. Different aging patterns were detected between sebaceous-rich and their eccrine-rich counterparts for all light conditions using regression analysis. All eccrine-rich fingermarks exhibited little (or minimal) change in IA over time, whereas sebaceous-rich samples showed varied patterns, from significant decreases to slight increases. These findings confirm and expand previous observations on the potential use of MI and IA as metrics to study latent fingermark degradation patterns that could eventually be used to estimate the age of a fingermark.
RESUMO
Members of the anaerobic gut fungi (AGF) reside in rumen, hindgut, and feces of ruminant and non-ruminant herbivorous mammals and reptilian herbivores. No protocols for gene insertion, deletion, silencing, or mutation are currently available for the AGF, rendering gene-targeted molecular biological manipulations unfeasible. Here, we developed and optimized an RNA interference (RNAi)-based protocol for targeted gene silencing in the anaerobic gut fungus Pecoramyces ruminantium strain C1A. Analysis of the C1A genome identified genes encoding enzymes required for RNA silencing in fungi (Dicer, Argonaute, Neurospora crassa QDE-3 homolog DNA helicase, Argonaute-interacting protein, and Neurospora crassa QIP homolog exonuclease); and the competency of C1A germinating spores for RNA uptake was confirmed using fluorescently labeled small interfering RNAs (siRNA). Addition of chemically-synthesized siRNAs targeting D-lactate dehydrogenase (ldhD) gene to C1A germinating spores resulted in marked target gene silencing; as evident by significantly lower ldhD transcriptional levels, a marked reduction in the D-LDH specific enzymatic activity in intracellular protein extracts, and a reduction in D-lactate levels accumulating in the culture supernatant. Comparative transcriptomic analysis of untreated versus siRNA-treated cultures identified a few off-target siRNA-mediated gene silencing effects. As well, significant differential up-regulation of the gene encoding NAD-dependent 2-hydroxyacid dehydrogenase (Pfam00389) in siRNA-treated C1A cultures was observed, which could possibly compensate for loss of D-LDH as an electron sink mechanism in C1A. The results demonstrate the feasibility of RNAi in anaerobic fungi, and opens the door for gene silencing-based studies in this fungal clade.