Assessing agreement among crime scene measurement methods.

A critical concern with crime scene documentation is the accuracy with which a crime scene can be reconstructed. Here, we discuss the accuracy of eight documentation methods as a function of measurement distance between reference ground targets in an outdoor scene. The relative accuracy of each documentation method was assessed with respect to a widely accepted and well-established standard method for land surveying, Total Station, from which measurements served as "ground truth" or reference data. For the majority of methods, the actual relative difference between measurements when compared to Total Station was small (less than a quarter of an inch). Measurements from FARO LiDAR agreed the most with to those of Total Station, while drone without the use of ground control points (GCPs) agreed the least. GCPs or a reference scale were also found to be important in mitigating increasing imprecision with increasing distance when measuring between two targets ~9-85 ft apart via drone and orthomosaic methods. Additionally, there were no statistical differences in the use of 2D (horizontal) or 3D (slope) measurement configurations for the Total Station. Overall, linear regression of difference plots did not reveal meaningful correlation between increasing distance measured and the error of a method when compared to Total Station. As more measurement methods become available, and the need for training and validating new tools become a necessity, these results point to the importance of establishing a ground truth or known distance range on which crime scene measurement methods can be validated.

Assessing Single-Source Reproducibility of Human Head Hair Peptide Profiling from Different Regions of the Scalp.

Neither microscopical hair comparisons nor mitochondrial DNA sequencing alone, or together, constitutes a basis for personal identification. Due to these limitations, a complementary technique to compare questioned and known hair shafts was investigated. Recently, scientists from Lawrence Livermore National Laboratory's Forensic Science Center and other collaborators developed a peptide profiling technique, which can infer non-synonymous single nucleotide polymorphisms (SNPs) preserved in hair shaft proteins as single amino acid polymorphisms (SAPs). In this study, peptide profiling was evaluated to determine if it can meet forensic expectations when samples are in limited quantities with the possibility that hair samples collected from different areas of a single donor's scalp (i.e., single source) might not exhibit the same SAP profile. The average dissimilarity, percent differences in SAP profiles within each source, ranged from 0% difference to 29%. This pilot study suggests that more work is needed before peptide profiling of hair can be considered for forensic comparisons.

A quantitative method for selecting a hair for nulear DNA analysis.

This study evaluated a quantitative method to predict the success of nuclear DNA (nuDNA) typing for head hair roots, using the minor-groove DNA binding dye, 4', 6-diamidino-2-phenylindole (DAPI). The procedure was successful in staining nuclear material in hair roots, regardless of soft tissue presence or growth phase. We found that the dye can even reveal an abundance of visible nuclei in hairs that were previously assumed to be unsuitable for nuDNA analysis (e.g., telogen hairs). The value of DAPI staining is particularly evident when considering the STR typing results for telogen hairs. Here, telogen hairs with greater than 100 visible nuclei frequently produced full or high-partial STR profiles, while telogen hairs with fewer than 100 visible nuclei rarely resulted in >20 % STR allele recovery. In addition, our findings indicated no interference by DAPI in the forensic examination of hair evidence, including preparation of hairs on microscopic slides, microscopic examination, DNA extraction, quantitative PCR, and short tandem repeat (STR) typing. Furthermore, the method remained steadfast for hairs washed by sonication as well as hairs retrieved from Permount™ mounting medium. When validated, this simple, quick, and quantitative screening method can be used in casework to select a hair for nuDNA analysis, especially for hairs that were previously sent directly for mitochondrial (mt) DNA analysis based on the lack of adhering soft tissue, regardless of growth phase. Conversely, nuDNA degradation may exist in hairs which exhibit microscopic characteristics typically associated with a potential to generate successful nuclear DNA profile including stretched roots with attached root sheath. DAPI staining of hairs gives forensic examiners the ability to have more information, other than growth phase, when selecting a hair or hairs for possible nuDNA analysis.

A voltage-dependent K+ channel in the lysosome is required for refilling lysosomal Ca2+ stores.

The resting membrane potential (Δψ) of the cell is negative on the cytosolic side and determined primarily by the plasma membrane's selective permeability to K+ We show that lysosomal Δψ is set by lysosomal membrane permeabilities to Na+ and H+, but not K+, and is positive on the cytosolic side. An increase in juxta-lysosomal Ca2+ rapidly reversed lysosomal Δψ by activating a large voltage-dependent and K+-selective conductance (LysoKVCa). LysoKVCa is encoded molecularly by SLO1 proteins known for forming plasma membrane BK channels. Opening of single LysoKVCa channels is sufficient to cause the rapid, striking changes in lysosomal Δψ. Lysosomal Ca2+ stores may be refilled from endoplasmic reticulum (ER) Ca2+ via ER-lysosome membrane contact sites. We propose that LysoKVCa serves as the perilysosomal Ca2+ effector to prime lysosomes for the refilling process. Consistently, genetic ablation or pharmacological inhibition of LysoKVCa, or abolition of its Ca2+ sensitivity, blocks refilling and maintenance of lysosomal Ca2+ stores, resulting in lysosomal cholesterol accumulation and a lysosome storage phenotype.

MCOLN1 is a ROS sensor in lysosomes that regulates autophagy.

Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes 'host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca(2+)-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca(2+) release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell.

Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation.

Upon nutrient starvation, autophagy digests unwanted cellular components to generate catabolites that are required for housekeeping biosynthesis processes. A complete execution of autophagy demands an enhancement in lysosome function and biogenesis to match the increase in autophagosome formation. Here, we report that mucolipin-1 (also known as TRPML1 or ML1), a Ca(2+) channel in the lysosome that regulates many aspects of lysosomal trafficking, plays a central role in this quality-control process. By using Ca(2+) imaging and whole-lysosome patch clamping, lysosomal Ca(2+) release and ML1 currents were detected within hours of nutrient starvation and were potently up-regulated. In contrast, lysosomal Na(+)-selective currents were not up-regulated. Inhibition of mammalian target of rapamycin (mTOR) or activation of transcription factor EB (TFEB) mimicked a starvation effect in fed cells. The starvation effect also included an increase in lysosomal proteostasis and enhanced clearance of lysosomal storage, including cholesterol accumulation in Niemann-Pick disease type C (NPC) cells. However, this effect was not observed when ML1 was pharmacologically inhibited or genetically deleted. Furthermore, overexpression of ML1 mimicked the starvation effect. Hence, lysosomal adaptation to environmental cues such as nutrient levels requires mTOR/TFEB-dependent, lysosome-to-nucleus regulation of lysosomal ML1 channels and Ca(2+) signaling.