20 research outputs found
Attenuated Total Reflectance-FT-IR Imaging for Rapid and Automated Detection of Gunshot Residue
An
alternative approach for the nondestructive, rapid and selective
detection of gunshot residue (GSR) was investigated. A cloth substrate
containing GSR particles expelled during a firearm discharge was used
as an analog for the clothing of a shooting victim or a suspect discharging
a firearm. An established and efficient procedure for GSR collection
(tape lifting) was utilized to recover GSR particles from the cloth
substrate. Microscopic-attenuated total reflectance (ATR) Fourier
transform (FT) infrared (IR) spectroscopic imaging rapidly and automatically
scanned large areas of the tape collection substrate and detected
varying morphologies (microscopic and macroscopic) and chemical compositions
(organic and inorganic) of GSR. The āspectroscopic fingerprintā
of each GSR type provided unique virbrational modes, which were not
characteristic of the tape collection substrate or the cloth debris
which was also recovered. ATR images (maps) targeted the detection
of these unique chemical markers over the mapped area. The hues of
the ATR images were determined by the intensity of the signal for
the chemical marker of each analyte. The spatial resolution of the
technique was determined to be 4.7 Ī¼m. Therefore, all GSR particles
sized 4.7 Ī¼m or larger will be resolved and detected on the
tape substrate using micro-ATR imaging
Differentiating Donor Age Groups Based on Raman Spectroscopy of Bloodstains for Forensic Purposes
Developments in analytical
chemistry technologies and portable
instrumentation over the past decade have contributed significantly
to a variety of applications ranging from point of care testing to
industrial process control. In particular, Raman spectroscopy has
advanced for analyzing various types of evidence for forensic purposes.
Extracting phenotypic information (e.g., sex, race, age, etc.) from
body fluid traces is highly desirable for criminal investigations.
Identifying the chronological age (CA) of a blood donor can provide
significant assistance to detectives. In this proof-of-concept study,
Raman spectroscopy and chemometrics have been used to analyze blood
from human donors, and differentiate between them based on their CA
[i.e., newborns (CA of <1 year), adolescents (CA of 11ā13
years), and adults (CA of 43ā68 years)]. A support vector machines
discriminant analysis (SVMDA) model was constructed, which demonstrated
high accuracy in correctly predicting blood donorsā age groups
where the lowest cross-validated sensitivity and specificity values
were 0.96 and 0.97, respectively. Overall, this preliminary study
demonstrates the high selectivity of Raman spectroscopy for differentiating
between blood donors based on their CA. The demonstrated capability
completes our suite of phenotype profiling methodologies including
the determination of sex and race. CA determination has particular
importance since this characteristic cannot be determined through
DNA profiling unlike sex and race. When completed, the developed methodology
should allow for phenotype profiling based on dry traces of body fluids
immediately at the scene of a crime. The availability of this information
within the first few hours since the crime discovery could be invaluable
for the investigation
Attenuated Total Reflectance-FT-IR Spectroscopy for Gunshot Residue Analysis: Potential for Ammunition Determination
The ability to link a suspect to
a particular shooting incident
is a principal task for many forensic investigators. Here, we attempt
to achieve this goal by analysis of gunshot residue (GSR) through
the use of attenuated total reflectance (ATR) Fourier transform infrared
spectroscopy (FT-IR) combined with statistical analysis. The firearm
discharge process is analogous to a complex chemical process. Therefore,
the products of this process (GSR) will vary based upon numerous factors,
including the specific combination of the firearm and ammunition which
was discharged. Differentiation of FT-IR data, collected from GSR
particles originating from three different firearmāammunition
combinations (0.38 in., 0.40 in., and 9 mm calibers), was achieved
using projection to latent structures discriminant analysis (PLS-DA).
The technique was cross (leave-one-out), both internally and externally,
validated. External validation was achieved via assignment (caliber
identification) of unknown FT-IR spectra from unknown GSR particles.
The results demonstrate great potential for ATR-FT-IR spectroscopic
analysis of GSR for forensic purposes
Structural Organization of Insulin Fibrils Based on Polarized Raman Spectroscopy: Evaluation of Existing Models
Many different proteins undergo misfolding
and self-assemble into
amyloid fibrils, resulting in a range of neurodegenerative diseases.
The limitations of conventional methods of structural biology for
fibril characterization have led to the use of polarized Raman spectroscopy
for obtaining quantitative structural information regarding the organization
of amyloid fibrils. Herein, we report the orientation of selected
chemical groups and secondary structure elements in aligned insulin
fibrils, including Ī²-sheets, which possess a high level of orientation
in the cross-Ī² core, and Ī±-helices in the disordered portions
of the fibrils. Strong orientation of disulfide bonds in amyloid fibrils
was also revealed, indicating their association with the fibril core.
The determined orientation of chemical groups provides strong constraints
for modeling the overall structure of amyloid fibrils, including the
core and disordered parts. The developed methodology allows for the
validation of structural models proposed in the literature for amyloid
fibrils. Specifically, the polarized Raman data obtained herein strongly
agreed with two insulin fibril models (JimeĢnez et al., <i>Proc. Natl. Acad. Sci. U. S. A.</i> <b>2002</b>, <i>99</i>, 9196ā9201 and Ivanova et al., <i>Proc. Natl.
Acad. Sci. U. S. A.</i> <b>2009</b>, <i>106</i>, 18990ā18995) yet revealed significant qualitative and quantitative
differences. This work demonstrates the great potential of polarized
Raman spectroscopy for structural characterization of anisotropic
biological species
Raman Spectroscopy of Blood for Species Identification
The
species identification of a blood stain is an important and
immediate challenge for forensic science, veterinary purposes, and
wildlife preservation. The current methods used to identify the species
of origin of a blood stain are limited in scope and destructive to
the sample. We have previously demonstrated that Raman spectroscopy
can reliably differentiate blood traces of human, cat, and dog (Virkler et al. Anal. Chem. 2009, 81, 7773ā7777) and, most recently, built a binary model
for differentiating human vs animal blood for 11 species integrated
with human existence (McLaughlin
et al. Forensic Sci. Int. 2014, 238, 91ā95). Here we report a satisfactory classification of blood obtained
from 11 animal classes and human subjects by statistical analysis
of Raman spectra. Classification of blood samples was achieved according
to each sampleās species of origin, which enhanced previously
observed discrimination ability. The developed approach does not require
the knowledge of a specific (bio)Āchemical marker for each individual
class but rather relies on a spectroscopic statistical differentiation
of various components. This approach results in remarkable classification
ability even with intrinsically heterogeneous classes and samples.
In addition, the obtained spectroscopic characteristics could potentially
provide information about specific changes in the (bio)Āchemical composition
of samples, which are responsible for the differentiation
Structure and Composition of Insulin Fibril Surfaces Probed by TERS
Amyloid fibrils associated with many neurodegenerative
diseases
are the most intriguing targets of modern structural biology. Significant
knowledge has been accumulated about the morphology and fibril-core
structure recently. However, no conventional methods could probe the
fibril surface despite its significant role in the biological activity.
Tip-enhanced Raman spectroscopy (TERS) offers a unique opportunity
to characterize the surface structure of an individual fibril due
to a high depth and lateral spatial resolution of the method in the
nanometer range. Herein, TERS is utilized for characterizing the secondary
structure and amino acid residue composition of the surface of insulin
fibrils. It was found that the surface is strongly heterogeneous and
consists of clusters with various protein conformations. More than
30% of the fibril surface is dominated by Ī²-sheet secondary
structure, further developing Dobsonās model of amyloid fibrils
(Jimenez et al. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 9196ā9201). The propensity
of various amino acids to be on the fibril surface and specific surface
secondary structure elements were evaluated. Ī²-sheet areas are
rich in cysteine and aromatic amino acids, such as phenylalanine and
tyrosine, whereas proline was found only in Ī±-helical and unordered
protein clusters. In addition, we showed that carboxyl, amino, and
imino groups are nearly equally distributed over Ī²-sheet and
Ī±-helix/unordered regions. Overall, this study provides valuable
new information about the structure and composition of the insulin
fibril surface and demonstrates the power of TERS for fibril characterization
Deconstruction of Stable Cross-Beta Fibrillar Structures into Toxic and Nontoxic Products Using a Mutated Archaeal Chaperonin
Our group recently determined that
a mutant archaeal chaperonin
(Hsp 60) exhibited substantially enhanced protein folding activity
at low temperatures and was able to deconstruct refractory protein
aggregates. ATP dependent conversion of fibril structures into amorphous
aggregates was observed in insulin amyloid preparations (Kurouski
et al. <i>Biochem. Biophys. Res. Commun.</i> 2012). In the
current study, mechanistic insights into insulin fibril deconstruction
were obtained by examination of early stage complexes between Hsp60
and fibrils in the absence of ATP. Activity of the Hsp60 was significantly
curtailed without ATP; however, some fibril deconstruction occurred,
which is consistent with some models of the folding cycle that predict
initial removal of unproductive protein folds. Chaperonin molecules
adsorbed on the fibril surface and formed chaperonin clusters with
no ATP present. We propose that there are specific locations on the
fibril surface where chaperonin can unravel the fibril to release
short fragments. Spontaneous coagulation of these fibril fragments
resulted in the formation of amorphous aggregates without the release
of insulin into solution. The addition of ATP significantly increased
the toxicity of the insulin fibril-chaperonin reaction products toward
mammalian cells
Disulfide bonds preserve their conformation upon insulin fibrillation.
<p>Raman spectra of native insulin (red) and insulin fibrils (black) have a peak at 510 cm<sup>ā1</sup>, corresponding to the gauche-gauche-gauche (g-g-g) conformation of disulfide bonds (schematically represented in the inset).</p
The majority of H/D exchange-protected amino acids are located in the B-chain of insulin.
<p>The amino acids in the 3D structure (A) and primary sequence of insulin fibrillar monomer (B) are colored according to the following degree of H/D exchange protection: yellow, I<sub>D2O</sub>/I<sub>H2O</sub>ā„0.75; red, 0.75>I<sub>D2O</sub>/I<sub>H2O</sub>ā„0.675; and blue, 0.675>I<sub>D2O</sub>/I<sub>H2O</sub>ā„0.60. Gray (in the 3D structure) and white colors (in the primary sequence) indicate unprotected (exchanged) amino acids. (C) 2D <sup>1</sup>H,<sup>1</sup>H-TOCSY spectrum of insulin fibrillar monomer in DMSO and 0.05% TFA at 30Ā°C after a 7-day D<sub>2</sub>O exchange at room temperature. NMR data were acquired at room temperature on a Bruker Avance II 500 MHz NMR spectrometer equipped with a cryoprobe.</p
The Ī±-helix and Ī²-sheet character of simulated insulin monomer and dimer.
<p>The Ī±-helix and Ī²-sheet character of simulated insulin monomer and dimer.</p