Genetic individualization of Cannabis sativa by a short tandem repeat multiplex system

Cannabis sativa is the most frequently used of all illicit drugs in the United States. Cannabis has been used throughout history for its stems in the production of hemp fiber, for its seed for oil and food, and for its buds and leaves as a psychoactive drug. Short tandem repeats (STRs), were chosen as molecular markers because of their distinct advantages over other genetic methods. STRs are co-dominant, can be standardized such that reproducibility between laboratories can be easily achieved, have a high discrimination power and can be multiplexed. ^ In this study, six STR markers previously described for Cannabis were multiplexed into one reaction. The multiplex reaction was able to individualize 98 Cannabis samples (14 hemp and 84 marijuana, authenticated as originating from 33 of the 50 United States) and detect 29 alleles averaging 4.8 alleles per loci. The data did not relate the samples from the same state to each other. This is the first study to report a single reaction six-plex and apply it to the analysis of almost 100 Cannabis samples of known geographic collection site. ^

Neanderthal admixture in current human populations

In the present body of work two primary subjects have been addressed, both individually and in their correspondence, namely (1) the potential for Neanderthals to have contributed to the Modern Human population, and (2) the genetic diversity of one of the most prehistorically impactful human popuations, the Armenians. The first subject is addressed by assessing 1000 mutations in 384 current humans, particularly for those mutations which appear to derive from the Neanderthal lineage. Additionally, the validity of the Neanderthal sequences themselves is evaluated through alignment analysis of fragementary DNA derived from the Vindija Cave sample. Armenian genetic diversity is analyzed through the autosomal short tandem repeats, y-chromsome single nucleotide polymorphisms, and y-chromosome short tandem repeats. The diversity found indicates that Armenians are a diverse group which has been genetically influenced by the various migrations and invasions which have entered their historic lands. Further, we find evidence that Armenians may be closely associated with the peopling of Europe.

Microvariation at the human D1S80 locus

Microvariant allelic polymorphisms have been known since 1966 when Harris, Hubby and Lewontin described the huge store of genetic variation detectable at the polypeptide level. Later Jeffreys used MVR (minisatellite variant repeat) analysis to describe the variation hidden within minisatellite VNTRs and to propose a mutational mechanism.^ The questions I have asked follow these traditions: (1) How much microvariant polymorphism exists at the discrete allele minisatellite D1S80 locus? (2) Do alleles or groups of alleles associate randomly with the flanking markers to form haplotypes? (3) What mechanisms might explain mutations at this locus? What are the phylogenetic relationships among the alleles?^ The minisatellite locus D1S80 (1p35-36), GenBank sequence (Accession # D28507), is a highly polymorphic Variable Number of Tandem Repeat (VNTR) based on a 16 base core. D1S80 alleles are electrophoretically separable into discontinuous sets of equivalent length alleles. Sequence variation or minor length variation within these classes was expected: I have sought to determine the nature of this microvariant heterogeneity by sequencing nominal and variant alleles.^ Alleles were analyzed by Single-Strand Conformation Polymorphism (SSCP) analysis. Sequences were determined to ascertain whether sequence variation or size variation is the major cause of altered electrophoretic migration of microvariant D1S80 alleles. Twenty three alleles from 14 previously typed individuals were sequenced. The individuals were from African American, Caucasian, or Hispanic databases.^ A Tsp509 I restriction site, previously reported as a Hinf I flanking polymorphism, and a 3$\sp\prime$ flanking region BsoF I restriction site polymorphism were identified. There appears to be a strong association of the 5$\sp\prime$ flanking region Hinf I(+) and Tsp509 I(-) site and the 3$\sp\prime$ flanking region BsoF I(-) site with the 18 allele, while the 24 tends to be associated with the Hinf I(-), Tsp509 I(+) and BsoF I(+) sites.^ The general conclusion for this locus is clearly the closer you look, the more you find. D1S80 allelic polymorphisms are primarily due to variation in the number of repeat units and to sequence variation among repeats. The sequenced based gene tree depicts two major classes of alleles which conform to the two most common alleles, reflecting either equivalent age or population size bottlenecks. ^

The application of reduced-size short tandem repeat primer sets in the analysis of human DNA from degraded and compromised samples

The purpose of this research was to demonstrate the applicability of reduced-size STR (Miniplex) primer sets to challenging samples and to provide the forensic community with new information regarding the analysis of degraded and inhibited DNA. The Miniplex primer sets were validated in accordance with guidelines set forth by the Scientific Working Group on DNA Analysis Methods (SWGDAM) in order to demonstrate the scientific validity of the kits. The Miniplex sets were also used in the analysis of DNA extracted from human skeletal remains and telogen hair. In addition, a method for evaluating the mechanism of PCR inhibition was developed using qPCR. The Miniplexes were demonstrated to be a robust and sensitive tool for the analysis of DNA with as low as 100 pg of template DNA. They also proved to be better than commercial kits in the analysis of DNA from human skeletal remains, with 64% of samples tested producing full profiles, compared to 16% for a commercial kit. The Miniplexes also produced amplification of nuclear DNA from human telogen hairs, with partial profiles obtained from as low as 60 pg of template DNA. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for forensic analysis of degraded DNA from human skeletal remains, telogen hairs, and other challenging samples. In the evaluation of inhibition by qPCR, the effect of amplicon length and primer melting temperature was evaluated in order to determine the binding mechanisms of different PCR inhibitors. Several mechanisms were indicated by the inhibitors tested, including binding of the polymerase, binding to the DNA, and effects on the processivity of the polymerase during primer extension. The data obtained from qPCR illustrated a method by which the type of inhibitor could be inferred in forensic samples, and some methods of reducing inhibition for specific inhibitors were demonstrated. An understanding of the mechanism of the inhibitors found in forensic samples will allow analysts to select the proper methods for inhibition removal or the type of analysis that can be performed, and will increase the information that can be obtained from inhibited samples.

Assessment of the occurrence and potential risks of antibiotics and their metabolites in South Florida waters using liquid chromatography tandem mass spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. ^ The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can't be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications.^ Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. ^ Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.^

Instability of CTG Repeats is Governed by the Position of a DNA Base Lesion through Base Excision Repair

Trinucleotide repeat (TNR) expansions and deletions are associated with human neurodegeneration and cancer. However, their underlying mechanisms remain to be elucidated. Recent studies have demonstrated that CAG repeat expansions can be initiated by oxidative DNA base damage and fulfilled by base excision repair (BER), suggesting active roles for oxidative DNA damage and BER in TNR instability. Here, we provide the first evidence that oxidative DNA damage can induce CTG repeat deletions along with limited expansions in human cells. Biochemical characterization of BER in the context of (CTG)20 repeats further revealed that repeat instability correlated with the position of a base lesion in the repeat tract. A lesion located at the 59-end of CTG repeats resulted in expansion, whereas a lesion located either in the middle or the 39-end of the repeats led to deletions only. The positioning effects appeared to be determined by the formation of hairpins at various locations on the template and the damaged strands that were bypassed by DNA polymerase b and processed by flap endonuclease 1 with different efficiency. Our study indicates that the position of a DNA base lesion governs whether TNR is expanded or deleted through BER.

Online solid phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) method for the determination of sucralose in reclaimed and drinking waters and its photo degradation in natural waters from South Florida

Background Sucralose has gained popularity as a low calorie artificial sweetener worldwide. Due to its high stability and persistence, sucralose has shown widespread occurrence in environmental waters, at concentrations that could reach up to several μg/L. Previous studies have used time consuming sample preparation methods (offline solid phase extraction/derivatization) or methods with rather high detection limits (direct injection) for sucralose analysis. This study described a faster and sensitive analytical method for the determination of sucralose in environmental samples. Results An online SPE-LC–MS/MS method was developed, being capable to quantify sucralose in 12 minutes using only 10 mL of sample, with method detection limits (MDLs) of 4.5 ng/L, 8.5 ng/L and 45 ng/L for deionized water, drinking and reclaimed waters (1:10 diluted with deionized water), respectively. Sucralose was detected in 82% of the reclaimed water samples at concentrations reaching up to 18 μg/L. The monthly average for a period of one year was 9.1 ± 2.9 μg/L. The calculated mass loads per capita of sucralose discharged through WWTP effluents based on the concentrations detected in wastewaters in the U. S. is 5.0 mg/day/person. As expected, the concentrations observed in drinking water were much lower but still relevant reaching as high as 465 ng/L. In order to evaluate the stability of sucralose, photodegradation experiments were performed in natural waters. Significant photodegradation of sucralose was observed only in freshwater at 254 nm. Minimal degradation (<20%) was observed for all matrices under more natural conditions (350 nm or solar simulator). The only photolysis product of sucralose identified by high resolution mass spectrometry was a de-chlorinated molecule at m/z 362.0535, with molecular formula C12H20Cl2O8. Conclusions Online SPE LC-APCI/MS/MS developed in the study was applied to more than 100 environmental samples. Sucralose was frequently detected (>80%) indicating that the conventional treatment process employed in the sewage treatment plants is not efficient for its removal. Detection of sucralose in drinking waters suggests potential contamination of surface and ground waters sources with anthropogenic wastewater streams. Its high resistance to photodegradation, minimal sorption and high solubility indicate that sucralose could be a good tracer of anthropogenic wastewater intrusion into the environment.

Method development for the analysis of smokeless powders and organic gunshot residue by ultra performance liquid chromatography with tandem mass spectrometry

The goal of this project was to develop a rapid separation and detection method for analyzing organic compounds in smokeless powders and then test its applicability on gunshot residue (GSR) samples. In this project, a total of 20 common smokeless powder additives and their decomposition products were separated by ultra performance liquid chromatography (UPLC) and confirmed by tandem mass spectrometry (MS/MS) using multiple reaction monitoring mode (MRM). Some of the targeted compounds included diphenylamines, centralites, nitrotoluenes, nitroglycerin, and various phthalates. The compounds were ionized in the MS source using simultaneous positive and negative electrospray ionization (ESI) with negative atmospheric pressure chemical ionization (APCI) in order to detect all compounds in a single analysis. The developed UPLC/MS/MS method was applied to commercially available smokeless powders and gunshot residue samples recovered from the hands of shooters, spent cartridges, and smokeless powder retrieved from unfired cartridges. Distinct compositions were identified for smokeless powders from different manufacturers and from separate manufacturing lots. The procedure also produced specific chemical profiles when tested on gunshot residues from different manufacturers. Overall, this thesis represents the development of a rapid and reproducible procedure capable of simultaneously detecting the widest possible range of components present in organic gunshot residue.^

Assessment of the Occurrence and Potential Risks of Antibiotics and their Metabolites in South Florida Waters Using Liquid Chromatography Tandem Mass Spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can’t be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications. Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.