Jodi Lynn Barta

Clovis Age Western Stemmed Projectile Points and Human Coprolites at the Paisley Caves

They Walked Together Paisley Cave in Oregon provides some of the earliest evidence for humans in North America. Jenkins et al. (p. 223) provide a wide variety of additional evidence of early human occupation of this site, including a series of radiocarbon ages extending back to nearly 12,500 radiocarbon years ago (about 14,500 calendar years ago). The find includes examples of projectile points representative of the Western Stemmed Tradition dating to about 11,100 radiocarbon years ago. The Western Stemmed Tradition has been thought to have evolved after the dominant Clovis technology, but the find suggests that the two cultures overlapped in time. The age of a Western Stemmed projectile point implies that this culture overlapped with the Clovis culture in North America. The Paisley Caves in Oregon record the oldest directly dated human remains (DNA) in the Western Hemisphere. More than 100 high-precision radiocarbon dates show that deposits containing artifacts and coprolites ranging in age from 12,450 to 2295 14C years ago are well stratified. Western Stemmed projectile points were recovered in deposits dated to 11,070 to 11,340 14C years ago, a time contemporaneous with or preceding the Clovis technology. There is no evidence of diagnostic Clovis technology at the site. These two distinct technologies were parallel developments, not the product of a unilinear technological evolution. “Blind testing” analysis of coprolites by an independent laboratory confirms the presence of human DNA in specimens of pre-Clovis age. The colonization of the Americas involved multiple technologically divergent, and possibly genetically divergent, founding groups.

Low wintertime vitamin D levels in a sample of healthy young adults of diverse ancestry living in the Toronto area: associations with vitamin D intake and skin pigmentation

BackgroundVitamin D plays a critical role in bone metabolism and many cellular and immunological processes. Recent research indicates that concentrations of serum 25-hydroxyvitamin D [25(OH)D], the main indicator of vitamin D status, should be in excess of 75 nmol/L. Low levels of 25(OH)D have been associated with several chronic and infectious diseases. Previous studies have reported that many otherwise healthy adults of European ancestry living in Canada have low vitamin D concentrations during the wintertime. However, those of non-European ancestry are at a higher risk of having low vitamin D levels. The main goal of this study was to examine the vitamin D status and vitamin D intake of young Canadian adults of diverse ancestry during the winter months.MethodsOne hundred and seven (107) healthy young adults self-reporting their ancestry were recruited for this study. Each participant was tested for serum 25(OH)D concentrations and related biochemistry, skin pigmentation indices and basic anthropometric measures. A seven-day food diary was used to assess their vitamin D intake. An ANOVA was used to test for significant differences in the variables among groups of different ancestry. Linear regression was employed to assess the impact of relevant variables on serum 25(OH)D concentrations.ResultsMore than 93% of the total sample had concentrations below 75 nmol/L. Almost three-quarters of the subjects had concentrations below 50 nmol/L. There were significant differences in serum 25(OH)D levels (p < 0.001) and vitamin D intake (p = 0.034) between population groups. Only the European group had a mean vitamin D intake exceeding the current Recommended Adequate Intake (RAI = 200 IU/day). Total vitamin D intake (from diet and supplements) was significantly associated with 25(OH)D levels (p < 0.001). Skin pigmentation, assessed by measuring skin melanin content, showed an inverse relationship with serum 25(OH)D (p = 0.033).ConclusionWe observe that low vitamin D levels are more prevalent in our sample of young healthy adults than previously reported, particularly amongst those of non-European ancestry. Major factors influencing 25(OH)D levels were vitamin D intake and skin pigmentation. These data suggest a need to increase vitamin D intake either through improved fortification and/or supplementation.

Serum 25-Hydroxyvitamin D Concentrations Fluctuate Seasonally in Young Adults of Diverse Ancestry Living in Toronto

Previous research indicates that circulating vitamin D levels are low in many otherwise healthy adults and that there is considerable seasonal variation in 25-hydroxyvitamin D [25(OH)D] concentrations at high latitudes. We examined seasonal variation in 25(OH)D levels in a sample of young adults of diverse ancestry living in the Greater Toronto Area. Three hundred and fifty-one (351) healthy young adults completed both a fall and winter visit during this study. The study was conducted over 2 y (y 1: fall 2007 to winter 2008 and y 2: fall 2008 to winter 2009). At both visits, each participants serum 25(OH)D concentration was measured. Information was also obtained on skin pigmentation (measured via reflectometer), vitamin D intake, and extent of sun exposure. Overall, the serum 25(OH)D concentration was 54.4 ± 1.3 nmol/L in the fall and 38.4 ± 1.1 nmol/L in the winter. Concentrations differed among ancestral groups at both visits (P < 0.001), with South Asians and East Asians having substantially lower concentrations than Europeans. Skin pigmentation (r(2) = 0.14; P < 0.001), supplemental vitamin D intake (r(2) = 0.09; P < 0.001), sun exposure (r(2) = 0.04; P < 0.001), and study year (r(2) = 0.02; P = 0.017) were predictors of fall 25(OH)D concentrations. During the wintertime, serum 25(OH)D concentrations were associated with concentrations taken in the fall (r(2) = 0.45; P < 0.001), supplemental (r(2) = 0.15; P < 0.001) and dietary vitamin D intake (r(2) = 0.06; P < 0.001), and with study year (r(2) = 0.02; P = 0.009). Our study confirms that serum 25(OH)D concentrations undergo strong seasonal variation at high latitudes and are influenced by vitamin D intake, skin pigmentation, and sun exposure.

The development and evaluation of a food frequency questionnaire used in assessing vitamin D intake in a sample of healthy young Canadian adults of diverse ancestry

Little data exist on vitamin D deficiency related with intake, especially for the Canadian population. The purpose of this study was to develop and evaluate a food frequency questionnaire (FFQ) with 37 items for rapid assessment of vitamin D intake in healthy young adults of diverse ancestry. We recruited 107 subjects in Southern Ontario during the late winter of 2007 who completed an FFQ twice (FFQ-1 and FFQ-2, repeated for reproducibility assessment) and a 7-day food diary (for validation). Serum 25-hydroxyvitamin D (25(OH)D), the major biomarker of vitamin D nutritional status, and skin melanin were determined. The FFQ results were highly correlated with 7-day diary results and with serum 25(OH)D concentrations (r = 0.529, P < .001; r = 0.481, P < .001, respectively). Modifications to the FFQ, by redefining the large serving size and excluding the fortified orange juice category, improved the validity of the FFQ (r = 0.602, P < .001; r = 0.520, P < .001, respectively). The FFQ results were highly correlated (r = 0.663, P < .001), but the mean intakes were different (P < .05). Using results from a modified version of FFQ-1, we examined dietary intakes in 3 predominant groups: East Asian (n = 27), European (n = 31), and South Asian (n = 32). The European group had higher total vitamin D intake (P < .05) and the highest serum 25(OH)D concentrations (P < .05), with a trend for dairy products being responsible for this (P < .10). Because Canadians are reliant on dietary intakes of vitamin D in the wintertime, especially those with higher skin melanin, our FFQ can monitor and provide information on intake and food group consumption.

To clone or not to clone: Method analysis for retrieving consensus sequences in ancient DNA samples

The challenges associated with the retrieval and authentication of ancient DNA (aDNA) evidence are principally due to post-mortem damage which makes ancient samples particularly prone to contamination from “modern” DNA sources. The necessity for authentication of results has led many aDNA researchers to adopt methods considered to be “gold standards” in the field, including cloning aDNA amplicons as opposed to directly sequencing them. However, no standardized protocol has emerged regarding the necessary number of clones to sequence, how a consensus sequence is most appropriately derived, or how results should be reported in the literature. In addition, there has been no systematic demonstration of the degree to which direct sequences are affected by damage or whether direct sequencing would provide disparate results from a consensus of clones. To address this issue, a comparative study was designed to examine both cloned and direct sequences amplified from ∼3,500 year-old ancient northern fur seal DNA extracts. Majority rules and the Consensus Confidence Program were used to generate consensus sequences for each individual from the cloned sequences, which exhibited damage at 31 of 139 base pairs across all clones. In no instance did the consensus of clones differ from the direct sequence. This study demonstrates that, when appropriate, cloning need not be the default method, but instead, should be used as a measure of authentication on a case-by-case basis, especially when this practice adds time and cost to studies where it may be superfluous.

Further evaluation of the efficacy of contamination removal from bone surfaces

Studies of low copy number (LCN) and degraded DNA are prone to contamination from exogenous DNA sources that in some cases out-compete endogenous DNA in PCR amplification, thus leading to false positives and/or aberrant results. Particularly problematic is contamination that is inadvertently deposited on the surfaces of bones through direct handling. Whereas some previous studies have shown that contamination removal is possible by subjecting samples to sodium hypochlorite prior to DNA extraction, others caution that such treatment can destroy a majority of the molecules endogenous to the sample. To further explore this topic, we experimentally contaminated ancient northern fur seal (Callorhinus ursinus) ribs with human DNA and treated them with sodium hypochlorite to remove that contamination. Our findings are consistent with previous studies that found sodium hypochlorite to be highly efficient (~81-99%) at contamination removal; however, there emerged no treatment capable of removing 100% of the contamination across all of the experiments. Moreover, the ability to estimate the degree of damage to endogenous northern fur seal molecules was compromised due to the inherent variability of preserved mtDNA across the bones, and the presence of co-extracted PCR inhibitors.

How Much DNA is Lost? Measuring DNA Loss of Short-Tandem-Repeat Length Fragments Targeted by the PowerPlex 16® System Using the Qiagen MinElute Purification Kit

ABSTRACT The success in recovering genetic profiles from aged and degraded biological samples is diminished by fundamental aspects of DNA extraction, as well as its long-term preservation, that are not well understood. While numerous studies have been conducted to determine whether one extraction method was superior to others, nearly all of them were initiated with no knowledge of the actual starting DNA quantity in the samples prior to extraction, so they ultimately compared the outcome of all methods relative to the best. Using quantitative PCR to estimate the copy count of synthetic standards before (i.e., “copies in”) and after (i.e., “copies out”) purification by the Qiagen MinElute PCR Purification Kit, we documented DNA loss within a pool of 16 different-sized fragments ranging from 106 to 409 bp in length, corresponding to those targeted by the PowerPlex 16 System (Promega, Madison, WI). Across all standards from 104 to 107 copies/&mgr;L, loss averaged between 21.75% and 60.56% (mean, 39.03%), which is not congruent with Qiagens claim that 80% of 70 bp to 4 kb fragments are retained using this product (i.e., 20% loss). Our study also found no clear relationship either between DNA strand length and retention or between starting copy number and retention. This suggests that there is no molecule bias across the MinElute column membrane and highlights the need for manufacturers to clearly and accurately describe on what their claims are based, and should also encourage researchers to document DNA retention efficiencies of their own methods and protocols. Understanding how and where to reduce loss of molecules during extraction and purification will serve to generate clearer and more accurate data, which will enhance the utility of ancient and low-copy-number DNA as a tool for closing forensic cases or in reconstructing the evolutionary history of humans and other organisms.