After removal of spots with a signal to noise ratio less than 3,

After removal of spots with a signal to noise ratio less than 3, a total of 24,387 genes were included in the statistical analysis. Effects of treatment were analyzed through contrasts of treatment groups. Contrasts measured tested effect of time for each treatment group, comparisons of each group to control, effect of vaccination, and effect of pathology. Large numbers (>100) of significantly

differentially learn more expressed genes (q value<0.05) were detected in 4 contrasts of interest, all involving the NV–C severe groups. The largest number of differentially expressed genes (1914 genes) was found in the contrast of NV–C severe day 5 vs. NV–C mild day 5. The second largest number of differentially expressed genes (1097 genes) was found in the contrast of NV–C severe group vs. NV–NC control group on day 1. This number was reduced to 506 differentially expressed genes for the same contrast on day 5. The number of differentially expressed Apoptosis inhibitor genes in the contrast of NV–C severe day

1 vs. NV–C severe 5 was 107 genes. Numbers of shared and unique differentially expressed genes between 3 contrasts analyzing differences in treatment group on the same day are displayed in Fig. 2. A total of 417 differentially expressed genes found in the contrast of NV–C severe day 5 vs. NV–NC control day 5 were also differentially expressed in the NV–C severe day 5 vs. NV–C mild day 5 contrast; 280 genes had increased expressed

in the severe group in both contrasts, 137 genes had decreased expressed in the severe group in both contrasts. In all of the shared genes between the contrasts represented in Fig. 2, 99% of genes were expressed in the same direction relative to the severe group; either all more highly expressed in the severe group across all contrasts or more lowly expressed among all contrasts. The differences between internal lesion scores of vaccinated and non-vaccinated, challenged birds were tested through 6-phosphogluconolactonase two-sample t-tests for each day. There was a significant (P value<0.001) reduction in internal lesions among vaccinated, challenged birds compared to those non-vaccinated, challenged birds on both days, however, no differentially expressed genes due to vaccination effect were detected. On day 1, the mean±standard deviation of lesion scores for vaccinated birds was 1.50±1.27 (N=76) and unvaccinated birds 3.06±1.68 (N=87). On day 5, the mean±standard deviation of lesion scores for vaccinated birds was 2.94±2.08 (N=80) and unvaccinated birds 4.28±1.94 (N=85). Vaccination effect on gene expression was tested both through the V–NC group vs. NV–NC contrast on both days and through a contrast utilizing a combination of all treatment groups, non-challenged and challenged, on both days. Samples from individuals with severe pathology or collected 5 day post-infection exhibited more gene induction than repression.

Studies report osteocyte apoptosis following mechanical stimulati

Studies report osteocyte apoptosis following mechanical stimulation in the ulnar physical load model [151] and in the experimental tooth movement model in rats [130] and [152]. In addition, Aguirre et al. [153] demonstrated that osteocyte apoptosis was induced in the murine model of unloading by tail suspension. We previously reported that apoptosis of compressive force-loaded osteocytes showed the

typical histological features of nuclear condensation and fragmentation [130]. Osteocyte apoptosis occurs at sites of microdamage, where the dying osteocyte may send signals to osteoclasts for targeted removal RO4929097 in vivo of bone [154]. It was found that Bax, an proapoptotic gene product, was elevated in osteocytes immediately at the microcrack site, whereas Bcl-2, an anti-apoptotic gene product, was expressed in cells 1–2 mm away from the microcrack [154]. This suggests that damaged osteocytes send signals for bone resorption, whereas osteocytes not engaged in apoptosis are prevented from

doing so by active protective mechanisms [154]. We found significant increases in apoptosis-related gene expression, including caspase-3, -8, -9 and Bcl-2 in osteocytes during compressive force loading, indicating that both death-receptor and mitochondrial pathways were activated in osteocytes under compressive force loading [155]. In addition, others have reported that osteocyte apoptotic bodies can initiate osteoclastogenesis [156]. It is still unclear whether signals of resorption sent by dying osteocytes are the same as those sent by living osteocytes. Further investigation is necessary to elucidate the mechanism of the initiation of bone AZD5363 nmr resorption controlled by osteocytes under compressive force loading. CCN proteins induce apoptosis in some cell types

[119] and [157]. Juric et al. [157] reported that CCN1 and CCN2 synergize with FAS ligand, a TNF family member, to induce human skin fibroblast apoptosis. In human breast cancer cells [119] and aortic smooth-muscle cells [158], CTGF/CCN2 induces apoptosis by decreasing Bcl-2 protein expression and the activation of caspase 3, respectively. In contrast, CTGF/CCN2 inhibits apoptosis in human rhabdomyosarcoma cells [159]. However, in osteocytes, it was previously unclear whether CCN2 induced osteocyte apoptosis. Cell press We showed that in the mouse experimental tooth movement model, CCN2 mRNA expression in osteocytes increased on the pressured side of alveolar bone, which induced osteocyte apoptosis and osteoclastic bone resorption [130]. Furthermore, recently we demonstrated that recombinant human (rh)CCN2 induced osteocyte apoptosis in primary chick osteocytes in culture [155]. Thus, we speculate that the enhanced expression of CCN2 after compressive force loading could be related to osteocyte apoptosis, which might be an important phenomenon in the dynamic responses of these cells to mechanical stimuli to induce bone resorption.

As shown in the Table 2, V-Primer (VTD) was introduced for the

As shown in the Table 2, V-Primer (VTD) was introduced for the

first time as a thione-based primer. In addition, Metaltite (MTU-6) and Alloy Primer (VTD and MDP) are currently used for bonding cast restorations and FPDs. After try-in procedure, the surface to be bonded of resin-bonded FPDs and restorations was air-borne particle abraded with alumina (50–70 μm grain-seized), and then primed with one of the sulfur-based primers. Minimally buy AT13387 reduced enamel was etched with 37–40% phosphoric acid, rinsed with water, and air-dried. More than ten clinical reports have been published concerning bonding of Ag–Pd–Cu–Au alloy castings. Except for one case, the castings were bonded with the 4-META/MMA-TBB resins (Super-Bond or C&B Metabond). After observation periods of 3 to 11 years, they are functioning

satisfactorily [18], [19], [20], [21], [22], [23], [25], [27], [28], [29] and [31]. Figure 2, Figure 3 and Figure 4 show clinical course of a resin-bonded FPD originally reported by Monya et al. [18]. The FPD is functioning for more than 10 years. The noble metal bonding systems were also applied to seating splinting devices to enamel structure. Similar technique can be applied to fixing Stem Cell Compound Library mobile teeth using cast retainers [32], [33] and [34]. Overcasting technique is a unique approach to partially preserve existing long-spanned FPDs. Preparation is applied to either restoration or pontic, in which veneering porcelain or composite material has been broken. Indirect restoration for repairing is laboratory fabricated. After try-in, both the intra- and extra-oral metal surfaces to be bonded are air-borne-particle abraded with different type abraders, and primed with sulfur-based agent. The surfaces are bonded with TBB-based adhesive. Clinical course of overcastting technique is excellent Loperamide [23], [35] and [36]. Hikage et al. evaluated clinical longevity of resin-bonded FPDs seated with the V-Primer and the Super-Bond adhesive [37]. They reported that ten prostheses had functioned

satisfactorily for 8–11 years, although six of the 26 FPDs had become detached. Tanoue et al. [38] assessed clinical performance of resin-bonded FPDs. They reported that five retainers of the 81 resin-bonded FPDs were failed. Also, they reported that the observation duration and corresponding survival ratio for complete survival were 165 months and 43.9%, and those for functional survival were 178 months and 87.7%, respectively. This work was supported in part by Grants-in-Aid for Scientific Research (C 20592302 and C 21592474) from the Japan Society for the Promotion of Science (JSPS), Grant from Dental Research Center, Nihon University School of Dentistry (A 2009), and Sato Fund, Nihon University School of Dentistry (2009).

, 2009) and antioxidant activity of kaempferol (Park, Rho, Kim, &

, 2009) and antioxidant activity of kaempferol (Park, Rho, Kim, & Chang, 2006), besides improving the bioavailability of hesperidin (Nielsen et al., 2006) and of flavonoid glycosides in fruit juices and green tea (Gonzáles-Barrio et al., 2004). The use of enzymes to modify the structure and improve the physicochemical and biological properties of flavonoids has been of great scientific and industrial

interest due to their wide availability, high selectivity, low cost and their promotion of efficient reactions with few by-products. α-l-Rhamnosidases [E. C.] are glycosyl hydrolases which cleave terminal α-l-rhamnose from natural glycosides. Only two commercial preparations of α-l-rhamnosidases (naringinase and hesperidinase) are available, and both are SCH 900776 chemical structure from fungal sources. Hesperidinase is obtained from species of Penicillium and Aspergillus Y-27632 mouse niger and naringinase is obtained from Penicillium decumbens. All these preparations

also show significant β-d-glucosidase activity that catalyzes the hydrolysis of terminal non-reducing residues with glucose release ( Yadav, Yadav, & Yadav, 2010). Thus the activity of the α-l-rhamnosidases in the rutin substrate produces two derivatives: quercetin-3-glucoside (isoquercetrin) and quercetin, in proportions that depend on the reaction conditions. Although there is a structural similarity to rutin, quercetin-3-glucoside and quercetin, there are some noticeable differences in physical, chemical

and biological properties. Quercetin glycosides show higher solubility in water than quercetin due to the hydrophilicity of the sugar moieties (Aherne & ÓBrien, 2002). In comparison with rutin and quercetin, quercetin-3-glucoside is better absorbed, suggesting that conjugation with glucose enhances quercetin absorption in small intestine (Arts, Sesink, Faassen-Peters, & Hollman, 2004). Indeed, previous reports have shown that quercetin-3-glucoside has a more potent antiproliferative effect than quercetin Amoxicillin or rutin (You, Ahn, & Ji, 2010). Thus, the synthesis of mono-glycosylated quercetin from rutin by the enzymatic hydrolysis method seems to be a good alternative for obtaining compounds with enhanced functional properties. Hesperidinase or naringinase with inactivated β-d-glucosidase activity and expressing α-l-rhamnosidase activity allow the production of very expensive flavonoid glycosides, quercetin-3-glucoside, in an easy and cheap bioprocess starting from rutin. In the present work, the enzymatic hydrolysis of rutin by two commercial heat-treated glycosyl hydrolases (hesperidinase and naringinase) was investigated in order to obtain partially hydrolyzed rutin with enhanced functional properties.

, 1996) Accumulated thermal time is measured in day-degrees

, 1996). Accumulated thermal time is measured in day-degrees

(DD). It is calculated UMI-77 purchase by adding the values for daily mean temperature. This concept is widely used in horticultural crop production to predict harvest dates and decide when to sow and plant. Based on previous experiments (data not shown), we set a target value of 400 DD (starting on the day of transfer into growth chamber) to obtain marketable lettuce heads of 200–250 g at the end of this experiment. Most crops have a “base temperature” below which no growth occurs. Based on previous experiments, we assumed a base temperature of 2 °C which is subtracted from the daily mean temperature in the calculations. The warm treatment reached the set day-degrees 26 days after planting (406 DD), the cool treatment 52 days after planting (395 DD). Some plants were exchanged after they reached half of the day-degrees (203 and 198 DD, after 13 and 26 days in the warm and cool treatment, respectively) and harvested after 39 days. On day 13 and 26 after planting, some Selleck NLG919 plants were harvested from the warm and the cool treatment. Thus, at the end we had information about lettuce plants from the following six conditions and stages: small

heads grown warm or cool (ca. 200 DD), as well as mature heads cultivated warm, cool, first cool then warm and first warm then cool (ca. 400 DD; see harvest schedule, Fig. 1). For all samples, only above ground organs (lettuce heads) were harvested. At all harvest dates, three heads per cultivar, O-methylated flavonoid treatment, and replicate were weighed to obtain the mean head mass. Values for head mass are given in gram fresh matter (FM). To obtain dry matter content, weight before and after lyophilization was compared. Values for dry matter content are given as milligram dry matter per gram fresh matter. A mixed sample from six heads was prepared for each cultivar, treatment, and replicate only limp or deteriorated outer leaves were removed. Within 30 min after harvesting, the

plants were cut in smaller pieces, mixed and frozen at -20 °C until lyophilized (Christ Beta 1-16, Osterode, Germany) and ground with an ultracentrifuge mill (hole size: 0.25 mm; ZM 200, Retsch, Haan, Germany). The well-established HPLC-DAD-ESI-MS method for the determination of flavonol glycosides and phenolic acids in kale, reported by (Neugart et al., 2012) was optimized for lettuce. Best results were obtained by extracting 0.5 g of lyophilized, pulverized lettuce powder with 25 ml of aqueous methanol (50% MeOH) at room temperature. The suspension was kept in motion with a magnetic stirrer for 1.5 h and then centrifuged (Labofuge 400R, Heraeus Instruments, Thermo Fisher Scientific, Waltham, USA) for 15 min at 4500 rcf (relative centrifugal force). The supernatant was filtered with PTFE-syringe filters (0.25 μm, polytetrafluoroethylene; Roth, Karlsruhe, Germany) transferred to a glass vial and analyzed via HPLC-DAD-ESI-MS3.

001), although an associated discrete decrease in the number of b

001), although an associated discrete decrease in the number of bone marrow cells (P = 0.076) KRX-0401 ic50 and a significant reduction in

the total number of spleen cells (P = 0.017) compared to the CON group ( Table 2) were also observed, indicating that the bone marrow maintained its capacity to restore the erythroid pool. There are no differences in the chemical component (moisture, protein, ether extract and total ash) levels among the diets. However, there are differences in dietary Fe concentrations, with FS diet showing the lowest value (53.0, 60.4, 64.7 and 61.7 for FS, FP, YF and RAF diets, respectively). No significant differences were observed in body weight among the groups at days 0, 7 and 14 of the repletion period. Moreover, total food intake did not vary among the groups after 7 (105.2 g for FS group)

and 14 days (222.6 g for FS group). However, on day 7, the FP rats (FP, YF and RAF groups) showed a higher Fe intake than those in the FS group (P = 0.03), whereas on day 14 the Fe intake was similar between the groups. In general, no statistically C59 wnt significant differences were observed in the haematological parameters among the experimental groups at the end of the repletion period, except for the higher reticulocyte count observed in the FP group when compared to the other groups (3.6% in FP group; P = 0.010). Haemoglobin values, Hb Fe pool, HRE and RBV during the 14-day repletion period are shown in Fig. 1. Considering that the groups presented similar Hb concentrations on day 0 (P = 0.347), the mean Hb concentration

in YF group increased by 18% (P < 0.001) and 7% in comparison to the RAF group and 40% and 24% (P < 0.001) in comparison to the FP group on days 7 and 14 of the repletion period, respectively ( Fig. 1-A). These data reinforce the reticulocyte count results in the FP group which, after the repletion period, still remained above the values of the control group. The efficiency of Hb recovery reflects the ratio of dietary Fe conversion into Hb to the amount of ingested Fe over the course of the repletion period. In the present study, only YF animals showed higher Tolmetin HRE values compared to those in the FP group and similar HRE values compared to FS group, on day 7 of the repletion period (P < 0.001; Fig. 1C). The Hb concentration observed at the end of the repletion period in the FS animals was considered the reference. Hence, the FS group was taken as a reference for expressing the bioavailability of Fe from FP (FS is assigned the value 100%) (RBV). The RBVs of the FP in the YF-supplemented group were 84% and 97% on days 7 and 14 of the repletion period, respectively. These values were significantly higher than those of FP and RAF groups on day 7 (P < 0.001; Fig. 1D). Moreover, at this time, no significant difference was observed between the RAF and FP groups in terms of RBV.

Nevertheless we can rely on the

Nevertheless we can rely on the Z-VAD-FMK lowest observable adverse effect level (WHO, 1987a and WHO, 2000a) in either short-term or annual average exposures as an alternative to dependency on longer term outcome measures. This approach also emphasizes the necessity of deriving an explicit

annual guideline for the other criteria pollutants, SO2 and O3, to support the evaluation of health effects from long-term exposure. Based on the present study, the estimated annual limits for SO2 and O3 can be regarded as hypotheses pending for confirmation from long-term studies. Nevertheless, our present estimations of 4.6 μg/m3 and 27.0 μg/m3 were supported by epidemiologic evidence. For examples attributable to SO2, the daily mortality in Finland was shown with an annual average concentration of 7 μg/m3 (Penttinen et al., 2004) and the reduced fetal growth in Australia with annual average concentration of 3 μg/m3 (Hansen et al., 2008). For example attributable to O3, the asthma hospital admissions in Finland were demonstrated LY294002 in vivo with an annual average concentration

of 22 μg/m3 (Ponka and Virtanen, 1996). Ensuring a valid relationship between the short-term and annual AQG will enhance the immediate and regular periodic evaluation of any new pollution control policy because efforts in ensuring compliance, with the short-term AQG being monitored in real-time, can be used to predict in advance whether the annual AQG will be achieved, providing either prompt warnings of the need for remedial measures or assurance for effective policies with improved public health protection and accountability. On the other hand, provision of an estimated annual limits based on the short-term AQG will allow consistent

comparative health impact assessment and economic evaluation for new policy implementations. The inclusion of more cities could improve the generalizability of findings. However our study provides a cross-sectional view of air quality over Farnesyltransferase seven years from 2004 to 2010, and the key parameters, dμ and dm, which we assumed to be constant in the model were very stable regardless of the variation in the annual mean in different years. While our findings require further support and no similar studies or alternative approaches have been conducted, indirect evidence of the discordance of the current NO2 guideline limits was also revealed in the EU in that 90% of the monitoring stations had violated the directive for the NO2 annual limit of 40 μg/m3 but complied with the one-hour limit of 200 μg/m3 ( ETCACC, 2010). This observation also indicates that the short-term limit should be lowered in order to be an effective measure to provide early indication that the annual limit will be violated.

The retained aspen might also be easier to colonize by


The retained aspen might also be easier to colonize by

wind dispersed spores simply because the surrounding forest that previously could have been a physical obstacle to dispersal now is removed. In the ISA we found 36 species to be characteristic of young forest. From information on lichen species ecology in the literature and our own field experience, we propose that Caloplaca cerina, C. flavorubescens, C. holocarpa, C. jemtlandica, Leptogium saturninum, Melanelia exasperata, Phaeophyscia ciliata, Physcia aipolia, P. tenella, Rinodina septentrionalis and Xanthoria parietina, all aspen specialists, were present in the old forest, but then higher up or in the crown, and had after logging migrated downwards. On SCH 900776 mw the other hand, the generalist species Bryoria spp., Catinaria atropurpurea, Cladonia coniocraea, C. fimbriata, Hypogymnia physodes, Lecanora expallens, L. circumborealis, L. pulicaris, PI3K inhibitor L. symmicta, Lecidea nylanderi, Ochrolechia androgyna, Parmeliopsis ambigua, P. hyperopta, Tuckermanopsis chlorophylla, Usnea

spp. and Vulpicida pinastri are probably new on the aspens, but could have existed in the forest on other tree species. Lecidea albofuscesens was the only species that indicated clearcuts, i.e. old forest. This species is known to grow in shady and moist environments (F. Jonsson, personal observation), and is evidently sensitive to the exposure after logging ( see Appendix for more details). We found 195 species, which was more than twice as much as we expected based on earlier reports. Another published study on the complete lichen flora (foliose, fruticose and crustose species) on aspens

trees was performed by Ellis and Coppins (2007) who found 273 species in 93 aspen stands in Scotland. Although the importance of aspen for lichen biodiversity has been emphasized earlier (e.g. Kuusinen, 1996 and Kouki et al., Amoxicillin 2004), our study clearly demonstrates the great diversity of the lichen community connected to aspen in boreal Northern Europe. Despite the high number of recorded species (85% of the regional species pool), new species should have been added successively with increasing sampling effort, and among these very likely a number of rare, red-listed ones. On the Red List for the counties of Västernorrland and Jämtland, where the study was conducted, there are 30 lichen species that could grow on aspen (Gärdenfors, 2010), i.e. 19 more than we found in our study. Aspens retained at harvest have a great potential to enrich future forest landscapes and to contribute to the persistence of biodiversity connected to this habitat. We found red-listed lichen species also in the young forest, and the total species richness was even higher on the aspens exposed for 10–16 years. This indicates that opening up of stands with aspens could be a positive conservation management action, for instance in connection with thinnings, but also in protected forests.

The natural distribution of P radiata is limited to a handful of

The natural distribution of P. radiata is limited to a handful of remaining

populations in Mexico and the USA where it has no role in commercial forestry ( Rogers, 2004). The species was introduced into Australia in the 1850s for ornamental plantings and R&D work started there one hundred years later, resulting in significantly improved germplasm ( Wu et al., 2007). Today, P. radiata is widely planted in diverse countries including Chile and New Zealand, in addition to Australia ( Rogers, 2004). Germplasm transfer of currently widely-used tropical and subtropical plantation trees such as Acacia, Eucalyptus Bcl-2 inhibitor and Pinus spp. started soon after their native ranges were colonised by Europeans ( Bennett, 2011). The development Erastin cost of their historical transfer patterns is similar to that of the temperate and boreal species:

large-scale tree planting efforts first created demand for germplasm transfer for production purposes and, later, germplasm was also transferred increasingly for R&D. By the 19th century, collection and export of Acacia and Eucalyptus spp. seed from Australia was well organized. During the same century, eucalypts, including E. camaldulensis, E. globulus and E. tereticornis, were widely planted throughout the temperate and Mediterranean-like climatic regions of the world ( FAO, 1979 and Freeman et al., 2007). Acacias such as A. saligna, A. cyclops and A. longifolia were similarly exported to southern Africa ( Carruthers et al., 2011). Exploration, collection and assessment of these species

and the transfer of their germplasm for production purposes were intensified in the 20th century, and more systematic R&D work was initiated around 50 years ago. Eucalyptus camaldulensis and E. globulus, for example, have been introduced from Australia to 91 and 37 countries, respectively ( Table 1). Of the more than 600 Eucalyptus species, just nine cover 90% of the planted eucalypt area globally: E. camaldulensis, E. dunnii, E. grandis, E. globulus, E. nitens, E. pellita, E. saligna, E. tereticornis and PRKACG E. urophylla ( Harwood, 2011). Of the 1,012 Australian Acacia species, it is estimated that 386 have been introduced by humans outside Australia ( Richardson et al., 2011), though R&D efforts in the last decades have largely focused on just a few tropical species, most notably A. mangium and A. crassicarpa. Today, A. mangium is estimated to be planted in 25 countries outside its native range ( Table 1). In addition to Acacia and Eucalyptus species, the germplasm of several fast-growing pines, predominantly from Central America, Mexico and the southern Unites States, has been transferred for establishing plantations throughout the tropics and subtropics. In Mexico, one of the first collections of Pinus patula seed was carried out in the early 20th century and the material was transferred to South Africa for establishing the first pine plantations in the country ( Butterfield, 1990).

This approach allows one system to be used for both casework and

This approach allows one system to be used for both casework and database activities. Figure options Download full-size image Download high-quality image (256 K) Download as PowerPoint slide Developmental validation was performed to demonstrate the quality BGB324 mw and robustness of the PowerPlex® Fusion System across a number of variables. SWGDAM, 2004 guidelines [5] were followed, although the results also meet the 2012 guidelines [6]. Consistent and high-quality results were compiled using data from 12 separate forensic and research laboratories including the Michigan State Police

casework and CODIS units, US Army Criminal Investigation Laboratory casework and database units, Arkansas State Crime Laboratory, Kansas Bureau of Investigation, Texas Department of Public Safety, Kentucky State Police, selleck inhibitor New Hampshire State Police, Washington State Patrol Crime Laboratory Division, Los Angeles County Sheriff’s Department, Oklahoma State Bureau of Investigation, National Institute of Standards and Technology (NIST), and Promega Corporation. Studies evaluated robustness and performance with case-type samples, sensitivity samples, inhibitors, mixtures, and several PCR conditions. The conclusions reported here support the fact that the PowerPlex®

Fusion System is suitable for human identification in casework and database applications. Extracted DNA and solid support materials including FTA® card punches (GE Healthcare), cotton swabs, and nonFTA Bode Buccal DNA Collectors™ (Bode Technology Group) were evaluated. Each laboratory examined its own extracted DNA samples for the majority of the studies.

However, the sensitivity, mixture, and reproducibility DNA samples were prepared by a single site and distributed. DNA was purified by organic extraction for all studies except case-type samples, which were extracted using EZ1® reagents and instrumentation Avelestat (AZD9668) (Qiagen) in addition to organic extraction. Unless otherwise noted, 500 pg of extracted DNA was amplified. Laboratories evaluated buccal Indicating FTA® cards or Bode Buccal DNA Collectors™ from their own collections for all studies outside of the reproducibility studies. For the reproducibility studies, single donors collected a series of cotton swabs, Indicating FTA® cards, or Bode Buccal Collectors™ for distribution to multiple sites. Single 1.2 mm punches from buccal Indicating FTA® cards were added directly to reactions. Swabs and nonFTA punches required a pretreatment step prior to addition into amplification reactions. Buccal cotton swab samples were incubated in SwabSolution™ Reagent (Promega Corporation) as directed in the technical manual [7], and 2 μl of the resulting lysate was added as the template to amplification reactions. Single 1.